NZ758109B2 - Methods of treatment using a jak inhibitor compound - Google Patents
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- NZ758109B2 NZ758109B2 NZ758109A NZ75810918A NZ758109B2 NZ 758109 B2 NZ758109 B2 NZ 758109B2 NZ 758109 A NZ758109 A NZ 758109A NZ 75810918 A NZ75810918 A NZ 75810918A NZ 758109 B2 NZ758109 B2 NZ 758109B2
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Abstract
The invention relates to methods of treating ocular diseases and certain respiratory diseases using the compound 5-ethyl-2-fluoro-4-(3-(5-(1-methylpiperidin-4-yl)-4,5,6,7- tetrahydro-1H-imidazo[4,5-c]pyridin-2-yl)-1H-indazol-6-yl)phenol or a pharmaceutically-acceptable salt thereof.
Description
METHODS OF TREATMENT USING A JAK INHIBITOR COMPOUND
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is generally directed to methods for treating ocular and certain
respiratory diseases using a particular JAK inhibitor compound or a pharmaceutically-
acceptable salt thereof.
State of the Art
Cytokines are intercellular signaling molecules which include chemokines,
interferons, interleukins, lymphokines, and tumour necrosis factor. Cytokines are critical for
normal cell growth and immunoregulation but also drive immune-mediated diseases and
contribute to the growth of malignant cells. Elevated levels of many cytokines have been
implicated in the pathology of a large number of diseases or conditions, particularly those
diseases characterized by inflammation. Many of the cytokines implicated in disease act
through signaling pathways dependent upon the Janus family of tyrosine kinases (JAKs),
which signal through the Signal Transducer and Activator of Transcription (STAT) family of
transcription factors.
The JAK family comprises four members, JAK1, JAK2, JAK3, and tyrosine kinase 2
(TYK2). Binding of cytokine to a JAK-dependent cytokine receptor induces receptor
dimerization which results in phosphorylation of tyrosine residues on the JAK kinase,
effecting JAK activation. Phosphorylated JAKs, in turn, bind and phosphorylate various
STAT proteins which dimerize, internalize in the cell nucleus and directly modulate gene
transcription, leading, among other effects, to the downstream effects associated with
inflammatory disease. The JAKs usually associate with cytokine receptors in pairs as
homodimers or heterodimers. Specific cytokines are associated with specific JAK pairings.
Each of the four members of the JAK family is implicated in the signaling of at least one of
the cytokines associated with inflammation.
Inflammation plays a prominent role in many ocular diseases, including uveitis,
diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular
degeneration, and atopic keratoconjunctivitis. Uveitis encompasses multiple intraocular
inflammatory conditions and is often autoimmune, arising without a known infectious
trigger. The condition is estimated to affect about 2 million patients in the US. In some
patients, the chronic inflammation associated with uveitis leads to tissue destruction, and it is
the fifth leading cause of blindness in the US. Cytokines elevated in uveitis patients' eyes that
signal through the JAK-STAT pathway include IL-2, IL-4, IL-5, IL-6, IL-10, IL-23, and
IFN-γ. (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744; Ooi et al, Clinical
Medicine and Research, 2006, 4, 294-309). Existing therapies for uveitis are often
suboptimal, and many patients are poorly controlled. Steroids, while often effective, are
associated with cataracts and increased intraocular pressure/glaucoma.
Diabetic retinopathy (DR) is caused by damage to the blood vessels in the retina. It is
the most common cause of vision loss among people with diabetes. Angiogenic as well as
inflammatory pathways play an important role in the disease. Often, DR will progress to
diabetic macular edema (DME), the most frequent cause of visual loss in patients with
diabetes. The condition is estimated to affect about 1.5 million patients in the US alone, of
whom about 20 % have disease affecting both eyes. Cytokines which signal through the
JAK-STAT pathway, such as IL-6, as well as other cytokines, such as IP-10 and MCP-1
(alternatively termed CCL2), whose production is driven in part by JAK-STAT pathway
signaling, are believed to play a role in the inflammation associated with DR/DME
(Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12; Sohn et al., American Journal of
Opthalmology, 2011, 152, 686-694; Owen and Hartnett, Curr Diab Rep, 2013, 13, 476-480;
Cheung et al, Molecular Vision, 2012, 18, 830-837; Dong et al, Molecular Vision, 2013, 19,
1734-1746; Funatsu et al, Ophthalmology, 2009, 116, 73-79). The existing therapies for
DME are suboptimal: intravitreal anti-VEGF treatments are only effective in a fraction of
patients and steroids are associated with cataracts and increased intraocular pressure.
Dry eye disease (DED) is a multifactorial disorder that affects approximately 5
million patients in the US. Ocular surface inflammation is believed to play an important role
in the development and propagation of this disease. Elevated levels of cytokines such as IL-1,
IL-2, IL-4, IL-5, IL-6, and IFN-γ have been noted in the ocular fluids of patients with DED.
(Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and the levels often correlated with
disease severity. Age-related macular degeneration and atopic keratoconjunctivitis are also
thought to be associated with JAK-dependent cytokines.
Given the number of cytokines elevated in inflammatory diseases and that each
cytokine is associated with a particular JAK pairing, it would be desirable to provide a
chemical inhibitor with pan-activity against all members of the JAK family for the treatment
of ocular disease. However, the broad anti-inflammatory effect of such inhibitors could
suppress normal immune cell function, potentially leading to increased risk of infection. It
would be desirable, therefore, to provide an inhibitor that can be locally delivered to the site
of action in the eye, thereby limiting the potential for adverse systemic immunosuppression.
It is an object of this invention to go at least some way to meeting this need; and/or to at least
provide the public with a useful choice.
Commonly assigned U.S. Application Serial No. 15/341,226, filed November 02,
2016 discloses diamino compounds useful as JAK inhibitors. In particular, the compound
5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-
c]pyridinyl)-1H-indazolyl)phenol (compound 1)
is specifically disclosed in the application as a potent pan-JAK inhibitor. This application
discloses various uses of compound 1, in particular treatment of respiratory diseases
including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis,
interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute
respiratory distress syndrome, bronchitis, emphysema and bronchiolitis obliterans. However,
this application does not disclose the use of compound 1 for the treatment of ocular disease.
In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that
such documents, or such sources of information, in any jurisdiction, are prior art, or form part
of the common general knowledge in the art.
The invention is defined in the claims. However, the disclosure preceding the claims
may refer to additional methods and other subject matter outside the scope of the present
claims. This disclosure is retained for technical purposes.
SUMMARY OF THE INVENTION
Described herein are methods of treating ocular diseases or symptoms thereof using
the JAK inhibitor 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro-1H-
imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol or a pharmaceutically-acceptable salt
thereof.
In a first aspect, the invention relates to use of 5-ethylfluoro(3-(5-(1-
methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazol
yl)phenol, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament
for the treatment of an ocular disease in a mammal.
In a second aspect, the invention relates to use of 5-ethylfluoro(3-(5-(1-
methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazol
yl)phenol, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament
for the treatment of a respiratory disease in a mammal, wherein the respiratory disease is a
lung infection, a helminthic infection, pulmonary arterial hypertension, sarcoidosis,
lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonary disease.
In a third aspect, the invention relates to use of 5-ethylfluoro(3-(5-(1-
methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazol
yl)phenol, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament
for the treatment of a respiratory disease in a mammal, wherein the respiratory disease is
drug-induced pneumonitis, fungal induced pneumonitis, allergic bronchopulmonary
aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis,
idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,
hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliterans organizing
pneumonia, or immune-checkpoint-inhibitor induced pneumonitis.
Described herein is a method of treating an ocular disease in a human patient, the
method comprising administering to the eye of the patient, the compound 5-ethylfluoro
(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-
indazolyl)phenol of formula
hereinafter compound 1, or a pharmaceutically-acceptable salt thereof.
In one embodiment the ocular disease is uveitis, diabetic retinopathy, diabetic
macular edema, dry eye disease, age-related macular degeneration, or atopic
keratoconjunctivitis. In particular, the ocular disease is uveitis or diabetic macular edema.
Described herein is a pharmaceutical composition of 5-ethylfluoro(3-(5-(1-
methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazol
yl)phenol (compound 1) or a pharmaceutically-acceptable salt thereof, wherein the
pharmaceutical composition is suitable for administration directly to the eye of a patient.
Described herein are methods of using compound 1 to treat certain specific
respiratory diseases.
Described herein is a method of treating a respiratory disease in a mammal, the
method comprising administering to the mammal a pharmaceutical composition comprising
compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically-acceptable
carrier, wherein the respiratory disease is a lung infection, a helminthic infection, pulmonary
arterial hypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, or an
infiltrative pulmonary disease.
Described herein is a method of treating a respiratory disease in a mammal, the
method comprising administering to the mammal a pharmaceutical composition comprising
compound 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically-acceptable
carrier, wherein the respiratory disease is drug-induced pneumonitis, fungal induced
pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis,
eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia,
idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Löffler syndrome,
bronchiolitis obliterans organizing pneumonia, or immune-checkpoint-inhibitor induced
pneumonitis.
DETAILED DESCRIPTION OF THE INVENTION
Chemical structures are named herein according to IUPAC conventions as
implemented in ChemDraw software (PerkinElmer, Inc., Cambridge, MA).
Furthermore, the imidazo portion of the tetrahydroimidazopyridine moiety in the
structure of the present compound exists in tautomeric forms. The compound could
equivalently be represented as
According to the IUPAC convention, these representations give rise to different numbering
of the atoms of the imidazopyridine portion. Accordingly this structure is designated 5-ethyl-
2-fluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridinyl)-
1H-indazolyl)phenol. It will be understood that although structures are shown, or named,
in a particular form, the invention also includes the tautomer thereof.
Definitions
When describing the present invention, the following terms have the following
meanings unless otherwise indicated.
The singular terms “a,” “an” and “the” include the corresponding plural terms unless
the context of use clearly dictates otherwise.
The term “about” means ± 5 percent of the specified value.
The term “therapeutically effective amount” means an amount sufficient to effect
treatment when administered to a patient in need of treatment, e.g., the amount needed to
obtain the desired therapeutic effect.
The term “treating” or “treatment” means preventing, ameliorating or suppressing the
medical condition, disease or disorder being treated (e.g., a respiratory disease) in a patient
(particularly a human); or alleviating the symptoms of the medical condition, disease or
disorder.
The term "unit dosage form" or “unit doses” means a physically discrete unit suitable
for dosing a patient, i.e., each unit containing a predetermined quantity of a therapeutic agent
calculated to produce a therapeutic effect either alone or in combination with one or more
additional units. Examples include capsules, tablets and the like.
All other terms used herein are intended to have their ordinary meaning as understood
by persons having ordinary skill in the art to which they pertain.
The term "pharmaceutically-acceptable" means acceptable for administration to a
patient (e.g., having acceptable safety for the specified usage).
The term “pharmaceutically-acceptable salt” means a salt prepared from an acid and a
base (including zwitterions) that is acceptable for administration to a patient (e.g., a salt
having acceptable safety for a given dosage regime).
Representative pharmaceutically acceptable salts include salts of acetic, ascorbic,
benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, edisylic, fumaric, gentisic,
gluconic, glucoronic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic,
lactobionic, maleic, malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,
naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, oxalic,
pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic and xinafoic
acid, and the like.
The term “salt thereof” means a compound formed when the hydrogen of an acid is
replaced by a cation, such as a metal cation or an organic cation and the like.
The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification and claims which include
the term “comprising”, other features besides the features prefaced by this term in each
statement can also be present. Related terms such as “comprise” and “comprises” are to be
interpreted in similar manner.
Compound 1
The present method employs 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-
4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol (compound 1)
or a pharmaceutically-acceptable salt thereof.
Compound 1 may be prepared as described in U.S. Application Serial No. 15/341,226
or in the appended examples.
Compound 1 is employed in the form of a crystalline freebase hydrate characterized
by a powder X-ray diffraction (PXRD) pattern having significant diffraction peaks, among
other peaks, at 2θ values of 6.20±0.20, 9.58±0.20, 17.53±0.20, 19.28±0.20, and 21.51±0.20.
The preparation of the crystalline hydrate is also described in U.S. Serial No. 15/341,226 and
in the examples below.
Pharmaceutical Compositions
The present compound, 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol (1) and pharmaceutically-
acceptable salts thereof is typically used in the form of a pharmaceutical composition or
formulation. Such pharmaceutical compositions may advantageously be administered to a
patient by any acceptable route of administration including, but not limited to, oral,
inhalation, optical injection, topical (including transdermal), rectal, nasal, and parenteral
modes of administration.
The pharmaceutical compositions utilized in the invention typically contain a
therapeutically effective amount of compound 1. Those skilled in the art will recognize,
however, that a pharmaceutical composition may contain more than a therapeutically
effective amount, i.e., bulk compositions, or less than a therapeutically effective amount, i.e.,
individual unit doses designed for multiple administration to achieve a therapeutically
effective amount. When discussing compositions and uses, compound 1 may also be referred
to herein as 'active agent'.
Typically, pharmaceutical compositions will contain from about 0.01 to about 95%
by weight of the active agent; including, for example, from about 0.05 to about 30% by
weight; and from about 0.1 % to about 10% by weight of the active agent.
Any conventional carrier or excipient may be used in the pharmaceutical
compositions utilized in the invention. The choice of a particular carrier or excipient, or
combinations of carriers or excipients, will depend on the mode of administration being used
to treat a particular patient or type of medical condition or disease state. In this regard, the
preparation of a suitable pharmaceutical composition for a particular mode of administration
is well within the scope of those skilled in the pharmaceutical arts. Additionally, the carriers
or excipients used in the pharmaceutical compositions described are commercially-available.
By way of further illustration, conventional formulation techniques are described in
Remington: The Science and Practice of Pharmacy, 20th Edition, Lippincott Williams &
White, Baltimore, Maryland (2000); and H.C. Ansel et al., Pharmaceutical Dosage Forms
and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland
(1999).
Representative examples of materials which can serve as pharmaceutically acceptable
carriers include, but are not limited to, the following: sugars, such as lactose, glucose and
sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline
cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and
cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter
and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as
glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide;
alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate
buffer solutions; and other non-toxic compatible substances employed in pharmaceutical
compositions.
Pharmaceutical compositions are typically prepared by thoroughly and intimately
mixing or blending the active agent with a pharmaceutically-acceptable carrier and one or
more optional ingredients. The resulting uniformly blended mixture can then be shaped or
loaded into tablets, capsules, pills and the like using conventional procedures and equipment.
In one aspect, the pharmaceutical composition is suitable for ocular injection. In this
aspect, the compound may be formulated as a sterile aqueous suspension or solution. Useful
excipients that may be included in such an aqueous formulation include polysorbate 80,
carboxymethylcellulose, potassium chloride, calcium chloride, magnesium chloride, sodium
acetate, sodium citrate, histidine, α-α-trehalose dihydrate, sucrose, polysorbate 20,
hydroxypropyl-β-cyclodextrin, and sodium phosphate. Benzyl alcohol may serve as a
preservative and sodium chloride may be included to adjust tonicity. In addition,
hydrochloric acid and/or sodium hydroxide may be added to the solution for pH adjustment.
Aqueous formulations for ocular injection may be prepared as preservative-free.
In another aspect, the pharmaceutical composition is suitable for inhaled
administration. Pharmaceutical compositions for inhaled administration are typically in the
form of an aerosol or a powder. Such compositions are generally administered using inhaler
delivery devices, such as a dry powder inhaler (DPI), a metered-dose inhaler (MDI), a
nebulizer inhaler, or a similar delivery device.
In a particular embodiment, the pharmaceutical composition is administered by
inhalation using a dry powder inhaler. Such dry powder inhalers typically administer the
pharmaceutical composition as a free-flowing powder that is dispersed in a patient's air-
stream during inspiration. In order to achieve a free-flowing powder composition, the
therapeutic agent is typically formulated with a suitable excipient such as lactose, starch,
mannitol, dextrose, polylactic acid (PLA), polylactide-co-glycolide (PLGA) or combinations
thereof. Typically, the therapeutic agent is micronized and combined with a suitable carrier
to form a composition suitable for inhalation.
A representative pharmaceutical composition for use in a dry powder inhaler
comprises lactose and the compound useful in the invention in micronized form. Such a dry
powder composition can be made, for example, by combining dry milled lactose with the
therapeutic agent and then dry blending the components. The composition is then typically
loaded into a dry powder dispenser, or into inhalation cartridges or capsules for use with a
dry powder delivery device.
Dry powder inhaler delivery devices suitable for administering therapeutic agents by
inhalation are described in the art and examples of such devices are commercially available.
For example, representative dry powder inhaler delivery devices or products include Aeolizer
(Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler (GlaxoSmithKline);
Diskus/Accuhaler (GlaxoSmithKline); Ellipta (GlaxoSmithKline); Easyhaler (Orion
Pharma); Eclipse (Aventis); FlowCaps (Hovione); Handihaler (Boehringer Ingelheim);
Pulvinal (Chiesi); Rotahaler (GlaxoSmithKline); SkyeHaler/Certihaler (SkyePharma);
Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler (Aventis); and the like.
In another particular embodiment, the pharmaceutical composition is administered by
inhalation using a metered-dose inhaler. Such metered-dose inhalers typically discharge a
measured amount of a therapeutic agent using a compressed propellant gas. Accordingly,
pharmaceutical compositions administered using a metered-dose inhaler typically comprise a
solution or suspension of the therapeutic agent in a liquefied propellant. Any suitable
liquefied propellant may be employed including hydrofluoroalkanes (HFAs), such as 1,1,1,2-
tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227); and
chlorofluorocarbons, such as CCl F. In a particular embodiment, the propellant is
hydrofluoroalkanes. In some embodiments, the hydrofluoroalkane formulation contains a co-
solvent, such as ethanol or pentane, and/or a surfactant, such as sorbitan trioleate, oleic acid,
lecithin, and glycerin.
A representative pharmaceutical composition for use in a metered-dose inhaler
comprises from about 0.01% to about 5% by weight of the compound useful in the invention;
from about 0% to about 20% by weight ethanol; and from about 0% to about 5% by weight
surfactant; with the remainder being an HFA propellant. Such compositions are typically
prepared by adding chilled or pressurized hydrofluoroalkane to a suitable container
containing the therapeutic agent, ethanol (if present) and the surfactant (if present). To
prepare a suspension, the therapeutic agent is micronized and then combined with the
propellant. The composition is then loaded into an aerosol canister, which typically forms a
portion of a metered-dose inhaler device.
Metered-dose inhaler devices suitable for administering therapeutic agents by
inhalation are described in the art and examples of such devices are commercially available.
For example, representative metered-dose inhaler devices or products include AeroBid
Inhaler System (Forest Pharmaceuticals); Atrovent Inhalation Aerosol (Boehringer
Ingelheim); Flovent (GlaxoSmithKline); Maxair Inhaler (3M); Proventil Inhaler (Schering);
Serevent Inhalation Aerosol (GlaxoSmithKline); and the like.
In another particular aspect, the pharmaceutical composition is administered by
inhalation using a nebulizer inhaler. Such nebulizer devices typically produce a stream of
high velocity air that causes the pharmaceutical composition to spray as a mist that is carried
into the patient's respiratory tract. Accordingly, when formulated for use in a nebulizer
inhaler, the therapeutic agent can be dissolved in a suitable carrier to form a solution.
Alternatively, the therapeutic agent can be micronized or nanomilled and combined with a
suitable carrier to form a suspension.
A representative pharmaceutical composition for use in a nebulizer inhaler comprises
a solution or suspension comprising from about 0.05 μg/mL to about 20 mg/mL of the
compound useful in the invention and excipients compatible with nebulized formulations. In
one embodiment, the solution has a pH of about 3 to about 8.
Nebulizer devices suitable for administering therapeutic agents by inhalation are
described in the art and examples of such devices are commercially available. For example,
representative nebulizer devices or products include the Respimat Softmist Inhaler
(Boehringer Ingelheim); the AERx Pulmonary Delivery System (Aradigm Corp.); the PARI
LC Plus Reusable Nebulizer (Pari GmbH); and the like.
The pharmaceutical compositions described may alternatively be prepared in a dosage
form intended for oral administration. Suitable pharmaceutical compositions for oral
administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees,
powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or
as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each
containing a predetermined amount of a compound useful in the present invention as an
active ingredient.
When intended for oral administration in a solid dosage form, the pharmaceutical
compositions described will typically comprise the active agent and one or more
pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate.
Optionally or alternatively, such solid dosage forms may also comprise: fillers or extenders,
binders, humectants, solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, coloring agents, and buffering agents. Release agents, wetting agents,
coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants
can also be present in the pharmaceutical compositions described.
Alternative formulations may also include controlled release formulations, liquid
dosage forms for oral administration, transdermal patches, and parenteral formulations.
Conventional excipients and methods of preparation of such alternative formulations are
described, for example, in the reference by Remington, supra.
The following non-limiting examples illustrate representative pharmaceutical
compositions described.
Aqueous formulation for ocular injection
Each mL of a sterile aqueous suspension includes from 5 mg to 50 mg of compound
1, sodium chloride for tonicity, 0.99 % (w/v) benzyl alcohol as a preservative, 0.75 %
carboxymethylcellulose sodium, and 0.04 % polysorbate. Sodium hydroxide or hydrochloric
acid may be included to adjust pH to 5 to 7.5.
Aqueous formulation for ocular injection
A sterile preservative-free aqueous suspension includes from 5 mg/mL to 50 mg/mL
of compound 1 in 10 mM sodium phosphate, 40 mM sodium chloride, 0.03 % polysorbate
, and 5 % sucrose.
Dry Powder Composition
Micronized compound 1 (1 g) is blended with milled lactose (25 g). This blended
mixture is then loaded into individual blisters of a peelable blister pack in an amount
sufficient to provide between about 0.1 mg to about 4 mg of the compound of formula I per
dose. The contents of the blisters are administered using a dry powder inhaler.
Dry Powder Composition
Micronized compound 1 (1 g) is blended with milled lactose (20 g) to form a bulk
composition having a weight ratio of compound to milled lactose of 1:20. The blended
composition is packed into a dry powder inhalation device capable of delivering between
about 0.1 mg to about 4 mg of the compound of formula I per dose.
Metered-Dose Inhaler Composition
Micronized compound 1 (10 g) is dispersed in a solution prepared by dissolving
lecithin (0.2 g) in demineralized water (200 mL). The resulting suspension is spray dried and
then micronized to form a micronized composition comprising particles having a mean
diameter less than about 1.5 μm. The micronized composition is then loaded into metered-
dose inhaler cartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amount
sufficient to provide about 0.1 mg to about 4 mg of the compound of formula I per dose
when administered by the metered dose inhaler.
Nebulizer Composition
Compound 1 (25 mg) is dissolved in a solution containing 1.5-2.5 equivalents of
hydrochloric acid, followed by addition of sodium hydroxide to adjust the pH to 3.5 to 5.5
and 3% by weight of glycerol. The solution is stirred well until all the components are
dissolved. The solution is administered using a nebulizer device that provides about 0.1 mg
to about 4 mg of the compound of formula I per dose.
Utility
The present compound, 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, (compound 1), has been
shown to be a potent inhibitor of the JAK family of enzymes: JAK1, JAK2, JAK3, and
TYK2.
Ocular Diseases
Many ocular diseases have been shown to be associated with elevations of
proinflammatory cytokines that rely on the JAK-STAT pathway. Since the compound useful
in the invention exhibits potent inhibition at all four JAK enzymes, it is expected to potently
inhibit the signaling and pathogenic effects of numerous cytokines (such as IL-6, IL-2 and
IFN-γ), that signal through JAK, as well as to prevent the increase in other cytokines (such as
MCP-1 and IP-10), whose production is driven by JAK-STAT pathway signaling.
In particular, the present compound exhibited pIC values of 6.7 or greater (IC
50 50
values of 200 nM or less) for inhibition of IL-2, IL-4, IL-6, and IFNγ signaling in the cellular
assays described in Assays 3 to 7, including assays registering inhibition of the downstream
effects of cytokine elevation.
The pharmacokinetic study of Assay 8 demonstrated sustained exposure in rabbit
eyes after a single intravitreal injection and a concentration in plasma at least three orders of
magnitude lower than that observed in vitreous tissue.
Furthermore, intravitreal dosing of the compound described has demonstrated
significant inhibition of IL-6 induced pSTAT3 in the rat retina/choroid tissue as well as
significant inhibition of IFN-γ induced IP-10 in the rabbit vitreous as well as retina/choroid
tissues.
It is expected that sustained ocular JAK inhibition in the absence of significant
systemic levels will result in potent, local anti-inflammatory activity in the eye without
systemically-driven adverse effects. The compound described is expected to be beneficial in
a number of ocular diseases that include, but are not limited to, uveitis, diabetic retinopathy,
diabetic macular edema, dry eye disease, age-related macular degeneration, and atopic
keratoconjunctivitis.
In particular, uveitis (Horai and Caspi, J Interferon Cytokine Res, 2011, 31, 733-744),
diabetic retinopathy (Abcouwer, J Clin Cell Immunol, 2013, Suppl 1, 1-12), diabetic macular
edema (Sohn et al., American Journal of Opthamology, 2011, 152, 686-694), dry eye disease
(Stevenson et al, Arch Ophthalmol, 2012, 130, 90-100), and age-related macular
degeneration (Knickelbein et al, Int Ophthalmol Clin, 2015, 55(3), 63-78) are characterized
by elevation of certain pro-inflammatory cytokines that signal via the JAK-STAT pathway.
Accordingly, compounds described herein may be able to alleviate the associated ocular
inflammation and reverse disease progression or provide symptom relief.
Retinal vein occlusion (RVO) is a highly prevalent visually disabling disease.
Obstruction of retinal blood flow can lead to damage of the retinal vasculature, hemorrhage,
and tissue ischemia. Although the causes for RVO are multifactorial, both vascular as well as
inflammatory mediators have been shown to be important (Deobhakta et al, International
Journal of Inflammation, 2013, article ID 438412). Cytokines which signal through the JAK-
STAT pathway, such as IL-6 and IL-13, as well as other cytokines, such as MCP-1, whose
production is driven in part by JAK-STAT pathway signaling, have been detected at elevated
levels in ocular tissues of patients with RVO (Shchuko et al, Indian Journal of
Ophthalmology, 2015, 63(12), 905-911). Accordingly, compound 1 may be able to alleviate
the associated ocular inflammation and reverse disease progression or provide symptom
relief in this disease. While many patients with RVO are treated by photocoagulation, this is
an inherently destructive therapy. Anti-VEGF agents are also used, but they are only
effective in a fraction of patients. Steroid medications that reduce the level of inflammation
in the eye (Triamcinolone acetonide and dexamethasone implants) have also been shown to
provide beneficial results for patients with certain forms of RVO, but they have also been
shown to cause cataracts and increased intraocular pressure/glaucoma.
Described herein is a method of treating an ocular disease in a mammal, the method
comprising administering 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol or a pharmaceutically-
acceptable salt thereof to the eye of the mammal. In one embodiment, the ocular disease is
uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular
degeneration, retinal vein occlusion, or atopic keratoconjunctivitis. In one embodiment, the
method comprises administering the present compound by intravitreal injection.
Respiratory Diseases
The present compound, 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol (1) has demonstrated
inhibition of T cell activation, inhibition of cytokines associated with inflammation, and
activity in rodent lung eosinophilia and neutrophilia assays. Therefore, the compound is
believed to be useful for the treatment of certain specific respiratory diseases.
Eosinophilic airway inflammation is a characteristic feature of diseases collectively
termed eosinophilic lung diseases (Cottin et al., Clin. Chest. Med., 2016, 37(3), 535-56).
Eosinophilic diseases have been associated with IL-4, IL-13 and IL-5 signaling.
Eosinophilic lung diseases include infections (especially helminthic infections), drug-induced
pneumonitis (induced for example by therapeutic drugs such as antibiotics, phenytoin,
or l-tryptophan), fungal-induced pneumonitis (e.g. allergic bronchopulmonary aspergillosis),
hypersensitivity pneumonitis and eosinophilic granulomatosis with polyangiitis (formerly
known as Churg-Strauss syndrome). Eosinophilic lung diseases of unknown etiology include
idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia,
hypereosinophilic syndrome, and Löffler syndrome. Compound 1 has been shown to
significantly reduce lung eosinophilia in the rodent airway model of Assay 13 and to potently
inhibit IL-13, IL-4, and IL-2 signaling in cellular assays.
A polymorphism in the IL-6 gene has been associated with elevated IL-6 levels and
an increased risk of developing pulmonary arterial hypertension (PAH) (Fang et al., J Am Soc
Hypertens., Interleukin-6 -572C/G polymorphism is associated with serum interleukin-6
levels and risk of idiopathic pulmonary arterial hypertension, 2017, ahead of print).
Corroborating the role of IL-6 in PAH, inhibition of the IL-6 receptor chain gp130
ameliorated the disease in a rat model of PAH (Huang et al., Can J Cardiol., 2016, 32(11),
1356.e1-1356.e10). The compound described has been shown to inhibit IL-6 signaling.
Cytokines such as IFNγ, IL-12 and IL-6 have been implicated in a range of non-
allergic lung diseases such as sarcoidosis, and lymphangioleiomyomatosis (El-Hashemite et
al., Am. J. Respir. Cell Mol. Biol., 2005, 33, 227-230, and El-Hashemite et al., Cancer Res.,
2004, 64, 3436-3443 ). The compound described has also been shown to inhibit IL-6 and
IFNγ signaling.
Bronchiectasis and infiltrative pulmonary diseases are diseases associated with
chronic neutrophilic inflammation. The compound described has been shown to inhibit
airway neutrophilia in a rodent model.
Pathological T cell activation is critical in the etiology of multiple respiratory
diseases. Autoreactive T cells play a role in bronchiolitis obliterans organizing pneumonia
(also termed COP [cryptogenic organizing pneumonia]). More recently, immune-checkpoint
inhibitor induced pneumonitis, another T cell mediated lung disease emerged with the
increased use of immune-checkpoint inhibitors. In cancer patients treated with these T cell
stimulating agents, fatal pneumonitis can develop. The compound described has been shown
to inhibit activation in T cells isolated from human peripheral blood mononuclear cells.
Described herein is a method of treating a respiratory disease in a mammal (e.g., a
human), the method comprising administering to the mammal 5-ethylfluoro(3-(5-(1-
methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazol
yl)phenol or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition
comprising a pharmaceutically-acceptable carrier and the compound described, or a
pharmaceutically acceptable salt thereof, wherein the respiratory disease is a lung infection, a
helminthic infection, pulmonary arterial hypertension, sarcoidosis,
lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonary disease.
Described herein is a method of treating a respiratory disease in a mammal (e.g., a
human), the method comprising administering to the mammal the compound described, or a
pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and the compound described, or a pharmaceutically
acceptable salt thereof, wherein the respiratory disease is drug-induced pneumonitis, fungal
induced pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis,
eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia,
idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Löffler syndrome,
bronchiolitis obliterans organizing pneumonia, or immune-checkpoint-inhibitor induced
pneumonitis.
JAK-signaling cytokines also play a major role in the activation of T cells, a sub-type
of immune cells that is central to many immune processes. Pathological T cell activation is
critical in the etiology of multiple respiratory diseases. Autoreactive T cells play a role in
bronchiolitis obliterans organizing pneumonia (also termed COS). Similar to COS the
etiology of lung transplant rejections is linked to an aberrant T cell activation of the
recipients T cells by the transplanted donor lung. Lung transplant rejections may occur early
as Primary Graft Dysfunction (PGD), organizing pneumonia (OP), acute rejection (AR) or
lymphocytic bronchiolitis (LB) or they may occur years after lung transplantation as Chronic
Lung Allograft Dysfunction (CLAD). CLAD was previously known as bronchiolitis
obliterans (BO) but now is considered a syndrome that can have different pathological
manifestations including BO, restrictive CLAD (rCLAD or RAS) and neutrophilic allograft
dysfunction. Chronic lung allograft dysfunction (CLAD) is a major challenge in long-term
management of lung transplant recipients as it causes a transplanted lung to progressively
lose functionality (Gauthier et al., Curr Transplant Rep., 2016, 3(3), 185–191). CLAD is
poorly responsive to treatment and therefore, there remains a need for effective compounds
capable of preventing or treating this condition. Several JAK-dependent cytokines such as
IFNγ and IL-5 are up-regulated in CLAD and lung transplant rejection (Berastegui et al, Clin
Transplant. 2017, 31, e12898). Moreover, high lung levels of CXCR3 chemokines such as
CXCL9 and CXCL10 which are downstream of JAK-dependent IFN signaling, are linked to
worse outcomes in lung transplant patients (Shino et al, PLOS One, 2017, 12 (7), e0180281).
Systemic JAK inhibition has been shown to be effective in kidney transplant rejection
(Vicenti et al., American Journal of Transplantation, 2012, 12, 2446-56). Therefore, JAK
inhibitors have the potential to be effective in treating or preventing lung transplant rejection
and CLAD. Similar T cell activation events as described as the basis for lung transplant
rejection also are considered the main driver of lung graft-versus-host disease (GVHD)
which can occur post hematopoietic stem cell transplants. Similar to CLAD, lung GVHD is a
chronic progressive condition with extremely poor outcomes and no treatments are currently
approved. A retrospective, multicenter survey study of 95 patients with steroid-refractory
acute or chronic GVHD who received the systemic JAK inhibitor ruxolitinib as salvage
therapy demonstrated complete or partial response to ruxolitinib in the majority of patients
including those with lung GVHD (Zeiser et al, Leukemia, 2015, 29, 10, 2062-68). As
systemic JAK inhibition is associated with serious adverse events and a small therapeutic
index, the need remains for an inhaled lung-directed, non-systemic JAK inhibitor to prevent
and/or treat lung transplant rejection or lung GVHD.
Accordingly, the disclosure further describes a method of treating the additional
respiratory conditions described above in a mammal, the method comprising administering to
the mammal compound 1, or a pharmaceutically acceptable salt thereof.
Gastrointestinal inflammatory disease
As a JAK inhibitor, compound 1, or a pharmaceutical salt thereof, may also be useful
to treat gastrointestinal inflammatory diseases that include, but are not limited to,
inflammatory bowel disease, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative
proctitis and left-sided colitis), Crohn’s disease, collagenous colitis, lymphocytic colitis,
Behcet’s disease, celiac disease, immune checkpoint inhibitor induced colitis, ileitis,
eosinophilic esophagitis, graft versus host disease-related colitis, and infectious colitis.
Ulcerative colitis (Reimund et al., J Clin Immunology, 1996, 16, 144-150), Crohn’s disease
(Woywodt et al., Eur J Gastroenterology Hepatology, 1999, 11, 267-276), collagenous
colitis (Kumawat et al., Mol Immunology, 2013, 55, 355-364), lymphocytic colitis (Kumawat
et al., 2013), eosinophilic esophagitis (Weinbrand-Goichberg et al., Immunol Res, 2013, 56,
249-260), graft versus host disease-related colitis (Coghill et al., Blood, 2001, 117, 3268-
3276), infectious colitis (Stallmach et al., Int J Colorectal Dis, 2004, 19, 308–315), Behcet’s
disease (Zhou et al., Autoimmun Rev, 2012, 11, 699-704), celiac disease (de Nitto et al.,
World J Gastroenterol, 2009, 15, 4609-4614), immune checkpoint inhibitor induced colitis
(e.g., CTLA-4 inhibitor-induced colitis; (Yano et al., J Translation Med, 2014, 12, 191), PD-
1- or PD-L1-inhibitor-induced colitis), and ileitis (Yamamoto et al., Dig Liver Dis, 2008, 40,
253-259) are characterized by elevation of certain pro-inflammatory cytokine levels. As
many pro-inflammatory cytokines signal via JAK activation, compounds described in this
application may be able to alleviate the inflammation and provide symptom relief.
Inflammatory skin disease
Atopic dermatitis and other inflammatory skin diseases have been associated with
elevation of proinflammatory cytokines that rely on the JAK-STAT pathway. Therefore
compound 1, or a pharmaceutical salt thereof, may be beneficial in a number of dermal
inflammatory or pruritic conditions that include, but are not limited to atopic dermatitis,
alopecia areata, vitiligo, psoriasis, dermatomyositis, cutaneous T cell lymphoma
(Netchiporouk et al., Cell Cycle. 2014; 13, 3331-3335) and subtypes (Sezary syndrome,
mycosis fungoides, pagetoid reticulosis, granulomatous slack skin, lymphomatoid papulosis,
pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, CD30+
cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non-mycosis
fungoides CD30− cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert
lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blastic NK-cell
lymphoma), prurigo nodularis, lichen planus, primary localized cutaneous amyloidosis,
bullous pemphigoid, skin manifestations of graft versus host disease, pemphigoid, discoid
lupus, granuloma annulare, lichen simplex chronicus, vulvar/scrotal/perianal pruritus, lichen
sclerosus, post herpetic neuralgia itch, lichen planopilaris, and foliculitis decalvans. In
particular, atopic dermatitis (Bao et al., JAK-STAT, 2013, 2, e24137), alopecia areata (Xing et
al., Nat Med. 2014, 20, 1043-1049), vitiligo (Craiglow et al, JAMA Dermatol. 2015, 151,
1110-1112), prurigo nodularis (Sonkoly et al., J Allergy Clin Immunol. 2006, 117, 411-417),
lichen planus (Welz-Kubiak et al., J Immunol Res. 2015, ID:854747), primary localized
cutaneous amyloidosis (Tanaka et al., Br J Dermatol. 2009, 161, 1217-1224), bullous
pemphigoid (Feliciani et al., Int J Immunopathol Pharmacol. 1999, 12, 55-61), and dermal
manifestations of graft versus host disease (Okiyama et al., J Invest Dermatol. 2014, 134,
992-1000) are characterized by elevation of certain cytokines that signal via JAK activation.
Accordingly, compound 1, or a pharmaceutically acceptable salt thereof, may be able to
alleviate associated dermal inflammation or pruritus driven by these cytokines.
Other diseases
Compound 1, or a pharmaceutically acceptable salt thereof, may also be useful to
treat other diseases such as other inflammatory diseases, autoimmune diseases or cancers.
Compound 1, or a pharmaceutically acceptable salt thereof, may be useful to treat one or
more of arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection,
xerophthalmia, psoriatic arthritis, diabetes, insulin dependent diabetes, motor neurone
disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus
erythematosus, leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute
lymphoblastic leukemia, acute myelogenous leukemia, ankylosing spondylitis, myelofibrosis,
B-cell lymphoma, hepatocellular carcinoma, Hodgkins disease, breast cancer, Multiple
myeloma, melanoma, non-Hodgkin lymphoma, non-small-cell lung cancer, ovarian clear cell
carcinoma, ovary tumor, pancreas tumor, polycythemia vera, Sjoegrens syndrome, soft tissue
sarcoma, sarcoma, splenomegaly, T-cell lymphoma, and thalassemia major.
Combination therapy
Compound 1 of the disclosure or a pharmaceutically acceptable salt thereof may be
used in combination with one or more agents which act by the same mechanism or by
different mechanisms to treat a disease. The different agents may be administered
sequentially or simultaneously, in separate compositions or in the same composition. Useful
classes of agents for combination therapy include, but are not limited to, a beta 2
adrenoceptor agonist, a muscarinic receptor antagonist, a glucocorticoid agonist, a G-protein
coupled receptor-44 antagonist, a leukotriene D4 antagonist, a muscarinic M3 receptor
antagonist, a histamine H1 receptor antagonist, an immunoglobulin E antagonist, a PDE 4
inhibitor, an IL-4 antagonist, a muscarinic M1 receptor antagonist, a histamine receptor
antagonist, an IL-13 antagonist, an IL-5 antagonist, a 5-Lipoxygenase inhibitor, a beta
adrenoceptor agonist, a CCR3 chemokine antagonist, a CFTR stimulator, an immunoglobulin
modulator, an interleukin 33 ligand inhibitor, a PDE 3 inhibitor, a phosphoinositide-3 kinase
delta inhibitor, a thromboxane A2 antagonist, an elastase inhibitor, a Kit tyrosine kinase
inhibitor, a leukotriene E4 antagonist, a leukotriene antagonist, a PGD2 antagonist, a TNF
alpha ligand inhibitor, a TNF binding agent, a complement cascade inhibitor, an eotaxin
ligand inhibitor, a glutathione reductase inhibitor, an histamine H4 receptor antagonist, an
IL-6 antagonist, an IL2 gene stimulator, an immunoglobulin gamma Fc receptor IIB
modulator, an interferon gamma ligand, an interleukin 13 ligand inhibitor, an interleukin 17
ligand inhibitor, a L-Selectin antagonist, a leukocyte elastase inhibitor, a leukotriene C4
antagonist, a Leukotriene C4 synthase inhibitor, a membrane copper amine oxidase inhibitor,
a metalloprotease-12 inhibitor, a metalloprotease-9 inhibitor, a mite allergen modulator, a
muscarinic receptor modulator, a nicotinic acetylcholine receptor agonist, a nuclear factor
kappa B inhibitor, a p-Selectin antagonist, a PDE 5 inhibitor, a PDGF receptor antagonist, a
phosphoinositide-3 kinase gamma inhibitor, a TLR-7 agonist, a TNF antagonist, an Abl
tyrosine kinase inhibitor, an acetylcholine receptor antagonist, an acidic mammalian chitinase
inhibitor, an ACTH receptor agonist, an actin polymerization modulator, an adenosine A1
receptor antagonist, an adenylate cyclase stimulator, an adrenoceptor antagonist, an
adrenocorticotrophic hormone ligand, an alcohol dehydrogenase 5 inhibitor, an alpha 1
antitrypsin stimulator, an alpha 1 proteinase inhibitor, an androgen receptor modulator, an
angiotensin converting enzyme 2 stimulator, an ANP agonist, a Bcr protein inhibitor, a beta 1
adrenoceptor antagonist, a beta 2 adrenoceptor antagonist, a beta 2 adrenoceptor modulator, a
beta amyloid modulator, a BMP10 gene inhibitor, a BMP15 gene inhibitor, a calcium
channel inhibitor, a cathepsin G inhibitor, a CCL26 gene inhibitor, a CCR3 chemokine
modulator, a CCR4 chemokine antagonist, a cell adhesion molecule inhibitor, a chaperonin
stimulator, a chitinase inhibitor, a collagen I antagonist, a complement C3 inhibitor, a CSF-1
antagonist, a CXCR2 chemokine antagonist, a cytokine receptor common beta chain
modulator, a cytotoxic T-lymphocyte protein-4 stimulator, a deoxyribonuclease I stimulator,
a deoxyribonuclease stimulator, a dipeptidyl peptidase I inhibitor, a DNA gyrase inhibitor, a
DP prostanoid receptor modulator, an E-Selectin antagonist, an EGFR family tyrosine kinase
receptor inhibitor, an elastin modulator, an Endothelin ET-A antagonist, an Endothelin ET-B
antagonist, an epoxide hydrolase inhibitor, a FGF3 receptor antagonist, a Fyn tyrosine kinase
inhibitor, a GATA 3 transcription factor inhibitor, a Glucosylceramidase modulator, a
Glutamate receptor modulator, a GM-CSF ligand inhibitor, a Guanylate cyclase stimulator, a
H+ K+ ATPase inhibitor, an hemoglobin modulator, an Heparin agonist, an Histone
deacetylase inhibitor, an Histone deacetylase-2 stimulator, an HMG CoA reductase inhibitor,
an I-kappa B kinase beta inhibitor, an ICAM1 gene inhibitor, an IL-17 antagonist, an IL-17
receptor modulator, an IL-23 antagonist, an IL-4 receptor modulator, an Immunoglobulin G
modulator, an Immunoglobulin G1 agonist, an Immunoglobulin G1 modulator, an
Immunoglobulin epsilon Fc receptor IA antagonist, an Immunoglobulin gamma Fc receptor
IIB antagonist, an Immunoglobulin kappa modulator, an Insulin sensitizer, an Interferon beta
ligand, an Interleukin 1 like receptor antagonist, an Interleukin 18 ligand inhibitor, an
Interleukin receptor 17A antagonist, an Interleukin-1 beta ligand inhibitor, an Interleukin-5
ligand inhibitor, an Interleukin-6 ligand inhibitor, a KCNA voltage-gated potassium
channel-3 inhibitor, a Kit ligand inhibitor, a Laminin-5 agonist, a Leukotriene CysLT1
receptor antagonist, a Leukotriene CysLT2 receptor antagonist, a LOXL2 gene inhibitor, a
Lyn tyrosine kinase inhibitor, a MARCKS protein inhibitor, a MDR associated protein 4
inhibitor, a Metalloprotease-2 modulator, a Metalloprotease-9 modulator, a
Mineralocorticoid receptor antagonist, a Muscarinic M2 receptor antagonist, a Muscarinic
M4 receptor antagonist, a Muscarinic M5 receptor antagonist, a Natriuretic peptide receptor
A agonist, a Natural killer cell receptor modulator, a Nicotinic ACh receptor alpha 7 subunit
stimulator, a NK cell receptor modulator, a Nuclear factor kappa B modulator, an opioid
growth factor receptor agonist, a P-Glycoprotein inhibitor, a P2X3 purinoceptor antagonist, a
p38 MAP kinase inhibitor, a Peptidase 1 modulator, a phospholipase A2 inhibitor, a
phospholipase C inhibitor, a plasminogen activator inhibitor 1 inhibitor, a platelet activating
factor receptor antagonist, a PPAR gamma agonist, a prostacyclin agonist, a protein tyrosine
kinase inhibitor, a SH2 domain inositol phosphatase 1 stimulator, a signal transduction
inhibitor, a sodium channel inhibitor, a STAT-3 modulator, a Stem cell antigen-1 inhibitor, a
superoxide dismutase modulator, a T cell surface glycoprotein CD28 inhibitor, a T-cell
surface glycoprotein CD8 inhibitor, a TGF beta agonist, a TGF beta antagonist, a
thromboxane synthetase inhibitor, a thymic stromal lymphoprotein ligand inhibitor, a
thymosin agonist, a thymosin beta 4 ligand, a TLR-8 agonist, a TLR-9 agonist, a TLR9 gene
stimulator, a Topoisomerase IV inhibitor, a Troponin I fast skeletal muscle stimulator, a
Troponin T fast skeletal muscle stimulator, a Type I IL-1 receptor antagonist, a Type II TNF
receptor modulator, an ion channel modulator, a uteroglobin stimulator, and a VIP agonist.
Specific agents that may be used in combination with the present JAK inhibitor
compound 1 include, but are not limited to rosiptor acetate, umeclidinium bromide,
secukinumab, metenkefalin acetate, tridecactide acetate, fluticasone propionate, alpha-
cyclodextrin-stabilized sulforaphane, tezepelumab, mometasone furoate, BI-1467335,
dupilumab, aclidinium, formoterol, AZD-1419, HI-1640V, rivipansel, CMP-001, mannitol,
ANB-020, omalizumab, tregalizumab, Mitizax, benralizumab, golimumab, roflumilast,
imatinib, REGN-3500, masitinib, apremilast, RPL-554, Actimmune, adalimumab,
rupatadine, parogrelil, MK-1029, beclometasone dipropionate, formoterol fumarate,
mogamulizumab, seratrodast, UCB-4144, nemiralisib, CK-2127107, fevipiprant, danirixin,
bosentan, abatacept, EC-18, duvelisib, dociparstat, ciprofloxacin, salbutamol HFA,
erdosteine, PrEP-001, nedocromil, CDX-0158, salbutamol, enobosarm, R-TPR-022,
lenzilumab, fluticasone furoate, vilanterol trifenatate, fluticasone propionate, salmeterol, PT-
007, PRS-060, remestemcel-L, citrulline, RPC-4046, nitric oxide, DS-102, gerilimzumab,
Actair, fluticasone furoate, umeclidinium, vilanterol, AG-NPP709, Gamunex, infliximab,
Ampion, acumapimod, canakinumab, INS-1007, CYP-001, sirukumab, fluticasone
propionate, mepolizumab, pitavastatin, solithromycin, etanercept, ivacaftor, anakinra, MPC-
300-IV, glycopyrronium bromide, aclidinium bromide, FP-025, risankizumab,
glycopyrronium, formoterol fumarate, Adipocell, YPL-001, tiotropium bromide,
glycopyrronium bromide, indacaterol maleate, andecaliximab, olodaterol, esomeprazole, dust
mite vaccine, mugwort pollen allergen vaccine, vamorolone, gefapixant, revefenacin,
gefitinib, ReJoin, tipelukast, bedoradrine, SCM-CGH, SHP-652, RNS-60, brodalumab, BIO-
11006, umeclidinium bromide, vilanterol trifenatate, ipratropium bromide, tralokinumab,
PUR-1800, VX-561, VX-371, olopatadine, tulobuterol, formoterol fumarate, triamcinolone
acetonide, reslizumab, salmeterol xinafoate, fluticasone propionate, beclometasone
dipropionate, formoterol fumarate, tiotropium bromide, ligelizumab, RUTI, bertilimumab,
omalizumab, glycopyrronium bromide, SENS-111, beclomethasone dipropionate, CHF-
5992, LT-4001, indacaterol, glycopyrronium bromide, mometasone furoate, fexofenadine,
glycopyrronium bromide, azithromycin, AZD-7594, formoterol, CHF-6001, batefenterol,
OATD-01, olodaterol, CJM-112, rosiglitazone, salmeterol, setipiprant, inhaled interferon
beta, AZD-8871, plecanatide, fluticasone, salmeterol, eicosapentaenoic acid monoglycerides,
lebrikizumab, RG-6149, QBKPN, Mometasone, indacaterol, AZD-9898, sodium pyruvate,
zileuton, CG-201, imidafenacin, CNTO-6785, CLBS-03, mometasone, RGN-137, procaterol,
formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone, MV-130, GC-1112,
Allergovac depot , MEDI-3506, QBW-251, ZPL-389, udenafil, GSK-3772847,
levocetirizine, AXP-1275, ADC-3680, timapiprant, abediterol, AZD-7594, ipratropium
bromide, salbutamol sulfate, tadekinig alfa, ACT-774312, dornase alfa, iloprost,
batefenterol, fluticasone furoate, alicaforsen, ciclesonide, emeramide, arformoterol, SB-010,
Ozagrel, BTT-1023, Dectrekumab, levalbuterol, pranlukast, hyaluronic acid, GSK-2292767,
Formoterol, NOV-14, Lucinactant, salbutamol, prednisolone, ebastine, dexamethasone
cipecilate, GSK-2586881, BI-443651, GSK-2256294, VR-179, VR-096, hdm-ASIT+,
budesonide, GSK-2245035, VTX-1463, Emedastine, dexpramipexole, levalbuterol, N-6022,
dexamethasone sodium phosphate, PIN-201104, OPK-0018, TEV-48107, suplatast, BI-
1060469, Gemilukast, interferon gamma, dalazatide, bilastine, fluticasone propionate,
salmeterol xinafoate, RP-3128, bencycloquidium bromide, reslizumab, PBF-680, CRTH2
antagonist, Pranlukast, salmeterol xinafoate, fluticasone propionate, tiotropium bromide
monohydrate, masilukast, RG-7990, Doxofylline, abediterol, glycopyrronium bromide, TEV-
46017, ASM-024, fluticasone propionate, glycopyrronium bromide, salmeterol xinafoate,
salbutamol, TA-270, Flunisolide, sodium chromoglycate, Epsi-gam, ZPL-521, salbutamol,
aviptadil, TRN-157, Zafirlukast, Stempeucel, pemirolast sodium, nadolol, fluticasone
propionate + salmeterol xinafoate, RV-1729, salbutamol sulfate, carbon dioxide +
perfluorooctyl bromide, APL-1, dectrekumab + VAK-694, lysine acetylsalicylate, zileuton,
TR-4, human allogenic adipose-derived mesenchymal progenitor cell therapy, MEDI-9314,
PL-3994, HMP-301, TD-5471, NKTT-120, pemirolast, beclomethasone dipropionate,
trantinterol, monosodium alpha luminol, IMD-1041, AM-211, TBS-5, ARRY-502,
seratrodast, recombinant midismase, ASM-8, deflazacort, bambuterol, RBx-10017609,
ipratropium + fenoterol, fluticasone + formoterol, epinastine, WIN-901X, VALERGEN-
DS,OligoG-COPD-5/20, tulobuterol, oxis Turbuhaler, DSP-3025, ASM-024, mizolastine,
budesonide + salmeterol, LH-011, AXP-E, histamine human immunoglobulin, YHD-001,
theophylline, ambroxol + erdosteine, ramatroban, montelukast, pranlukast, AG-1321001,
tulobuterol, ipratropium + salbutamol, tranilast, methylprednisolone suleptanate, colforsin
daropate, repirinast, and doxofylline.
Also described, herein, is a pharmaceutical composition comprising compound 1, or a
pharmaceutically acceptable salt thereof and one or more other therapeutic agents. The
therapeutic agent may be selected from the class of agents specified above and from the list
of specific agent described above. In some embodiments, the pharmaceutical composition is
suitable for delivery to the lungs. In some embodiments, the pharmaceutical composition is
suitable for inhaled or nebulized administration. In some embodiments, the pharmaceutical
composition is a dry powder or a liquid composition.
Described herein is a method of treating a disease or disorder in a mammal
comprising administering to the mammal compound 1 or a pharmaceutically acceptable salt
thereof and one or more other therapeutic agents.
When used in combination therapy, the agents may be formulated in a single
pharmaceutical composition, or the agents may be provided in separate compositions that are
administered simultaneously or at separate times, by the same or by different routes of
administration. Such compositions can be packaged separately or may be packaged together
as a kit. The two or more therapeutic agents in the kit may be administered by the same route
of administration or by different routes of administration.
The compound described has been demonstrated to be a potent inhibitor of the JAK1,
JAK2, JAK3, and TYK2 enzymes in enzyme binding assays, to have potent functional
activity without cytotoxicity in cellular assays, and to exert the pharmacodynamic effects of
JAK inhibition in preclinical models, as described in the following examples.
EXAMPLES
The following synthetic and biological examples are offered to illustrate the
invention, and are not to be construed in any way as limiting the scope of the invention. In
the examples below, the following abbreviations have the following meanings unless
otherwise indicated. Abbreviations not defined below have their generally accepted
meanings.
ACN = acetonitrile
DCC = dicyclohexylcarbodiimide
DIPEA = N,N-diisopropylethylamine
DMF = N,N-dimethylformamide
EtOAc = ethyl acetate
HATU= N,N,N',N'-tetramethyl-O-(7-azabenzotriazolyl)uronium
hexafluorophosphate
LDA = lithium diisopropylamide
min = minute(s)
MTBE = methyl tert-butyl ether
NBS = N-bromosuccinimide
RT = room temperature
THF = tetrahydrofuran
bis(pinacolato)diboron = 4,4,5,5,4',4',5',5'-octamethyl-
[2,2']bi[[1,3,2]dioxaborolanyl]
Pd(dppf)Cl -CH Cl = dichloro(1,1’-bis(diphenylphosphino)-ferrocene)-
2 2 2
dipalladium(II) complex with dichloromethane
Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka,
Sigma, etc.), and used without further purification. Progress of reaction mixtures was
monitored by thin layer chromatography (TLC), analytical high performance liquid
chromatography (anal. HPLC), and mass spectrometry. Reaction mixtures were worked up
as described specifically in each reaction; commonly they were purified by extraction and
other purification methods such as temperature-, and solvent-dependent crystallization, and
precipitation. In addition, reaction mixtures were routinely purified by column
chromatography or by preparative HPLC, typically using C18 or BDS column packings and
conventional eluents. Typical preparative HPLC conditions are described below.
Characterization of reaction products was routinely carried out by mass and H-NMR
spectrometry. For NMR analysis, samples were dissolved in deuterated solvent ( such as
CD OD, CDCl , or d -DMSO), and H-NMR spectra were acquired with a Varian Gemini
3 3 6
2000 instrument (400 MHz) under standard observation conditions. Mass spectrometric
identification of compounds was performed by an electrospray ionization method (ESMS)
with an Applied Biosystems (Foster City, CA) model API 150 EX instrument or a Waters
(Milford, MA) 3100 instrument, coupled to autopurification systems.
Preparative HPLC Conditions
Column: C18, 5 μm. 21.2 x 150 mm or C18, 5 μm 21 x 250 or
C14, 5 μm 21x150 mm
Column temperature: Room Temperature
Flow rate: 20.0 mL/min
Mobile Phases: A = Water + 0.05 % TFA
B = ACN + 0.05 % TFA,
Injection volume: (100-1500 µL)
Detector wavelength: 214 nm
Crude compounds were dissolved in 1:1 water:acetic acid at about 50 mg/mL . A
4 minute analytical scale test run was carried out using a 2.1 x 50 mm C18 column followed
by a 15 or 20 minute preparative scale run using 100 μL injection with the gradient based on
the % B retention of the analytical scale test run. Exact gradients were sample dependent.
Samples with close running impurities were checked with a 21 x 250 mm C18 column and/or
a 21 x 150 mm C14 column for best separation. Fractions containing desired product were
identified by mass spectrometric analysis.
Analytic HPLC Conditions
Method A
Column: Agilent Zorbax Bonus-RP C18, 150 x 4.60 nm, 3.5 micron
Column temperature: 40 ºC
Flow rate: 1.5 mL/min
Injection volume: 5 μL
Sample preparation: Dissolve in 1:1 ACN:1 M HCl
Mobile Phases: A = Water: TFA (99.95:0.05)
B = ACN:TFA (99.95:0.05)
Detector wavelength: 254 nm and 214 nm
Gradient: 26 min total (time (min)/ % B): 0/5, 18/90, 22/90, 22.5/90, 26/5
Method B
Column: Agilent Poroshell 120 Bonus-RP, 4.6 x 150 mm, 2.7 μm
Column temperature: 30 ºC
Flow rate: 1.5 mL/min
Injection volume: 10 μL
Mobile Phases: A = ACN:Water:TFA (2:98:0.1)
B = ACN:Water:TFA (90:10:0.1)
Sample preparation: Dissolve in Mobile phase B
Detector wavelength: 254 nm and 214 nm
Gradient: 60 min total (time (min)/ % B): 0/0, 50/100, 55/100, 55.1/0, 60/0
Preparation 1 : 1-benzylimino-1,4-dihydropyridinamine
A mixture of pyridine-3,4-diamine (445 g, 4.078 mol) and ACN (11.0 L) was stirred
for 80 min from 25 ºC to 15 °C. Benzyl bromide (485 mL, 4.078 mol) was added over 20
min and the reaction mixture was stirred at 20 ºC overnight. The reaction mixture was
cooled to 10 ºC and filtered. To the reactor was added ACN (3 L), which was cooled to 10
ºC. The cake was washed with the reactor rinse and washed again with ACN (3 L) warmed to
ºC. The solid was dried on the filter for 24 h under nitrogen, at 55 ºC under vacuum for 2
h and then at RT overnight and for 4 d to provide the HBr salt of the title compound (1102.2
g, 3.934 mol, 96 % yield). HPLC Method A Retention time 4.12 min.
Preparation 2 : 5-Benzyl(6-bromo-1H-indazolyl)-5H-imidazo[4,5-
c]pyridine
(a) 5-Benzyl(6-bromo-1H-indazolyl)-5H-imidazo[4,5-c]pyridine
A solution of 6-bromo-1H-indazolecarbaldehyde (550 g, 2.444 mol), 1-benzyl
imino-1,4-dihydropyridinamine HBr (721 g, 2.333 mol) and DMAc (2.65 L) was stirred
for 60 min and sodium bisulfite (257 g, 2.468 mol) was added. The reaction mixture was
heated to 135 °C and held for 3 h, and allowed to cool to 20 ºC and held at 20 ºC overnight.
Acetonitrile (8 L) was added and the reaction mixture was stirred for 4 h at 15 ºC. The slurry
was filtered on a pressure filter at medium filtration rate. To the reactor was added ACN (1
L) The cake was washed with the ACN reactor wash and dried under nitrogen overnight and
then under vacuum at 50 ºC for 24 h to provide the HBr salt of the title compound (1264 g,
2.444 mol, 100 % yield, 94 % purity) as a dense wet beige/brown solid. HPLC Method A
Retention time 8.77 min.
A mixture of the product of the previous step (1264 g, 2.444 mol), MeTHF (6 L) and
water (2.75 L) was heated to 65 ºC and sodium hydroxide 50 wt % (254 g, 3.177 mol) was
added over 5 min and the reaction mixture was stirred at 65 ºC for 1 h, cooled to RT, then to
ºC and held for 2 h. The slurry was filtered and the reactor and cake were washed with
MeTHF (1 L). The resulting beige to yellow solid was dried on the filter under nitrogen for 3
d to provide the title compound (475 g, 1.175 mmol, 48 % yield) as a beige/yellow solid.
The mother liquor (about 8 L) was concentrated to about 2 L, whereupon solids began to
crash out., The slurry was heated to 50 ºC, held for 2 h, cooled to 5 ºC over 2 h, stirred
overnight, and filtered. The cake was washed with MeTHF (100 mL) and dried overnight
under vacuum at 40 ºC to provide additional title compound (140 g, 0.346 mol, 14 % yield).
A mixture of the total product of the previous step, combined with the product of a
second batch at the same scale (1500 g, 3.710 mol) and MeTHF (4 L) was stirred at 20 ºC for
2 h and filtered. The reactor and cake were washed with MeTHF (1.5 L). The resulting
beige to yellow solid was dried under nitrogen for 3 d to provide the title compound as a
beige yellow solid (1325 g, 3.184 mol, 86 % yield (overall 68 % yield), 97 % purity). HPLC
Method A Retention time 8.77 min
Preparation 3 : 5-benzyl(6-bromo-1H-indazolyl)-4,5,6,7-tetrahydro-1H-
imidazo[4,5-c]pyridine
To a 15 L flask was added 5-benzyl(6-bromo-1H-indazolyl)-5H-imidazo[4,5-
c]pyridine (440 g, 1.088 mol) followed by MeTHF (4.5 L), methanol (2.25 L) and water
(1.125 L). The slurry was cooled to 20 ºC, stirred for 1 h, and NaBH4 (247 g, 6.530 mol) was
added. The reaction mixture was stirred at 25 °C for 18 h. Water (1.125 L) was added
followed by 20 wt %. sodium chloride solution (1.125 L) and the mixture was stirred for 30
min and the layers allowed to separate. The aqueous layer was drained. A premixed solution
of NaOH (522 g) and water (5 L) was added and the reaction mixture was stirred for 60 min;
the layers were allowed to separate and the aqueous layer was drained. Two additional
batches at the same scale were prepared.
The organic layer from one batch was concentrated under reduced pressure in a 15 L
jacketed reactor with the jacket set at 50 ºC, internal temperature 20 ºC. The additional
batches were added to the reactor and concentrated one at a time resulting in a slurry about 6
L in volume. The slurry was heated to 50 ºC, IPAc (6 L) was added and the mixture was
held at 60 ºC for 1.5 h, cooled to 20 ºC for 10 h, heated to 60 ºC for 50 h, cooled to 20 ºC in 5
h, then cooled to 5 ºC and held for 3 h. The mixture was filtered and the reactor and cake
was washed with a premixed solution of IPAc (1 L) and MeTHF (1 L), precooled to 5 ºC.
The solids were dried under nitrogen on the filter at 40 ºC for 3 d to provide the title
compound (1059 g, 2.589 mol, 79 % yield) as an off-white solid. The material was further
dried in a vacuum oven at 50-60 ºC for 8 h and at 27 ºC for 2 d to provide the title compound
(1043 g, 2.526 mol, 77 % yield, 99 % purity). HPLC Method A Retention time 6.73 min.
Preparation 4: (4-(Benzyloxy)ethylfluorophenyl)trifluoroborate,
potassium
(a) 2-(4-(Benzyloxy)ethylfluorophenyl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane
A mixture of 1-(benzyloxy)bromoethylfluorobenzene (520 g, 1682 mmol)
and dioxane (5193 mL) was purged with nitrogen and then bis(pinacolato)diboron (641 g,
2523 mmol) was added followed by potassium acetate (495 g, 5046 mmol). The reaction
mixture was purged with nitrogen; Pd(dppf)Cl (41.2 g, 50.5 mmol) was added; the reaction
mixture was purged with nitrogen, heated at 103 ºC under nitrogen for 5 h; and cooled to RT.
The reaction mixture was concentrated by vacuum distillation and partitioned between ethyl
acetate (5204 mL) and water (5212 mL). The reaction mixture was filtered through Celite;
the organic layer was washed with brine (2606 mL) followed by solvent removal by vacuum
distillation to provide crude product as a thick black oil (~800 g).
The crude product was dissolved in DCM (1289 mL) and purified by silica gel
chromatography (2627 g silica preslurried in hexane, eluted with 20 % ethyl acetate in
hexanes (10.35 L)). Solvent was removed by vacuum distillation to yield a light yellow oil
(600 g). HPLC Method B Retention time 33.74 min.
(b) (4-(benzyloxy)ethylfluorophenyl)trifluoroborate, potassium
The product of the previous step (200 g, 561 mmol) was mixed with acetone (1011
mL) until complete dissolution and methanol (999 mL) was added followed by
3 M potassium hydrogen difluoride (307 g, 3930 mmol) dissolved in water (1310 mL). The
reaction mixture was stirred for 3.5 h. Most of the organic solvent was removed by vacuum
distillation. Water (759 mL) was added and the resulting thick slurry was stirred for 30 min
and filtered. The cake was washed with water (506 mL) and the solids were dried on the
filter for 30 min. The solids were slurried in acetone (1237 mL) and stirred for 1 h. The
resulting slurry was filtered and the solids washed with acetone (247 mL). The acetone
solution was concentrated by vacuum distillation, and a constant volume (2 L) was
maintained by slow addition of toluene (2983 mL) until all acetone and water had been
distilled. The toluene solution was distilled to a thick yellow slurry by rotary evaporation,
during which time the products precipitated as white solids. An additional portion of toluene
(477 mL) was added to the mixture and stirred for 1 h. The mixture was then filtered and
rinsed with toluene (179 mL) and dried under vacuum at 50 ºC for 24 h to provide the title
compound (104 g, 310 mmol, 55 % yield) as a free-flowing, fluffy, slightly off-white solid.
HPLC Method B Retention time 27.71 min.
Preparation 5: 5-Benzyl(6-(4-(benzyloxy)ethylfluorophenyl)-1H-indazol-
3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine
(a) 5-Benzyl(6-(4-(benzyloxy)ethylfluorophenyl)-1H-indazolyl)-4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridine
A mixture of bis(pinacolato)diboron (250 g, 984 mmol) and IPA (1.88 L) was stirred
to dissolution and then a solution of potassium hydrogen difluoride (538 g, 6.891 mol) in
water (2.31 L) was added portion-wise over 10 min. The reaction mixture was stirred for 1 h
and filtered. The gel-like solids were slurried with water (1.33 L) until the mixture formed a
clear hydrogel and then for another 45 min. The resulting solids/gel were filtered, then
reslurried in acetone (1.08 L), filtered, air dried on the filter for 30 min and dried overnight to
provide a fluffy white solid (196.7 g).
To a 5 L flask was added 5-benzyl(6-bromo-1H-indazolyl)-4,5,6,7-tetrahydro-
1H-imidazo[4,5-c]pyridine (135 g, 331 mmol), (4-(benzyloxy)ethylfluorophenyl)-
trifluoroborate, potassium (133 g, 397 mmol), and the white solid product of the previous
step (40.5 g) followed by MeTHF (1.23 L) and MeOH (1.75 L). The resulting slurry was
degassed three times with nitrogen. To the slurry was added a degassed solution of cesium
carbonate (431 g, 1.323 mol) in water (1.35 L). The slurry was degassed twice, Pd
(amphos) Cl (11.71 g, 16.53 mmol) was added, the slurry was again degassed twice and the
reaction mixture was stirred at 67 ºC overnight and cooled to 20 ºC. The layers were
separated and back extracted with MeTHF (550 mL). The organic layers were combined and
concentrated by rotary evaporation until solids precipitated. MeTHF (700 mL) was added
and the reaction mixture was stirred at 65 ºC. The layers were separated and the aqueous
phase back extracted with MeTHF (135 mL). The organic phases were combined and
concentrated to about 300 mL resulting in a thick orange slurry. To the slurry was added
MeOH (270 mL) followed by 1M HCl (1.325 L) at 20 ºC with rapid stirring. The reaction
mixture was stirred for 5 min and water (1 L) was added and the resulting slurry was stirred
for 1 h. The solids were filtered, washed with water (150 mL), dried on the filter for 10 min
and at 45 ºC under nitrogen for 16 h to provide the 2 HCl salt of the title compound (221.1 g,
351 mmol, 92.2 % purity) as a light yellow solid. HPLC Method B retention time 23.41
min.
Preparation 6: 5-ethylfluoro(3-(4,5,6,7-tetrahydro-1H-imidazo[4,5-
c]pyridinyl)-1H-indazolyl)phenol
To a 1 L flask was added 5-benzyl(6-(4-(benzyloxy)ethylfluorophenyl)-1H-
indazolyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine, 2 HCl (40 g, 63.4 mmol) as a
slurry in ethanol (348 mL) and 1.25 M HCl in MeOH (101 mL) and water (17.14 mL). The
reaction mixture was degassed with nitrogen for 5 min and 10 wt %Pd/C, 50 wt% H O (4.05
g, 1.903 mmol) was added. The reactor was sealed, purged with H pressurized to 1-2 psi.
warmed to 50 ºC, and the reaction mixture was stirred overnight and filtered through Celite.
The reactor and filter were washed with methanol (100 mL).
The filtered solution was combined with the product of a second batch at the 98 mmol
scale and concentrated to 390 g. EtOAc (2.04 L) was added slowly with stirring and then the
solution was cooled to 5 ºC with stirring. Solids were filtered, washed with EtOAc (510 mL),
and dried overnight at 45 ºC under nitrogen to provide the 2 HCl salt of the title compound
(58 g, 80 % yield) as an off-white solid. HPLC Method B retention time 12.83 min.
Example 1: Crystalline hydrate of 5-ethylfluoro(3-(5-(1-methylpiperidin
yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol
To a 3 L flask was added NMP (239 mL) and 5-ethylfluoro(3-(4,5,6,7-
tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, 2 HCl (74.5 g,
165 mmol) with stirring followed by NMP (74 mL). Acetic acid (31.3 mL) was added and
the reaction mixture was warmed to 55 °C for 10 min and then cooled to 25 °C.
1-methylpiperidinone (61.0 mL, 496 mmol) was added in a single portion and the reaction
mixture was stirred at 25 °C for 30 min and cooled to 15 °C. Sodium triacetoxyborohydride
(98 g, 463 mmol) was added and the external jacket was set to 20 °C after 5 min. After 3 h,
ammonium hydroxide (365 mL, 5790 mmol) was added dropwise over 45 min maintaining
the temperature below 25 °C. The reaction mixture was stirred for 1.5 h at 20 °C, forming an
off-white slurry. Methanol (709 mL) was added and the reaction mixture was stirred slowly
overnight at 55 °C. Water (1.19 L) was added over 30 min at 55 °C and the mixture was
cooled to 10 °C, stirred for 2 h, and filtered. The cake was washed with 1:1 MeOH:water
(334 mL), dried on the filter for 20 min and at 45 °C under vacuum with nitrogen bleed to
provide yellow solids (87 g).
To the solids was added 5 % water/acetone (1.5 L) at 55 °C with slow stirring and the
reaction mixture was heated at 55 °C for 6 h, cooled to 10 °C, filtered, and washed with 5 %
water/acetone (450 mL). The solids were dried overnight at 50 °C under vacuum with
nitrogen bleed, equilibrated in air for 20 h, dried in the vacuum oven for 48 h and
equilibrated with air to provide the title compound (71.3 g, 91 % yield) as a free flowing pale
yellow solid. HPLC Method B Retention time 12.29 min.
Example 2: Powder X-Ray Diffraction
The powder X-ray diffraction (PXRD) pattern of the product of Example 1 was
obtained with a Bruker D8-Advance X-ray diffractometer using Cu-K α radiation
(λ = 1.54051 Å) with output voltage of 45 kV and current of 40 mA. The instrument was
operated in Bragg-Brentano geometry with incident, divergence, and scattering slits set to
maximize the intensity at the sample. For measurement, a small amount of powder
(5-25 mg) was gently pressed onto a sample holder to form a smooth surface and subjected to
X-ray exposure. The samples were scanned in 2θ-2θ mode from 2° to 40° in 2θ with a step
size of 0.02° and a scan speed of 0.30°seconds per step. The data acquisition was controlled
by Bruker DiffracSuite measurement software and analyzed by Jade software (version 7.5.1).
The instrument was calibrated with a corundum standard, within ±0.02° two-theta angle.
Observed PXRD two-theta peak positions and d-spacings are shown in Table1.
Table 1: PXRD Data for the Crystalline Hydrate
2-Theta d(Å) Area A%
14.24 81639 45.70
6.20
9.58 9.22 178629 100.00
.34 8.55 30022 16.80
.65 8.30 12801 7.20
11.54 7.66 27220 15.20
12.77 6.93 27705 15.50
13.01 6.80 48785 27.30
13.39 6.61 9261 5.20
16.94 5.23 40031 22.40
17.53 5.05 83718 46.90
18.67 4.75 9542 5.30
19.28 4.60 152922 85.60
.02 4.43 22391 12.50
.61 4.31 30308 17.00
21.51 4.13 92875 52.00
22.10 4.02 37495 21.00
3.90 13802 7.70
22.79
23.22 3.83 12117 6.80
.16 3.54 13792 7.70
28.80 3.10 14487 8.10
29.62 3.01 14810 8.30
.20 2.96 9709 5.40
Biological Assays
-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-
c]pyridinyl)-1H-indazolyl)phenol (compound 1) has been characterized in the
following biological assays.
Assay 1: Biochemical JAK Kinase Assays
A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were
carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM
MgCl , and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged
STAT1 peptide substrate were obtained from Life Technologies.
The serially diluted compound was pre-incubated with each of the four JAK enzymes
and the substrate in white 384-well microplates (Corning) at ambient temperature for 1h.
ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1%
DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM,
and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM,
1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase
reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL
preparation of EDTA (10mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody
(Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life
Technologies) was added. The plates were allowed to incubate at ambient temperature for 1h
before being read on the EnVision reader (Perkin Elmer). Emission ratio signals
(520nm/495nm) were recorded and utilized to calculate the percent inhibition values based
on DMSO and background controls.
For dose-response analysis, percent inhibition data were plotted vs. compound
concentrations, and IC values were determined from a 4-parameter robust fit model with
the Prism software (GraphPad Software). Results were expressed as pIC (negative
logarithm of IC ) and subsequently converted to pK (negative logarithm of dissociation
50 i
constant, Ki) using the Cheng-Prusoff equation.
The compound useful in the invention exhibited the following enzyme potency.
Table 2
JAK 1 JAK 2 JAK 3 Tyk2
pK pK pK pK
i i i i
.2 10.8 9.7 9.8
Assay 2: Cellular JAK Potency Assay: Inhibition of IL-13
The AlphaScreen JAKI cellular potency assay was carried out by measuring
interleukin-13 (IL-13, R&D Systems) induced STAT6 phosphorylation in BEAS-2B human
lung epithelial cells (ATCC). The anti-STAT6 antibody (Cell Signaling Technologies) was
conjugated to AlphaScreen acceptor beads (Perkin Elmer), while the anti-pSTAT6 (pTyr641)
antibody (Cell Signaling Technologies) was biotinylated using EZ-Link Sulfo-NHS-Biotin
(Thermo Scientific).
BEAS-2B cells were grown at 37°C in a 5% CO humidified incubator in 50%
DMEM/50% F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 100
U/mL penicillin, 100 µg/mL streptomycin (Life Technologies), and 2 mM GlutaMAX (Life
Technologies). On day 1 of the assay, cells were seeded at a 7,500 cells/well density in white
poly-D-lysine-coated 384-well plates (Corning) with 25µL medium, and were allowed to
adhere overnight in the incubator. On day 2 of the assay, the medium was removed and
replaced with 12 μL of assay buffer (Hank's Balanced Salt Solution/HBSS, 25mM HEPES,
and 1 mg/ml bovine serum albumin/BSA) containing dose-responses of test compounds. The
compound was serially diluted in DMSO and then diluted another 1000-fold in media to
bring the final DMSO concentration to 0.1%. Cells were incubated with test compounds at
37°C for 1 h, and followed by the addition of 12 μl of pre-warmed IL-13 (80 ng/mL in assay
buffer) for stimulation. After incubating at 37°C for 30 min, the assay buffer (containing
compound and IL-13) was removed, and 10 μL of cell lysis buffer (25 mM HEPES, 0.1 %
SDS, 1 % NP-40, 5 mM MgCl , 1.3 mM EDTA, 1 mM EGTA, and supplement with
Complete Ultra mini protease inhibitors and PhosSTOP from Roche Diagnostics). The plates
were shaken at ambient temperature for 30min before the addition of detection reagents. A
mixture of biotin-anti-pSTAT6 and anti-STAT6 conjugated acceptor beads was added first
and incubated at ambient temperature for 2h, followed by the addition of streptavidin
conjugated donor beads (Perkin Elmer). After a minimum of 2 h incubation, the assay plates
were read on the EnVision plate reader. AlphaScreen luminescence signals were recorded
and utilized to calculate the percent inhibition values based on DMSO and background
controls.
For dose-response analysis, percent inhibition data were plotted vs. compound
concentrations, and IC values were determined from a 4-parameter robust fit model with the
Prism software. Results may also be expressed as the negative logarithm of the IC value,
pIC . The compound useful in the invention exhibited a pIC value of 8.2 in this assay.
50 50
Assay 3: Cellular JAK Potency Assay: Inhibition of IL-2/anti-CD3 Stimulated
IFNγ in human PBMCs
The potency of the test compound for inhibition of interleukin-2 (IL-2)/anti-CD3
stimulated interferon gamma (IFNγ) was measured in human peripheral blood mononuclear
cells (PBMCs) isolated from human whole blood (Stanford Blood Center). Because IL-2
signals through JAK, this assay provides a measure of JAK cellular potency.
(1) Human peripheral blood mononuclear cells (PBMC) were isolated from human
whole blood of healthy donors using a ficoll gradient. Cells were cultured in a 37 °C, 5 %
CO humidified incubator in RPMI (Life Technologies) supplemented with 10 % Heat
Inactivated Fetal Bovine Serum (FBS, Life Technologies), 2 mM Glutamax (Life
Technologies), 25 mM HEPES (Life Technologies) and 1X Pen/Strep (Life Technologies).
Cells were seeded at 200,000 cells/well in media (50 µL) and cultured for 1 h. Compounds
were serially diluted in DMSO and then diluted another 500-fold (to a 2x final assay
concentration) in media. Test compounds (100 µL/well) were added to cells, and incubated at
37 °C, 5 % CO for 1 h, followed by the addition of IL-2 (R&D Systems; final concentration
100 ng/mL) and anti-CD3 (BD Biosciences; final concentration 1 μg/mL) in pre-warmed
assay media (50 μL) for 24 h.
(2) After cytokine stimulation, cells were centrifuged at 500 g for 5 min and
supernatants removed and frozen at -80 ºC. To determine the inhibitory potency of the test
compound in response to IL-2/anti-CD3, supernatant IFNγ concentrations were measured via
ELISA (R&D Systems). IC values were determined from analysis of the inhibition curves
of concentration of IFNγ vs compound concentration. Data are expressed as pIC (negative
decadic logarithm IC ) values. The compound useful in the invention exhibited a pIC value
50 50
of about 7.3 in this assay.
Assay 4: Cellular JAK Potency Assay: Inhibition of IL-2 Stimulated pSTAT5 in
CD4+ T cells
The potency of the test compound for inhibition of interleukin-2 (IL-2)/anti-CD3
stimulated STAT5 phosphorylation was measured in CD4-positive (CD4+) T cells in human
peripheral blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford
Blood Center) using flow cytometry. Because IL-2 signals through JAK, this assay provides
a measure of JAK cellular potency.
CD4+ T cells were identified using a phycoerythrobilin (PE) conjugated anti-CD4
antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor 647 conjugated anti-
pSTAT5 antibody (pY694, Clone 47, BD Biosciences) was used to detect STAT5
phosphorylation.
(1) The protocol of Assay 3 paragraph (1) was followed with the exception that the
cytokine stimulation with anti-CD3 was performed for 30 min instead of 24 h.
(2) After cytokine stimulation, cells were fixed with pre warmed fix solution (200 µL;
BD Biosciences) for 10 min at 37 °C, 5 % CO , washed twice with DPBS buffer (1 mL, Life
Technologies), and resuspended in ice cold Perm Buffer III (1000 µL, BD Biosciences) for
min at 4 °C. Cells were washed twice with 2 % FBS in DPBS (FACS buffer), and then
resuspended in FACS buffer (100 µL) containing anti-CD4 PE (1:50 dilution) and anti-CD3
anti-CD3Alexa Fluor 647 (1:5 dilution) for 60 min at room temperature in the dark. After
incubation, cells were washed twice in FACS buffer before being analyzed using a LSRII
flow cytometer (BD Biosciences). To determine the inhibitory potency of test compounds in
response to IL-2/anti-CD3, the median fluorescent intensity (MFI) of pSTAT5 was measured
in CD4+ T cells. IC values were determined from analysis of the inhibition curves of MFI
vs compound concentration. Data are expressed as pIC (negative decadic logarithm IC )
50 50
values. The compound useful in the invention exhibited a pIC value of about 7.7 in this
assay.
Assay 5: Cellular JAK Potency Assay: Inhibition of IL-4 Stimulated pSTAT6 in
CD3+ T cells
The potency of the test compound for inhibition of interleukin-4 (IL-4) stimulated
STAT6 phosphorylation was measured in CD3-positive (CD3+) T cells in human peripheral
blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford Blood
Center) using flow cytometry. Because IL-4 signals through JAK, this assay provides a
measure of JAK cellular potency.
CD3+ T cells were identified using a phycoerythrobilin (PE) conjugated anti-CD3
antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor 647 conjugated anti-
pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences) was used to detect STAT6
phosphorylation.
Human peripheral blood mononuclear cells (PBMC) were isolated from human whole
blood of healthy donors as in Assays 3 and 4. Cells were seeded at 250,000 cells/well in
media (200 µL), cultured for 1 h and then resuspended in assay media (50 µL) (RPMI
supplemented with 0.1% bovine serum albumin (Sigma), 2mM Glutamax, 25mM HEPES
and 1X Penstrep) containing various concentrations of test compounds. Compounds were
serially diluted in DMSO and then diluted another 500-fold (to a 2x final assay
concentration) in assay media. Test compounds (50 μL) were incubated with cells at 37°C,
% CO for 1 h, followed by the addition of IL-4 (50 μL) (R&D Systems; final concentration
20 ng/mL) in pre-warmed assay media for 30 min. After cytokine stimulation, cells were
fixed with pre-warmed fix solution (100 µL) (BD Biosciences) for 10 min at 37°C, 5% CO ,
washed twice with FACS buffer (1 mL) (2% FBS in DPBS), and resuspended in ice cold
Perm Buffer III (1000 µL) (BD Biosciences) for 30 min at 4°C. Cells were washed twice
with FACS buffer, and then resuspended in FACS buffer (100 µL) containing anti-CD3 PE
(1:50 dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5 dilution) for 60 min at room
temperature in the dark. After incubation, cells were washed twice in FACS buffer before
being analyzed using a LSRII flow cytometer (BD Biosciences).
To determine the inhibitory potency of the test compound in response to IL-4, the
median fluorescent intensity (MFI) of pSTAT6 was measured in CD3+ T cells. IC values
were determined from analysis of the inhibition curves of MFI vs compound concentration.
Data are expressed as pIC (negative decadic logarithm IC ). The compound useful in the
50 50
invention exhibited a pIC value of 8.1 in this assay.
Assay 6: Cellular JAK Potency Assay: Inhibition of IL-6 Stimulated pSTAT3 in
CD3+ T cells
A protocol analogous to that of Assay 5 was used to determine the potency of the test
compound for inhibition of interleuken-6 (IL-6) stimulated STAT3 phosphorylation. An
Alexa Fluor 647 conjugated anti-pSTAT3 antibody (pY705, Clone 4/P, BD Biosciences) was
used to detect STAT3 phosphorylation.
The compound useful in the invention exhibited a pIC value of 7.4 in this assay.
Assay 7: Cellular JAK Potency Assay: Inhibition of IFNγ-Induced pSTAT1
The potency of the test compound for inhibition of interferon gamma (IFNγ)
stimulated STAT1 phosphorylation was measured in CD14-positive (CD14+) monocytes
derived from human whole blood (Stanford Blood Center) using flow cytometry. Because
IFNγ signals through JAK, this assay provides a measure of JAK cellular potency.
Monocytes were identified using a fluorescein isothiocyanate (FITC) conjugated anti-
CD14 antibody (Clone RM052, Beckman Coulter), and an Alexa Fluor 647 conjugated anti-
pSTAT1 antibody (pY701, Clone 4a, BD Biosciences) was used to detect STAT1
phosphorylation.
Human peripheral blood mononuclear cells (PBMC) were isolated from human whole
blood of healthy donors using a ficoll gradient. Cells were cultured in a 37 °C, 5 % CO
humidified incubator in RPMI (Life Technologies) supplemented with 10 % Fetal Bovine
Serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life
Technologies) and 1X Pen/Strep (Life Technologies). Cells were seeded at 250,000
cells/well in media (200 µL), cultured for 2 h and resuspended in assay media (50 µL)
(RPMI supplemented with 0.1 % bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM
HEPES and 1X Penstrep) containing various concentrations of test compounds. The
compound was serially diluted in DMSO and then diluted another 1000-fold in media to
bring the final DMSO concentration to 0.1 %. Test compound dilutions were incubated with
cells at 37 °C, 5 % CO for 1 h, followed by the addition of pre-warmed IFNγ (R&D
Systems) in media (50 µL) at a final concentration of 0.6 ng/mL for 30 min. After cytokine
stimulation, cells were fixed with pre-warmed fix solution (100 µL) (BD Biosciences) for 10
min at 37 °C, 5 % CO , washed twice with FACS buffer (1 mL) (1% BSA in PBS),
resuspended in 1:10 anti-CD14 FITC:FACS buffer (100 µL), and incubated at 4 °C for
min. Cells were washed once, and then resuspended in ice cold Perm Buffer III (BD
Biosciences) (100 µL) for 30 min at 4 °C. Cells were washed twice with FACS buffer, and
then resuspended in 1:10 anti-pSTAT1 Alexa Fluor 647:FACS buffer (100 µL) for 30 min at
RT in the dark, washed twice in FACS buffer, and analyzed using a LSRII flow cytometer
(BD Biosciences).
To determine the inhibitory potency of the test compound, the median fluorescent
intensity (MFI) of pSTAT1 was measured in CD14+ monocytes. IC values were
determined from analysis of the inhibition curves of MFI vs compound concentration. Data
are expressed as pIC (negative decadic logarithm IC ) values The compound useful in the
50 50
invention exhibited a pIC value of about 7.5 in this assay.
Assay 8: Ocular Pharmacokinetics in Rabbit Eyes
The objective of this assay was to determine the pharmacokinetics of the test
compound in rabbit ocular tissues.
Solution formulation
The compound useful in the invention, 5-ethylfluoro(3-(5-(1-methylpiperidin
yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol (1) was
dissolved in either 10 % 2-hydroxypropyl-β-cyclodextrin to attain a target concentration of
4 mg/mL or in purified water to attain a target concentration of 1 mg/mL . Bilateral
intravitreal injection (50 µL/eye) of the solution of test compound was administered to New
Zealand white rabbits in two dose groups, 200 µg/eye and 50 µg/eye, respectively, for the
cyclodextrin and water vehicle formulations, respectively. The test compound concentration
was measured in ocular tissues: vitreous, aqueous, retina/choroid and iris-ciliary body at pre-
determined time points post injection (30 min, 4 h, 1 d, 3 d, 7 d, 14 d). Two rabbits (four
eyes) were dosed for each time point. In the vitreous tissue, compound 1 exhibited a two-
phase decrease in concentration characterized by an initial decrease in concentration with a
half-life of approximately 12 hours and finally a terminal half-life of approximately 3.6 days.
The compound was found to distribute quickly into the retinal and choroidal region as well
and shows a similar pharmacokinetic profile as in the vitreous tissue.
Suspension formulation
A suspension formulation was prepared by combining crystalline compound 1 of
Example 1 with 0.5 % hydroxypropyl methylcellulose (HPMC E5) + 0.02 % Tween 80 to
attain a target concentration of 10 mg/mL. Bilateral intravitreal injection (50 µL/eye) of the
suspension of test compound was administered to New Zealand white rabbits (500 µg/eye).
The test compound concentration was measured in ocular tissues as in the suspension
formulation assay at 30 min, 2 wks, 4 wks, 6 wks, and 8 wks post injection. The compound
showed a linear decrease in drug concentration in the vitreous from 30 min to 6 weeks with a
clearance rate of approximately 3 µg/mL/day. The behavior is consistent with the solubility
of compound 1 in the vehicle and the ocular pharmacokinetic behavior in the solution
formulation. The drug concentration in plasma was measured and found to be at least 3
orders of magnitude lower than the concentration in vitreous tissue.
Assay 9: Pharmacodynamic Assay: Inhibition of IL6-induced pSTAT3 in Rats
The ability of a single intravitreal administration of test compound to inhibit IL-6
induced pSTAT3 was measured in rat retina/choroid homogenates.
Suspension formulations were prepared by combining crystalline compound 1 of
Example 1 with 0.5 % hydroxypropyl methylcellulose (HPMC E5 LV), 0.02 % Tween 80,
and 0.9 % sodium chloride in purified water to attain target concentrations of 3 mg/mL and
mg/mL.
Female Lewis rats were intravitreally (IVT) dosed (5 µL per eye) with the suspension
formulations or with the drug vehicle. Three days later, IL-6 (Peprotech; 0.1 mg/mL; 5 µL
per eye) or vehicle was intravitreally administered to induce pSTAT3. Ocular tissues were
dissected one hour after the second IVT injection with IL-6. The retina/choroid tissues were
homogenized and pSTAT3 levels were measured using an ELISA (Cell Signaling
Technology). The percent inhibition of ILinduced pSTAT3 was calculated in comparison
to the vehicle/vehicle and vehicle/IL-6 groups. Inhibition of greater than 100 % reflects a
reduction of pSTAT3 levels to below those observed in the vehicle/vehicle group.
With a 3 day pre-treatment prior to IL-6 challenge, the 15 µg dose and the 50 µg dose
of the compound useful in the invention administered by the suspension formulation
inhibited ILinduced pSTAT3 by 33 % and 109 %, respectively in the retina/choroid
tissues.
Assay 10: Pharmacodynamic Assay: Inhibition of IFNγ-induced IP-10 in Rabbits
The ability of a single intravitreal administration of test compound to inhibit
interferon-gamma (IFNγ) induced IP-10 protein levels was measured in rabbit vitreous and
retina/choroid tissues.
Solution formulations at concentrations of 1 mg/mL and 4 mg/mL of compound 1 of
Example 1 were prepared as in Assay 8. A suspension formulation was prepared by
combining crystalline compound 1 of Example 1 with 0.5 % hydroxypropyl methylcellulose
(HPMC E5), 0.02 % Tween 80, and 9 mg/mL sodium chloride in purified water to attain a
target concentration of 20 mg/mL.
Male, New Zealand White rabbits (Liveon Biolabs, India) were used for the studies.
Animals were acclimated after arrival at the research facilities (Jubilant Biosys Ltd., India).
Each rabbit was given a total of two intravitreal (IVT) injections with a total dose volume of
50 µL per eye. The first IVT injection (45 µL per eye) delivered test compound or vehicle at
a prescribed time point (i.e. 24 hours for the solution formulations or 1 week for the
suspension formulation). The second IVT injection (5 µL per eye) delivered IFNγ (1 µg/eye;
Stock solution 1 mg/mL; Kingfisher Biotech) or vehicle for the induction of IP-10. In brief,
on the day of the injections, rabbits were anesthetized with an intramuscular injection of
ketamine (35 mg/kg) and xylazine (5 mg/kg). Once deeply anesthetized, each eye was rinsed
with sterile saline and IVT injections were performed using a 0.5 mL insulin syringe (50
units=0.5 mL) with a 31-gauge needle at the supra-nasal side of the both eyes by marking the
position with a Braunstein fixed caliper (2 3/4”) 3.5 mm from the rectus muscle and 4 mm
from the limbus.
Tissues were collected 24 hours after the second IVT injection with IFNγ. Vitreous
humor (VH) and retina/choroid tissues (R/C) were collected and homogenized, and IP-10
protein levels were measured using a rabbit CXCL10 (IP-10) ELISA kit (Kingfisher
Biotech). The percent inhibition of IFNγ-induced IP-10 was calculated in comparison to the
vehicle/vehicle and vehicle/IFNγ groups.
When dosed as a solution, with a 24 hour pre-treatment prior to the IFNγ challenge,
45 µg of compound 1 inhibited IFNγ-induced IP-10 by 70% and 86% in the vitreous humor
and retina/choroid tissue, respectively, while 180 µg of the compound inhibited IFNγ-
induced IP-10 by 91% and 100% in the vitreous humor and retina/choroid tissue,
respectively.
With a 1 week pre-treatment prior to the IFNγ challenge, the crystalline suspension
formulation of compound 1 inhibited IFNγ-induced IP-10 by 100% in both the vitreous
humor and retina/choroid tissues.
Assay 11: Pharmacokinetics in Plasma and Lung in Mouse
Plasma and lung levels of the test compound and the ratio thereof was determined in
the following manner. BALB/c mice from Charles River Laboratories were used in the
assay. Test compounds were individually formulated in 20% propylene glycol in pH 4
citrate buffer at a concentration of 0.2 mg/mL and 50 uL of the dosing solution was
introduced into the trachea of a mouse by oral aspiration. At various time points (typically
0.167, 2, 6, 24hr) post dosing, blood samples were removed via cardiac puncture and intact
lungs were excised from the mice. Blood samples were centrifuged (Eppendorf centrifuge,
5804R) for 4 minutes at approximately 12,000 rpm at 4°C to collect plasma. Lungs were
padded dry, weighed, and homogenized at a dilution of 1:3 in sterile water. Plasma and lung
levels of test compound were determined by LC-MS analysis against analytical standards
constructed into a standard curve in the test matrix. A lung to plasma ratio was determined as
the ratio of the lung AUC in µg hr/g to the plasma AUC in µg hr/mL, where AUC is
conventionally defined as the area under the curve of test compound concentration vs. time.
The compound useful in the invention exhibited exposure in lung about 55 times
greater than exposure in plasma in mouse.
Assay 12: Murine (Mouse) model of IL-13 induced pSTAT6 induction in lung
tissue
Il-13 is an important cytokine underlying the pathophysiology of asthma (Kudlacz et
al. Eur. J. Pharmacol, 2008, 582,154-161). IL-13 binds to cell surface receptors activating
members of the Janus family of kinases (JAK) which then phosphorylate STAT6 and
subsequently activates further transcription pathways. In the described model, a dose of IL-
13 was delivered locally into the lungs of mice to induce the phosphorylation of STAT6
(pSTAT6) which is then measured as the endpoint.
Adult balb/c mice from Harlan were used in the assay. On the day of study, animals
were lightly anesthetized with isoflurane and administered either vehicle or test compound
(0.5 mg/mL, 50 μL total volume over several breaths) via oral aspiration. Animals were
placed in lateral recumbency post dose and monitored for full recovery from anesthesia
before being returned to their home cage. Four hours later, animals were once again briefly
anesthetized and challenged with either vehicle or IL-13 (0.03 μg total dose delivered, 50 μL
total volume) via oral aspiration before being monitored for recovery from anesthesia and
returned to their home cage. One hour after vehicle or IL-13 administration, lungs were
collected for both pSTAT6 detection using an anti-pSTAT6 ELISA (rabbit mAb
capture/coating antibody; mouse mAb detection/report antibody: anti-pSTAT6-pY641;
secondary antibody: anti-mouse IgG-HRP) and analyzed for total drug concentration as
described above in Assay 11.
Activity in the model is evidenced by a decrease in the level of pSTAT6 present in
the lungs of treated animals at 5 hours compared to the vehicle treated, IL-13 challenged
control animals. The difference between the control animals which were vehicle- treated, IL-
13 challenged and the control animals which were vehicle-treated, vehicle challenged
dictated the 0% and 100% inhibitory effect, respectively, in any given experiment. The
compound useful in the invention exhibited about 60 % inhibition of STAT6 phosphorylation
at 4 hours after IL-13 challenge.
Assay 13: Murine model of Alternaria alternata-induced eosinophilic
inflammation of the lung
Airway eosinophilia is a hallmark of human asthma. Alternaria alternata is a fungal
aeroallergen that can exacerbate asthma in humans and induces eosinophilic inflammation in
the lungs of mice (Havaux et al. Clin Exp Immunol. 2005, 139(2):179-88). In mice, it has
been demonstrated that alternaria indirectly activates tissue resident type 2 innate lymphoid
cells in the lung, which respond to (e.g. IL-2 and IL-7) and release JAK-dependent cytokines
(e.g. IL-5 and IL-13) and coordinate eosinophilic inflammation (Bartemes et al. J Immunol.
2012, 188(3):1503-13).
Seven- to nine-week old male C57 mice from Taconic were used in the study. On the
day of study, animals were lightly anesthetized with isoflurane and administered either
vehicle or test compound (0.1 – 1.0 mg/mL, 50 μL total volume over several breaths) via
oropharyngeal aspiration. Animals were placed in lateral recumbency post dose and
monitored for full recovery from anesthesia before being returned to their home cage. One
hour later, animals were once again briefly anesthetized and challenged with either vehicle or
alternaria extract (200 ug total extract delivered, 50 μL total volume) via oropharyngeal
aspiration before being monitored for recovery from anesthesia and returned to their home
cage. Forty-eight hours after alternaria administration, bronchoalveolar lavage fluid (BALF)
was collected and eosinophils were counted in the BALF using the Advia 120 Hematology
System (Siemens).
Activity in the model is evidenced by a decrease in the level of eosinophils present in
the BALF of treated animals at forty-eight hours compared to the vehicle treated, alternaria
challenged control animals. Data are expressed as percent inhibition of the vehicle treated,
alternaria challenged BALF eosinophils response. To calculate percent inhibition, the
number of BALF eosinophils for each condition is converted to percent of the average
vehicle treated, alternaria challenged BALF eosinophils and subtracted from one-hundred
percent. The compound useful in the invention exhibited about 88 % inhibition of BALF
eosinophil counts at forty-eight hours after alternaria challenge.
Assay 14: Murine model of LPS/G-CSF/IL-6/IFNγ cocktail-induced airway
neutrophilic inflammation of the lung model
Airway neutrophilia is a hallmark of a range of respiratory disease in humans.
Compound 1 was tested in a model of neutrophilic airway inflammation using a LPS/G-
CSF/IL-6/IFNγ cocktail to induce airway neutrophilia.
Seven- to nine-week old male Balb/C (wildtype) mice from Jackson Laboratory were
used in the study. On the day of study, animals were lightly anesthetized with isoflurane and
administered either vehicle or test compound (1.0 mg/mL, 50 μL total volume over several
breaths) via oropharyngeal aspiration. Animals were placed in lateral recumbency post dose
and monitored for full recovery from anesthesia before being returned to their home cage.
One hour later, animals were once again briefly anesthetized and challenged with either
vehicle or LPS; 0.01 mg/kg/G-CSF; 5 μg/IL-6; 5 μg/IFNγ; 5 μg (100 μL total volume) via
oropharyngeal aspiration (OA). Twenty-four hours after the LPS/G-CSF/IL-6/IFNγ cocktail
administration, bronchoalveolar lavage fluid (BALF) was collected and neutrophils were
counted.
Upon OA treatment with compound 1, there was a statistically significant reduction
of the airway neutrophils (84% compared to vehicle treated mice), demonstrating that the
blockade of JAK-dependent signaling has effects on neutrophilic airway inflammation.
Assay 15: Inhibition of IFNγ and IL-27 induced chemokines CXCL9 and
CXCL10 in human 3D airway cultures
EpiAirway tissue cultures were obtained from Mattek (AIR-100). Cultures were
derived from asthmatic donors. In a cell culture insert, human derived tracheal/bronchial
epithelial cells were grown and differentiated on a porous membrane support, allowing an
air-liquid interface with warmed culture medium below the cells and a gaseous test
atmosphere above. Tissues were cultured in maintenance media (Mattek, AIRMM) in a
37°C, 5% CO2 humidified incubator. Four donors were tested. On Day 0, tissue cultures
were treated with test compounds at 10µM, 1µM and/or 0.1µM. Compounds were diluted in
dimethyl sulfoxide (DMSO, Sigma) to a final concentration of 0.1%. DMSO at 0.1% was
used as a vehicle control. Test compounds were incubated with cultures for 1 hour at 37°C,
5% CO , followed by the addition of pre-warmed media containing IFNγ (R&D Systems) or
IL-27 (R&D Systems) at a final concentration at 100ng/ml. Tissue cultures were maintained
for 8 days. Media was replaced every 2 days with fresh media containing compounds and
IFNγ or IL-27. On Day 8, tissue cultures and supernatants were collected for analysis.
Supernatant samples were assayed for CXCL10 (IP-10) and CXCL9 (MIG) using luminex
analysis (EMD Millipore). Data is expressed as % Inhibition +/- standard deviation
(±STDV). Percent inhibition was determined by compound inhibitory potency against IFNγ
or IL-27 induced CXCL10 or CXCL9 secretion compared to vehicle treated cells. Data is the
average from 3 or 4 donors. Compound 1 was able to inhibit IFNγ induced CXCL10
secretion by 99% ±2.0 (at 10µM), 71% ±19 (at µM) and 17% ±12 (at 0.1µM) when
compared to vehicle control. Compound 1 was able to inhibit IFNγ induced CXCL9
secretion by 100% ±0.3 (at 10µM), 99% ±0.9 (at 1µM) and 74% ±17 (at 0.1µM) when
compared to vehicle. Compound 1 was able to inhibit IL-27 induced CXCL10 secretion by
108% ±11 (at 10µM), 98% ±10 (at 1µM) and 73% ±8.5 (at 0.1µM) when compared to
vehicle control. Compound 1 was able to inhibit IL-27 induced CXCL9 secretion by 100%
±0 (at 10µM), 95% ±3.7 (at 1µM) and 75% ±3.5 (at 0.1µM) when compared to vehicle
control.
Assay 16: IL-5 mediated eosinophil survival assay
The potency of the test compound for IL-5 mediated eosinophil survival was
measured in human eosinophils isolated from human whole blood (AllCells). Because IL-5
signals through JAK, this assay provides a measure of JAK cellular potency.
Human eosinophils were isolated from fresh human whole blood (AllCells) of healthy
donors. Blood was mixed with 4.5% Dextran (Sigma-Aldrich) in a 0.9% sodium chloride
solution (Sigma-Aldrich). Red blood cells were left to sediment for 35 minutes. The
leukocyte rich upper layer was removed and layered over Ficoll-Paque (GE Healthcare) and
centrifuged at 600g for 30 minutes. The plasma and mononuclear cell layers were removed
before the granulocyte layer was lysed with water to remove any contaminating red blood
cells. Eosinophils were further purified using a human eosinophil isolation kit (Miltenyi
Biotec). A fraction of the purified eosinophils were incubated with anti-CD16 FITC
(Miltenyi Biotec) for 10 minutes at 4°C in the dark. Purity was analyzed using a LSRII flow
cytometer (BD Biosciences).
Cells were cultured in a 37°C, 5% CO humidified incubator in RPMI 1640 (Life
Technologies) supplemented with 10% Heat Inactivated Fetal Bovine Serum (FBS, Life
Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies) and
1X Pen/Strep (Life Technologies). Cells were seeded at 10,000 cells/well in media (50 µL).
The plate was centrifuged at 300g for 5 minutes and supernatants removed. Compounds
were serially diluted in DMSO and then diluted another 500-fold to a 2x final assay
concentration in media. Test compounds (50 µL/well) were added to cells, and incubated at
37 °C, 5 % CO for 1 hour, followed by the addition of IL-5 (R&D Systems; final
concentrations 1 ng/mL and 10 pg/ml) in pre-warmed assay media (50 μL) for 72 hours.
After cytokine stimulation, cells were centrifuged at 300 g for 5 min and washed
twice with cold DPBS (Life Technologies). To access viability and apoptosis, cells were
incubated with Propidium Iodide (Thermo Fisher Scientific) and APC Annexin V (BD
Biosciences) and analyzed using a LSRII flow cytometer (BD Biosciences). IC values
were determined from analysis of the viability curves of percent cell viability vs compound
concentration. Data are expressed as pIC (negative decadic logarithm IC ) values.
50 50
Compound 1 exhibited a pIC value of 7.9±0.5 in the presence of 10 pg/ml IL-5 and a pIC
50 50
value of 6.5±0.2 in the presence of 1 ng/ml IL-5.
Assay 17: Pharmacodynamic Assay: Inhibition of IFNγ-induced pSTAT1 in
Rabbit Eyes
The ability of a single intravitreal administration of test compound to inhibit
interferon-gamma (IFNγ) induced phosphorylation of STAT1 protein (pSTAT1) was
measured in rabbit retina/choroid tissue.
A suspension formulation was prepared by combining compound 1 of Example 1,
with 0.5 % hydroxypropyl methylcellulose (HPMC E5), 0.02 % Tween 80, and 9 mg/mL
sodium chloride in purified water to attain a target concentration of 20 mg/mL.
Male, New Zealand White rabbits (Liveon Biolabs, India) were used for the studies.
Animals were acclimated after arrival at the research facilities (Jubilant Biosys Ltd., India).
Each rabbit was given a total of two intravitreal (IVT) injections with a total dose volume of
50 µL per eye. The first IVT injection (45 µL per eye) delivered 0.9 mg of test compound or
vehicle. One week later, a second IVT injection (5 µL per eye) delivered IFNγ (1 µg/eye;
stock solution 1 mg/mL; Kingfisher Biotech) or vehicle for the induction of IP-10. On the
day of the injections, rabbits were anesthetized with an intramuscular injection of ketamine
(35 mg/kg) and xylazine (5 mg/kg). Once deeply anesthetized, each eye was rinsed with
sterile saline and IVT injections were performed using a 0.5 mL insulin syringe
(50 units=0.5 mL) with a 31-gauge needle at the supra-nasal side of the both eyes by marking
the position with a Braunstein fixed caliper (2 3/4”) 3.5 mm from the rectus muscle and 4
mm from the limbus.
Tissues were collected 2 hours after the second IVT injection with IFNγ.
Retina/choroid tissues (R/C) were collected and homogenized, and pSTAT1 levels were
measured by quantitative Western Blot on the ProteinSimple WES instrument. The percent
inhibition of IFNγ-induced pSTAT1 was calculated in comparison to the vehicle/vehicle and
vehicle/IFNγ groups.
With a 1 week pre-treatment prior to the IFNγ challenge, the suspension formulation
of compound 1 of Example 1 inhibited IFNγ-induced pSTAT1 by 85%.
While the present invention has been described with reference to specific aspects or
embodiments thereof, it will be understood by those of ordinary skilled in the art that various
changes can be made or equivalents can be substituted without departing from the true spirit
and scope of the invention. Additionally, to the extent permitted by applicable patent statutes
and regulations, all publications, patents and patent applications cited herein are hereby
incorporated by reference in their entirety to the same extent as if each document had been
individually incorporated by reference herein.
Claims (13)
1. Use of 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro- 1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, or a pharmaceutically-acceptable 5 salt thereof, in the manufacture of a medicament for the treatment of an ocular disease in a mammal.
2. The use of Claim 1, wherein the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, or atopic 10 keratoconjunctivitis.
3. The use of Claim 2, wherein the ocular disease is uveitis or diabetic macular edema. 15 4. The use of Claim 1 wherein 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-
4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, or a pharmaceutically-acceptable salt thereof, is administered by injection.
5. Use of 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro- 20 1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament for the treatment of a respiratory disease in a mammal, wherein the respiratory disease is a lung infection, a helminthic infection, pulmonary arterial hypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, or an infiltrative pulmonary disease.
6. The use of claim 5 wherein the medicament is suitable for administration by inhalation.
7. The use of claim 6 wherein the medicament is suitable for administration by a 30 nebulizer inhaler.
8. The use of claim 6 wherein the medicament is suitable for administration by a dry powder inhaler.
9. Use of 5-ethylfluoro(3-(5-(1-methylpiperidinyl)-4,5,6,7-tetrahydro- 5 1H-imidazo[4,5-c]pyridinyl)-1H-indazolyl)phenol, or a pharmaceutically-acceptable salt thereof, in the manufacture of a medicament for the treatment of a respiratory disease in a mammal, wherein the respiratory disease is drug-induced pneumonitis, fungal induced pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, 10 idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Löffler syndrome, bronchiolitis obliterans organizing pneumonia, or immune-checkpoint-inhibitor induced pneumonitis.
10. The use of claim 9 wherein the medicament is suitable for administration by 15 inhalation.
11. The use of claim 10 wherein the medicament is suitable for administration by a nebulizer inhaler. 20
12. The use of claim 10 wherein the medicament is suitable for administration by a dry powder inhaler.
13. The use according to any one of claims 1 to 12, substantially as herein described with reference to any example thereof.
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US201762492568P | 2017-05-01 | 2017-05-01 | |
US62/492,568 | 2017-05-01 | ||
PCT/US2018/030140 WO2018204233A1 (en) | 2017-05-01 | 2018-04-30 | Methods of treatment using a jak inhibitor compound |
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NZ758109A NZ758109A (en) | 2021-09-24 |
NZ758109B2 true NZ758109B2 (en) | 2022-01-06 |
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