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WO2019117813A1 - Agents thérapeutiques ciblant une kinase d'adhésion focale pour le traitement du glaucome et de la fibrose - Google Patents

Agents thérapeutiques ciblant une kinase d'adhésion focale pour le traitement du glaucome et de la fibrose Download PDF

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WO2019117813A1
WO2019117813A1 PCT/SG2018/050612 SG2018050612W WO2019117813A1 WO 2019117813 A1 WO2019117813 A1 WO 2019117813A1 SG 2018050612 W SG2018050612 W SG 2018050612W WO 2019117813 A1 WO2019117813 A1 WO 2019117813A1
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Prior art keywords
fak
amino
methyl
fibrosis
compound
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PCT/SG2018/050612
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English (en)
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David M. Epstein
Tzee Ling Tina WONG
Permeen A. YUSOFF
Zhu Li YAP
Pamela BL GOH
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National University Of Singapore
Singapore Health Services Pte Ltd
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Publication of WO2019117813A1 publication Critical patent/WO2019117813A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/485—Morphinan derivatives, e.g. morphine, codeine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

Definitions

  • the present invention relates to a treatment for diseases associated with eye- related scarring and post-surgical scarring of glaucoma patients. More particularly, the invention relates to compositions and methods of treating eye-related scarring and fibrosis by inhibiting the active focal adhesion kinase using a small molecule inhibitor.
  • Glaucoma is a leading cause of irreversible blindness in Singapore as well as worldwide with an estimated 70 million people affected, of whom over 6 million suffer from bilateral blindness.
  • current surgical outcomes are limited due to long-term graft failure and rejection, resulting in increased morbidity.
  • Ocular fibrosis and scarring are responsible for the pathogenesis or failure of treatment of almost all the major blinding diseases worldwide.
  • the existing methods to prevent fibrosis after glaucoma surgery use the antimetabolites, Mitomycin C (MMC) and 5-fluorouracil (5-FU). Although these antimetabolites improve the surgical outcome of glaucoma filtration surgery, they have non-specific cytotoxicity which potentially leads to blinding complications like tissue damage, breakdown and infection. In addition, not all patients are responsive to this anti metabolite therapy.
  • FAK focal adhesion kinase
  • TQRb transforming growth factor b
  • COL1A1 collagen1a1
  • aSMA a-smooth muscle actin
  • Focal adhesion kinase is a 125-kDa non-receptor cytoplasmic tyrosine kinase, which is known to play a role in many physiological processes such as regulating cell migration, proliferation and survival of a variety of cell types.
  • FAK Focal adhesion kinase
  • the present invention described herein involves the use of inhibitors of the active focal adhesion kinase (FAK) to reduce post-surgery fibrosis in subjects treated for glaucoma.
  • FAK active focal adhesion kinase
  • an example of one such inhibitor is the small molecule Aminopyrimidine Pyrimidine, Compound 96; diethyl (3-methoxy-4- ⁇ [4-( ⁇ 2- methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3-dihydro-1 H-isoindol-4- yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2-yl]amino ⁇ benzyl)phosphonate.
  • This small molecule was shown to inhibit the active focal adhesion kinase (Phospho-FAK (Tyr397 and Tyr925)) and reduce the expression of the pro-fibrotic markers collagen I, collagen 3, a smooth muscle actin (aSMA) and SPARC (secreted protein, acidic, rich in cysteine).
  • Phospho-FAK Phospho-FAK (Tyr397 and Tyr925)
  • aSMA smooth muscle actin
  • SPARC secreted protein, acidic, rich in cysteine
  • the present invention provides a composition comprising at least one FAK inhibitor for inhibiting scarring and fibrosis of the eye of a subject.
  • Suitable FAK inhibitors include those disclosed in WO2010/141406, the contents of which is incorporated herein by reference. Such compounds have the general formula 1 :
  • R is an optionally substituted aryl or heteroaryl
  • R1 is halogen, CF 3 , CCH, or other suitable substituent
  • X 2 is -(CR 7 R 8 )O-2-
  • each R 7 and R 8 is independently halogen, C 0-3 aliphatic, or -OC 0-3 aliphatic, either of which is optionally halogen substituted, except that in the case of X 2 , R 7 and R 8 are not halogen or -OCo- 3 aliphatic
  • R6 is halogen, -OC 0-3 aliphatic, or C 0-3 aliphatic, either optionally substituted by one or more halogen or by -OCF 3 ;
  • Q1 to Q 4 are independently >CH, >CF, >N, or >N-oxide; except that at least one of Q 2 to Q 4 includes a substituent that includes a phosphinate, phosphonate, or phosphine oxide.
  • the compound has formula 1a:
  • Xi and X 2 are independently -(CR 7 R 8 )o-r;
  • A is CH or N
  • R1 is halogen, -CF 3 , or -CCH
  • R2 is -P(0)R 9 R 1 °
  • R3 is -S(0) 2 R 11 , -S(0) 2 NR 11 R 12 , -C(0)NR 11 R 12 , -C(0)0R 11 , -NR 11 S(0) 2 R 12 , or - NR 11 R 12 ;
  • R4 is -x-y-z, wherein: x is 4-6cyclic; y is absent or ⁇ heterocyclic; and z is absent or Ci-3alkyl optionally substituted by 1-2 hydroxy or Ci- 6 alkoxy groups, or z is hydroxy or -C(0)0- Co-3alkyl; or
  • -x-y-z is Co- 6 alkoxy, C 2-6 alkenyl, C 2-6 alkynyl, or Co- 6 alkyl, each optionally substituted by 1-2 hydroxy groups;
  • R5 is Co-2alkyl optionally substituted by 1-3 independent hydroxy or halogen groups
  • R6 is halo, -Co-3alkoxy, -Co-3alkyl optionally substituted by halo, or -OCFs; each R 7 and R 8 is independently H or -CH3;
  • R 9 and R 10 are independently hydroxy, Ci- 6 alkoxy, or Co- 6 alkyl, any of which can be taken together at any of their atoms to form a ring, or aryl, -O-aryl, or ⁇ heterocyclic, wherein any of the foregoing can be further substituted by ⁇ cyclic;
  • R 11 and R 12 are independently Co- 6 alkyl, which can be taken together at any of their atoms to form a ring containing 1-3 heteroatoms; R 11 or R 12 can independently be taken at any of their atoms with the ring to which R3 is attached to form a ring.
  • composition of any aspect of the invention comprises pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of said at least one FAK inhibitor.
  • the at least one FAK inhibitor is selected from any one of Examples 1-329 disclosed in W02010/141406, or a pharmaceutically acceptable salt or solvate thereof.
  • a suitable FAK inhibitor exemplified herein includes Compound 96; diethyl (3- methoxy-4- ⁇ [4-( ⁇ 2-methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3- dihydro-1 H-isoindol-4-yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2- yl]amino ⁇ benzyl)phosphonate; N-methyl-4-(4-((3-(N-methylmethan-3-ylsulfonamido)pyrazin-2-yl)methylamino)-5-
  • Benzamide 2-[[5-chloro-2-[[2-methoxy-4-(4-morpholinyl)phenyl]amino]-4- pyrimidinyl]amino]-N-methyl-; or a pharmaceutically acceptable salt or solvate thereof for inhibiting scarring and fibrosis of the eye of a subject.
  • the compound is Compound 96; diethyl (3-methoxy-4- ⁇ [4-( ⁇ 2- methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3-dihydro-1 H-isoindol-4- yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2-yl]amino ⁇ benzyl)phosphonate or a pharmaceutically acceptable salt or solvate thereof for inhibiting scarring and fibrosis of the eye of a subject.
  • the scarring and fibrosis occurs post-surgery for glaucoma in the subject.
  • the composition inhibits focal adhesion kinase (FAK) and/or the expression of at least one pro-fibrotic factor.
  • FAK focal adhesion kinase
  • the composition inhibits the phosphorylation of FAK to form FAK pTyr925 and/or FAK pTyr397.
  • the at least one pro-fibrotic factor is selected from the group comprising the pro-fibrotic markers collagen1a1 (COL1A1), a smooth muscle actin (aSMA) and SPARC (secreted protein, acidic, rich in cysteine).
  • COL1A1 pro-fibrotic markers collagen1a1
  • aSMA smooth muscle actin
  • SPARC secreted protein, acidic, rich in cysteine
  • the composition further comprises at least one anti metabolite such as mitomycin C (MMC) and/or 5-fluorouracil (5-FU).
  • MMC mitomycin C
  • 5-fluorouracil 5-fluorouracil
  • the composition further comprises one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the present invention provides the use of at least one FAK inhibitor or variant thereof for the manufacture of a medicament for inhibiting scarring and fibrosis of the eye of a subject.
  • the present invention provides the use of compounds of formula 1 , preferably of formula 1a, or pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for inhibiting scarring and fibrosis of the eye of a subject.
  • the present invention provides the use of a compound selected from the group comprising Compound 96; diethyl (3-methoxy-4- ⁇ [4- ( ⁇ 2-methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3-dihydro-1 H- isoindol-4-yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2-yl]amino ⁇ benzyl)phosphonate;
  • the compound is Compound 96; diethyl (3-methoxy-4- ⁇ [4-( ⁇ 2- methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3-dihydro-1 H-isoindol-4- yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2-yl]amino ⁇ benzyl)phosphonate or pharmaceutically acceptable salt or solvate thereof.
  • the medicament is for inhibiting the scarring and fibrosis that occurs post-surgery for glaucoma in a subject.
  • the medicament is formulated for subconjunctival or intravenous delivery.
  • the medicament inhibits focal adhesion kinase (FAK) and/or the expression of at least one pro-fibrotic factor.
  • FAK focal adhesion kinase
  • the medicament inhibits phosphorylation of FAK to form FAK pTyr925 and/or FAK pTyr397.
  • the at least one pro-fibrotic factor is selected from the group comprising collagenlal (COL1A1), a smooth muscle actin (aSMA) and SPARC (secreted protein, acidic, rich in cysteine).
  • the medicament is formulated for administration below the conjunctiva or intravenously.
  • the present invention provides a method of prophylaxis or treatment of eye related scarring and fibrosis, comprising administering to a subject in need thereof an effective amount of a composition according to any embodiment of the first aspect.
  • the administration is before, during or after surgery for treating glaucoma in the subject.
  • the composition is administered below the conjunctiva or intravenously.
  • the composition is administered to reduce fibrosis caused by an implanted drainage device.
  • the drainage device may be valved or non-valved. It would be understood that the composition could be administered separately from the device or be incorporated with the device for slow release.
  • Figure 1 shows the toxicity of Compound 96 on 3D cultured rabbit Tenon’s fibroblast cells (RTFs).
  • RTFs fibroblast cells
  • FIG. 2 shows dose-response of Compound 96 inhibition of pFAK [Y397] Human Tenon’s fibroblast cells (HTFs) were cultured as described by Seet et al. , J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 11 to 10 6 M. After 48 hr of treatment cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) (Cell signaling technology #3283), the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 3 shows dose-response of Compound 96 inhibition of Collagen.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 -6 M. After 48 hr of treatment cell lysates were prepared and subjected to western analysis. Blots were probed using anti-Collagen 1 NB600-408 antibody (Nevus Biologicais), the loading control was done using the anti-FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 4 shows dose-response of Compound 96 inhibition of Collagen 1 mRNA expression.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 -6 M. After 48 hr of treatment total RNA was extracted and Collagen 1 mRNA level was determined using quantitative real-time PCR.
  • Figure 5 shows dose-response of Compound 96 inhibition of aSMA mRNA expression.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 -6 M. After 48 hr of treatment total RNA was extracted and aSMA mRNA level was determined using quantitative real-time PCR.
  • Figure 6 shows dose-response of Compound 96 inhibition of SPARC mRNA expression. HTFs were cultured as described by Seet et al. , J Cell Mol Med.
  • FIG. 7 shows dose-response of Compound 96 inhibition of pFAK [Y397]
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 7 M. Post 48 hr of treatment cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) (Cell signaling technology #3283), the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 8 shows dose-response of Compound 96 inhibition of Collagen 1.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 7 M. After 48 hr of treatment cell lysates were prepared and subjected to western analysis. Blots were probed using anti-Collagen 1 NB600-408 antibody (Novus Bioiogicais), the loading control was done using the anti-beta Actin antibody AC-15 (Santa Cruz Biotechnology).
  • Figure 9 shows dose-response of Compound 96 inhibition of aSMA mRNA expression.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 7 M. After 48 hr of treatment total RNA was extracted and aSMA mRNA level was determined using quantitative real-time PCR.
  • Figure 10 shows dose-response of Compound 96 inhibition of Collagen 1 mRNA expression.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012). Cells were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis and Compound 96 was added to the cells at various concentrations from 10 -11 to 10 7 M. After 48 hr of treatment total RNA was extracted and Collagen 1 mRNA level was determined using quantitative real-time PCR.
  • Figure 11 shows the effect of Compound 96 when added at 0 and 12 to 48 hr before inducing fibrosis.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at various concentrations from 10 -9 to 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2.
  • Figure 12 shows the effect on Collagen 1 mRNA expression of Compound 96 when added at 0 and 12 to 48 hr before attempting to induce fibrosis.
  • RTFs were cultured as described by Seet et al. , J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment total RNA was extracted and Collagen 1 mRNA level was determined using quantitative real-time PCR.
  • Figure 13 shows the effect on Collagen 3 mRNA expression of Compound 96 when added at 0 and 12 to 48 hr before attempting to induce fibrosis.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment total RNA was extracted and Collagen 3 mRNA level was determined using quantitative real-time PCR.
  • Figure 14 shows the effect on aSMA mRNA expression of Compound 96 when added at 0 and 12 to 48 hr before attempting to induce fibrosis.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment total RNA was extracted and aSMA mRNA level was determined using quantitative real-time PCR.
  • Figure 15 shows the effect of Compound 96 on the expression of fibrosis proteins pFAK(Y397) and collagen 1a when added at 0 and 12 to 48 hr before inducing fibrosis.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245- 59 (2012).
  • Compound 96 was added to the cells at various concentrations from 10 -9 to 10- 7 M before inducing fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment cell lysates were prepared and subjected to western analysis.
  • Figure 16 shows the effect on aSMA mRNA expression of Compound 96 when added 12 to 48 hr before inducing fibrosis.
  • HTFs were cultured as described by Seet et al. , J Cell Mol Med. 16(6): 1245-59 (2012) and Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2.
  • total RNA was extracted and aSMA mRNA level was determined using quantitative real-time PCR.
  • Figure 17 shows the effect of Compound 96 on the expression of fibrosis markers after TGF-beta-induced fibrosis.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment cell lysates were prepared and subjected to western analysis.
  • Figure 18 shows the effect of Compound 96 on pro-fibrotic Collagen 1 mRNA expression, after TGF-beta-induced fibrosis.
  • RTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment total RNA was extracted and Collagenl mRNA level was determined using quantitative real-time PCR.
  • Figure 19 shows the effect of Compound 96 on fibrosis after TGF-beta-induced fibrosis.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245- 59 (2012).
  • Compound 96 was added to the cells at 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment cell lysates were prepared and subjected to western analysis.
  • Lane 3 is used as control where Compound 96 is added together with TGF-beta and HTFs were harvested 48 hr after treatment, while in lane 4 Compound 96 was added 24 hr post-fibrosis and HTFs were harvested 48 hr post-fibrosis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), anti-Collagen 1 NB600-408 antibody (Novus Bioiogicais) and the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 20 shows the effect of Compound 96 on pro-fibrotic Collagen 1 gene expression, after TGF-beta-induced fibrosis.
  • HTFs were cultured as described by Seet et al. , J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at 10- 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment total RNA was extracted and Collagen 1 mRNA level was determined using quantitative real-time PCR.
  • Figure 21 shows the effect of Compound 96 on pro-fibrotic Collagen 3 gene expression, after TGF-beta-induced fibrosis.
  • HTFs were cultured as described by Seet et al., J Cell Mol Med. 16(6): 1245-59 (2012).
  • Compound 96 was added to the cells at 10 7 M before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment total RNA was extracted and Collagen 3 mRNA level was determined using quantitative real-time PCR.
  • Figure 22 shows the effect of FAK inhibitors on preventing the on-set of fibrosis in TGF-beta-treated HTFs.
  • HTFs were cultured as described in Example 2.
  • Compound 96, PF-045 (abbreviated as P04 or P045), PF-00562271 (abbreviated as P005) or GSK2256098 (abbreviated as GSK or GSK2) were added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2.
  • cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • FIG 23 shows that other FAK inhibitors are able to inhibit pFAK(Y397) and Collagen in Human embryonic kidney 293 (HEK293) cells.
  • HEK 293 cells were cultured under the same conditions as described in Example 2. Cells were cultured in 10 cm tissue culture plates, upon reaching 70% confluency. Cells were treated simultaneously with 2 ng/ml recombinant TGF-beta2, to induce fibrosis, and FAK inhibitors at various concentrations. After 48 hr of treatment cell lysates were prepared and subjected to western analysis. A first set of blots were probed using the Phospho-FAK (Tyr397) antibody (CelHO signaling technology #3283); the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 24 shows the plasma concentration-time curve of Compound 96 in rabbits following intravenous injection of 2 mg/ml_ of Compound 96.
  • Figure 25 shows in vivo confocal microscopy images (Heidelberg Rentina Tomography HRT3, Heidelberg Engineering GmbH, Germany) of treated and control eyes at 22 and 29 days post-treatment. Filtering bleb stroma with loose collagen like meshwork and large cystic spaces (yellow stars) were seen in FAKi treated eyes. Condensed, hyper-reflective and blurred stroma with less microcysts were observed in controls (larger darker spaces observed here are mostly blood vessels, indicated in red circles).
  • Figure 26 shows that Compound 96 reduced collagen deposition at the surgical site in rabbits in which glaucoma surgery was performed.
  • Hematoxylin and eosin (H&E) staining showed a flattened conjunctiva at the day 28 control operated site (Y7, bottom panels, arrows) whereas the Compound 96-treated operated site subconjunctival space was expanded with an almost clear matrix (Y2, top panels, arrows) at day 30.
  • H&E Hematoxylin and eosin
  • Figure 27 shows that Picrosirius red staining of the deposition of scar tissue and densely packed collagen fibers in the subconjunctival space, marked with **, in the vehicle-treated eyes (Y7 bottom panels) was significantly greater than in the Compound 96-treated eyes (Y2 top panels) at day 30.
  • Figure 28 shows reduced collagen, keratin and erythrocyte accumulation in operated rabbit eyes treated with Compound 96.
  • Compound 96-treated eyes (Y4, top panels) were noted to have less collagen deposition with Masson Trichrome stain compared to vehicle-treated eyes (Y8, bottom panels) as well as reduced keratin, erythrocytes and muscle fibers at day 30.
  • Figure 29 shows that Compound 96 reduced expression of pro-fibrotic marker at the surgical site in operated rabbit eyes.
  • Figure 30 shows that C96 when dissolved in SRP80 formulation and tested in vivo doesn’t cause any irritation, swelling or redness. This was also the case when DMSO was used as excipient. No redness or increased vascularization was seen in all the experimental samples at day 7, 14, 21 and 30 (black arrows). Data for day 14 is shown.
  • Figure 31 shows Hematoxylin and Eosin-stained sections of sub-conjunctival tissue at day 30 of treatment with DMSO, SRP80 and combinations of DMSO or SRP80 with Compound 96.
  • the presence of goblet cells in the superficial epithelium (black arrows) is a critical parameter that reflects the overall health of the ocular surface.
  • the results show that SRP80 on its own or as an excipient for C96 doesn’t adversely affect the goblet cell number.
  • Figure 32 shows Masson’s trichrome stained sections of sub-conjunctival tissue at day 30 of treatment with DMSO, SRP80 and combinations of DMSO or SRP80 with Compound 96. Images show selective staining of connective tissue (white arrows) which is the collagen stroma. A rabbit treated with 100 mM of the compound in DMSO shows disrupted expression of collagenous stroma (top right image; marked with asterisks *). The drug preparation with the SRP80 shows regular arranged collagenous stroma and a rich layer of conjunctival goblet (red staining with black arrows) cells which are required for protection from external factors. The higher dose of the compound in DMSO shows decreased density of conjunctival goblet cells which may have been the result from chronic irritation produced with this formulation.
  • Figure 33 shows immunofluorescence staining for a-SMA on sections of sub conjunctival tissue at day 30 of treatment with DMSO, SRP80 and combinations of DMSO or SRP80 with Compound 96. Staining shows a significantly lower proportion of cells than would normally be seen with scarring were found to express alpha-SMA in all the samples indicating that addition of the drug with either DMSO or SRP80 does not result in trans-differentiation of the epithelial cells to myofibroblasts.
  • Figure 34 shows (A) XEN ® gel stent; (B) preloaded injector and correct handling.
  • the term “comprising” or“including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof.
  • the term “comprising” or “including” also includes “consisting of”.
  • the variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as“include” and“includes”, have correspondingly varied meanings.
  • Compound 96 and‘C96’ are used herein interchangeably and refer to diethyl (3-methoxy-4- ⁇ [4-( ⁇ 2-methyl-7-[fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3- oxo-2, 3-dihydro-1 H-isoindol-4-yl ⁇ amino)-5-(trifluoromethyl)pyrimidin-2- yl]amino ⁇ benzyl)phosphonate.
  • references herein (in any aspect or embodiment of the invention) to said FAK inhibitor compounds includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
  • Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula 1 with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a serotonergic compound in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • acid addition salts include acid addition salts formed with acetic, 2,2-dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1 , 5-disulphonic and p-toluenesulphonic), ascorbic (e.g.
  • L-glutamic L-glutamic
  • a-oxoglutaric glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic
  • lactic e.g. (+)-L-lactic and ( ⁇ )-DL-lactic
  • lactobionic maleic, malic (e.g.
  • salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids
  • metals such as sodium, magnesium, or preferably, potassium and calcium.
  • FAK inhibitor compounds are any solvates of the compounds and their salts.
  • Preferred solvates are solvates formed by the incorporation into the solid state structure (e
  • solvating solvent examples include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide.
  • Solvates can be prepared by recrystallizing the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
  • TGE thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography X-ray crystallography
  • the solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.
  • Antagonist refers to a molecule which decreases the amount or the duration of the effect of the biological activity of FAK.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or small molecules which decrease the effect of FAK.
  • Compounds of the present invention will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable adjuvant diluent or carrier
  • Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability.
  • Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science 249, 1527-1533 (1990). Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
  • any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of a compound in the formulation may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 1 to 99% (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • a controlled release tablet may in addition contain from 0 to 90% (w/w) of a release-controlling polymer.
  • a parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50% (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • a liquid or semisolid carrier or vehicle e.g. a solvent such as water
  • one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • compounds may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated.
  • treatment refers to prophylactic, ameliorating, therapeutic or curative treatment.
  • Aqueous 3M HCI (25 mL, 75 mmol, 4.5 eq. ) was added in one portion to a room temperature solution of 7-amino-4-(1 ,4-dioxaspiro[4.5]dec-8-yl)-2-methyl-2,3- dihydro-1 H-isoindol-1-one (5 g, 16.5 mmol) in THF (180 mL). A thick suspension resulted. Stirring was continued overnight. A clear amber solution was observed the following morning. Some conversion to the desired ketone was observed by LCMS. Additional water (45 mL) was added and stirring was continued for 4d after which time no acetal remained as determined by LCMS.
  • PF- 045 Defactinib (VS-6063, PF-04554878) [N-methyl-4-(4-((3-(N- methylmethan-3-ylsulfonamido)pyrazin-2-yl)methylamino)-5-(trifluoromethyl)pyrimidin- 2-ylamino)benzamide] is a selective, and orally active FAK inhibitor, purchased from Selleck Chemicals LCC.
  • benzenesulfonate is the benzenesulfonate salt of PF-562271 , purchased from Selleck Chemicals LCC
  • Netarsudil (trade name Rhopressa) is a drug for the treatment of glaucoma. In the United States, the Food and Drug Administrationhas approved a 0.02% ophthalmic solution for the lowering of elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Netarsudil primarily targets cells in the conventional outflow tract, efficiently decreasing IOP in both human and non-human primate eyes. In addition, netarsudil has been shown to increase outflow facility in non-human primate eyes and to decrease episcleral venous pressure in rabbit eyes [Li G et al 2016 Eur J Pharmacol Sept 15, 787: 20-31]
  • Netarsudil ophthalmic solution 0.02% is supplied as a sterile, isotonic, buffered aqueous solution of netarsudil dimesylate with a pH of approximately 5 and an osmolality of approximately 295 mOsmol/kg. It is intended for topical application in the eye.
  • Each mL of netarsudil contains 0.2 mg of netarsudil (equivalent to 0.28 mg of netarsudil dimesylate).
  • Benzalkonium chloride 0.015%, is added as a preservative.
  • N-TAE2266 N-TAE2266
  • Benzamide, 2-[[5-chloro-2-[[2-methoxy-4-(4- morpholinyl)phenyl]amino]-4-pyrimidinyl]amino]-N-methyl-] was purchased from Selleck Chemicals LCC.
  • Frozen aliguots of the Tenon fibroblast cells were cultured in DMEM with 10% FBS, 37°C, 5% CO2 using standard tissue culture plates. Cells were plated onto 10 cm tissue culture plates at 1X10 6 cells/plate and cells were allowed to grow up to 70% confluency before being treated with TGF-b and test compounds, including C96, PF- 04554878, PF-00562271 , GSK2256098, AR-13324, PF-562271 HCI, PND-1186 or TAE226. Fibrosis was induced using TGF-b at 2ng/ml for 48hr and test compound was added simultaneously during induction of fibrosis.
  • Non-treated cells and TGF-b treated alone were used as controls for each experiment.
  • Cells treated for 48hr were the baseline for pre-fibrosis and post-fibrosis experiments.
  • cells were harvested in 20 mM HEPES (pH 7.4), 137 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM EGTA, 10% (v/v) glycerol, 1 % Triton X-100, a mixture of protease inhibitors (Roche Molecular Biochemicals), and 0.2 mM sodium orthovanadate.
  • Cell lysates were precleared by centrifugation at 16000xg for 15 min.
  • mRNA levels of pro-fibrotic genes were determined via quantitative real time PCR (qPCR) to assess the effect of Compound 96 or other FAK inhibitors on COL1 , aSMA and SPARC expression in RTFs and HTFS.
  • qPCR quantitative real time PCR
  • total RNA was isolated using Rneasy MiniKit (Qiagen) according to the manufacturer’s instructions.
  • cDNA was synthesized with 1 pg total RNA extract with iScriptTM cDNA synthesis kit (Bio-rad) according to the manufacturer’s instructions.
  • Quantitative real-time PCR was performed in a total volume of 10 pi in 96-well microtiter plates with SsoFastTM EvaGreen ® Supermix (Bio-rad) according to the manufacturer’s instructions (Table 1) and primers indicated in Tables 2 and 3. The resulting amplification and melt curves were analyzed to ensure the identity of the specific PCR product. Threshold cycle values were used to calculate the fold change in the transcript levels by using the 2 _AACt method. The relative mRNA expression levels were normalized to the beta-actin gene.
  • the animals were examined before receiving the injections at set intervals for 28 days after surgery. Clinically, bleb appearance, size, height, and surrounding vascularity were recorded in addition to the intraocular pressure (IOP), on days 1 , 7, 14, 21 , 28. The animals were killed on day 28. Both eyes were enucleated and histologically analyzed. The non-surgical right eyes served as the normal control for histologic comparison.
  • IOP intraocular pressure
  • Two animals were used for pharmacokinetics/ pharmacodynamics analysis to quantify drug efficacy.
  • One was given an intravenous dose of the compound and venous blood was collected at specified time points.
  • the other was given a sub conjunctival injection of the compound and venous blood was collected at specified time points.
  • Example 2 Non-toxic effect of Compound 96 on 3D cultured Rabbit Tenon’s Fibroblast cells (RTFs).
  • Example 3 Dose-response of Compound 96 inhibition of pFAK(Y397) and Collagen in Human Tenon’s Fibroblast cells (HTFs)
  • HTFs were cultured as described above.
  • Cells were cultured in 10 cm tissue culture plates, upon reaching 70% confluency. Cells were treated simultaneously with 2 ng/ml recombinant TGF-beta2, to induce fibrosis, and Compound 96 at various concentrations from 10 -11 to 10 -6 M. After 48 hr of treatment cell lysates were prepared and subjected to western analysis and quantitative polymerase chain reaction (qPCR).
  • qPCR quantitative polymerase chain reaction
  • Example 4 Compound 96 inhibits Collagen 1, aSMA and SPARC mRNA expression in HTFs
  • HTFs were cultured as described in Example 3 and were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis.
  • Compound 96 was added to the cells at various concentrations from 10 11 to 10 6 M. After 48 hr of treatment, total RNA was extracted and Collagen 1 mRNA level (Figure 4), aSMA mRNA level ( Figure 5) and SPARC mRNA level ( Figure 6) were determined using quantitative real-time PCR. It can be seen that TGF beta induced collagen 1 expression by over 4-fold and Compound 96 at 10 6 M reduced the TGF beta-induced collagen 1 expression by more than 50% (Figure 4).
  • RTFs were cultured as described in Example 1 and were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis.
  • Compound 96 was added to the cells at various concentrations from 10 11 to 10 7 M. After 48 hr of treatment cell lysates were prepared and subjected to western analysis.
  • HTFs were cultured as described in Example 3.
  • Compound 96 or other FAK inhibitors were added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2.
  • cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • Figure 22 shows that PF-04554878, PF- 00562271 and GSK2256098 are effective in the inhibition of FAK phosphorylation at Tyr397. The levels of inhibition of phosphorylation by these compounds increased with increasing times of incubation with the compounds.
  • the inhibitors were purchased from Selleck Chemicals LCC.
  • the IC50 of each tested compound is shown in Table 4.
  • Table 4 IC50 of each tested compound on HTF cells.
  • Example 7 Compound 96 inhibits aSMA and Collagen 1 mRNA expression in RTFs
  • RTFs were cultured as described in Example 1 and were treated with 2 ng/ml of TGF-beta 2 to induce fibrosis.
  • Compound 96 was added to the cells at various concentrations from 10 -11 to 10 7 M.
  • RNA was extracted and a-SMA mRNA level ( Figure 9) and Collagen 1 mRNA level ( Figure 10) were determined using quantitative real-time PCR. Collagen 1 and a-SMA are expressed in fibrotic tissues and the expression level of these fibrosis-related genes was reduced in samples treated with C96.
  • Example 8 Effect of Compound 96 on fibrosis in RTFs when added 12 to 48 hr before inducing fibrosis
  • RTFs were cultured as described in Example 1. Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M before inducing fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment, cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), anti-Collagen 1 NB600-408 antibody (Novus Biologicals); the loading control was tested using the FAK SC-557 A-17 antibody and GAPDH BF7 antibody (Santa Cruz Biotechnology) ( Figure 11).
  • the inhibition is shown in a dose-dependent manner, the strongest inhibition being seen when C96 is added at 10 7 M 12 to 48 hours pre-fibrosis.
  • This data supports the notion that C96 can be given pre-operatively as a treatment to prevent fibrosis.
  • W is the wash-out sample where the treated sample was rinsed with buffer and harvested 24 hr post-treatment. It shows that the inhibition effect of the collagen and FAK activation is lost in this sample. Hence, the effect of C96 is reversible, which is an important factor when developing drugs for safe use.
  • Example 9 Effect of Compound 96 on Collagen 1 mRNA, Collagen 3 mRNA and aSMA mRNA when added 12 to 48 hr before inducing fibrosis
  • RTFs were cultured as described in Example 1.
  • Compound 96 was added to the cells at various concentrations from 10 -9 to 10 7 M either simultaneously or 12, 24 or 48 hours before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2.
  • total RNA was extracted and Collagen 1 mRNA level (Figure 12), Collagen 3 mRNA level ( Figure 13) and aSMA mRNA level ( Figure 14) were determined using quantitative real-time PCR.
  • the data from Figs. 12 , 13 and 14 show that C96 is able to inhibit expression of fibrotic markers when added to the cells 12 hr to 48 hr before fibrosis. The inhibition is shown in a dose-dependent manner, the strongest inhibition being seen when C96 is added at 10 7 M 12 to 48 hours pre-fibrosis. This data also supports the notion that C96 may be used pre-operative treatment to prevent fibrosis.
  • Example 10 Effect of Compound 96 on fibrosis in HTFs when added 12 to 48 hrs before inducing fibrosis
  • HTFs were cultured as described in Example 3. Compound 96 was added to the cells at various concentrations from 10 9 to 10 7 M either simultaneously, 12, 24 or 48 hours before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2. After 48 hr of treatment, cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), anti-Collagen 1 NB600-408 antibody ( ovus Biologicals); the loading control was tested using the FAK SC-557 A-17 antibody and GAPDH BF7 antibody (Santa Cruz Biotechnology) (Figure 15).
  • HTFs were cultured as described in Example 3, Compound 96 was added to the cells at various concentrations from 10 -9 to 10 7 M either simultaneously or 12, 24 or 48 hours before attempting to induce fibrosis with 2 ng/ml of TGF-beta 2.
  • total RNA was extracted and aSMA mRNA level was determined using quantitative real-time PCR (Figure 16).
  • Figure 16 The data shows that C96 is able to inhibit activation of FAK and collagen synthesis when added to the cells 12 hr to 48 hr before fibrosis. The inhibition is shown in a dose-dependent manner and the inhibition effect is seen even up to 2 days treatment with C96 before inducing fibrosis. This data supports the notion that C96 can be given as a pre-operative treatment to prevent fibrosis.
  • Example 12 Compound 96 prevents on-set of fibrosis in TGF-beta-treated RTFs
  • RTFs were cultured as described in Example 1.
  • Compound 96 was added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment, cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), anti-Collagen 1 NB600-408 antibody ( ovus Biologicals); the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology) (Figure 17). The data shows that C96 is able to inhibit activation of FAK and collagen synthesis in the fibrosis-induced cells even up to 7 days without new addition of drug.
  • Example 13 Compound 96 reduces Collagen 1 gene expression, in TGF-beta- treated RTFs
  • RTFs were cultured as described in Example 1.
  • Compound 96 was added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment, total RNA was extracted and Collagen 1 mRNA level was determined using quantitative real-time PCR (Figure 18). The data shows that C96 is able to inhibit expression of Collagen 1 mRNA in the fibrosis-induced cells even up to 7 days of fibrosis without addition of new drug.
  • Example 14 Compound 96 reduces indicators of fibrosis in TGF-beta-treated HTFs
  • HTFs were cultured as described in Example 3, Compound 96 was added to the cells at 10 7 M together with 2ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment, cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), anti-Collagen 1a NB600-408 antibody (Novus Bioiogicals); the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology) (Figure 19). The data shows that C96 is able to inhibit activation of FAK and collagen synthesis in the fibrosis-induced HTF cells even up to 7 days of fibrosis without addition of new C96.
  • Example 15 Compound 96 reduces pro-fibrotic gene Collagen 1 mRNA expression, in TGF-beta-treated HTFs
  • HTFs were cultured as described in Example 3, Compound 96 was added to the cells at 10 7 M together with 2ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment, total RNA was extracted and Collagenl mRNA level was determined using the quantitative real-time PCR (Figure 20). The data shows that C96 is able to inhibit expression of Collagen 1 mRNA in the fibrosis-induced HTFs cells even up to 7 days of fibrosis without addition of new drug.
  • Example 16 Effect of Compound 96 on pro-fibrotic genes Collagen 3, in TGF- beta-treated HTFs
  • HTFs were cultured as described in Example 3.
  • Compound 96 was added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2. After 2 days, 5 days and 7 days of treatment, total RNA was extracted and Collagen 3 mRNA level was determined using the quantitative real-time PCR (Figure 21). The data shows that C96 is able to inhibit expression of Collagen 3 mRNA in the fibrosis-induced HTFs cells even up to 7 days of fibrosis without addition of new drug.
  • HTFs were cultured as described in Example 3.
  • Compound 96 or other FAK inhibitors described in the materials and methods were added to the cells at 10 7 M together with 2 ng/ml of TGF-beta 2.
  • cell lysates were prepared and subjected to western analysis. Blots were probed using the Phospho-FAK (Tyr397) antibody (Cell signaling technology #3283), the loading control was tested using the FAK SC-557 A-17 antibody (Santa Cruz Biotechnology).
  • the data ( Figure 22) shows that the FAK inhibitors are not as effective as the C96 at inhibiting the pFAK on the first day of fibrosis (0 hr). However, as the time of contact increases, these other FAK inhibitors were all able to inhibit the pFAK level back to the basal level.
  • Example 18 Other FAK inhibitors are able to inhibit pFAK(Y397) and Collagen in HEK293 cells
  • Hek 293 were cultured as described previously for RTF cells. Cells were cultured in 10 cm tissue culture plates, upon reaching 70% confluency. Cells were treated simultaneously with 2 ng/ml recombinant TGF-beta2, to induce fibrosis, and FAK inhibitors at various concentrations. After 48 hr of treatment cell lysates were prepared and subjected to western analysis. A first set of blots were probed using the Phospho-FAK (Tyr397) antibody (CelHO signaling technology #3283); the loading control was tested using the FAK SC-557 A- 17 antibody (Santa Cruz Biotechnology).
  • FIG. 23 shows that the PF-04554878 Defactinib (VS-6063) [lanes 7 and 8], PF-00562271 -benzenesulfonate salt of PF-562271 [lanes 10 and 11], and GSK2256098 [lanes 13 and 14] are able to inhibit pFAK at 10 7 M as well as at 10 6 M.
  • Example 19 In vivo animal data
  • Two New Zealand white rabbits were used for Pharmacokinetics/pharmaco dynamics analysis to quantify drug efficacy.
  • the compound prepared in 0.1% DMSO/PBS pH 7.2 was injected locally.
  • One rabbit was given an intravenous dose of the compound and venous blood was collected at specified time points.
  • the other was given a subconjunctival injection of the compound and venous blood was collected at specified time points.
  • Table 5 Plasma concentration (ng/ml) of C96 in New Zealand white rabbits over time following subconjunctival and intravenous injection of 2 mg/ml C96
  • the C96 is successfully quantified till 6 hours after intravenous injection.
  • a microvitreoretinal (MVR) blade was used to make a partial-thickness scleral incision 3 to 4 behind the limbus, and a scleral tunnel to the corneal stroma was fashioned.
  • a 22-gauge, 25-mm intravenous cannula (Venflon 2; Beckton Dickinson, Oxford, UK) was passed through a scleral tunnel anteriorly until the cannula needle was visible in the clear cornea. Entry into the anterior chamber was made with a cannula needle, which was then withdrawn as the cannula was advanced to the midpupillary area. The cannula was trimmed and beveled at its scleral end so that it protruded 1 mm from the insertion point, and a 10-0 nylon suture was placed to fix the tube to the scleral surface.
  • the conjunctival incision was closed with two interrupted sutures and a central, mattress-type 10-0 nylon suture attached to a needle (B/V 100-4; Ethicon) to give a water-tight closure.
  • a needle B/V 100-4; Ethicon
  • Example 20 Loose sub-epithelial connective tissue spaces associated with bleb filtration are seen in Compound 96-treated eyes but not control eyes
  • the rationale for this dose regimen is to attempt to achieve local availability of the inhibitor at the critical time of pre- and post operative wound repair and remodeling, as observed in other studies.
  • the animals were examined before receiving the injections at set intervals for 28 days after surgery.
  • the intraocular pressure (IOP) was recorded on days 1 , 7, 14, 21 and 28.
  • the animals were sacrificed on day 28. Both eyes were enucleated and histologically analyzed.
  • the non-surgical right eyes served as the normal control for histologic comparison.
  • Example 21 Tissue Sections from mock glaucoma-operated rabbit eyes
  • Masson Trichrome staining - Red - keratin, erythrocytes and muscle fibres, Blue or green - collagen and bone, light red/pink cytoplasm and dark brown to black cell nuclei.
  • Compound 96-treated eyes (Y4, top panel) were noted to have less collagen deposition with Masson Trichrome stain compared to vehicle- treated eyes (Y8, bottom panel) as well as reduced keratin, erythrocytes and muscle fibers at day 30 ( Figure 28).
  • Compound C96 was tested in eye formulations from a commercial source (CRODA).
  • Polysorbates are hydrophilic, nonionic surfactants widely used as emulsifying agents in the preparation of stable oil-in-water pharmaceutical emulsions. They are also excellent solubilisers, stabilisers, wetting and dispersing agents. However, like other excipients, standard pharmaceutical polysorbates possess impurities that cause formulation challenges especially when working with oxidatively unstable Active Pharmaceutical Ingredients (APIs). As with the Super Refined PEGs, CRODA has Super Refined a range of polysorbates, removing or reducing polar and oxidative impurities. The formula tested is called Super Refined Polysorbate 80 (SRP80).
  • SRP80 Super Refined Polysorbate 80
  • sub-conjunctival tissue at day 30 was dissected and histology/immunohistochemistry was performed on tissue sections.
  • Sub-conjunctival tissue sections at Day 30 were stained for:
  • a-SMA smooth muscle actin
  • Picrosirius red-stained sections showed that the deposition of total scar tissue and densely packed collagen fibers in the subconjunctival space were significantly greater in the DMSO-treated eyes and in the drug dissolved in DMSO-treated eyes than in the SRP80-treated eyes and the drug dissolved in SRP80 treated eye at day 30.
  • the green-yellow staining seen in the DMSO formulation-treated eyes is indicative of damaged tissue undergoing scarring. Even though the DMSO combination did not produce any redness in the rabbit eye, the internal morphology of the conjunctival tissue appeared to be going through some pathophysiological changes not observed with the SRP80 formulation.
  • Example 23 Adjunct use of FAK inhibitors to reduce implanted drainage device- induced fibrosis
  • Drainage device implants have shown great promise in reducing IOP as well as having an increased safety profile. Similar to conventional glaucoma filtration surgeries, Adjunct Mitomycin C is commonly used to reduce device-induced fibrosis, with some ophthalmologists using the cytotoxin even post-surgery for needlings. As such, it is important that we find an alternative medication that reduces the fibrosis while being less toxic to the ocular structures.
  • the FAK inhibitors detailed in this invention can serve as non-cytotoxic alternatives to Mitomycin C.
  • Two such devices are the XEN ® GEL Implant (AqueSys Inc., Aliso Viejo, CA, USA) and the InnFocus Microshunt ® (InnFocus Inc, Miami, FL, USA) which both drain into the subconjunctival space.
  • the XEN ® gel stent is a hydrophilic tube made of a porcine gelatin cross-linked with glutaraldehyde to achieve permanence in tissue. This material is used for a variety of medical applications because of its well-established biocompatibility, and it does not cause a foreign-body reaction (Figure 34A).
  • the XEN ® 45 is the only one currently available and has an inside diameter of ⁇ 45 pm, an outside diameter of 150 pm, and is 6 mm long. The implant is hard when dry and becomes soft within 1-2 min when hydrated, adapting to the tissue shape, thus avoiding migration and potential erosion.
  • the procedure can be performed under local or topical anesthesia.
  • a FAK inhibitor of the invention is injected with a 30-gauge needle in the subconjunctival supero-temporal quadrant space to obtain a bubble that is gently rolled toward the supero-nasal quadrant. This induces a hydroexpansion, which reduces tissue resistance, preparing the space for the implant and supporting the bleb formation. Blood vessels should be avoided during the introduction of the needle into the subconjunctival space as bleeding may compromise visibility required for the stent implantation.
  • the surgical procedure for stent placement is described in De Gregorio A, et al., Clin Ophthalmol, 12: 773-782 (2016).
  • the InnFocus Microshunt (InnFocus Inc, Miami, FL, USA), formerly known as the MIDI Arrow, is an aqueous drainage shunt designed to be implanted ab externo. It is implanted after making a fornix based conjunctival flap, creating a scleral pocket and applying mitomycin-c. This approach creates a bleb space in preparation for the fluid that will be directed from the anterior chamber through the InnFocus MicroShunt ® to the bleb.
  • a small molecule inhibitor, Compound 96 [diethyl (3-methoxy-4- ⁇ [4-( ⁇ 2-methyl-7- [fra/is-4-(4-methylpiperazin-1-yl)cyclohexyl]-3-oxo-2,3-dihydro-1 H-isoindol-4-yl ⁇ amino)- 5-(trifluoromethyl)pyrimidin-2-yl]amino ⁇ benzyl)phosphonate] that targets the Y397 and Y925 sites of FAK was synthesized and the effect of this specific FAK inhibitor in eye- related scarring and fibrosis was investigated.
  • PF-04554878 Defactinib (VS-6063, ), PF-00562271 -benzenesulfonate salt of PF-562271 , PF-562271 HCI is the hydrochloride salt of PF-562271 , GSK2256098 and TAE226 (NVP-TAE226) to be developed as anti-fibrotic drugs.
  • FAK inhibitors can be used alone or as an adjunct when drainage devices are implanted in patients with glaucoma.
  • Bevacizumab is a recombinant humanized monoclonal antibody against VEGF.
  • RNAi can have off-target effects and is immunogenic
  • gene therapy presents a challenge with the delivery of the gene into the target cell and could be oncogenic
  • nanoparticles have moderate specificity while potentially being cytotoxic and genotoxic
  • small molecule inhibitors such as Rho- kinase (ROCK) inhibitors have the disadvantage of causing ocular redness (conjunctival hyperemia).

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Abstract

La présente invention concerne un traitement pour des maladies associées à une cicatrisation liée à l'oeil et une cicatrisation post-chirurgicale de patients atteints de glaucome. Plus particulièrement, l'invention concerne des compositions et des procédés de traitement de cicatrisation et de fibrose liées à l'oeil par inhibition de la kinase d'adhésion focale active à l'aide d'un inhibiteur à petites molécules.
PCT/SG2018/050612 2017-12-15 2018-12-14 Agents thérapeutiques ciblant une kinase d'adhésion focale pour le traitement du glaucome et de la fibrose WO2019117813A1 (fr)

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WO2024140671A1 (fr) * 2022-12-29 2024-07-04 南京昕瑞再生医药科技有限公司 Dérivé tricyclique fusionné, composition pharmaceutique le comprenant et son utilisation

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EP4126223A4 (fr) * 2020-03-26 2024-04-24 The Board of Trustees of the Leland Stanford Junior University Procédés de régénération tissulaire et kits associés
WO2024140671A1 (fr) * 2022-12-29 2024-07-04 南京昕瑞再生医药科技有限公司 Dérivé tricyclique fusionné, composition pharmaceutique le comprenant et son utilisation

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