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  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
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  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/70—Mechanisms involved in disease identification
  • G01N2800/7052—Fibrosis

Definitions

• This disclosure provides a conjugate and method of using thereof to image and/or treat idiopathic pulmonary fibrosis (IPF).
• IPF idiopathic pulmonary fibrosis
• FAP Fibroblast active protein
• Fibrotic diseases constitute a major health problem worldwide affecting a large number of individuals.
• fibrosis of the liver represents a paradigm for this disease, as it may be reversible at early stages but become irreversible as it progresses to cirrhosis, resulting in liver cancer in addition to end stage disease. It has multiple potentially preventable etiologies; they include HBV and HCV infection, obesity, alcoholism, and aflatoxin among others; each presents opportunities for and serious barriers to primary and/or secondary prevention.
• fibrotic diseases For many other fibrotic diseases, the underlying etiologies are less clear, although many are associated with chronic production of proteolytic enzymes, fibrogenic cytokines, growth factors, and angiogenic factors, presumably secondary to triggering irritants (e.g., radiation, chronic infections, toxins). Others are congenital or associated with autoimmunity. For fibrosis of all types, the point of irreversibility and the molecular mechanisms by which it occurs are not well defined. Organ failure is the end-result of uncontrolled fibrosis. Treatment of fibrotic diseases in these organs is necessary to prevent the eventual organ failure and morbidity.
• Idiopathic pulmonary fibrosis is a progressive fibrotic disease of lungs. It is believed to be caused by repetitive environment injury to the lining of the lungs and resulting abnormal wound-healing responses. When tissues of lung experience prolonged activation of wound healing responses, the result usually is permanent scarring, organ malfunction and more significantly, death.
• lung transplantation can be a final option, but finding an HLA match is often difficult and avoiding transplant rejection can be challenging.
• the conjugate comprises a targeting ligand to FAP (TL), a linker (L) and an effector (E), wherein the TL has a molecular weight below 10,000, the L is a non-releasable linker when the effector is an imaging agent or a radioactive therapeutic agent; the L is a releasable linker when the effector is a therapeutic drug.
• the linker is selected from the group consisting of pegylated, alkyl, sugar or peptide based dual linker.
• the aforementioned imaging agent is a fluorescent molecule.
• the aforementioned effector is a PET imaging agent.
• the aforementioned PET imaging agent comprises the structure of
• the aforementioned effector is a 99mTc imaging agent comprising a DOT A, NOT A, TETA or NODAGA chelating agent
• the aforementioned 99mTc imaging agent comprises the structure of .
• Ri and R 2 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen and C 1 -C 4 alkyl;
• R 3 is a C 1 -C 4 alkyl, nitrile, or isonitrile
• R 4 is H or -CH 3
• R 5 and R 6 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen, and C 1 -C 4 alkyl,
• R- 7 -R 9 are the same or different, and are each independently selected from the group consisting of hydrogen, methoxy, halogen, CF 3 and C 1 -C 4 alkyl
• the aforementioned Ri and R 2 is a halogen.
• each of Ri and R 2 is fluorine.
• the aforementioned effector is a kinase inhibitor for VEGFR1,
• VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR.
• the aforementioned effector is an SMAD inhibitor.
• the aforementioned effector is a cytotoxic agent.
• the aforementioned effector is a PI-3 kinase inhibitor.
• This disclosure further provides a PI-3 Kinase inhibitor comprising the structure of (PI3KI1).
• PI-3 kinase inhibitor comprising the structure below:
• X can be any of the following
• the aforementioned targeting ligand to FAP has a binding affinity to FAP in the range between about 1 nM and about 10 nM.
• This disclosure further provides a method of diagnosing IPF in a subject, comprising the following steps: obtaining the lung tissue from the subject, wherein said tissue may or may not express FAP in fibroblast cells;
• TL-L-I a targeting ligand A conjugate to target Fibroblast Activation Protein (FAP) expressing cells in fibrotic lung diseases, wherein said TL has a molecular weight below 10,000, said L is a non- releasable linker, said I is an imaging agent; and
• FAP Fibroblast Activation Protein
• the aforementioned TL is a small molecule having the structure of
• Ri and R 2 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen and C 1 -C 4 alkyl;
• R 3 is a C 1 -C 4 alkyl, nitrile, or isonitrile
• R 4 is H or -CH 3 ;
• R 5 and R 6 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen, and C 1 -C 4 alkyl;
• R 7 -R 9 are the same or different, and are each independently selected from the group consisting of hydrogen, methoxy, halogen, CF 3 and C 1 -C 4 alkyl.
• This disclosure further provides a method of treating IPF in a subject.
• the method comprising the steps of: providing to the IPF patient cells with a pharmaceutically effective amount of conjugate of TL-L-D, wherein TL is a targeting ligand to FAP that has a molecular weight below 10,000, L is a releasable linker and D is a therapeutic drug that has pan PI-3Kinase inhibitory effect; and
• Ri and R 2 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen and C 1 -C 4 alkyl;
• R 3 is a C 1 -C 4 alkyl, nitrile, or isonitrile
• R 4 is H or -CH 3 ;
• R 5 and R 6 are the same or different, and are each independently selected from the group consisting of hydrogen, halogen, and C 1 -C 4 alkyl,
• R-7-R9 are the same or different, and are each independently selected from the group consisting of hydrogen, methoxy, halogen, CF3 and C1-C4 alkyl.
• X can be any of the following:
• the aforementioned method uses a conjugate that reduces collagen I deposits on activated fibroblast cells.
• the aforementioned method uses a subject that is a mouse IPF model induced by intratracheal administration of bleomycin at about 0.75u/kg for consecutive 10 days.
• the aforementioned method administering a conjugate at about 0.2-10 umol/kg to the mouse IPF model for consecutive 10 days and the conjugate is FAPL_PI3KIl with the structure of .
• the aforementioned method reduces the hydroxyproline production of fibroblast cells.
• the aforementioned method uses a subject of a mouse IPF model induced by silica or radiation.
• FAPL-FITC targets human FAP in HLF1 cells.
• FIG. 3 Binding of FAPL-FITC by human IPF patient cell line aSMA and FAP predominantly expressed in IPF lung fibroblasts and targeted by FAP-FITC.
• A IPF fibroblasts and non IPF control fibroblast were seeded and stained with antibody against FAP or aSMA.
• B IPF cells were incubated with FAPL-FITC (10 nM) and analyzed by flow cytometry.
• Figure 4. Design and synthesis of PI3KI1 and FAPL-PI3KI1
• PI3KI1 inhibits Akt phosphorylation, proliferation, collagen secretion and collagen gel contraction in HLF-l fibroblasts.
• A PI3KI1 structure.
• B Confluent HLF-l fibroblasts were stimulated with TGFP (1 ng/mL) with indicated PI3KI1 or OMIPALISIB and lysates collected for Western blotting.
• PI3KI1 inhibits TGF-b (lng/ml for 24 hr) induced AKT phosphorylation with IC50 1.4 nM. Collagen 1 and aSMA expression was suppressed by 100 nM PI3KI1, but pSMAD2 was not affected by PI3K inhibitors.
• C MTT assay and Caspase 3 and 7 activity (Supplementary data) show PI3KI1 inhibited HLF-l proliferation at >100 nM.
• D HLF-l fibroblasts were stimulated with TGFP (1 ng/mL) for 3 days and level of secreted collagen in culture medium was determined by Sirius Red staining.
• E PI3KI1 (100 nM for 12 hours) disrupted fibroblasts reorganization and contraction, characteristics of activated fibrosis, by collagen gel contraction assay. Data are analysis by t-test; *p ⁇ 0.05, **p ⁇ 0.0l.
• PI3KI1 and PI3KI1-FAPL suppressed TGFP- induced collagen production in IPF fibroblasts.
• Confluent IPF fibroblasts were stimulated for 48 h with TGFP (1 ng/mL) with increasing concentrations of OMIPALISIB or PI3KI1 or PI3KI1-FAPL and Collagen I expression was assayed by molecular crowding assay with high content image analysis. Data are expressed as relative fluorescent intensity over TGFP treated IPF fibroblasts (C) and cell counts obtained from DAPI counterstaining (D).
• FAPL-FITC targets mouse FAP in mouse fibroblast cells. Fluorescence binding affinity study of FAPL_FITC in HLF-hFAP cells. (C) Binding of FAPL_FITC to TGFb induced mouse NIH-3T3 cell line.
• A Schematic representation of the experimental protocol for induction, treatment and examination of pulmonary fibrosis in a mouse model.
• B Survival probability of the FAPL_PI3KIl treated mice over the saline control.
• C Hydroxyproline content (ug/lung) of control (saline), bleomycin treated with and without FAPL_PI3KIl treatment at day 21.
• D Body weight change
• FIG. 12 TGF-b (1 ng/ml for 24 hr) induced FAP and aSMA expression in human lung fibroblast cell line (HLF-l). HLF-l cells were 0.4 % serum starved for 12 hr and then stimulated by TGF-b (1 ng/mL) for 24 hr. FAPL and aSMA expression were detected by immunofluorescence analysis.
• Figure 13 Chemical structure of Compound 3.
• 19A shows FAP targeted TC in various organs
• 19B shows FAP targeted
• IPF is a lethal, chronic, progressive disease, and increases with age, particularly in individuals over the age of 50. In US, IPF kills as many people (about 40,000 per year) as breast cancer, with most patients dying within 3-5 years of diagnosis.
• lung transplantation can be a final option, but finding an HLA match is often difficult and avoiding transplant rejection can be challenging.
• fibroblasts In response to stimulation by activated immune cells such as macrophages and T cells, fibroblasts become activated to myofibroblasts that then accumulate in areas called fibroblast foci where they produce the collagen that causes the fibrosis.
• activated immune cells such as macrophages and T cells
• myofibroblasts are readily distinguished from nonpathologic fibroblasts by their expression of a transmembrane protein, fibroblast activation protein (FAP) that is critical for the process of collagen remodeling.
• FAP fibroblast activation protein
• FAP farnesofibroblasts
• FAP fibroblast activation protein
• FAPL-PI3KI1 In vivo therapy with FAPL-PI3KI1 demonstrated increased survival and decreased hydroxyproline/collagen production compared to PBS -treated control mice induced to develop experimental lung fibrosis. These findings demonstrate, 1) FAPL-S0456 can target the fibrotic lungs with good specificity, 2) targeted delivery of Pi3K inhibitor (FAPL-PI3KI1) may be beneficial in treating lung fibrosis, as well as other diseases that are characterized by pathological inflammation and fibrosis.
• FAPL conjugated warhead can be used on other disease models as well, as long as the therapeutic drug conjugated to FAPL is specific and effective for the disease.
• FAP protein expression is restricted, occurring at high levels on mesenchymal cells during embryogenesis then repressed shortly after birth, and its expression is upregulated on activated fibroblast in conditions associated with wound healing, cancer and fibrosis. From the examples below, a person skill in the art will appreciate that the expression pattern of FAP qualifies it as a good marker for the targeted delivery of drugs or other effectors to fibrotic cells, a hall mark of fibrosis.
• a FAP ligand targeted PI-3 kinase therapy can be primarily applied to adults at times when they are not recovering from a serious lung or other tissue trauma, one would not expect to encounter significant off target toxicity with the use of this drug.
• fibrotic process share some similarities among different types of fibrosis, therefore, a targeted drug that might inactivate a myofibroblast and reprogram it to become a quiescence fibroblast might also prove useful in treating fibrosis of liver, kidney, heart, skin, and bladder organs. Without being restricted to any theory, this is because the basic pattern of disease progression in these several types of fibrosis may involve immune cell activation arising from unknown causes, leading to activation of fibroblasts to form myofibroblasts, thereby triggering the excessive production of collagen.
• a common feature in essentially all known fibrotic diseases is the production of collagen by the myofibroblasts, which invariably expresses FAP regardless of the organ in which the fibrosis occurs.
• kinase inhibitors to VEGFR1, VEGFR2, VEDFR3, FGFR1, FGFR2, or PDGFR are potential candidates for delivery.
• Other warheads such as kinase inhibitors for FAK or ROCK, other effectors such as SMAD inhibitor, or cytotoxic agent, are all within the contemplation of this disclosure.
• a potent pan PI-3 kinase inhibitor was selected in view of the many failures of prior PI-3 kinase inhibitors in the clinic, primarily due to its following advantages:
• PI-3 kinase pathway activation is reported in fibrotic foci, the cardinal lesions in IPF.
• PI- 3 Kinase isoforms exhibit increased expression in IPF tissue and fibroblast lines, with signaling activated downstream of several key profibrotic growth factors implicated in IPF, including platelet-derived growth factor and transforming growth factor (TGFj-b I .
• OMIPALISIB inhibited PI-3 Kinase signaling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells. Inhibition of PI-3Kinase pathway also effects normal cellular functions, including proliferation, apoptosis and metabolism.
• pan-PI3K/mTOR kinase inhibitor A targeted approach is essential to prevent the cumulative toxicity of a pan-PI3K/mTOR inhibitor.
• an isoform- specific inhibitor could have been selected for delivery to the myofibroblasts
• a pan-PI3K/mTOR kinase inhibitor was chosen to overcome any functional redundancy between isoforms and blocking potential crosstalk and feedback of compensatory mechanisms through inhibition of three key nodes (PI3K, mTORCl and mTORC2). 25 While such a general PI-3 kinase inhibitor will generally be more toxic, suppressing all PI-3 kinase dependent processes is not so undesirable, since the myofibroblasts are not essential to normal lung function and they generally undergo apoptosis during resolution of the disease.
• pan PI-3 kinase inhibitor war head can be conjugated to a targeting ligand to a specific disease marker, to exert its inhibitory effect in that particular disease model.
• a targeting ligand to a specific disease marker
• invasive bladder cancer treated with PI-3 kinase inhibitor can be improved with targeted ligand to bladder cancer marker Epidermal growth factor receptor (EPGR) conjugated with a choice of pan PI-3 kinase inhibitor.
• EPGR Epidermal growth factor receptor
• Our platform of pan PI-3 kinase warhead conjugated to a disease specific targeting ligand provides a therapeutic platform model for tackling various diseases that have unique surface marker expression on the diseased tissue.
• H-Cys(Trt)-2-Cl-Trt resin and protected amino acids were purchased from Chem- Impex Inti.
• 2-(Hydroxymethyl)pyridine-5-boronic acid, pinacol ester was purchased from Combi- Blocks.
• 6-bromo-4-iodoquinoline, 2-4-Diflurobenzene-l-sulfonyl-chloride and 5-bromo-2- methoxypyridine-3-amine was purchased from ArkPharm. All the other chemicals were purchased from SIGMA-Aldrich and Fisher Scientific and used as received.
• Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 TLC plates.
• Silica gel column chromatography was performed using silica gel (60-120 pm particle size).
• the high resolution mass measurements were recorded on a LTQ Orbitrap XL mass spectrometer utilizing electrospray ionization (ESI).
• FAP ligand (11), FAP-FITC and FAP-S0456 was synthesized following a previously published procedure. See WO2018111989A1.
• mice were purchased from Charles River and maintained on normal rodent chow. They were housed in a sterile environment on a standard 12 h light-and-dark cycle for the duration of the study. All animal procedures were approved by the Purdue Animal Care and Use Committee (PACUC) in accordance with NIH guidelines.
• PACUC Purdue Animal Care and Use Committee
• HLFl-hFAP cells were seeded in the glass-bottom dish and incubated with adequate amount of endosomes tracker (Rab7a-RFP, ThermoFisher) for overnight. Then cells were incubated with FAP-FITC (10 nM) for 1 hour at 4 °C. Followinged by staining with 5 nM DRAQ5 nucleus dye (ThermoFisher) and 3 times of PBS washes, spatial localization of FAP-FITC was monitor at given time under ambient temperature by confocal microscope (FV 1000, Olympus). Confocal images were further processed by FV10-ASW, Olympus software.
• endosomes tracker Rab7a-RFP, ThermoFisher
• HLF1 cells primary IPF fibroblasts and the non-IPF fibroblasts were cultured, fixed, and permeabilized on glass-bottom dishes for immuno fluorescent staining.
• Sirius red staining for secreted total collagen Confluent HLF1 cells were seeded in DMEM medium containing 10% FBS and then 0.4 % serum starvation overnight before stimulation of collagen secretion. TGFbl (0.1 ng/ml) were added into cells with or without PI3K inhibitors. At 2 days post co-incubation, culture medium was collected for determination of total secreted collagen level. Total collagen level was determined by Sirius Red Total Collagen Detection Kit (Chondrex, Inc). Basically, concentrated sample incubated with 500 ml of Sirius Red Solution with for 20 minutes at room temperature. Pellet was collect by centrifuge at 10,000 rpm for 3 minutes and followed by washing with 500 ml of washing solution for 3 times. Add 250 ml of Extraction Buffer to the Sirius Red stained pellet and read the OD at 510-550 nm.
• Serum starved confluent HLF1 cells were co-incubated in medium containing lng/ml TGFbl with or without designate concentrations of PI3K inhibitors for 24 hours. Cells were harvested and lysed for Western blot analysis. Following sodium dodecyl sulphate polyacrylamide gel electrophoresis and blocking, membranes were incubated with antibodies to detect pSMAD2 Ser465/ 467 (#3 ] Q p
• mice 8 to lO-week old C57BL/6-NCrl (Strain Code: 027) male mice (Charles River) were anesthetized (mixture of xylazine/ketamine), followed by a single intratracheal injection of freshly prepared 0.75 mg/Kg of bleomycin sulfate (Cayman Chemicals, Cat N13877) in 50 DL of sterile phosphate- buffered saline (PBS). Control mice were injected with 50 DL of sterile PBS. Body weights were monitored throughout the study. To access the FAP expression and fibrosis in the longitudinal study the lungs were harvested at Days 7, 14 and 21 post-bleomycin instillation. For the therapy with FAP-PI3K inhibitor, same procedure was carried out to administer bleomycin and the lungs were harvested at Day 21 to access the therapeutic efficacy (Day 0 was accounted as the day of bleomycin administration).
• Total lung collagen was determined by analysis of hydroxyproline as previously described. 30 The right lung was consistently dedicated for this assay to allow comparison. Briefly, harvested right lung was homogenized in PBS (PH 7.4), digested with 12N HC1 at 120° C for 3 hr. Citrate/acetate buffer (PH 6.0) and Chloramine-T solution were added at room temperature for 20 minutes and the samples were incubated with Ehrlich’s solution for 15 min at 65°C. Samples were cooled to room temperature and read at 550 nm. Hydroxyproline standards (Sigma, MO) at concentrations between 0 to 100 pg/ml were used to construct a standard curve.
• PBS PH 7.4
• Citrate/acetate buffer PH 6.0
• Chloramine-T solution were added at room temperature for 20 minutes and the samples were incubated with Ehrlich’s solution for 15 min at 65°C. Samples were cooled to room temperature and read at 550 nm. Hydroxyproline standards (Sigma, MO) at concentrations
• the left lung was inflated and fixed with 4% paraformaldehyde. Lung tissues were embedded in paraffin, and 10- Dm sections were prepared and stained using H&E and Masson’s Trichrome. The severity of bleomycin-induced fibrosis was determined by semiquantitative histopathological scoring, at the indicated dates after bleomycin administration. 29
• mice were treated via tail vein (i.v.) injection with 5 nmol of FAP targeted NIR dye conjugate (FAP-S0456) and imaged 2 hr post-injection using a Spectral AMI optical imaging system.
• FAP-S0456 FAP targeted NIR dye conjugate
• Spectral AMI optical imaging system For competition experiments, a 100- fold excess of base the FAP ligand was used. The settings were as follows: Object height, 1.5; excitation, 745 nm; emission, 790 nm; FOV, 25; binning, 2; f-stop, 2; acquisition time, ls.
• animals were dissected, and selected organs were collected and imaged again for complete biodistribution. The conditions remained same for the longitudinal imaging study, except the mice were imaged at day 7, day 14, and day 21 post-bleomycin administration.
• Micro-CT analysis of the whole lung was performed at day 7, day 14, and day 21 post-bleomycin administration Briefly animals were anesthetized with isoflurane and fixed in prone position. Micro-CT images were acquired on a Quantum FX micro-CT system (Perkin Elmer, Waltham, MA) with cardiac gating (without respiratory gating), using the following parameters: 90 kV; 160mA; FOV, 60 x 60 x 60 mm; spatial resolution, 0.11 mm, resulting in a total acquisition time of 4-5 minutes
• an imaging agent conjugate comprising FAP targeting ligand (FAPL) and a fluorescein such as FITC was generated according to the scheme shown in Figure 1.
• FAP targeting ligand FAP targeting ligand
• FITC conjugate was incubated with a FAP transfected cell line HLF1 (human fibroblast cells), confocal microscopy and flow cytometry are used to observe the conjugate’s specific targeting to FAP expressing cell line, and its subsequent endocytosis in the cell line. See Figure 2 and its legend, which shows FAPL-FITC binds well with good competition in the hFAP-HLFl cell line.
• FAP ligand can recognize and target FAP with good specificity.
• FAPL_FITC conjugates are internalized upon receptor engagement. This shows the conjugate is likely to deliver a payload of effectors to the activated fibroblast, for example, the imaging agent or, a therapeutic drug.
• PI3KI1 pan PI-3Kinase-mTOR inhibitor
• This potential IPF drug has a good handle to incorporate releasable linkers to conjugate to a targeting ligand, for example, a FAP ligand in example 2-3.
• the novel PI-3Kinase inhibitor PI3KI1 inhibits Akt phosphorylation better than its GSK counterpart OMIPALISIB.
• the latter is in a clinical trial for IPF therapeutic drug.
• the PI3KI1 also suppresses collagen secretion and collagen gel contraction in HLF-l cell line. Because the novel PI-3Kinase inhibitor PI3KI1 has a free hydroxyl group, it allows facile conjugation to a FAP ligand, show in the example 2 and 3.
• the novel drug does not have toxicity and it behaves similarly to GSK drug OMIPALISIB. See Figure 6 and its Legend.
• FAP targeted PI-3 kinase inhibitor FAP_PI3KIl suppresses TGFP induced collagen secretion at low drug concentration, better than free PI3KI1 and free GSK drug OMIPALISIB in IPF patient cells. See Figure 7 and its legend.
• FAPL_FITC can recognize mouse FAP. Briefly, NIH- 3T3 cells were induced with TGFP to express FAP (mimicking the pathogenesis of fibrosis in mice). FAPL_FITC can recognize almost 98% of the cell population that binds to a specific monoclonal antibody against mouse FAP. See Figure 8 and its legends.
• EXAMPLE 7 Evaluation of FAP targeting of IPF lung in mouse model of IPF in vivo
• FAPL conjugated with drug or other effector specifically target fibrotic lungs in mice day 14 after acute lung injury. Briefly, both the saline treated and fibrotic lungs with FAP competition showed minimum retention of the NIR signal while the diseased lung without competition showed high uptake of the FAP_S0456 dye. Both micro CT and NIR imaging at different time points (day 7, 14, 21) after acute lung injury, showed progression of fibrosis.
• EXAMPLE 8 Evaluation of myofibroblast inactivation with FAPL-targeted PI-3 kinase inhibitor in vivo

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