WO2023230593A1

WO2023230593A1 - Ptp1b inhibitors for treating lung injury

Info

Publication number: WO2023230593A1
Application number: PCT/US2023/067530
Authority: WO
Inventors: Nicholas Tonks; Dongyan Song
Original assignee: Cold Spring Harbor Laboratory
Priority date: 2022-05-27
Filing date: 2023-05-26
Publication date: 2023-11-30

Abstract

Methods and compositions for treating lung injury include administering an effective amount of an PTP1B inhibitor to a subject in need thereof. The lung injury may, for example, be acute lung injury, antibody-induced acute lung, acute lung injury associated with inflammation, ARDS, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID-19, or lung injury resulting from WHIM syndrome.

Description

PTP1B Inhibitors For Treating Lung Injury

Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application Number 63/346,476, which was filed May 27, 2023, the entire content of which is hereby incorporated herein in its entirety.

Technical Field

[0002] This disclosure relates to Protein-Tyrosine Phosphatase IB (PTP1B) inhibitors and their use in treating lung injury. More specifically, in aspects this disclosure relates to the use of PTP1B inhibitors to treat acute lung injury, such as, antibody-induced lung injury. Even more specifically, in aspects this disclosure relates to the use of PTP1B inhibitors to treat Acute Respiratory Distress Syndromes, including those induced in Coronavirus disease 2019 (COVID- 19).

BACKGROUND

[0003] Acute Respiratory Distress Syndrome (ARDS) arises from a variety of causes, including Transfusion-Related Acute Lung Injury (TRALI), sepsis, and Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection. Patients suffer acute lung injury such that, even if they recover, they may face the challenge of permanent lung damage, including for example pulmonary fibrosis. It is not clear what initiates and propagates the features of ARDS, including after severe COVID- 19 infections; however, a drug that attenuated the hyperinflammatory state of ARDS would be of major benefit to patients. Initial approaches to repurposing existing drugs to target COVID-19 ARDS have brought limited success. The UKbased RECOVERY clinical trial illustrated that anti-inflammatory corticosteroids, such as dexamethasone, reduced mortality by up to -12% in patients with advanced COVID-19 who were being treated with mechanical ventilation. Among those receiving oxygen without mechanical ventilation, the reduction in mortality was only -3%. Although subsequent trials also pointed to benefits of corticosteroids in general, timing of administration appears to be critical. Dexamethasone is likely to be more effective late in infection, in the hyperinflammatory state, whereas early, when the body is responding to the virus, it may actually be harmful. Broad spectrum anti-viral agents, such as Remdesivir (VEKLURY®), an inhibitor of the RNA- dependent RNA polymerase, produce a modest reduction in time to recovery for COVID-19 patients. Attempts to target the inflammatory cytokine IL-6, such as with antibodies, have met with limited success and currently are not recommended. Therefore, it would be desirable to identify therapeutic targets through which to address ARDS, including COVID- 19 ARDS.

SUMMARY

[0004] In an aspect, provided is a method of treating lung injury in a subject in need thereof including administering a PTP1B inhibitor to the subject. In an example, the lung injury includes one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID-19, and lung injury resulting from WHIM syndrome. In another example, the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. In yet another example, the PTP1B inhibitor is administered from one to two times a day. In still another example, administering is accomplished via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

[0005] In another aspect, provided is a method of prophylactically inhibiting symptoms of lung injury in a subject at risk of developing lung injury including administering a PTP1B inhibitor to the subject. In an example, the subject is at risk of developing symptoms of lung injury associated with one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID-19, and lung injury resulting from WHIM syndrome. In another example, the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. In yet another example, the PTP1B inhibitor is administered from one to two times a day. In still another example, administering is accomplished via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

[0006] In still another aspect, provided is a pharmaceutical composition including a PTP1B inhibitor and a carrier, wherein the PTP1B inhibitor is present in an amount sufficient to reduce lung injury in a subject or inhibit symptoms of lung injury in a subject at risk of developing lung injury. In an example, the PTP1B inhibitor is MST-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. In still another example, the carrier permits administration of the pharmaceutical composition via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

[0007] In yet another aspect, provided is a method of treating lung injury in a subject in need thereof including administering a PTP1B inhibitor to the subject, wherein the lung injury is at least one of acute lung injury, antibody -induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS- CoV-2 infection, lung injury resulting from COVID- 19, or lung injury resulting from WHIM syndrome and the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. In an example, the lung injury includes acute lung injury. In another example, the lung injury includes antibody-induced acute lung injury. In still another example, the lung injury includes acute lung injury associated with inflammation. In yet another example, the lung injury includes ARDS. In another example, the lung injury includes lung injury resulting from sepsis. In a further example, the lung injury includes lung injury resulting from SARS- CoV-2 infection. In still a further example, the lung injury results from WHIM syndrome. In another example, the lung injury includes lung injury resulting from sepsis. In still a further example, the lung injury includes lung injury resulting from COVID-19.

[0008] In another example, the lung injury includes acute lung injury and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes acute lung injury and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In yet another example, the lung injury includes antibody-induced acute lung injury and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In a further example, the lung injury includes antibody-induced acute lung injury and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In still a further example, the lung injury includes acute lung injury associated with inflammation and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In yet a further example, the lung injury includes acute lung injury associated with inflammation and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes ARDS and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes ARDS and the PTP1B inhibitor is DPM-1003 or a pharm ceutically acceptable salt thereof. Tn yet another example, the lung injury includes lung injury resulting from SARS-CoV-2 infection and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In a further example, the lung injury includes lung injury resulting from SARS-CoV-2 infection and the PTP1B inhibitor is DPM- 1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes lung injury resulting from sepsis and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes lung injury resulting from sepsis and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In still a further example, the lung injury includes lung injury resulting from COVID- 19 and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In yet a further example, the lung injury includes lung injury resulting from COVID- 19 and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes lung injury resulting from WHIM syndrome and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In yet another example, the lung injury includes lung injury resulting from WHIM syndrome and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

[0009] In a further aspect, provided is a method of prophylactically inhibiting symptoms of lung injury in a subject at risk of developing lung injury including administering a PTP1B inhibitor to the subject, wherein the subject is at risk of developing symptoms of lung injury associated with one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID- 19, and lung injury associated with WHIM syndrome and the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. In an example, the lung injury includes acute lung injury. In another example, the lung injury includes antibody-induced acute lung injury. In still another example, the lung injury includes acute lung injury associated with inflammation. In yet another example, the lung injury includes ARDS. In another example, the lung injury includes lung injury resulting from sepsis. In a further example, the lung injury includes lung injury resulting from SARS-CoV-2 infection. In another example, the lung injury includes lung injury resulting from sepsis. In still a further example, the lung injury includes lung injury resulting from COVTD-19. Tn another example, the lung injury includes lung injury resulting from WHIM syndrome.

[0010] In another example, the lung injury includes acute lung injury and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes acute lung injury and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In yet another example, the lung injury includes antibody-induced acute lung injury and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In a further example, the lung injury includes antibody-induced acute lung injury and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In still a further example, the lung injury includes acute lung injury associated with inflammation and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In yet a further example, the lung injury includes acute lung injury associated with inflammation and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes ARDS and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes ARDS and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In yet another example, the lung injury includes lung injury resulting from SARS-CoV-2 infection and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In a further example, the lung injury includes lung injury resulting from SARS-CoV-2 infection and the PTP1B inhibitor is DPM- 1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes lung injury resulting from sepsis and the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes lung injury resulting from sepsis and the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In still a further example, the lung injury includes lung injury resulting from COVID- 19 and the PTPIB inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In yet a further example, the lung injury includes lung injury resulting from COVID- 19 and the PTPIB inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. In another example, the lung injury includes lung injury resulting from WHIM syndrome and the PTPIB inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof. In still another example, the lung injury includes lung injury resulting from WHIM syndrome and the PTPIB inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof. [001 1 ] In aspects of this disclosure, PTP1B inhibitors attenuate CXCR4 signaling and induce an aged neutrophil phenotype to protect against lethality in a mouse model of acute lung injury. These data highlight PTP1B as a therapeutic target for Acute Respiratory Distress Syndromes, including those induced in COVID-19.

[0012] In aspects, PTP1B inhibitors are used in accordance with methods of the present disclosure to attenuate lung injury and increase survival in a classical Transfusion-Related Acute Lung Injury (TRALI) model. Treatment with PTP1B inhibitors in accordance with the present disclosure also attenuates neutrophil function, associated with release of myeloperoxidase, suppression of Neutrophil Extracellular Trap (NET) formation, and inhibition of neutrophil migration. By attenuating signaling through the CXCR4 chemokine receptor, particularly to the activation of mT0RC2, PTP1B inhibition promotes an aged neutrophil phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.s 1A-1P show PTP1B inhibitors improved survival and ameliorated lung damage in the TRALI mouse model. (A) Schematic illustration of the TRALI induction and PTP1B inhibition protocol. (B) The structural formulae of PTP1B inhibitor MSI- 1436. (C) The survival curves of TRALI mice treated with increasing concentrations of the PTP1B inhibitor MSL1436 or saline (n=20 mice, two independent biological repeats). (D) The survival curves of TRALI mice treated with increasing concentrations of the PTP1B inhibitor DPM-1003 or saline (n=20-25 mice, two independent biological repeats). (E) The structural formulae of PTP1B inhibitor DPM-1003. (F) Top panel: representative images of H&E-stained lung tissue from no treatment mice (NT), and TRALI mice after administering saline, MSI-14362 mg/kg, or MSL 1436 10 mg/kg. Arrows indicate alveolar damage. Asterisks indicate edema or hyaline membranes. Scale bars: 25pm. Magnification: 40x. Bottom panel: lung injury scores for each treatment group. (n=4 mice per group) (G) The protein concentrations in the bronchoalveolar lavage fluid (BALF) collected from NT, TRALI mice treated with either saline or 10 mg/kg MSI-1436 (n=6 mice per group). (H) Representative 3D-rendered images of lung volumes from CT scans of mice from the saline- and MSI-1436-treated TRALI mice. Cyan color represents hyperdense areas of edema and vasculature (HU (Hounsfield units) > 0), grey /lavender color for hypodense regions of airspace i.e. viable lung (HU < 0). (I) Top panel: the representative transverse CT images from TRALI mice treated with either saline or MSI-1436. Dashed lines indicate the boundaries of the total lung ROT. Bottom panel: the mean lung radiodensity measured by longitudinal CT scans in two experimental groups (n=7-8 mice per group). HU 0 = density of water and -1,000 = density of air. (J) The percentage of viable lung volume (volume of viable lung/volume of total lung) calculated from longitudinal CT scans in two experimental groups as in (F). (n=7-8 mice per group) (K) The survival curves of CLP-induced sepsis model treated either with saline or 5 mg/kg MSI-1436 two hours before surgery (n=18 mice per group). Data are presented as mean ± SEM. (L) The survival curves of CLP-induced sepsis model treated either with saline or 5 mg/kg MSI-1436, six hours after surgery (n=21 mice per group). (M) Schematic illustration of LPS-induced sepsis model. (N) The survival curves of 30 mg/kg LPS challenged sepsis mice treated with either MSI- 1436 or saline (n=10 mice, two independent biological repeats). (O) The survival curves of 15 mg/kg LPS challenged sepsis mice treated with either MSI- 1436 or saline (n=10 mice, two independent biological repeats). (Q) Representative H&E staining images of lung tissues harvested from mice treated with saline only, saline 2 hours prior to LPS, and MSI-1436 2 hours prior to LPS administration. Statistical analysis forC, D, and K by Log-rank (Mantel-Cox) test; for F by nonparametric two-tailed Mann-Whitney test; for G by one-way ANOVA; for J by two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001, ****pO .0001. Statistical analysis for I by two-way ANOVA; F, N, and O by Log-rank (Mantel- Cox) test; ****p<0.0001.

[0014] FIGs. 2A-2M show Treatment with PTP1B inhibitors in vivo induced neutrophilia. (A) Pie charts of flow cytometry analysis to measure infdtration of nine immune cell populations into lung tissues. The numbers are average abundance of each immune cell subset (% of CD45+ cells, n=5). For saline- and MSI-1436 (10 mg/kg)-treated groups, the lungs were harvested 30 min after TRALI induction. (B) The gating strategy used to identify the nine immune cell populations from the single cell suspension of lung tissue. (C) The percentages of WBC subsets measured in the circulating blood collected from either NT or TRALI mice. For TRALI mice, the blood was collected 30 min after anti- MHC I antibody injection, and pretreated with either saline or 10 mg/kg MSI-1436 (n=5). Statistical analysis by two-way ANOVA with Tukey’s multiple comparisons test (D) Left panel: representative flow cytometry plots for neutrophils infdtrated into lung tissues. Right panel: quantification of percentage of neutrophil population out of myeloid cells. Statistical analysis by one-way ANOVA, Tukey’s multiple comparisons test; *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) Percentage of neutrophils (relative to total WBCs) in peripheral blood from mice treated with saline, or 10 mg/kg MSI-1436 for 2.5 hours (n=5). Statistical analysis by two-tailed student’ s ttest; *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (F) Percentage of neutrophils (relative to total WBCs) in peripheral blood from mice treated with saline, or 20 mg/kg DPM-1003 for 2.5 hours (n=5). Data are presented as mean ± SEM. Statistical analysis by two-tailed student’s t-test; **p<0.01, ****p_<0.0001. (G, H) Cytokine arrays generated from serum (G) or lung tissue (H) collected from NT or TRALI mice treated with saline or MSI-1436. Each group contained an equal amount of serum or lung tissue homogenate pooled from 5 mice. (I, J) CXCL1 levels in serum (I) and matched lung tissue (J) from NT, and TRALI mice treated with saline or MSI-1436 at the indicated doses (n=5). Statistical analysis by one-way ANOVA, Tukey’s multiple comparisons test; *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (K) CXCL2 levels in lung tissue from NT, and TRALI mice treated with saline or MSI-1436 at the indicated doses (n=5). Data are presented as mean ± SEM. Statistical analysis by one-way ANOVA, Tukey’s multiple comparisons test; *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (L, M) CXCL1 was measured in the plasma of mice treated with saline, 10 mg/kg MSI- 1436 (L) or 20 mg/kg DPM- 1003 (M). Data are presented as mean ± SEM, where n=5 mice per group. Statistical analysis for M and N by two-tailed student’s t-test; ****p<0.0001.

[0015] FIGs. 3A-3F show Treatment with MSI-1436 in vivo induced an aged neutrophil phenotype.. (A) Reactome pathway analysis performed using g:Profiler for genes up-regulated upon MSI-1436 treatment. (B) Representative confocal immunofluorescence microscopy images and quantification of neutrophils isolated from peripheral blood 2.5 hours after injection of saline or MSI- 1436, and stained with anti-MPO (green) and DAPI (blue). Scale bars: 10pm, n=4 mice per group. Statistical analysis by two-tailed student’ s ttest; **p<0.01, ***p<0.001, ****p<0.0001. (C) Neutrophils stained for MPO-containing granules from mice treated with saline or MSI- 1436 for 2.5 hours. MPO signals were quantified as MFI (Mean Fluorescence Intensity) (n=4). Statistical analysis by two-tailed student’s ttest; **p<0.01, ***p<0.001, ****p<0.0001. (D) The flow cytometry analysis of MPO in the neutrophils following treatment with saline or DPM-1003 for 2.5 hours. MPO was quantified by MFI. Statistical analysis by two- tailed student’s t-test; *p<0.05, **p<0.01. (E) Surface expression of CD62L and CXCR2 on neutrophils from peripheral blood collected 2.5 hours after saline or MSI-1436 treatment, quantified as MFI (n=5). Data are presented as mean ± SEM. Statistical analysis by two-tailed student’ s ttest; **p<0.01, ***p<0.001, ****p<0.0001. (F) The flow cytometry analysis of fresh neutrophil markers CD62L and CXCR2 following treatment with saline or DPM-1003 for 2.5 hours, quantified as MFI. Data are presented as mean ± SEM. Statistical analysis by two-tailed student’ s t-test; *p<0.05, **p<0.01.

[0016] FIGs. 4A-4F show Treatment with MSI-1436 suppressed NET formation ex vivo and in vivo.. (A) Representative immunofluorescence microscopy images and quantification, showing formation of NETs upon PMA treatment ex vivo. Arrows indicate NETs visualized by colocalization of DAPI (blue), citH3 (red) and MPO (green) staining. Higher magnifications of selected regions are shown in the lower squares. Scale bars: 100 pm, n=4. The zoomed-in details is shown for neutrophils treated with saline together with PMA (lower left panel) with three individual channels to show colocalization of DAPI, citH3 and MPO. Statistical analysis by oneway ANOVA with Dunnet’s multiple comparison test; *P<0.05, ***p<0.001, ****p<0.0001. (B) Representative confocal images and quantification, showing the NETosis frequency in response to PTP1B inhibitor and PMA stimulation. Scale bars: 50 pm, n=4 mice per group with total 20 random fields for quantification. Statistical analysis by one-way ANOVA with Sidak’s multiple comparisons test; *P<0.05, ***p<0.001, ****p<0.0001. (C) Representative confocal images and associated quantification, showing the NET frequency in response to PMA (left panels) or MSI- 1436+PMA (right panel) stimulation of human primary neutrophils. Scale bars: 100 qm, n=3 healthy volunteers. Data are presented as mean ± SEM. Statistical analysis by two-tailed student’s t-test, **p<0.01. (D) Whole-mount staining of lung tissue from NT, and TRALI mice administered saline, 2 mg/kg or 10 mg/kg MSI-1436, with quantification of the frequencies of NETs. Arrows indicate NETs, visualized as in (A). Scale bars: 100 pm, n=4. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test, *P<0.05, ***p<0.001, ****p<0.0001. (E) Immunoblot analyses showing Akt signaling changes using primary neutrophils isolated from bone marrow. Representative immunoblot of three independent experiments. Data are presented as mean ± SEM. (F) Neutrophils isolated from bone marrow (BM) were stimulated with 100 nM PMA for the indicated time. Immunoblot analysis showing DPM-1003 attenuated Akt and Erkl/2 phosphorylation. The numbers on the left indicate molecular weight standards.

[0017] FIGs. 5A-5K show PTP1B inhibitors attenuated PI3Kg-mediated CXCR4 signaling. (A) Quantitation of MPO from neutrophils isolated from mice treated with vehicle + saline, vehicle + MST-1436, AZD5069 + MSI-1436, and AZD5069 + saline. n=5 mice for each group. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test;

*P<0.05, ***p<0.001, ****p<0.0001. (B) Percentage of neutrophils, designated as Ly6Ghi population, relative to total WBCs, from the indicated treatment groups. n=5 mice for each group. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test;

*P<0.05, ***p<0.001, ****p<0.0001. (C) Immunoblot analyses showing the effect of MSI-1436 on CXCR4 signaling upon CXCL12 stimulation from primary neutrophils isolated from bone marrow. Representative immunoblot of four independent experiments. (D) Immunoblot analyses of DPM-1003 treated neutrophils showing dose-dependent inhibitions of CXCR4-mediated AKT signaling. (E) Immunoblot analyses showing the AKT signaling in response to PI3K isoform specific inhibitors in HeLa, HL-60, and mouse neutrophils. Inhibitors used: a-specific (HS-173, IpM); b-specific (GSK2636771, lOpM); d-specific (Nemiralisib, 100 nM); g-specific (Eganelisib, 200nM); pretreated 1 hour before CXCL12 stimulation. Representative immunoblot of three independent experiments. (F) Immunoblot analyses showing the impact of pretreatment with MSI-1436 on AKT signaling in HeLa, HL-60 and mouse primary neutrophils.

Representative immunoblot of three independent experiments. Data are presented as mean ± SEM. (G) Immunoblot analyses of the effect of DPM-1003 on AKT signaling in HeLa, HL-60, and mouse primary neutrophils upon CXCL12 stimulation. (H, I) Immunoblot analyses showing the decrease of CXCL2-stimulated AKT and ERK1/2 phosphorylation upon MSI-1436 (H) and DPM-1003 (I) treatment. (J, K) Immunoblot analyses showing the increase of fMLP stimulated AKT and ERK1/2 signaling upon MSI-1436 (J) and DPM-1003 (K) treatment. The numbers on the left indicate molecular weight standards.

[0018] FIGs. 6A-6H show the effect of mTOR inhibitor on the survival of TRALI model and induction of aged neutrophil phenotype. (A) Neutrophil migration towards CXCL12 examined using Transwell assays. n=3 independent biological repeats. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test; *P<0.05, **p<0.01. (B, C) The impact of PTP1B inhibitors MSI-1436 (B) and DPM-1003 (C) on neutrophil migration towards fMLP examined using Transwell assays. n=4-5 independent biological repeats. (D) Immunoblot analyses showing the effect of P529 on mTOR signaling upon CXCL12 stimulation from primary neutrophils isolated from bone marrow. Representative immunoblot of three independent experiments. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test; *P<0.05, **p<0.01 . (E) Survival curve of TRALT mice treated with vehicle or 25 mg/kg P529 (n = 19 mice, two independent biological repeats). Statistical analysis by Logrank (Mantel-Cox) test. *P<0.05, **p<0.01. (F) P529 prolonged survival in LPS-induced sepsis model. Data are presented as mean ± SEM. (G) Surface expression of CD62L, CXCR2, and CXCR4 on neutrophils from peripheral blood collected 2.5 hours after vehicle or P529 treatment, quantified as MFI (n = 6 mice for each group). (H) Schematic representation of the proposed mechanism.

DETAILED DESCRIPTION

[0019] The functions of protein kinases and phosphatases, which promote the addition and removal of phosphate groups, respectively, are coordinated in signal transduction pathways to mediate the cellular response to environmental stimuli, including cytokines. These pathways are of fundamental importance to control of cellular homeostasis and their disruption frequently underlies major human diseases. Consequently, the ability to modulate such pathways selectively with drugs holds enormous therapeutic potential. There have been successes with kinase-directed drugs in various indications. Nevertheless, although attempts to repurpose kinase inhibitors have produced some encouraging results, to date they have had little impact in COVID-19. What is needed are additional, novel targets through which to exploit pathological changes in signaling. Like the kinases, the protein phosphatases are also regulators of signaling; however, they are viewed as challenging targets and remain largely an untapped resource for drug development. PTP1B, the prototypic protein tyrosine phosphatase (PTP), plays a role in down-regulating insulin and leptin signaling and is a validated therapeutic target for diabetes and obesity.

Furthermore, PTP IB also plays a positive role in promoting signaling events associated with HER2-induced breast tumorigenesis and has been validated as a target in cancer. The identification of PTPs as therapeutic targets led to the generation of active site-directed inhibitors that were potent and selective but, due to the architecture of the active site and the mechanism of PTP-mediated catalysis, these molecules were highly charged and limited in drug development potential. Allosteric inhibitors of PTP1B have been developed that have circumvented the problems at the PTP active site. Also, by testing analogues of the prototypic allosteric inhibitor, MSL1436, also known as trodusquemine, additional PTP1B inhibitors have been identified and validated with improved drug-like properties. It has been reported that, in addition to diabetes, obesity and cancer, PTP1B also serves an important regulatory function in immunity and host defense. It has now been found that in accordance with the presently disclosed methods PTP1B inhibitors provide a mechanism to address COVID- 19 acute lung injury and ARDS.

[0020] With respect to an anti-inflammatory effect of PTP1B inhibitors, deletion of PTP1B protects against cardiovascular inflammation associated with septic shock, inhibition of PTP1B induces M2 macrophage polarization and a potential anti-inflammatory effect, PTP1B has been suggested to be a therapeutic target for neuroinflammatory diseases, and PTP1B deficiency protects against hepatic fibrosis. In addition, PTPIB-deficient mice clear P. aeruginosa more efficiently from their lungs than wild type mice; however, this effect is associated with increased cytokine production. Nevertheless, there are conflicting data suggesting that B-cell-specific PTP1B knockout results in systemic autoimmunity (Medgyesi, et al., Journal of Experimental Medicine, 211 :427-440, 2014), deletion ofPTPlB exacerbates lung damage in response to respiratory syncytial virus (Foronjy, et al., Mucosal immunology 9: 1317- 1329, 2016), and loss of PTP1B increases the effects of pro-inflammatory stimuli in both human and rodent macrophages (Traves, et al., Cell death & disease, 5:el l25-el l25, 2014).

Furthermore, PTPIB-deficient neutrophils display enhanced bacterial phagocytosis and killing, with increased TLR4 signaling. These studies make use of PTP1B knockout animals in which expression of the phosphatase is abrogated throughout their development and do not necessarily correlate to a situation, such as in COVID-19, where a short dose of PTP1B inhibitor is administered. Furthermore, advanced COVID 19 patients already have high levels of cytokines, such as IL6, and one target of PTP1B is the JAK family of protein tyrosine kinases that transduce the signaling response of cytokine receptors. A function of PTP1B is to attenuate JAK activity, such as in the context of leptin receptor signaling. Consequently, the possibility that a PTP1B inhibitor would activate JAKs further under hyperinflammatory conditions must also be considered. Therefore, in accordance with aspects of the present disclosure a suitable model of ARDS has been developed to test the impact of PTP1B inhibitors.

[0021] The activation of neutrophils and the formation of neutrophil extracellular traps (NETs) impact the pathophysiology of ARDS, and the roles of neutrophils and NETs are documented in TRALI. A classical two-step TRALI model involves immune priming with a low dose of lipopolysaccharide (LPS) then, ~24 hours later, initiation of an acute phase caused by injecting Major Histocompatibility Complex-I (MHC-I) antibodies. In accordance with aspects of the present disclosure, the administration of two distinct allosteric inhibitors of PTP1B, MST- 1436 and DPM-1003 is provided, and their effect on the induction of TRALI in this animal model. As elevated levels of neutrophils and excess NET formation are features of severe SARS- CoV-2 infection, the methods of the present disclosure provide a new treatment of ARDS, including in COVID- 19.

[0022] WHIM syndrome (warts, hypogamma-globulinemia, infections, and myelokathexis), a rare immunodeficiency disorder, arises from gain-of-function mutations of CXCR4 (Hernandez et al., 2003; Gulino et al., 2004). Patients display greater than normal susceptibility to life-threatening infections. WHIM syndrome involves panleukopenia, which underlies the increased susceptibility to bacterial and viral infection. In addition, myelokathexis, resulting from neutrophil retention in bone marrow, leads to neutropenia.

[0023] As disclosed herein, administrating a PTP1B inhibitors effectively increases the number of neutrophils in the peripheral blood by approximately threefold and inhibits CXCR4 signaling (Song et al, 2022). However, as disclosed herein, PTP1B inhibitor administration attenuates aberrant neutrophil function that drives Acute Lung Injury and was associated with release of myeloperoxidase, suppression of Neutrophil Extracellular Trap (NET) formation, and inhibition of neutrophil migration. As disclosed herein, reduced signaling through the CXCR4 chemokine receptor, for example to the activation of PI3Ky/AKT/mTOR, may be involved in PTP1B inhibition to promoting an aged-neutrophil phenotype, without being limited to any particular mechanism of action.

[0024] In the context of acute lung injury and sepsis, it has been shown that knock-in mice expressing WHIM-derived activating mutations in CXCR4 display increased lung infiltration of myeloid cells following LPS challenge, and that neutrophils expressing Cxcr4WHIM exhibit constitutive elevations of CD62L, a marker of fresh neutrophils. Fresh neutrophils are more prone to forming neutrophil extracellular traps (NETs) and causing lung damage in mouse models of acute lung injury.

[0025] As disclosed herein, administration of PTP1B inhibitors induces an aged neutrophil phenotype, which provides protection against lung damage. PTP1B inhibitor administration may therefore attenuate the effects of gain-of-function mutations of CXCR4 for the treatment of WHIM syndrome, and may be a treatment for WHIM syndrome and lung injury associated with r resulting from WHIM syndrome. [0026] Acute lung injury (ALT) remains a significant source of morbidity and mortality in critically ill patient populations. Defined by a constellation of clinical criteria (acute onset of bilateral pulmonary infiltrates with hypoxemia without evidence of hydrostatic pulmonary edema), ALT has a high incidence (200,000 per year in the US) and overall mortality remains high. Pathogenesis of ALT is explained by injury to both the vascular endothelium and alveolar epithelium. Essentially, ALT is a disorder of acute inflammation that causes disruption of the lung endothelial and epithelial barriers. The alveolar-capillary membrane includes the microvascular endothelium, interstitium, and alveolar epithelium. Cellular characteristics of ALT include loss of alveolar-capillary membrane integrity, excessive transepithelial neutrophil migration, and release of pro-inflammatory, cytotoxic mediators. Biomarkers found on the epithelium and endothelium and that are involved in the inflammatory and coagulation cascades predict morbidity and mortality in ALT include those presented in Table 1.

Table 1. Biomarkers of ALT and ARDS

[0027] As used herein the term "acute lung injury" means acute hypoxemic respiratory failure with bilateral pulmonary infdtrates that is associated with both pulmonary and nonpulmonary risk factors and that is not primarily due to left atrial hypertension. ARDS is a subtype of acute lung injury characterized by more severe hypoxemia. For example: in embodiments, the acute lung injury may be evidenced by the development of microvascular thrombi; in embodiments, the acute lung injury may be evidenced by hyaline membrane formation; in embodiments, the acute lung injury may be evidenced by injury of the alveolar epithelium and endothelium; in embodiments, the acute lung injury is evidenced by neutrophilic alveolitis; in embodiments, the acute lung injury is associated with inflammation; in embodiments, the acute lung injury is associated with sepsis; in embodiments, the acute lung injury is associated with ARDS; in embodiments the acute lung injury is associated with SARS- CoV-2 infection; in embodiments, the acute lung injury is associated with COVID-19. In embodiments, acute lung injury is characterized by one or more symptoms including pulmonary edema, sepsis, mi crovascul ar thrombi, hyaline membrane formation, or neutrophilic alveolitis. [0028] Any PTP1B inhibitor that provides a therapeutic benefit with respect to lung injury may be employed in accordance with the present disclosure. Suitable PTP1B inhibitors include, but are not limited to, MSI-1436 and DPM-1003.

[0029] The structure of MSI-1436 is:

[0030] The structure of DPM-1003 is:

[0031] Other PTP1B inhibitors are known to skilled persons and could be used in place of one or both of MSI-1436 and DPM-1003 for all methods, uses, purposes, treatments, and clinical and other indications disclosed throughout this application, without restriction or limitation, and all such PTP1B inhibitors are hereby explicitly included in this disclosure for all such purposes. References disclosing other such PTP1B inhibitors include, without limitation, U.S. Patent Number 9,365,608, U.S. Patent Number 9,546, 194, U.S. Patent Number 10,556,923, U.S Patent Application Publication Number 2020/0376006 Al , the entire contents of all of which are hereby incorporated by reference in their entireties.

[0032] A non-limiting, non-exhaustive, listing of illustrative examples of such PTP1B inhibitors includes MSI 1241, MSI 1255, MSI 1256, MSI 1271, MSI 1272, MSI 1303, MSI 1304, MSI 1317, MSI 1320, MSI 1321, MSI 1322, MSI 1336, MSI 1352, MSI 1370, MSI 1371, MSI 1409, MSI 1413, MSI 1431, MSI 1432, MSI 1433, MSI 1436, MSI 1437, MSI 1448, MSI 1459, MSI 1466, MSI 1469, MSI 1470, MSI 1486, MSI 1487, MSI 1520, MSI 1521, MSI 1561,

MSI 1562, MSI 1569, MSI 1597, MSI 1598, MSI 1678, MSI 1701, MSI 1718, MSI 1751, MSI 1755, MSI 1768, MSI 1777, MSI 1783, MSI 1804, MSI 1805, MSI 1810, MSI 1811, MSI 1812, MSI 1814, MSI 1830, MSI 1839, MSI 1873, MSI 1875, MSI 1876, MSI 1877, MSI 1888, MSI 1892, MSI 1893, MSI 1894, MSI 1909, MSI 1911, MSI 1913, MSI 1920, MSI 2347, MSI 2348, MSI 2349, MSI 2351, MSI 2352, MSI 2353, MSI 2354, MSI 2355, MSI 2356, MSI 2357, MSI 2358, MSI 2360, MSI 2361, MSI 2363, MSI 2364, MSI 2365, MSI 2367, MSI 2368, MSI 2369, MSI 2370, MSI 2371, MSI 2374, MSI 2375, MSI 2450, MSI 2451, MSI 2459, MSI 2464, MSI

2465, MSI 2484, MSI 2490, MSI 2491, MSI 2492, MSI 2493, MSI 2494, MSI 2495, MSI 2496,

MSI 2497, MSI 2498, MSI 2522, MSI 2526, MSI 2520, MSI 2521, MSI 2524, MSI 2518, MSI 2519, MSI 2527, MSI 2523, MSI 2514, MSI 2507, MSI 2511, MSI 2512, MSI 2515, MSI 2528, MSI 2510, MSI 2529, MSI 2506, MSI 2516, MSI 2532, MSI 2517, MSI 2531, MSI 2530, MSI 2504, MSI 2505, MSI 2500, MSI 2501, MSI 2502, MSI 2503, MSI 2508, MSI 2509, MSI 2513, MSI 2525, MSI 2533, MSI 2534, MSI 2535, MSI 2536, MSI 2537, MSI 2538, MSI 2539, MSI

2540, MSI 2541, MSI 2542, MSI 2543, MSI 2544, MSI 2545, MSI 2546, 2547, MSI 2548, MSI

2549, MSI 2550, MSI 2551, MSI 2552, MSI 2553, MSI 2554, MSI 2555, MSI 2556, MSI 2557,

MSI 2558, DPM-1001, DPM-1003, DPM-1011, DPM-1013, DPM-1014, DPM-1015, DPM-

1016, and pharmaceutically acceptable salts thereof.

[0033] A non-limiting, non-exhaustive, listing of illustrative examples of such PTP1B inhibitors includes

salts thereof.

[0034] In embodiments, MSI-1436 or DPM-1003 may be provided as an acid addition salt, a zwitter ion hydrate, zwitter ion anhydrate, hydrochloride or hydrobromide salt, or in the form of the zwitter ion monohydrate. Acid addition salts, include but are not limited to, maleic, fumaric, benzoic, ascorbic, succinic, oxalic, bis-methylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic, palmitic, itaconic, glycolic, pantothenic, p-amino-benzoic, glutamic, benzene sulfonic or theophylline acetic acid addition salts, as well as the 8-halotheophyllines, for example 8-bromo-theophylline. In embodiments, inorganic acid addition salts, including but not limited to, hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, phosphoric or nitric acid addition salts may be used.

[0035] An effective amount of MSI-1436 or DPM-1003, or a respective pharmaceutically acceptable salt thereof for treatment of acute lung injury herein may advantageously be devoid of or exhibits less unwanted side-effects. In addition, an effective amount of MSI-1436 or DPM-1003, or a respective pharmaceutically acceptable salt thereof for treatment of lung injury is surprisingly effective despite the increase in the number of neutrophils induced by administration of the PTP1B inhibitor.

[0036] In embodiments, the terms “effective amount” or “therapeutically effective amount” may be used interchangeably and refer to an amount of a compound, material, composition, medicament, or other material that is effective to achieve reduction, elimination or prophylaxis of acute lung injury. For example, a pharmaceutical composition including an effective amount of a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof may contain from about 20 mg to about 25 mg, about 25 mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40 mg, about 40 mg to about 45 mg, about 45 mg to about 50 mg, about 50 mg to about 55 mg, about 55 mg to about 60 mg, about 60 mg to about 65 mg, about 65 mg to about 70 mg, about 70 mg to about 75 mg, about 75 mg to about 80 mg, about 80 mg to about 85 mg, about 85 mg to about 90 mg, about 90 mg to about 95 mg, about 95 mg to about 100 mg, about 100 mg to about 105 mg, about 105 mg to about 110 mg, about 110 mg to about 115 mg, about 115 mg to about 120 mg, about 120 mg to about 125 mg, about 125 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, about 450 mg to about 500 mg, about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg to about 950 mg, or about 950 mg to about 1 ,000 mg of a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof. In embodiments a pharmaceutical composition containing an effective amount of a PTP1B inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof includes 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg, 103 mg, 104 mg, 105 mg, 106 mg, 107 mg, 108 mg, 109 mg, 110 mg, 111 mg, 112 mg, 113 mg, 114 mg, 115 mg, 116 mg, 117 mg, 118 mg, 119 mg, 120 mg, 121 mg, 122 mg, 123 mg, 124 mg, or 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1,000 mg of a PTPlB inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof. Amounts below about 20 mg of a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof are less effective at relieving or eliminating acute lung injury than amounts in the effective range. Amounts above about 125 mg of a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof may exhibit increased side effects than amounts in the effective range.

[0037] In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is administered to a subject at about 25 mg/per day, 30 mg/per day, 35 mg/per day, 40 mg/per day, 45 mg/per day, 50 mg/per day, 60 mg/per day, 65 mg/per day, 70 mg/per day, 75 mg/per day, 80 mg/per day, 85 mg/per day, 90 mg/per day, 95 mg/per day, 100 mg/per day, 105 mg/per day, 110 mg/per day, 115 mg/per day, 120 mg/per day, 125 mg/per day, 130 mg/per day, 135 mg/per day, 140 mg/per day, 145 mg/per day, 150 mg/per day, 155 mg/per day, 160 mg/per day, 165 mg/per day, 170 mg/per day, 175 mg/per day, 180 mg/per day, 185 mg/per day, 190 mg/per day, 195 mg/per day, 200 mg/per day, 205 mg/per day, 210 mg/per day, 215 mg/per day, 220 mg/per day, 225 mg/per day, 230 mg/per day, 235 mg/per day, 240 mg/per day, 245 mg/per day, or 250 mg/per day, in one or more doses. In embodiments, the subject may be started at a low dose and the dosage is escalated over time. [0038] In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is administered to a subject experiencing symptoms of acute lung injury via a pharmaceutical composition. Pharmaceutical compositions herein encompass dosage forms. Dosage forms herein encompass unit doses. In embodiments, as discussed below, various dosage forms including conventional formulations and modified release formulations can be administered one or more times daily. In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is administered to a subject once or twice a day, (e.g., morning and/or evening). In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is administered to a subject at the start of an acute lung injury episode, whenever that may occur. Any suitable route of administration may be utilized, e.g., oral, rectal, nasal, pulmonary, vaginal, sublingual, transdermal, intravenous, intraarterial, intramuscular, intraperitoneal and subcutaneous routes. Suitable dosage forms include tablets, capsules, oral liquids, powders, aerosols, transdermal modalities such as topical liquids, patches, creams and ointments, parenteral formulations and suppositories. In embodiments, a PTPlB inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is used to manufacture a medicament for treatment of acute lung injury. [0039] In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 1 hour after administration to the subject. In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 2 hours after administration to the subject. In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 3 hours after administration to the subject. In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 4 hours after administration to the subject. In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 6 hours after administration to the subject. In embodiments, methods of treating acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury for more than 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours after administration to the subject. In embodiments, the pharmaceutical compositions provide improvement of next day functioning of the subject having acute lung injury. For example, the pharmaceutical compositions may provide improvement in symptoms of acute lung injury for more than about, e.g., 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours after administration and waking from a night of sleep.

[0040] In embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTPlB inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 1 hour after administration to the subject. In embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including (a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 2 hours after administration to the subject. Tn embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 3 hours after administration to the subject. In embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 4 hours after administration to the subject. In embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 6 hours after administration to the subject. In embodiments, methods of treating antibody induced acute lung injury are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in one or more symptoms of antibody induced acute lung injury for more than 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours after administration to the subject. In embodiments, the pharmaceutical compositions provide improvement of next day functioning of the subject experiencing antibody induced acute lung injury. For example, the pharmaceutical compositions may provide improvement in one or more symptoms of antibody induced acute lung injury for more than about, e.g., 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours after administration and waking from a night of sleep. [0041] In embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 1 hour after administration to the subject. Tn embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTPTB inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 2 hours after administration to the subject. In embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 3 hours after administration to the subject. In embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTPlB inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 4 hours after administration to the subject. In embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 6 hours after administration to the subject. In embodiments, methods of treating acute lung injury associated with inflammation are provided which include administering to a subject in need thereof a pharmaceutical composition including a PTPlB inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof wherein the composition provides improvement in symptoms of acute lung injury associated with inflammation for more than 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours after administration to the subject. In embodiments, the pharmaceutical compositions provide improvement of next day functioning of the subject having acute lung injury associated with inflammation. For example, the pharmaceutical compositions may provide improvement in symptoms of acute lung injury associated with inflammation for more than about, e g., 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours after administration and waking from a night of sleep.

[0042] In embodiments, as mentioned previously, pharmaceutical compositions herein may be provided with conventional release or modified release profiles. Pharmaceutical compositions may be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective. The “carrier” includes all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, and coating compositions. Those with skill in the art are familiar with such pharmaceutical carriers and methods of compounding pharmaceutical compositions using such carriers.

[0043] In embodiments, pharmaceutical compositions herein are modified release dosage forms which provide modified release profiles. Modified release profiles may exhibit immediate release, delayed release, or extended release profiles. Conventional (or unmodified) release oral dosage forms such as tablets, capsules, suppositories, syrups, solutions and suspensions typically release medications into the mouth, stomach or intestines as the tablet, capsule shell or suppository dissolves, or, in the case of syrups, solutions and suspensions, when they are swallowed. The pattern of drug release from modified release (MR) dosage forms is deliberately changed from that of a conventional dosage form to achieve a desired therapeutic objective and/or better patient compliance. Types of MR drug products include orally disintegrating dosage forms (ODDFs) which provide immediate release, extended release dosage forms, delayed release dosage forms (e.g., enteric coated), and pulsatile release dosage forms. [0044] An ODDF is a solid dosage form containing a medicinal substance or active ingredient which disintegrates rapidly, usually within a matter of seconds when placed upon the tongue. The disintegration time for ODDFs generally range from one or two seconds to about a minute. ODDFs are designed to disintegrate or dissolve rapidly on contact with saliva. This mode of administration can be beneficial to people who may have problems swallowing tablets whether it be from physical infirmity or psychiatric in nature. Subjects in pain may exhibit such behavior. ODDF’s can provide rapid delivery of medication to the blood stream through mucosa resulting in a rapid onset of action. Examples of ODDFs include orally disintegrating tablets, capsules and rapidly dissolving films and wafers. [0045] Extended release dosage forms (ERDFs) have extended release profdes and are those that allow a reduction in dosing frequency as compared to that presented by a conventional dosage form, e.g., a solution or unmodified release dosage form. ERDFs provide a sustained duration of action of a drug. Suitable formulations which provide extended release profiles are well-known in the art. For example, coated slow release beads or granules (“beads” and “granules” are used interchangeably herein) in which a PEP IB inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is applied to beads, e.g., confectioners nonpareil beads, and then coated with conventional release retarding materials such as waxes, enteric coatings and the like. In embodiments, beads can be formed in which a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is mixed with a material to provide a mass from which the drug leaches out. In embodiments, the beads may be engineered to provide different rates of release by varying characteristics of the coating or mass, e.g., thickness, porosity, using different materials, etc. Beads having different rates of release may be combined into a single dosage form to provide variable or continuous release. The beads can be contained in capsules or compressed into tablets.

[0046] In embodiments, modified dosage forms herein incorporate delayed release dosage forms having delayed release profiles. Delayed release dosage forms can include delayed release tablets or delayed release capsules. A delayed release tablet is a solid dosage form which releases a drug (or drugs) such as a PTP1B inhibitor, such as, for example MSI-1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof at a time other than promptly after administration. A delayed release capsule is a solid dosage form in which the drug is enclosed within either a hard or soft soluble container made from a suitable form of gelatin, and which releases a drug (or drugs) at a time other than promptly after administration. For example, enteric-coated tablets, capsules, particles and beads are well-known examples of delayed release dosage forms. Enteric coated tablets, capsules and particles and beads pass through the stomach and release the drug in the intestine. In embodiments, a delayed release tablet is a solid dosage form containing a conglomerate of medicinal particles that releases a drug (or drugs) at a time other than promptly after administration. In embodiments, the conglomerate of medicinal particles are covered with a coating which delays release of the drug. In embodiments, a delayed release capsule is a solid dosage form containing a conglomerate of medicinal particles that releases a drug (or drugs) at a time other than promptly after administration. Tn embodiments, the conglomerate of medicinal particles are covered with a coating which delays release of the drug. [0047] Delayed release dosage forms are known to those skilled in the art. For example, coated delayed release beads or granules in which a PTP1B inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is applied to beads, e.g., confectioners nonpareil beads, and then coated with conventional release delaying materials such as waxes, enteric coatings and the like. In embodiments, beads can be formed in which a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is mixed with a material to provide a mass from which the drug leaches out. In embodiments, the beads may be engineered to provide different rates of release by varying characteristics of the coating or mass, e g., thickness, porosity, using different materials, etc. In embodiments, enteric coated granules of a PTPlB inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof can be contained in an enterically coated capsule or tablet which releases the granules in the small intestine. In embodiments, the granules have a coating which remains intact until the coated granules reach at least the ileum and thereafter provide a delayed release of the drug in the colon. Suitable enteric coating materials are well known in the art, e.g., Eudragit® coatings such methacrylic acid and methyl methacrylate polymers and others. The granules can be contained in capsules or compressed into tablets.

[0048] In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is incorporated into porous inert carriers that provide delayed release profiles. In embodiments, the porous inert carriers incorporate channels or passages from which the drug diffuses into surrounding fluids. In embodiments, a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof is incorporated into an ion-exchange resin to provide a delayed release profile. Delayed action may result from a predetermined rate of release of the drug from the resin when the drug-resin complex contacts gastrointestinal fluids and the ionic constituents dissolved therein. In embodiments, membranes are utilized to control rate of release from drug containing reservoirs. In embodiments, liquid preparations may also be utilized to provide a delayed release profile. For example, a liquid preparation consisting of solid particles dispersed throughout a liquid phase in which the particles are not soluble. The suspension is formulated to allow at least a reduction in dosing frequency as compared to that drug presented as a conventional dosage form (e.g., as a solution or a prompt drug-releasing, conventional solid dosage form). For example, a suspension of ion-exchange resin constituents or microbeads.

[0049] In embodiments, pharmaceutical compositions described herein are suitable for parenteral administration, including, e.g., intramuscular (i.m ), intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), or intrathecal (i.t.). Parenteral compositions must be sterile for administration by injection, infusion or implantation into the body and may be packaged in either single-dose or multi -dose containers. In embodiments, liquid pharmaceutical compositions for parenteral administration to a subject include an active substance, e.g., a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof in any of the respective amounts described above. In embodiments, the pharmaceutical compositions for parenteral administration are formulated as a total volume of about, e.g., 10 ml, 20 ml, 25 ml, 50 ml, 100 ml, 200 ml, 250 ml, or 500 ml. In embodiments, the compositions are contained in a bag, a glass vial, a plastic vial, or a bottle.

[0050] Pharmaceutical compositions for parenteral administration provided herein may include one or more excipients, e.g. , solvents, solubility enhancers, suspending agents, buffering agents, isotonicity agents, stabilizers or antimicrobial preservatives. When used, the excipients of the parenteral compositions will not adversely affect the stability, bioavailability, safety, and/or efficacy of a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof used in the composition Thus, parenteral compositions are provided wherei n there is no incompatibility between any of the components of the dosage form.

[0051] In embodiments, parenteral compositions a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof include a stabilizing amount of at least one excipient. For example, excipients may be selected from the group consisting of buffering agents, solubilizing agents, tonicity agents, antioxidants, chelating agents, antimicrobial agents, and preservative. One skilled in the art will appreciate that an excipient may have more than one function and be classified in one or more defined group.

[0052] In embodiments, parenteral compositions include a PTP1B inhibitor, such as, for example MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof and an excipient wherein the excipient is present at a weight percent (w/v) of less than about, e.g., 10%, 5%, 2.5%, 1 %, or 0.5%. Tn embodiments, the excipient is present at a weight percent between about, e.g, 1.0% to 10%, 10% to 25%, 15% to 35%, 0.5% to 5%, 0.001% to 1%, 0.01% to 1%, 0.1% to 1%, or 0.5% to 1%. In embodiments, the excipient is present at a weight percent between about, e.g., 0.001% to 1%, 0.01% to 1%, 1.0% to 5%, 10% to 15%, or 1% to 15%. [0053] In embodiments, parenteral compositions of an active substance, e.g., a PTP1B inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof are provided, wherein the pH of the composition is between about 4.0 to about 8.0. In embodiments, the pH of the compositions is between, e.g., about 5.0 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0. In embodiments, the pH of the compositions is between, e.g., about 6.5 to about 7.5, about 7.0 to about 7.8, about 7.2 to about 7.8, or about 7.3 to about 7.6. In embodiments, the pH of the aqueous solution is, e.g., about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.7, about 7.8, about 8.0, about 8.2, about 8.4, or about 8.6. [0054] It should be understood that the dosage amounts of a PTP1B inhibitor, such as, for example MSI- 1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof that are provided herein are applicable to all the dosage forms described herein including conventional dosage forms, modified dosage forms, as well as the parenteral formulations described herein. Those skilled in the art will determine appropriate amounts depending on criteria such as dosage form, route of administration, subject tolerance, efficacy, therapeutic goal and therapeutic benefit, among other pharmaceutically acceptable criteria.

[0055] Clinical efficacy of treatment can be monitored using any method known in the art. Measurable parameters to monitor efficacy will depend on the condition being treated. For monitoring the status or improvement of acute lung injury, both subjective parameters (e.g., patient reporting) and objective parameters (e.g., arterial blood gas measurements, measurements of markers of oxidative injury in the lung, chest-radiography, etc.) can be used.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosure herein belongs.

[0057] The term "about" or "approximately" as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, and/or up to 1% of a given value.

[0058] “Improvement” refers to the treatment of symptoms of lung injury, including but not limited to pulmonary edema, sepsis, microvascular thrombi, hyaline membrane formation, or neutrophilic alveolitis.

[0059] “Improvement in next day functioning” or “wherein there is improvement in next day functioning” refers to improvement after waking from an overnight sleep period wherein the beneficial effect of administration of a PTPlB inhibitor, such as, for example MSI-1436, DPM- 1003, or respective a pharmaceutically acceptable salt thereof applies to symptoms of lung injury and is discernable, either subjectively by a subject or objectively by an observer, for a period of time, e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, etc. after waking. [0060] "PK" refers to the pharmacokinetic profile. Cmax is defined as the highest plasma drug concentration estimated during an experiment (ng/ml). Tmax is defined as the time when Cmax is estimated (min). AUCo-® is the total area under the plasma drug concentration-time curve, from drug administration until the drug is eliminated (ng*hr/ml or pg*hr/ml). The area under the curve is governed by clearance. Clearance is defined as the volume of blood or plasma that is totally cleared of its content of drug per unit time (ml/min).

[0061] "Treating", "treatment" or “treat” can refer to the following: reducing, improving, relieving, ameliorating, mitigating, inhibiting, reversing and/or alleviating symptoms of lung injury in a subject, or delaying the appearance of symptoms of lung injury (prophylaxis) in a subject. In embodiments, “treating”, “treat” or “treatment” may refer to preventing the appearance of clinical symptoms of a disease or condition in a subject that may be afflicted with or predisposed to the disease or condition, but does not yet experience or display clinical or subclinical symptoms of the disease or condition. "Treating", “treat” or "treatment" also refers to inhibiting or relieving symptoms of lung injury, e.g., causing regression of symptoms of lung injury or at least one of its clinical or subclinical symptoms. The benefit to a subject to be treated may be statistically significant, mathematically significant, or at least perceptible to the subject and/or the physician. Nonetheless, prophylactic (preventive) and therapeutic (curative) treatment are two separate embodiments of the disclosure herein.

[0062] "Pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In embodiments, this term refers to molecular entities and compositions approved by a regulatory agency of the federal or a state government, as the GRAS list under section 204(s) and 409 of the Federal Food, Drug and Cosmetic Act, that is subject to premarket review and approval by the FDA or similar lists, the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0063] “Co-administered with”, “administered in combination with”, “a combination of’ or “administered along with” may be used interchangeably and mean that two or more agents are administered in the course of therapy. The agents may be administered together at the same time or separately in spaced apart intervals. The agents may be administered in a single dosage form or in separate dosage forms.

[0064] “Subject in need thereof’ includes individuals that are experiencing symptoms of lung injury or are about to experience symptoms of lung injury with reasonable certainty. The methods and compositions including a PTP1B inhibitor, such as, for example MSI- 1436, DPM- 1003, or a respective pharmaceutically acceptable salt thereof may be provided to any individual including, e.g., wherein the subject is a neonate, infant, a pediatric subject (6 months to 12 years), an adolescent subject (age 12-18 years) or an adult (over 18 years). Subjects include mammals. “Patient” and “subject” may be used interchangeably herein.

[0065] The term “pharmaceutically acceptable salt”, as used herein, refers to derivatives of the compounds defined herein, wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include but are not limited to mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Such conventional non-toxic salts include but are not limited to those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, tolunesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic salts. The pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.

[0066] EXAMPLES

[0067] Materials and Methods

[0068] Mice. Male Balb/c mice (7-14-week-old) were obtained from Charles River and male C57BL/6J mice (7-10-week-old) were purchased from The Jackson Laboratory. All the mice were housed in the animal facilities of Cold Spring Harbor Laboratory. All experimental protocols were reviewed and approved by the Cold Spring Harbor Laboratory Institutional Animal Care and Use Committee and were conducted in accordance with the NIH’s Guide for the Care and Use of Laboratory Animals. Mice were housed five per cage and maintained on a 12h light/dark cycle and an ambient temperature of 25°C, with sterile food and water, in conventional space. All mice were acclimatized to the animal facility for a minimum of 7 days prior to enrollment in experiments.

[0069] TRALI induction. Male Balb/c mice (7-9-week-old) were given an intraperitoneal injection with 0.15 mg/kg lipopolysaccharide (LPS) in 100 pl physiological saline (0.9%). Twenty-six hours later, mice received an intravenous injection through retro-orbital venous sinus of 100 pl 1.5 mg/kg anti-mouse MHC Class I (BioXcell, clone 34-l-2s). Two hours before TRALI induction, the mice were treated either with saline or different doses of MSL1436 or DPM-1003, intraperitoneally. Due to the circadian oscillation of neutrophils, anti-MHC-I was administered at ZT13 (7 p.m.) Mice were observed for up to 2hrs during the acute phase of TRALI. Mice were euthanized when they appeared moribund by physical inspection as evidenced by change in mobility (endpoint of the experiment). Time to endpoint was used for statistical analysis of overall survival.

[0070] CLP-induced sepsis. Male C57BL/6J mice (8-10-week-old) were administered a subcutaneous injection in the flank of analgesic (72-hour sustained release Buprenorphine (1 mg/kg)) 15 min before surgery. Then, mice were anesthetized with isoflurane, and surgery were performed under aseptic survival conditions. A small midline incision was made and the cecum was exteriorized and ligated (2 cm) using non-ab sorbable 3-0 suture. A 23-gauge needle was then used to puncture one hole in the middle of the ligated segment, and a small amount of feces was extruded to ensure constant drainage of puncture. The ligated and punctured cecum was repositioned inside of abdominal cavity without the feces touching the incision to avoid infection of the surgical wound. The peritoneum was closed with surgical absorbable Vicryl sutures, and the skin was closed using sterile 7 mm wound clips. Survival was monitored for 10 days, and mice were euthanized once they became moribund and a humane endpoint was reached.

[0071] LPS-induced sepsis. Male C57BL/6J mice (7-9-week-old) were given LPS (E. coli Ol l i :B4) at the indicated concentrations through intraperitoneal injection. Two hours before LPS challenge, 10 mg/kg MSI-1436 or saline were administered intraperitoneally. Mice were observed for up to 5 days for survival, and mice were euthanized once they became moribund. [0072] Histology. Two lung fixation methods were used in this study. For both techniques, animals were euthanized with CO2 immediately prior to procedure. The first technique required exposing the trachea and lung, followed by making an incision in the trachea to allow insertion of a 20-gauge catheter (Exel Safelet catheter). The catheter and trachea were secured with sutures and the lungs were slowly inflated with approximately 1ml of 4% paraformaldehyde (PF A). The trachea was then tied to prevent deflation and the lungs were dissected and immersed in 4% PFA at room temperature for 24 hours to ensure thorough fixation. For the second technique, animals were first transcardially perfused with 30ml of physiological saline solution (0.9%), to flush out blood, using a 25-27GA needle. The lungs were then inflated by repositioning the needle from the left ventricle of the heart into the right ventricle and perfusing with an additional 3-5ml of saline. The lungs were dissected and drop fixed in 4% PFA at room temperature for 24 hours. After dehydration, the fixed lungs were embedded in paraffin and 5 pm sections were cut coronally, to represent all the lobes, and mounted on slides. Tissue sections were stained with hematoxyline and eosin (H&E), and scanned using Aperio ScanScope CS (Leica Biosystems).

[0073] All H&E staining slides were read blindly and scored for lung damage. H&E- stained sections were scored according to severity, distribution, amount and content of edema, alveolar damage, hyaline membranes and vessel damage. Severity was scored 0-4 based on the least to most affected lung in the study. Distribution was scored 1-4 based on the percentage of the lung involved (focal, multifocal, locally extensive, and diffuse). Edema scores were 0-4 based on the distribution, severity, and intensity of the edematous proteinaceous exudate in the alveolar space. Alveolar damage was scored 0-4 according to the degree of loss of alveolar wall integrity and alveolar pneumonocyte reaction (type II hyperplasia and sloughed cells in alveoli). Hyaline membranes were scored according to number and extent of membrane formation. Vessel damage was scored 0-4 according to the degree of endothelial damage. The scores of each lesion were added to give a final overall score.

[0074] Quantification of bronchoalveolar lavage fluid (BALF) protein. The BALF was collected from NT, saline or 10 mg/kg MSI- 1436 treated TRALI mice. Mice were euthanized in a CO2 chamber. For the TRALI group, BALF extraction was performed 45 minutes after MHC-I antibody injection, or postmortem if mice died earlier than 45 minutes. Each mouse was positioned front side up, with the trachea exposed. A silk thread (Perma-Hand silk, 3-0, Ethicon) was placed under the trachea. After the trachea were hemisected transversally, a 20G catheter (Exel Safelet catheter 20G 1”, Exelint) was inserted into the trachea, and tied firmly with silk thread. PBS (1ml) was injected into the lungs, and slowly recovered after ~ 1 minute. A total of 600 -700 pl BALF was recovered from each mouse, and kept on ice. BALF was centrifuged (300xg, 10 min, 4°C), and the protein concentrations in the supernatants were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

[0075] CT scan for lung edema measurement. Balb/c mice were anesthetized with 120 mg/kg ketamine and 8 mg/kg xylazine. After immobilization, mice were positioned prone on the imaging cradle of either a Mediso nanoScan PET/CT or SPECT/CT system (Mediso), and secured with tape and gauze to prevent movement. The scanner’s field of view was set using a 2D scout scan to cover the lungs and airways, and images were acquired using the following x- ray settings: beam energy of 50 kVp, exposure of 186 pAs, in an axial scan with 720 projections. After acquisition of each baseline CT scan, the mouse was injected with 1.5 mg/kg antibody against MHC-I retro-orbitally, immediately followed by sequential scans that were acquired at 5- minute intervals for 45 minutes, or until endpoint. To minimize the effect of circadian rhythm on edema accumulation, TRALI was induced for one saline- and one MSI-1436-treated mouse at similar times (6 p.m. - 10 p.m.), and placed alternately on either CT scanner.

[0076] Images were reconstructed using a back-projection algorithm with a Butterworth filter to an isotropic voxel size of 138 pm in Nucline software v3.0 (Mediso). Pre-processing and analyses were performed in VivoQuant v4.0 (Invicro). Briefly, a 3D region of interest (ROI) covering the entire lung volume was semi-manually contoured on the baseline scan and the first follow-up scan after antibody injection, with the latter ROI being used on all subsequent scans as the mouse was not moved. Total lung volume and mean image intensity in Hounsfield units (HU) were calculated from the ROT at each imaging timepoint. The viable lung volume was defined as the volume within the total lung ROT below a threshold of 0 HU, and percent viable lung volume was calculated by dividing this value by the total lung volume at each timepoint. [0077] Blood count. Blood was collected by cardiac puncture into a syringe freshly coated with 0.5 M EDTA, then transferred into EDTA-coated tubes (Microvette 500, Sarstedt). We ensured that blood used for functional analysis in this study was clot free, in order to avoid neutrophil activation. Blood (50 pl) was analyzed for differential counts using a ProCyte Dx Hematology Analyzer (Idexx Laboratories).

[0078] Lung immune cell infiltration analysis. Lung tissues were harvested from control and TRALI mice 30 minutes post MHC-I antibody challenge. Lungs were rinsed in cold PBS and mechanically dissociated into small pieces, and further enzymatically digested for 30 min at 37 °C in 5 ml of RPMI with 2% FCS and containing Dispase (2.5 U/nil, #07913, Stem Cell), Collagenase D (0.1 mg/ml, #11088866001, Sigma), DNase I (25 U/ml, #04536282001, Sigma), and Liberase DL (0.2 mg/ml, #05466202001, Sigma). The suspensions were then passed through a 70 pm cell strainer (#352340, BD Falcon), and centrifuged at 1,500 rpm for 5 minutes at 4 °C. After removing the red blood cells by incubating with 5 ml of ammonium-chlori depotassium (ACK) buffer for 3 min on ice, the suspensions were then centrifuged (1,500 rpm, 5 min) and resuspended in FACS buffer (1% FCS and 0.02% sodium azide in PBS). The singlecell suspensions were finally collected by passing through a 40 pm cell strainer. For flow cytometry analysis, lung single-cell suspensions (1,000,000 cells) from each sample were fixed in 2% of PFA for 10 min on ice. After washing with FACS buffer, the cells were blocked with Fc Receptor Blocker (Innovex Biosciences, Richmond, CA) for 15 min at 4 °C, followed by incubating with conjugated antibodies (listed in Table 2, below) for 30 min at 4 °C. Finally, the cells were washed once in FACS buffer and resuspended in 500 pl of FACS buffer and further analyzed by BD LSRFortessa DUAL flow cytometer (BD Biosciences). Data were analyzed by FlowJo software (V10.6.2). Immune cell subsets and corresponding markers: CD8+ T cells (CD45+CD3+CD8+); CD4+ T cells (CD45+CD3+CD4+); yo T cells (CD45+CD3+gdTCR); NKT cells (CD45+CD3+CD335+); B cells (CD45+CD3-CD19+), NK cells (CD45+CD3-CD335+); Neutrophils (CD45+CDl lb+Ly6G+Ly6C+); Macrophages (CD45+CD1 lb+Ly6G-F4/80+); and DC cells (CD45+CD1 lb+Ly6G-F4/80-CDl lc+).

Table 2. Lung immune cell infiltration analysis antibodies

[0079] Multiplex cytokine array. The cytokine arrays were performed with serum and lung tissue homogenates from NT (no treatment) and TRALI mice 30 min after MHC-I antibody injection. For the serum samples, blood was collected by cardiac puncture, then allowed to clot for 30min at room temperature. The serum was isolated by centrifuging at 1,500 x g for 15 minutes in a refrigerated centrifuge. For lung tissue homogenates, lungs were harvested and weighed, homogenized using a Precellys Evolution tissue homogenizer (Bertin Instruments) at 6,800 rpm, 0°C. The samples were homogenized for three cycles, 20 s per cycle, and 30 s pause after each cycle. Samples were analyzed with the Proteome Profiler mouse cytokine array kit, panel A (R&D systems, ARY006) according to the manufacturer’s instructions.

[0080] Enzyme-linked immunosorbent assay (ELISA). ELISA was performed with serum, plasma or lung homogenates to measure the levels of CXCL1 (R&D systems, DY453-05) and CXCL2 (R&D systems, DY452-05) following manufacturer’s instructions.

[0081] Isolation of bone marrow (BM) neutrophils. BM neutrophils were isolated by density gradient centrifugation, as previously described (1). Briefly, BM was flushed from tibias and femurs using HBSS. The cell pellet was resuspended in ACK buffer and passed through 100 pm cell strainer (Falcon). Different concentrations of Percoll (Cytiva, 17089102) were prepared according to previous publication (2). Neutrophils were enriched using gradient centrifugation at 1,300 x g for 20 min, and collected from the band at the interface between the 81 and 62% Percoll layers. Cells were washed with HBSS, and resuspended in RPMI at the desired concentration.

[0082] RNA isolation and RNA-seq library preparation. Neutrophils were isolated from bone marrow (BM), and treated with either saline or 10 pM MSI-1436 for 2 hours. Total RNA was extracted from cells using TRIzol reagent (Thermo Scientific, Cat# 15596018). Chloroform (200 pl) was added to 1 mL TRIzol and incubated at room temperature for 10 minutes. After centrifugation at 10,000 x g for 15 minutes in the cold, the aqueous phase was taken, mixed with an equal volume of isopropanol and supplemented with 0.5 pl glycogen, to increase RNA recovery. Samples were kept at -80°C for 30 min, followed by centrifugation at 10,000 x g for 10 minutes in the cold. The RNA pellet was washed once with 80% Ethanol and dissolved in DEPC-treated water. Poly-A-tailed mRNA was isolated with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490). The RNA-seq library was prepared with NEBNext Ultra™ II RNA Library Prep Kit (NET, E7770) for Illumina sequencing, following the manufacturer’s instructions. Samples were pooled together and sequenced on NextSeq with Single Read 75 bases.

[0083] RNA-seq analysis. The sequencing reads were aligned to customized mm 10 gtf- containing protein coding genes by using salmon 1.0.0 with default setting. Expressed genes (TPM > 0.5 in either control or treatment) were subjected to differential gene expression analysis with DESeq2. Genes were then ranked by their log2 fold change and upregulated genes were subjected to g:Profiler for Reactome analysis. (GEO accession: GSE184197)

[0084] Flow cytometry. Blood samples were treated with ACK buffer to lyse the red blood cells (RBCs), and washed with Hanks’ Balanced Salt solution (HBSS). Cells were incubated, for 30 min on ice, in FACS-sorting buffer (2 mM EDTA, 0.5% heat-inactivated fetal bovine serum (FBS) in PBS) containing neutrophil surface marker antibodies (Table 3). When only surface markers were to be measured, cells were examined using a BD LSRFortessa DUAL flow cytometer (BD Biosciences) and data were analyzed by FlowJo software (Tree Star). When intracellular staining for myeloperoxidase (MPO) was needed, cells were fixed with 4% PFA for 10 min at room temperature, permeabilized with 0.1% Triton-XlOO, and blocked with PBS containing 5% donkey serum and 0.1% Triton X-100 for 1 hour. Samples were next incubated at 4°C overnight, washed with PBS, and incubated with Donkey anti-Goat IgG, Alexa 488 (1 :400, Invitrogen, A-l 1055) for 1 hour at room temperature. To determine the levels of surface markers or MPO in the neutrophils, the scatter plots were gated on Ly6G^lu8h population, and the MFI of surface markers or MPO were calculated.

Table 3. Neutrophil Surface Marker Antibodies

[0085] Confocal immunofluorescence microscopy. Balb/c mice were injected either with saline or lOmg/kg MSI-1436, and blood was collected by cardiac puncture 2.5 hours after injection. ACK buffer was added to lyse the RBCs. After washing with HBSS, cells were resuspended in RPMI-1640, and plated onto poly -L-ly sine coated 8-well p.-slide (Ibidi) at 20,000 to 50,000 cells per well. The cell culture was incubated at 37 °C for 30min to allow attachment of neutrophils. To stain for MPO, the cells were first fixed with 4% PFA for lOmin, washed with PBS, blocked and permeabilized with 5% donkey serum, 0.1% Triton X-100 in PBS for 1 hour at room temperature, and incubated with anti-MPO (1:300, R&D systems, AF3667) overnight at 4°C. Next day, cells were washed with PBS, incubated with Donkey anti-Goat IgG, Alexa 488 (1:400) and DAPI (Abeam, ab228549) for 1 hour at room temperature, washed with PBS and then Ibidi mounting medium was added (Ibidi, 50001). Images were acquired using a Zeiss LSM 780 confocal laser scanning microscope with a Zeiss Plan-Apochromat 63x/1.4 NA Oil DIC M27 objective lens and Zen black acquisition software. The intensity mean of MPO per cell was quantified using the Surface tool in the Surpass and 3D View window of Imaris 3D/4D Visualization and Analysis Software (Bitplane, Version 9.6.1, Oxford Instruments). All the images were processed with the consistent parameter settings of thresholding at absolute intensity 200, and seed density diameter of 6.5 pm to split touching objects.

[0086] Neutrophil Extracellular Trap formation with mouse neutrophils. For the ex vivo study, mice were injected intraperitoneally with either saline, 2mg/kg MSI- 1436 or 10 mg/kg MSI-1436. Peripheral blood was collected through cheek bleeding 2.5 hours after compound administration, and 50 pl blood was required for each well. For the in vitro study, blood was obtained from untreated mice. Red Blood Cells (RBCs) were removed with ACK buffer, and the leukocytes were plated onto poly-L-lysine-coated 8-well p-slide. After 30 min incubation at 37°C to allow neutrophils to attach, DMSO or PMA (100 nM) was added for ex vivo test; DMSO, PMA, or PMA together with MSI-1436 was added for in vitro test. Two hours later, cells were fixed, permeabilized and stained for MPO, citH3 (Abeam, ab5103), DAPI, and were visualized using a Zeiss LSM 780 confocal laser scanning microscope. Quantitation was performed based on triple colocalization of DNA, MPO and citH3, using ImageJ and a custom- made macro, available in FigShare (DOI: 10.6084/m9. figshare.14401958).

[0087] Neutrophil Extracellular Trap formation with human neutrophils. Whole blood was taken from three healthy volunteers (20-40, male and females) with informed consent and approved by the IRB of CSHL (IRB-13-025). The blood was collected into BD Vacutainer K2EDTA tube using venipuncture. After lysing RBCs with ACK buffer, the neutrophils were isolated using Percoll density gradient centrifugation as done in isolation of mouse BM neutrophils. The NETosis stimulation and imaging were performed as with mouse neutrophils. [0088] Whole mount staining. To confirm the presence and abundance of NETs in the lungs of mice after TRALI induction, we performed whole-mount immunostaining and tissue clearing of excised lungs. Mice were subject to TRALI and euthanized with CO2 30 min after MHC-I antibody injection. Mice were then perfused with 20 ml of saline through the left ventricle of the heart, and the lungs were collected in cold PBS. Afterwards, lungs were fixed at 4 °C overnight in PBS with 4% PFA and 30% sucrose. After three washes of I h with PBS at room temperature, tissues were permeabilized in methanol gradients in PBS for 30 min (PBS > 50% MeOH > 80% MeOH > 100% MeOH). Then, tissues were bleached with Dent’s bleach (15% H2O2, 16.7% DMSO in MeOH) for 1 h at room temperature, and rehydrated through descending methanol gradients in PBS (80% MeOH> 50% MeOH > PBS). Then tissues were incubated with blocking buffer containing PBS with 0.3% Triton X-100, 0.2% BSA, 5% DMSO, 0.1% azide and 25% FBS overnight at 4 °C with shaking. Afterwards, lungs were stained with antibodies against cit-H3 (Abeam, ab5103), MPO (R&D, AF3667) and CD31 (BioLegend, 102502) for 2 days at 4 °C with shaking. After washing for 24 h in washing buffer (PBS with 0.2% Triton X-100 and 3% NaCl), the tissues were stained with secondary antibodies for 24 h at 4 °C with shaking. After 24 h, tissues were washed for 24 h in washing buffer and were dehydrated in MeOH gradients in dFLO using glass containers for 30 min in each step (50% MeOH > 70% MeOH > 90% MeOH > 3 x 100% MeOH). Finally, tissues were submerged for 30 min in MeOH with 50% BABB (benzyl alcohol :benzyl benzoate, 1 :2) and afterwards cleared in 100% BABB and imaged on a Leica SP8 X confocal microscopy system. Quantitation was performed with Imans (Bitplane), using spots on a triple-colocalization channel of DNA, MPO and citH3. Neutrophils were quantified using spots based on MPO signal. Frequency was calculated as the number of NETs / number of neutrophils in the 3D volume.

[0089] Administration of AZD-5069. The CXCR2 antagonist AZD-5069 (MedChemExpress, HY-19855) was given to mice orally (100 mg/kg). Before feeding AZD- 5069, mice were trained to consume 100 pl 10% sucrose through pipette tips for 3 days. On the day of oral administration of AZD-569, compound was freshly prepared in 10% DMSO, 40% PEG-300, 5% Tween-80, 45% physiological saline (0.9%) AZD-5069 was completely dissolved before adding next solvent. AZD-5069 solution (20 mg/ml) was kept in the 50°C water bath until ready to feed mice.

[0090] Immunoblots. After PMA or CXCL12 stimulation, the 6-well plates were place on ice and lysed with RIPA buffer, containing freshly added cOmplete™ EDTA-free protease inhibitors (Roche, 11836153001) and Halt phosphatase inhibitors (Thermo Fisher Scientific, 78428). Equal amounts of cell lysate were loaded onto SDS-PAGE and transferred to Amersham nitrocellulose membrane (Cytiva). The membranes were blocked in 5% BSA in Tris-buffered saline Tween-20 (TBST), followed by overnight incubation with primary antibodies at 4°C. All the primary antibodies were purchased from Cell Signaling Technology (AKT, 9272 and 4691; pAKT T308, 2965; pAKT S473, 9271; Rps6, 2217; pRps6 S235/236, 4858; ERK1/2, 9102; pERKl/2 T202/Y204, 9101; HSP90, 4877). After washing with TBST, secondary antibodies (Peroxidase AffmiPure Goat anti-Rabbit IgG (H+L), Jackson ImmunoResearch Laboratories) were applied and following a TBST wash, the blots were developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). [0091 ] Administration of Palomid 529. Palomid 529 (Selleck Chemicals, S2238) was freshly prepared in a micronized formulation in 8% DMSO, 40% PEG300, 5% Tween80, 47% ddFLO, in which the solvents were added individually and in the order in which they are listed. Before each injection, the tube was vortexed to retain suspension. The compound was administered IP in doses up to 25 mg/kg.

[0092] Chemotaxis assay. Chemotaxis assays were performed using Corning HTS Transwell-24 well permeable supports (6.5 mm diameter, 3 pm pore size, Sigma, CLS3398- 2EA) and Coming Ultra-Low attachment plates (Sigma, CLS3473-24EA). Either 600 pl of RPMI-1640 as a negative control, or RPMI-1640 containing 100 ng/ml CXCL12 (R&D systems, 460-SD-010), was added to the lower chambers. The upper chambers were seeded with neutrophils, 200 pl at 5 X 10⁶ cells/ml. For the treatment groups, the neutrophils were pre-treated with the indicated concentration of PTP1B inhibitors for 30 min, and then loaded into the upper chambers. After 1-2 hours incubation at 37°C, 5% CO2, EDTA was added to the lower chambers to a final concentration of 10 mM, for 10 min, to detach the cells. The number of neutrophils in the lower chamber were counted using Guava easyCyte.

[0093] Statistical analysis. Statistical analyses were performed with Prism software (GraphPad Software). All data are presented as mean ± standard error of the mean (SEM). Comparison between two groups were analyzed by unpaired two tailed t-test. Comparison for more than two groups were analyzed using one-way ANOVA followed by indicated post-hoc test. Statistical significance of Kaplan-Meier curves was determined by Log-rank (Mantel-Cox) test.

[0094] Example 1. PTP1B inhibitors improved survival and prevented lung damage in the TRALI and LPS-induced sepsis mouse models.

[0095] To investigate the function of PTP1B in the biology of ALLARDS, in particular its function in neutrophils, we examined the effect of PTP1B inhibitors in murine disease models that were driven by aberrant activation of neutrophils. First, we tested PTP1B inhibitors in an established TRALI model, summarized in Figure 1 A, in which neutrophils and neutrophil extracellular traps (NETs) play a crucial role (20, 21). Following pretreatment with lipopolysaccharide (LPS) as a priming step, we tested the effects of injecting single doses of PTP1B inhibitors, intraperitoneally, two hours prior to intravenous injection of anti-major histocompatibility complex-I antibodies. Timing and route of the injection were chosen so that the maximal dose of the compound in serum coincided with the initiation of the acute phase of lung injury. We tested the effect of MSI-1436 (Figure IB), an allosteric inhibitor of PTP1B for which efficacy and specificity has been demonstrated in animal models of HER2-positive breast cancer (19). Compared to the saline control group, MSI-1436 treatment resulted in a dosedependent increase in survival. At doses of 5 and 10 mg/kg, MSI-1436 resulted in 100% survival of mice at the 2-hour time point compared to 40% survival of mice in the control group (Figure 1C). Similar dose-dependent protective effects were observed following treatment with DPM- 1003, a structurally distinct inhibitor of PTP1B compared to MSI-1436 (Figures ID and IE). [0096] We compared the histopathological appearance of lung tissue taken from mice in which TRALI had not been induced (NT, No Treatment controls) with TRALI mice treated with saline, or with different doses of MSI-1436. These analyses revealed marked accumulation of alveolar edema and hyaline membranes following vehicle (saline) treatment of the TRALI mice, whereas after treatment with MSI-1436 at 10 mg/kg, the lungs appeared nearly normal (Figure IF). The 2mg/kg MSI-1436-treated group also displayed reduced edema and hyaline membranes in the alveolar space. To quantify lung damage, we defined a lung injury score that reflected a combination of gross examination of damage distribution (multifocal, locally extensive, diffuse), accumulation of edema and hyaline membrane, and alveolar and vessel damage (Figure IF). Consistent with the survival data, treatment with MSI-1436 at 2 mg/kg improved the lung injury score reflecting moderate injury, whereas 10 mg/kg-treated mice did not present signs of lung injury. To assess pulmonary permeability, we measured the protein leakage in the bronchoalveolar lavage fluid (BALF) and edema formation. In the saline-treated TRALI mice, the BALF protein concentration significantly increased compared to NT controls, whereas in the MSI-1436-treated TRALI mice, the levels were not significantly elevated compared to the control group (Figure 1G). We used longitudinal computed tomography (CT) scans to monitor the accumulation of edema after TRALI induction in the mice pre-treated with either saline or MSI-1436; viable lung airspace was defined as lung volume not occupied by edema. After anti- MHC-I antibody injection, there is an acute increase of pulmonary edema (Figures 1H and II); however, MSI-1436 treatment prevented progressive loss of viable lung volume compared to saline treatment (Figures 1G-1I). Overall, these observations demonstrate that PTP1B inhibitors are protective against lung damage, consistent with promoting survival in the TRALI model. [0097] Example 2. Treatment with PTP1B inhibitors improved survival in additional models of sepsis.

[0098] To generalize this observation, we examined also the effect of PTP1B inhibitors in the CLP-induced polymicrobial sepsis and LPS-induced lethal endotoxemia (sepsis) models, which recapitulate the systemic inflammation and shock-like state of bacterial septic challenge (22). The high lethality of sepsis is associated with dysregulation of the host inflammatory response, including the detrimental effects of aberrant activation of neutrophils (23-27). In the CLP sepsis model, which is a clinically relevant experimental model of septic peritonitis in humans, the cecum was ligated and perforated to cause drainage of cecal bacteria into peritoneal cavity (28). We administered either saline or 5 mg/kg MSL1436 2 hours before surgery. The saline-treated mice all died within 96 hours after CLP surgery, whereas -20% of mice were alive at day 10 in MSI-1436-pretreated group (Figure IK). In addition, we treated the mice with either saline or 5 mg/kg MSL1436 6 hours post-CLP surgery, which is the time at which we observed onset of symptoms, such as reduced activity and lethargy. Treatment with MSI- 1436 postsurgery did not exacerbate the symptoms, but did not significantly delay death (Figure IL).

[0099] In an additional model, we induced sepsis by intraperitoneal injection of LPS at two concentrations, and pretreated each condition with either saline or 10 mg/kg MSI- 1436 from 2 hours before LPS challenge. The high concentration of LPS (30 mg/kg) led to 100% lethality within 24 hours, whereas MSI- 1436 pretreatment significantly prolonged the survival time and increased the survival rate to 20% (Figures IM and IN). Following administration of LPS at 15 mg/kg, all the mice died within 50 hours in the saline control group; in contrast, 20% of the mice survived, and the death of the remaining mice was substantially delayed in the MSI-1436-treated group (Figure 10). Consistently, 24 hours after 15 mg/kg LPS challenge, we observed MSL 1436-treated mice displayed less pulmonary edema than the saline control (Figure IP). Taken together, PTP1B inhibitors have prophylactic function to improve survival in TRALI, CLP-and LPS-induced sepsis models. In light of the complicated inflammatory response involved in the sepsis models, we focused on the neutrophil-dependent TRALI model (20, 29, 30) for mechanistic analyses.

[0100] Example 3. Example 2. Treatment with PTP1B inhibitors induced neutrophilia. [0101 ] We profiled the accumulation of immune cells in lungs and the circulation 30 min after anti-MHC I antibody injection. In agreement with reports that neutrophils are critical for the initiation of TRALI (21, 30-32), the most dramatic increase we observed was in neutrophil numbers (Figures 2A-2C). Unexpectedly, we observed that following pretreatment with MSL 1436, CD1 lb+Ly6C+Ly6G+ neutrophil infiltration into lung tissues after TRALI induction was elevated compared to saline-treated mice (Figure 2D). This increase of pulmonary neutrophil accumulation prompted us to examine directly the effect of PTP1B inhibitors on neutrophils. We examined hematological parameters after treatment with PTP1B inhibitors and observed that the number of neutrophils in the peripheral blood increased ~3-fold following administration of either MSI-1436 or DPM-1003 (Figures 2E and 2F, and Table 4).

Table 4. Peripheral blood analyses after saline, MSI-1436 or DPM-1003 treatment.

Data are presented as mean±SEM (n=5 for each group). Statistical analysis by two-tailed student’s t-test to compare PTP1B inhibitor treatment and saline; *P<0.05, *** p<0.001, ****p<0.0001. [0102] To examine the mechanism underlying the neutrophilia that occurred following treatment with PTP1B inhibitors, we analyzed cytokine arrays for both serum and lung tissue in the no-treatment control and TRALI mice, injected with saline or MSI-1436; the results were then validated by ELISA (Figures 2G and 2H). Upon MSI-1436 treatment, the signal intensity of CXCL1 markedly increased in both serum and lung homogenates, and the level of CXCL2 was elevated in lung homogenates (Figures 2I-2K). Furthermore, we observed that the chemokine CXCL1 was elevated in plasma in response to both MSI-1436 and DPM-1003, independently of TRALT induction (Figures 2L and 2M). Consistent with our observation of neutrophilia, CXCL1 and CXCL2 are principal chemotactic cues for neutrophil migration (33).

[0103] Example 4. Treatment with PTP1B inhibitors induced an aged neutrophil phenotype in vivo.

[0104] Neutrophils display heterogeneity and plasticity, with the immune response determined more by the type of neutrophil subpopulation than by the number of neutrophils (34). Following administration of PTP1B inhibitors, TRALI-induced damage to the lung was minimal despite increased neutrophil infiltration; to explore the mechanistic basis for this observation, we performed RNA-seq to characterize phenotypic changes in neutrophils upon MSI- 1436 treatment. We conducted Reactome pathway analysis on up-regulated genes, which revealed that the most significantly altered pathway was neutrophil degranulation (Figure 3A, and Table 5). Table 5. Reactome pathway enrichment analysis of up-regulated genes upon MSI- 1436 treatment.

[0105] The ability of neutrophils to clear pathogens is conferred primarily by three processes, degranulation, formation of NETs and phagocytosis (35), which are modulated during neutrophil aging (8, 9). Neutrophil granules contain antimicrobial and proteolytic proteins, which facilitate digestion of microorganisms in response to infection, but have potential to cause harm to highly vascularized tissues, especially lungs, if not controlled appropriately. In the systemic circulation, neutrophils release granules in a controlled fashion, becoming less toxic and less able to cause tissue damage before they infiltrate the lungs (9). We harvested peripheral neutrophils 2.5 hours after treatment with either saline or MSI-1436 and stained for myeloperoxidase (MPO) as a marker of primary granules. We observed a pronounced decrease in immunofluorescence intensity of MPO in MSI-1436-treated neutrophils compared with saline-treated samples, suggesting the release of granules in vivo after treatment with the PTP1B inhibitor (Figure 3B). In addition, using flow cytometry, we demonstrated that the levels of MPO were decreased in the Y6G¹¹¹ neutrophil population isolated from MST-1436- or DPM-1003-treated animals compared to saline treated controls (Figures 3C and 3D).

[0106] There is a temporal heterogeneity, referred to neutrophil aging, in which fresh neutrophils are released from bone marrow, then undergo phenotypic changes to become aged neutrophils that are eventually eliminated from circulation (7). Compared to fresh neutrophils, intrinsically aged neutrophils display decreased granule contents, reduced ability to form NETs, and their predominance in the circulation coincides with diminished risk for damage to the vascular system (8-10, 36). As a consequence, neutrophil aging is a physiological strategy to dampen the toxic nature of neutrophils before they infiltrate the lung and to prevent tissue damage. Considering the degranulation phenotype we observed, we assessed the impact of treatment with PTP1B inhibitors on neutrophil aging by measuring the expression of surface markers for fresh neutrophils. Using flow cytometry, we demonstrated that in MSI-1436-treated mice, the expression of CD62L and CXCR2, two markers of fresh neutrophils, was downregulated (Figure 3E). Similarly, the markers of fresh neutrophils decreased upon DPM- 1003 treatment (Figure 3F). Collectively, these data suggest that PTP1B inhibitors promoted neutrophil aging in vivo and attenuated the neutrophil inflammatory response.

[0107] Example 5. Treatment with MSI-1436 suppressed formation of NETs ex vivo and in vivo.

[0108] NETs are formed in a neutrophil cell death pathway, referred to as NETosis. In the classical form of NETosis, NADPH oxidase-induced reactive oxygen species (ROS) stimulate MPO to promote the translocation of neutrophil elastase (NE), a serine protease, to the nucleus and the decondensation of chromatin. Having observed that treatment with PTP1B inhibitors decreased MPO-containing primary granules, we examined whether release of NETs was also impaired. We harvested neutrophils 2.5 hours after MSI-1436 administration, and stimulated them with phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator and trigger of ROS production, to generate NETs ex vivo. NETs, which consist of DNA decorated with citrullinated-histone H3 (citH3) and granule proteins, were designated by colocalization of DNA, citH3 and MPO, using confocal microscopy. As shown in Figure 4A, the PMA-induced formation of NETs was blocked by MSI-1436, compared to PMA treatment in the presence of DMSO control. [0109] We examined whether MSL1436 suppressed NETosis through an intrinsic or extrinsic mechanism. The neutrophils were isolated from mouse circulating blood and incubated with either MSI-1436 or saline together with PMA. The PMA-induced NET formation was impaired by MSI-1436 treatment, indicating this inhibitory effect was intrinsic to neutrophils (Figure 4B). Similarly, when we treated human primary neutrophils with MSI-1436 upon PMA stimulation, NET production was significantly reduced (Figure 4C).

[0110] To determine whether MSI-1436 could also limit the production of NETs in the TRALI model, we treated mice with either saline or MSI-1436, and collected lungs 30 minutes after injection of MHC-I antibody. We performed whole-mount immunostaining of the lung tissue for DAPI, citH3, and MPO. In the lungs of the TRALI mouse group treated with MSI- 1436 at 10 mg/kg, the production of NETs was almost abolished (Figure 4D). Furthermore, Akt signaling activated by NOX2-mediated ROS production is essential for NETosis (37); therefore, we tested the impact of PTP1B inhibitors on phosphorylation of Akt. We stimulated neutrophils isolated from bone marrow with PMA or vehicle control, and noted that both MSI-1436 and DPM-1003 suppressed the PMA-induced elevation of Akt phosphorylation (Figures 4E and 4F).

[0111] Example 6. The effect of PTP1B inhibitors on neutrophil aging was mediated via the CXCR4-CXCR2 signaling axis.

[0112] Our data indicate that treatment of TRALI mice with PTP1B inhibitors led to neutrophil aging, which, in turn, likely contributed to decreased lung damage compared to control animals. These observations phenocopied the ablation of CXCR4 from the myeloid lineage in mice (9), which led us to investigate whether there was a connection between inhibition of PTP1B and CXCR4 signaling.

[0113] The trafficking of neutrophils between bone marrow and the circulation is controlled by the CXCR4-CXCR2 signaling axis (38). Stromal cells express a high level of CXCLI2 (SDF-1), which interacts with CXCR4 and sequesters neutrophils in the bone marrow (38). CXCL1 and CXCL2, which activate CXCR2 signaling, promote the egress of neutrophils into the blood stream (39). During circulation, the level of surface CXCR4 is upregulated, leading to homing back to the bone marrow (40). To determine whether MSI-1436 acts through the CXCR4-CXCR2 axis, we tested whether AZD5069, a CXCR2 antagonist (41) could reverse the phenotype induced by MSI-1436. We administered AZD5069 orally, 2 hours prior to MSI- 1436 injection, and collected blood samples to quantify the level of MPO using flow cytometry. As before, we found that the MPO fluorescence intensity decreased significantly in the MST- 1436-treated mice compared to vehicle-treated controls; however, pretreatment with AZD5069 was sufficient to prevent this reduction of the MPO signal in MSI-1436-treated mice (Figure 5A). In addition, although MSI-1436 treatment increased the Ly6G^lu neutrophil population ~3- fold in serum, AZD5069 prevented this increase. Furthermore, treatment of mice with AZD5069 alone resulted in a lower percentage of Ly6G^hl neutrophils compared to controls, which is consistent with the known function of CXCR2 to increase neutrophil release (Figure 5B). Altogether, these results demonstrated opposing effects of the CXCR2 antagonist AZD5069 and the PTP1B inhibitor MSI-1436, consistent with a stimulatory effect of PTP1B on the CXCR4- CXCR2 signaling axis.

[0114] Example 7. Treatment with PTP1B inhibitors impaired CXCR4 signaling.

[0115] Since CXCR4 inhibits the signaling output of CXCR2, and treatment with PTP1B inhibitors phenocopied ablation of CXCR4 from myeloid cells in mice, we examined changes in signaling downstream of CXCR4. We purified neutrophils from bone marrow, stimulated with the CXCR4 ligand CXCL12, and examined signaling changes by immunoblotting with appropriate antibodies. Class I phosphatidylinositol 3 -kinases (PI3Ks) are activated downstream of G protein-coupled receptors (GPCRs), including C-X-C chemokine receptors (CXCRs), and play an important role in neutrophil inflammatory response (42, 43). We observed that treatment with CXCL12 led to enhanced phosphorylation of AKT on Thr³⁰⁸ and Ser⁴⁷³, which are response markers for the abundance of PIP3 that is generated by PI3K; upon treatment with PTP1B inhibitors, we observed that phosphorylation of AKT, RPS6 and ERK1/2 were suppressed (Figures 5C and 5D). This result was unexpected because it has been well-documented that PTP1B dephosphorylates the Insulin Receptor (IR) and thereby decreases AKT phosphorylation; consequently, activation of Akt might have been expected to accompany inhibition of PTP1B in this context (44, 45). Upon ligand binding to receptor tyrosine kinases or GPCRs, PI3Ks are recruited to plasma membrane to activate AKT signaling. The catalytic subunit of PI3K, pl 10, consists of four isoforms, among which pl 10a, -|3, and 5 are mainly activated by receptor tyrosine kinases, whereas pl lOy is activated by G protein 0y dimer (46). Therefore, since pl 10y is preferentially expressed in leukocytes, we hypothesized that PTP1B inhibitors may instead exert suppressive effects on pl lOy-dependent AKT signaling. We used PI3K isoform selective inhibitors to study the contribution of different pl 10 isoforms in regulating AKT downstream of CXCR4. We used the HL-60 promyelocytic cell line and mouse primary neutrophils to examine cells from myeloid lineage, and HeLa (PI3K^WT and PTEN^WT) as a non-leukocyte control. After CXCL12 stimulation of HeLa cells, the phosphorylation of AKT was suppressed by the PI3Ka inhibitor, HS-173 (47), whereas both HS-173 and the PI3Ky inhibitor Eganelisib (48), inhibited CXCL12-dependent AKT phosphorylation in neutrophils and HL-60 cells. In contrast, inhibition ofPI3K0, using GSK2636771 (49), and inhibition of PI3K5, using Nemiralisib (50), exerted much less effect (Figure 5E) in all of the cells tested. To examine whether the attenuation of CXCL12-dependent AKT phosphorylation in response to PTP1B inhibitors was mediated by PI3Ky, we pretreated cells with MSL1436 and DPM-1003. Both PTP1B inhibitors exerted minimal effect on CXCL12-induced AKT phosphorylation in HeLa cells. In contrast, they suppressed AKT signaling in response to CXCL12 in HL-60 cells and neutrophils, suggesting that PTP1B inhibitors impaired PI3Ky-mediated AKT signaling downstream of CXCR4 (Figures 5F and 5G).

[0116] To assess further the specificity of PTP1B inhibitors on CXCR4 signaling, we evaluated their effects on two additional GPCRs. Upon stimulation of CXCR2 with CXCL2, downstream signaling was activated, and PTP1B inhibitors attenuated AKT phosphorylation (Figures 5H and 51). In contrast, treatment with PTP1B inhibitors enhanced A-formyl- methionine-leucine-phenylalanine (fMLP)-induced AKT phosphorylation (Figures 51 and 5K), suggesting that the suppression of GPCR signaling was agonist and receptor-dependent.

[0117] Example 8. mTOR inhibitor improved survival in the TRALI model and induced an aged neutrophil phenotype.

[0118] In the studies above, we observed a pronounced suppression of AKT phosphorylation at residue Ser⁴⁷³ by PTP1B inhibitors; this phosphorylation is mediated by mammalian target of rapamycin complex 2 (mT0RC2) (51). AKT that has been phosphorylated at Thr³⁰⁸ indirectly activates mTORCl, which activates S6 kinase 1 to phosphorylate RPS6 (52). The decreased phosphorylation of AKT, RPS6 and ERK1/2 suggested PTP1B inhibitors suppressed mTOR-mediated CXCR4 signaling. It has been reported that mT0RC2 plays a role in chemoattractant-stimulated neutrophil chemotaxis by regulating myosin II in a RhoA- dependent manner (53). To evaluate whether inhibition of PTP1B also affected migration of neutrophils toward chemotactic cues, we used a Transwell assay to assess their response to CXCL12. Compared to the control, pretreatment of either MSI-1436 or DPM-1003 significantly impaired the mobility of neutrophils (Figure 6A). Consistent with the effect of PTP1B inhibitors promoting fMLP-dependent AKT signaling, both inhibitors enhanced the migration of neutrophil towards fMLP (Figures 6B and 6C).

[0119] Since PTP1B inhibitors promoted an aged neutrophil phenotype through suppression of mTOR, we examined whether mTOR inhibitors act similarly to PTP1B inhibitors. We observed that the CXCL12-stimulated phosphorylation of AKT Ser⁴⁷³ was attenuated following treatment with Palomid 529 (P529) (Figure 6D), an inhibitor of mTORCl and mT0RC2 complexes that has been reported to decrease phosphorylation of AKT on Ser⁴⁷³ (54). Furthermore, treatment with P529 two hours before TRALI induction improved overall survival at 2 hour to over 80%, compared to less than 50% in the vehicle-treated control group (Figure 6E). In the LPS-induced sepsis model, pretreatment of P5292 hours prior to LPS (15 mg/kg) challenge significantly prolonged the survival time compared to the vehicle control mice (Figure 6F). Similar to PTP1B inhibitor treatment, P529 treatment promoted an aged neutrophil phenotype, including downregulation of fresh neutrophil markers CD62L and CXCR2 (Figure 6G). Taken together these findings highlight inhibition of PI3Ky/AKT/mTOR-mediated CXCR4 signaling as a component of the mechanism by which PTP1B inhibitors protected against lung injury in the TRALI model and improved survival in LPS-induced sepsis model (Figure 6H).

[0120] Discussion

[0121] Neutrophils, which are the most abundant white blood cell type, play an important role in the innate immune response, providing protection from invading pathogens (5). These beneficial anti-microbial functions, which include phagocytosis, degranulation, and NET formation, have to be balanced with potentially deleterious inflammatory effects. This balance is achieved in part through a neutrophil aging process that follows a circadian rhythm and contributes to the homeostasis of neutrophil number and phenotypic status (7). Neutrophils, which are produced from hematopoietic stem cells in the bone marrow, differentiate into a mature form that is enriched in the granules and secretory vesicles that underlie the microbicidal function (55). Upon their controlled release into the bloodstream, the neutrophils circulate throughout the body and distribute to the sites of infection or inflammation in various tissues. Finally, upon homing back to bone marrow, they are eliminated by macrophages and dendritic cells (56). It has now been established that neutrophils undergo morphological changes, from when they leave the bone marrow as fresh neutrophils until they age and are cleared from circulation (7). Tn this current study, we have demonstrated that a single dose of either of two, distinct, allosteric inhibitors of PTP1B induced a phenotype that exhibited features of neutrophil aging. This coincided with attenuation of lung injury and decreased mortality in a murine, neutrophil-dependent TRALI model of ARDS and the LPS-induced endotoxemia model of sepsis. Furthermore, PTP1B inhibitor MSI-1436 also improved survival in the cecal ligation and puncture model (CLP)-induced model of sepsis.

[0122] Both MSI-1436 and DPM-1003 are allosteric inhibitors that target primarily the non-catalytic, disordered segment in the C-terminus of PTP1B. This segment is a unique portion of the PTP1B protein that is unrelated to TC-PTP, its closest relative, or to any other member of the PTP family (19). Consequently, we expect that such inhibitors have the potential to be highly specific for PTP IB over other members of the PTP family. In our initial study of the impact of MSI-1436 on PTP1B in models of breast cancer, we reported a double mutant, PTP1B- L192A/S372P, in which catalytic function was preserved but inhibition by MSI-1436 was abrogated (19). Following expression of this mutant in tumor cells, inhibition of cell migration and growth of tumor xenografts by MSI-1436 was markedly attenuated, consistent with PTP IB being a major target through which the compound exerted its effects. In this current study, we observed improvement of survival in the TRALI model at 2 mg/kg MSI-1436 and 5 mg/kg DPM-1003; the plasma concentrations of the compounds at these doses are also consistent with their effects being exerted through inhibition of PTP1B. Furthermore, the fact that both inhibitors produce consistent effects in this study also highlights the importance of PTP IB as a target. Nevertheless, this does not exclude a contribution of additional effects on other targets. At this time, due to technical challenges posed by differences in genetic backgrounds of the various mouse models, it is not possible to incorporate complementary genetic studies in PTPIB-ablated animals.

[0123] The process of neutrophil aging features a cell-intrinsic signaling module, in which chemokine receptors CXCR2 and CXCR4 functionally oppose one another. CXCR2 promotes mobilization of neutrophils into the blood stream, whereas CXCR4 retains neutrophils in the bone marrow, with CXCR4 playing a dominant role over CXCR2 (38). As neutrophils circulate in the blood, they upregulate the expression of CXCR4 to promote migration back to bone marrow, where the level of the chemokine ligand CXCL12 is constitutively high (57). In the context of neutrophil aging, the signaling module is driven by BMAL1, a transcription factor that regulates the circadian clock (58). BMAL1 controls the expression of CXCL2, a chemokine ligand of CXCR2, to promote neutrophil aging; in contrast, CXCR4 impairs the aging process (8). In fact, deletion of Cxcr4 from the myeloid lineage promotes the acquisition of an aged phenotype. It is interesting to note that treatment of mice with inhibitors of PTP1B phenocopies the loss of CXCR4, including neutrophilia, progressive loss of granule content, downregulation of fresh neutrophil markers CD62L and CXCR2, and decreased formation of NETs in the lung. This suggests that one mechanism by which our inhibitors of PTP1B exert their effects is through suppression of the inhibitor of aging, CXCR4.

[0124] CXCR4 is a G protein-coupled receptor (GPCR) that can activate diverse downstream signaling pathways (59). Among them, PI3K plays an important role in regulating neutrophil migration, ROS generation, and the respiratory burst (42). In leukocytes, PI3Ky is the preferentially expressed isoform, and its activity is regulated by G protein 0y heterodimers (46). Mice deficient in the p l lOy PI3K catalytic subunit showed a higher number of neutrophils in the circulation, impaired neutrophil migration and ROS generation, phenotypes similar to the mouse model of Akt2 deletion (42, 60, 61). In this study, following treatment with our PTP1B inhibitors, we observed neutrophilia, reduced phosphorylation of AKT, and attenuated neutrophil migration towards CXCL12. These observations suggest that PTP1B inhibitors function to inhibit PI3Ky/AKT signaling downstream of CXCR4. In accordance with the reduction in NET formation following suppression of AKT (37), we show that MSI-1436 is a potent inhibitor of PMA-mediated NETs in vitro and NET formation in the lung tissues during TRALI.

[0125] The phosphorylation of AKT on Thr³⁰⁸ is mediated by 3 -phosphoinositide- dependent protein kinase- 1 (PDK-1), whereas phosphorylation of Ser⁴⁷³ is mediated by mT0RC2 (43, 62). It has been shown that mTORC2 controls neutrophil chemotaxis by cAMP/RhoA dependent phosphorylation of Myo-II, which regulates tail retraction and adhesion (53, 63). In our study, we demonstrated an impaired chemotactic response to the CXCR4 ligand CXCL12 upon PTP1B inhibitor treatment. Furthermore, we observed an aged-neutrophil phenotype upon pharmacological inhibition of mTOR with P529. Our observation that P529 partially rescued mortality in the TRALI model and prolonged survival in LPS-induced sepsis model suggests that inhibitors of PTP1B function, at least in part, through suppressing AKT/mTOR-mediated CXCR4 signaling, to promote neutrophil aging. Compared to fresh neutrophils, intrinsically aged neutrophils have reduced propensity to migrate into inflamed tissues, less granule content and lower capacity to produce NETs (8, 9). As a consequence, neutrophil aging is a physiological strategy to dampen the toxic nature of neutrophils before they infiltrate into lungs, and to prevent tissue damage. Nevertheless, there are also reports suggesting that neutrophil aging mediated by extrinsic stimuli, such as microbiota, favor a pro-inflammatory phenotype (64, 65). The mechanism by which such extrinsic signals would be coordinated with the intrinsic program requires further investigation.

[0126] It is interesting to note that in the presence of multiple stimuli, the chemotactic response of neutrophils is processed in a hierarchical manner, prioritizing “end target” (such as fMLP) over “intermediary” (such as CXCL2, CXCL12) chemoattractants to determine the direction of migration (66). This preference is consistent with differences in the nature of the signaling events downstream of the cognate GPCRs and context-dependent effects of phosphatases, such as PTP1B, on signaling outcome (67). In our study, we highlight specificity in the effects of PTP1B inhibitors in suppressing signaling in response to CXCL2 and CXCL12, but enhancing fMLP-induced signaling. In response to fMLP, activation of p38 MAPK promotes neutrophil migration. PTP1B dephosphorylates p38 MAPK directly (13), which likely explains why PTP1B inhibition enhanced fMLP-mediated signaling. In contrast, intermediary chemokines primarily function through PI3K (68), consistent with a different point of action of PTP1B.

[0127] It is important to emphasize that the old ideas that phosphatases are merely housekeeping enzymes that are there simply to dephosphorylate, and antagonize the activity of, protein kinases are no longer borne out by the data (67, 69, 70). It is now well established that phosphatases are themselves critical regulators of signaling in their own right, with the potential to function both negatively and positively, depending upon the context (67). This has been established already for PTP1B. PTP1B can serve to dephosphorylate and inactivate the insulin receptor and the leptin receptor-associated kinase JAK2; this is what laid the foundation for excitement about PTP1B as a therapeutic target for diabetes and obesity (71, 72). Considering this precedent, it would have been expected that inhibition of PTP1B would promote PI3Koc- mediated AKT, rather than the attenuation of AKT signaling we observed in neutrophils. Nevertheless, it is already established that in different contexts PTP1B serves as a positive regulator of signaling, for example, downstream of the HER2 oncoprotein tyrosine kinase (73, 74). In such cases, inhibition of PTP1B would be expected to suppress signaling. In the context of G protein coupled receptors (GPCR), the G0Y heterodimer promotes the activation of PI3Ky. Tn fact, we observed that MST-1436 and DPM-1003 exerted minimal effect on CXCL12-induced AKT phosphorylation in HeLa cells. In contrast, they suppressed AKT signaling in response to CXCL12 in HL-60 cells and neutrophils, suggesting that PTP1B inhibitors impaired PI3Ky- mediated AKT signaling downstream of CXCR4. Our observations, which provide a further illustration of specificity in PTP1B function in a biological context, are consistent with previous studies reporting that PTP1B inhibition attenuates mTOR signaling through activating AMPK, which suppressed mTOR activity in mast cells, pancreatic cancer cells, hepatocytes (75-77). In addition, the phosphorylation of mTOR at Ser2448 is decreased in bone marrow-derived macrophages isolated from myeloid PTP IB -depleted mice (78). The identification of target substrates of any phosphatase is a formidable undertaking and, at this time, the identity of the direct substrate(s) of PTP1B that underlie the control of PI3Ky/AKT/mT0R signaling is unclear and requires further study.

[0128] It is important to note that PTP1B has been implicated in various immune responses, including attenuation of CD40 and B cell activating factor receptor pathways in B cells (13), negative regulation of JAK/STAT5 pathway in T cells (79), and modulation of macrophage activities (14-16). Neutrophils are important pathological drivers in ARDS and many other inflammatory diseases, such as multiple sclerosis, psoriasis, chronic obstructive pulmonary disease (80-82). Thus, manipulation of neutrophil aging to dampen the neutrophil activity may be an attractive anti-inflammatory therapeutic approach. In this study, we now demonstrate that PTP IB inhibitors prevent lung injury in the TRALI model and improve survival in CLP- and LPS-induced sepsis models via a mechanism to promote neutrophil aging. MSI- 1436, the prototypic allosteric PTP1B inhibitor, was well tolerated by patients enrolled in clinical trials for obesity and type 2 diabetes (T2D) (83) and breast cancer. This, coupled with the beneficial effect in ameliorating ALI, highlight PTP IB inhibitors as potential therapeutics for treating neutrophil-mediated lung damage, an indication that has risen to prominence recently due to the impact of lung injury in patients with advanced COVID- 19 disease.

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Claims

What is claimed is:

1. A method of treating lung injury in a subject in need thereof comprising administering a PTP1B inhibitor to the subject.

2. A method according to claim 1 wherein the lung injury comprises one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID- 19, and lung injury resulting from WHIM syndrome.

3. The method according to either claim 1 or claim 2, wherein the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof.

4. The method according to any of claims 1-3, wherein the PTP1B inhibitor is administered from one to two times a day.

5. The method according to any of claims 1-4, wherein administering is accomplished via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

6. A method of prophylactically inhibiting symptoms of lung injury in a subject at risk of developing lung injury comprising administering a PTP1B inhibitor to the subject.

7. A method according to claim 6 wherein the subject is at risk of developing symptoms of lung injury associated with one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID-19, and lung injury resulting from WHIM syndrome.

8. The method according to either claim 6 or claim 7, wherein the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof.

9. The method according to any of claims 6-8, wherein the PTP1B inhibitor is administered from one to two times a day.

10. The method according to any of claims 6-9, wherein administering is accomplished via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

1 1. A pharmaceutical composition comprising: a PTP1B inhibitor and a carrier, wherein the PTP1B inhibitor is present in an amount sufficient to reduce lung injury in a subject or inhibit symptoms of lung injury in a subject at risk of developing lung injury.

12. The pharmaceutical composition according to claim 11, wherein the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof.

13. The pharmaceutical composition according to claim 11 or claim 12, wherein the carrier permits administration of the pharmaceutical composition via a route selected from oral, buccal, sublingual, rectal, topical, intranasal, vaginal, or parenteral administration.

14. A method of treating lung injury in a subject in need thereof comprising administering a PTP1B inhibitor to the subject, wherein the lung injury is at least one of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID- 19, or lung injury resulting from WHIM syndrome and the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof.

15. The method of claim 14, wherein the lung injury comprises acute lung injury.

16. The method of claim 14 or 15, wherein the lung injury comprises antibody- induced acute lung injury.

17. The method of any one of claims 14-16, wherein the lung injury comprises acute lung injury associated with inflammation.

18. The method of any one of claims 14-17, wherein the lung injury comprises ARDS.

19. The method of any one of claims 14-18, wherein the lung injury comprises lung injury resulting from sepsis.

20. The method of any one of claims 14-19, wherein the lung injury comprises lung injury resulting from SARS-CoV-2 infection.

21. The method of any one of claims 14-20, wherein the lung injury comprises lung injury resulting from COVID- 19.

22. The method of any one of claims 14-21, wherein the lung injury comprises lung injury resulting from WHIM syndrome.

23. The method of claim 14, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

24. The method of claim 14, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

25. The method of claim 15, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

26. The method of claim 15, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

27. The method of claim 16, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

28. The method of claim 16, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

29. The method of claim 17, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

30. The method of claim 17, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

31. The method of claim 18, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

32. The method of claim 18, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

33. The method of claim 19, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

34. The method of claim 19, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

35. The method of claim 20, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

36. The method of claim 20, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

37. The method of claim 21, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

38. The method of claim 21, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

39. The method of claim 22, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

40. The method of claim 22, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

41 . A method of prophylactically inhibiting symptoms of lung injury in a subject at risk of developing lung injury comprising administering a PTP1B inhibitor to the subject, wherein the subject is at risk of developing symptoms of lung injury associated with one or more of acute lung injury, antibody-induced acute lung injury, acute lung injury associated with inflammation, ARDS, lung injury resulting from sepsis, lung injury resulting from SARS-CoV-2 infection, lung injury resulting from COVID- 19, and lung injury resulting from WHIM syndrome and the PTP1B inhibitor is MSI-1436, DPM-1003, or a respective pharmaceutically acceptable salt thereof.

42. The method of claim 41, wherein the lung injury comprises acute lung injury.

43. The method of claim 41 or 42, wherein the lung injury comprises antibody- induced acute lung injury.

44. The method of any one of claims 41-43, wherein the lung injury comprises acute lung injury associated with inflammation.

45. The method of any one of claims 41-44, wherein the lung injury comprises ARDS.

46. The method of any one of claims 41-45, wherein the lung injury comprises lung injury resulting from sepsis.

47. The method of any one of claims 41-46, wherein the lung injury comprises lung injury resulting from SARS-CoV-2 infection.

48. The method of any one of claims 41-47, wherein the lung injury comprises lung injury resulting from COVID-19.

49. The method of any one of claims 41-48, wherein the lung injury comprises lung injury resulting from WHIM syndrome.

50. The method of claim 41, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

51. The method of claim 41, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

52. The method of claim 42, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

53. The method of claim 42, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

54. The method of claim 43, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

55. The method of claim 43, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

56. The method of claim 44, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

57. The method of claim 44, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

58. The method of claim 45, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

59. The method of claim 45, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

60. The method of claim 46, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

61. The method of claim 46, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

62. The method of claim 47, wherein the PTP1B inhibitor is MST-1436 or a pharmaceutically acceptable salt thereof.

63. The method of claim 47, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

64. The method of claim 48, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

65. The method of claim 48, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.

66. The method of claim 49, wherein the PTP1B inhibitor is MSI-1436 or a pharmaceutically acceptable salt thereof.

67. The method of claim 49, wherein the PTP1B inhibitor is DPM-1003 or a pharmaceutically acceptable salt thereof.