Compositions and methods using tyrosine kinase inhibitors

Abstract

To provide a method of treating a cardiovascular disease or condition having aberrant protein tyrosine phosphorylation.SOLUTION: The method comprises administering a low-dosage of a tyrosine kinase inhibitor to a subject, where the tyrosine kinase inhibitor decreases aberrant levels of tyrosine phosphorylation and improves at least one cardiac function in the subject. Preferably, the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, and the like.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 250,052, filed on November 3, 2015, and U.S. Provisional Application No. 62 / 107,553, filed on January 26, 2015, the contents of which are hereby incorporated by reference in their entirety.

[0002] Description of Research and Development Funded by the Federal Government This invention was made with government support under Grant No. GM099801 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] Background of the Invention Cardiovascular disease remains the leading cause of death worldwide for both men and women, despite significant progress. According to a World Health Organization (WHO) report, it is estimated that by 2030, 23.6 million people will die each year from cardiovascular disease.

[0004] The RAS - MAPK pathway is of great significance for human growth and development. Abnormalities at various stages of this signaling cascade lead to neuro - cardiac - facial - cutaneous syndromes or RASopathies, a group of disorders with overlapping but distinct phenotypes. RASopathy patients exhibit varying degrees of intellectual disability, growth retardation, relative macrocephaly, ectodermal abnormalities, features of dysmorphia, and an increased risk of certain malignancies. Marked locus heterogeneity is observed in many RASopathies.

[0005] Congenital heart disease (CHD) is the most common defect found in newborns, occurring in about 1% of live births. In the United States, more than one million people have some form of CHD, the majority of whom require continuous monitoring and treatment to prevent a decline in cardiac function. AVCD involves various abnormalities of the atrioventricular valves as well as the atrial and ventricular septa. In the complete form, there is one common atrioventricular valve and an atrial septal defect (foramen primari) fused with a posterior ventricular septal defect at the ostium of the ventricular septum. In the incomplete form, there are two separate right and left atrioventricular valves, a clefted mitral valve, and an atrial septal defect (foramen primari), with no ventricular septal communication. A clefted mitral valve is considered a less severe form of AVCD. AVCD is also the most common congenital heart defect (CHD) found in children with Down syndrome and is one of the structural cardiac defects most frequently associated with extracardiac abnormalities in the context of chromosomal disorders and Mendelian genetic diseases. Distinctive anatomical features are found in AVCD associated with NS. In fact, the defect is usually incomplete, caused by abnormal insertion of the mitral valve due to accessory fibrous tissue and / or abnormal papillary muscles of the left ventricle, ultimately leading to subaortic stenosis.

[0006] Congenital heart disease (CHD) occurs in approximately 60–86% of patients affected by RAS opathy, i.e., disorders involving abnormalities in the RAS-MAPK pathway. Pulmonary valve stenosis (PVS) and hypertrophic cardiomyopathy are the most common defects that have shown a clear association with RAS opathy. The range of CHD is even broader in Noonan syndrome with lentigo polycarpa (NSML), and the family of atrioventricular canal defects (AVCD) is the third most common cardiac defect.

[0007] Most patients with cardiovascular disease and congenital heart defects associated with RAS opathy require treatment for many years. In particular, congenital heart defects associated with RAS opathy are typically associated with low mortality rates. Therefore, it is necessary to treat patients' cardiovascular disease using low-risk therapies that have the greatest effect on the heart condition. [Overview of the Initiative]

[0008] As described later, the present invention includes compositions and methods for inhibiting abnormal protein tyrosine phosphorylation, such as phosphorylation of Src family tyrosine kinases and their substrates.

[0009] In one aspect, the present invention provides a method for treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0010] In another aspect, the present invention includes a method for treating a congenital heart disease, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0011] In yet another aspect, the present invention provides a method for treating a cardiovascular disease or condition associated with RAS opathy having abnormal protein tyrosine phosphorylation, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0012] In yet another aspect, the present invention includes a composition comprising a low dose of a tyrosine kinase inhibitor, wherein the low dose of the tyrosine kinase inhibitor can reduce tyrosine phosphorylation and improve at least one cardiac function in a subject requiring such reduction.

[0013] In another aspect, the present invention includes a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable carrier.

[0014] In yet another aspect, the present invention includes the use of the compositions described herein in the manufacture of a pharmaceutical product for treating a cardiovascular disease or condition in a subject.

[0015] In various aspects of the above or any other aspect of the present invention described in detail herein, congenital heart disease is related to RAS opathy, selected from the group consisting of, for example, neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with lentigo polycarcinoma (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiac-facial-cutaneous syndrome, and Regius syndrome. In one aspect, the cardiovascular disease or pathology is congenital heart disease.

[0016] In another embodiment, the low dose ranges from about 1 / 175 to about 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor.

[0017] In another embodiment, cardiac function is selected from the group consisting of myofibrils organizing, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.

[0018] In another embodiment, the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cejiranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In yet another embodiment, tyrosine kinase inhibitors include Src family tyrosine kinase inhibitors, such as A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228 is a Src family tyrosine kinase inhibitor selected from the group consisting of artenusin, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and their analogs.

[0019] In another embodiment, the subjects are pediatric patients, for example, pediatric patients under 12 years of age. In yet another embodiment, the subjects are older than 18 years of age.

[0020] In another embodiment, abnormal levels of tyrosine phosphorylation include abnormal levels of tyrosine-phosphorylated protein zero-related (PZR) proteins. In such an embodiment, low doses of tyrosine kinase inhibitors reduce PZR tyrosine phosphorylation. In yet another embodiment, low doses of tyrosine kinase inhibitors induce an anti-fibrotic effect in cardiac tissue. In yet another embodiment, low doses of tyrosine kinase inhibitors reduce abnormal tyrosine phosphorylation of transmembrane glycoproteins such as transmembrane glycoprotein zero-related (PZR) proteins. In yet another embodiment, low doses of tyrosine kinase inhibitors induce an anti-fibrotic effect in cardiac tissue. [Invention 1001] A method for treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject. [Invention 1002] A method for treating a congenital heart defect, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject. [Invention 1003] A method for treating a cardiovascular disease or condition associated with RAS opathy having abnormal protein tyrosine phosphorylation, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject. [Invention 1004] The method of the present invention 1002, wherein congenital heart disease is related to RAS opathy. [Invention 1005] The method of the present invention 1003, wherein the cardiovascular disease or pathological condition is a congenital heart disease. [Invention 1006] The method of either 1003 or 1004 of the present invention, wherein RAS opathy is selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with lentigo polycarcinoma (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiac-facial-cutaneous syndrome, and Regius syndrome. [Invention 1007] A method according to any one of the present invention 1001 to 1003, wherein the low dose is in the range of approximately 1 / 175 to approximately 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor. [Invention 1008] A method according to any one of the present invention 1001 to 1003, wherein cardiac function is selected from the group consisting of myofibrils, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. [Invention 1009] A method according to any one of the present invention 1001 to 1003, wherein the tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cejiranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. [Invention 1010] A method according to any one of the present invention 1001 to 1003, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. [Invention 1011] Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL The method of the present invention 1010, selected from the group consisting of 228, artenusine, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and analogs thereof. [Invention 1012] A method according to any of the present invention 1001 to 1003, wherein the subject is a pediatric patient. [Invention 1013] The method of the present invention 1012, wherein the target of pediatrics is children under 12 years of age. [Invention 1014] A method according to any of the present invention 1001 to 1003, wherein the subject is 18 years of age or older. [Invention 1015] A method according to any one of the present invention 1001 to 1003, wherein the abnormal level of tyrosine phosphorylation includes an abnormal level of tyrosine phosphorylated protein zero-related (PZR). [Invention 1016] The method of the present invention 1015, wherein a low dose of a tyrosine kinase inhibitor reduces PZR tyrosine phosphorylation. [Invention 1017] A method according to any of the present invention 1001 to 1003, wherein a low dose of a tyrosine kinase inhibitor is used to induce an antifibrotic effect in cardiac tissue. [Invention 1018] A composition comprising a low dose of a tyrosine kinase inhibitor capable of reducing tyrosine phosphorylation and improving at least one cardiac function in a subject requiring such reduction. [Invention 1019] The composition of the present invention 1018, wherein a low dose of a tyrosine kinase inhibitor reduces abnormal tyrosine phosphorylation of transmembrane glycoproteins. [Invention 1020] The composition of the present invention 1019, wherein the transmembrane glycoprotein is a protein zero-related (PZR). [Invention 1021] The composition of the present invention 1018, wherein the low dose of the tyrosine kinase inhibitor is in the range of approximately 1 / 175 to approximately 1 / 250 of the chemotherapy dose of the tyrosine kinase inhibitor. [Invention 1022] The composition of the present invention 1018, wherein cardiac function is selected from the group consisting of myofibrils organizing, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. [Invention 1023] The composition of Invention 1018, wherein a low dose of a tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cejiranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. [Invention 1024] The composition of the present invention 1018, wherein the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor. [Invention 1025] Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL A composition of the present invention 1024, selected from the group consisting of 228, artenusine, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and analogs thereof. [Invention 1026] The composition of the present invention 1018, wherein a low dose of a tyrosine kinase inhibitor provides an antifibrotic effect in cardiac tissue. [Invention 1027] A pharmaceutical composition comprising the composition of the present invention 1018 and a pharmaceutically acceptable carrier. [Invention 1028] Use of the composition of the present invention 1018 in the manufacture of a pharmaceutical product for treating cardiovascular disease or pathological condition in a subject. [Brief explanation of the drawing]

[0021] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact same arrangements and means as those shown in the drawings.

[0022] [Figure 1A]This figure shows the proteomics analysis of differentially tyrosyl-phosphorylated proteins in the heart of Ptpn11D61G / + mice. It also shows the classification of low-tyrosyl-phosphorylated and high-tyrosyl-phosphorylated proteins in the heart of Ptpn11D61G / + mice. [Figure 1B] This graph shows the log2-converted ratios of each phosphotyrosine-containing peptide in the hearts of wild-type mice and Ptpn11D61G / + mice. [Figure 1C] This is a heatmap of differentially tyrosyl-phosphorylated peptides (phosphorylation sites were identified by MS and are indicated in parentheses). [Figure 1D] These are images of extracted ion chromatograms and peptide sequences of PZRs containing tyrosine 242 (top panel) and PZRs containing tyrosine 264 (bottom panel), obtained by differential proteomics. [Figure 1E] This shows the C-terminal amino acid sequences of protein zero-related (PZR) proteins in various vertebrates. The consensus sequence of the immune receptor tyrosine-based repression motif (ITIM; S / I / V / LXYXXI / V / L) is shown in bold, with tyrosine residues highlighted in red and labeled with appropriate amino acid numbers. The C-terminal sequences of PZR proteins from humans (Homo sapiens), mice (Mus musculus), rats (Rattus norvegicus), cattle (Bos taurus), dogs (Canis lupus familiaris), zebrafish (Danio rerio), and chickens (Gallus gallus) are shown. [Figure 2A]This is a panel of blots illustrating the characterization of PZR tyrosyl phosphorylation. C2C12 cells were co-transfected with either an empty vector or activated glutathione S-transferase (GST)-Shp2E76A, or one of the following: empty vector (vector), wild-type human PZR (WT), or PZR with a tyrosine 241 mutation (Y241F), PZR with a tyrosine 263 mutation (Y263F), or PZR with mutations in both (2YF). Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibody, anti-PZR antibody, or anti-Shp2 antibody. [Figure 2B] This is a panel of blots illustrating the characterization of PZR tyrosyl phosphorylation. HEK-293 cells were co-transfected with either an empty vector (vector) or activated Shp2E76A, or either an empty vector (vector), wild-type zebrafish PZR (WT), or a PZR mutant with tyrosine 236 mutation (Y241F), a PZR mutant with tyrosine 258 mutation (Y263F), or a PZR mutant with both mutations (2YF). Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibody, anti-PZR antibody, or anti-Shp2 antibody. ERK1 / 2 was used as a loading control. [Figure 2C] This is a panel of blots illustrating the characterization of PZR tyrosyl phosphorylation. HUVEC cells were infected with adenoviruses expressing either GFP, wild-type Shp2, or Shp2E76A as controls. Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibodies, anti-total PZR antibodies, and anti-Shp2 antibodies. [Figure 2D]This is a panel of blots illustrating the characterization of PZR tyrosyl phosphorylation. HEK-293 cells were transiently transfected with an empty vector, wild-type Shp2 (WT), or the Shp2 mutants shown in the figure (activated Shp2, E76A; Noonan syndrome (NS) mutant, N308D; or Noonan syndrome with multiple lentigo (NSML) mutants, Y279C and T468M). Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibody, anti-PZR antibody, and anti-Shp2 antibody. ERK1 / 2 was used as a loading control. [Figure 2E] This is a panel of blots illustrating the characterization of PZR tyrosyl phosphorylation. HEK-293T cells were transfected with HA-tagged zebrafish PZR along with an empty vector, wild-type Shp2, Shp2D61G (NS mutant), or Shp2A462T (NSML mutant). Cell lysates were immunoprecipitated using anti-HA antibodies, and immune complexes were immunoblotted using anti-Shp2 and anti-HA antibodies. Whole cell lysates (WCL) were blotted using anti-pPZR (Y241 and Y263) antibodies, anti-Shp2 antibodies, and anti-HA antibodies. [Figure 3A] This study shows PZR tyrosine phosphorylation in the hearts of Ptpn11D61G / + mice. Hearts were isolated from 5-week-old WT mice and Ptpn11D61G / + mice. Tissue lysates were immunoblotted using pPZR(Y263) antibody and total PZR antibody. Phosphorylation of tyrosine 264 in PZR was n=5 per genotype. All data represent mean ± standard error of the mean (SEM). **, P<0.01. [Figure 3B] This study shows PZR tyrosyl phosphorylation in the cortex of Ptpn11D61G / + mice. Cortical tissue was isolated from 5-week-old wild-type (WT) and Ptpn11D61G / + mice. Tissue lysates were immunoblotted using pPZR(Y263) antibody and total PZR antibody. Phosphorylation of tyrosine 264 in PZR was observed in n=5 for each genotype. All data represent mean ± standard error of the mean (SEM). ***, P<0.001. [Figure 3C]This study shows PZR tyrosine phosphorylation in the heart of Ptpn11Y279C / + mice. Hearts were isolated from 8-week-old WT mice and Ptpn11Y279C / + mice. Tissue lysates were immunoblotted with pPZR(Y263) antibody and total PZR antibody. Phosphorylation of tyrosine 264 in PZR was n=5 per genotype. All data are mean ± standard error of the mean (SEM). *, P<0.05. [Figure 3D] This study shows PZR tyrosine phosphorylation in the cortex of Ptpn11Y279C / + mice. Cortical tissue was isolated from 8-week-old wild-type (WT) and Ptpn11Y279C / + mice. Tissue lysates were immunoblotted using pPZR(Y263) antibody and total PZR antibody. Phosphorylation of tyrosine 264 in PZR was observed in n=5 for each genotype. All data are mean ± standard error of the mean (SEM). ***, P<0.001. [Figure 4A] This study shows PZR tyrosine phosphorylation in the liver of Ptpn11D61G / + mice. Liver tissue was isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted using anti-pPZR(Y263) antibody and anti-total PZR antibody. The phosphorylation level of tyrosine 263 in PZR was analyzed by concentration measurement. The results represent the mean ± SEM values ​​obtained from 5 mice per genotype. [Figure 4B] Images and graphs showing PZR tyrosine phosphorylation in the kidneys of Ptpn11D61G / + mice. Kidneys were isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted using anti-pPZR(Y263) antibody and anti-total PZR antibody. Concentration analysis of tyrosine 263 phosphorylation levels in PZR was performed. Results represent mean ± SEM values ​​obtained from 5 mice per genotype. **, P<0.01 (WT vs NS). [Figure 4C]Images and graphs showing PZR tyrosine phosphorylation in the spleen of Ptpn11D61G / + mice. Spleens were isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were immunoblotted using anti-pPZR(Y263) antibody and anti-total PZR antibody. Concentration analysis of tyrosine 263 phosphorylation levels in PZR was performed. Results represent the mean ± SEM values ​​obtained from 5 mice per genotype. ***, P<0.001 (WT vs NS). [Figure 5A] This is a panel of immunoblots showing ERK and Akt phosphorylation in the hearts of Ptpn11D61G / + mice. Hearts were isolated from 5-week-old wild-type and Ptpn11D61G / + mice (A and B). Tissue lysates were subjected to immunoblotting using anti-Shp2 antibody, anti-pERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-pAkt antibody, and anti-Akt antibody. Results represent the mean ± SEM of concentration metric analyses for pERK1 / 2 and pAkt obtained from 5 mice per genotype. [Figure 5B] This is a panel of images showing ERK and Akt phosphorylation in the cortex of Ptpn11D61G / + mice. Corticoplasm was isolated from 5-week-old wild-type and Ptpn11D61G / + mice. Tissue lysates were subjected to immunoblotting using anti-Shp2 antibody, anti-pERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-pAkt antibody, and anti-Akt antibody. Results represent the mean ± SEM of concentration metric analyses for pERK1 / 2 and pAkt obtained from 5 mice per genotype. [Figure 5C] This is a panel of images showing ERK and Akt phosphorylation in the hearts of Ptpn11Y279C / + mice. Hearts were isolated from 8-week-old wild-type and Ptpn11Y279C / + mice. Tissue lysates were subjected to immunoblotting using anti-Shp2 antibody, anti-pERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-pAkt antibody, and anti-Akt antibody. Results represent the mean ± SEM of concentration metric analyses for pERK1 / 2 and pAkt obtained from 5 mice per genotype. [Figure 5D] This is a panel of images showing ERK and Akt phosphorylation in the cortex of Ptpn11Y279C / + mice. Corticoplasm was isolated from 8-week-old wild-type and Ptpn11Y279C / + mice. Tissue lysates were subjected to immunoblotting using anti-Shp2 antibody, anti-pERK1 / 2 antibody, anti-total ERK1 / 2 antibody, anti-pAkt antibody, and anti-Akt antibody. Results represent the mean ± SEM values ​​of concentration metric analyses for pERK1 / 2 and pAkt obtained from 5 mice per genotype. [Figure 6A] This is a panel of blots showing the effects of Src family kinases on the phosphorylation of PZR Y241 and Y263. HEK-293 cells were transiently transfected with the Shp2 mutant shown in the figure and treated with either dimethyl sulfoxide (DMSO) or 5 μM SU6656 as a control. Cell lysates were immunoblotted with anti-Shp2 antibody, anti-pSrc (Y416) antibody, anti-Src antibody, anti-pPZR (Y241 or Y263) antibody, and anti-total PZR antibody. ERK1 / 2 was used as a loading control. [Figure 6B] This is a panel of blots showing the effects of Src family kinases on the phosphorylation of PZR Y241 and Y263. NIH 3T3 cells were infected with adenovirus expressing either GFP or constitutively active Shp2E76A as a control, in the presence of DMSO, PP2, or SU6656 at the concentrations shown in the figure. Cell lysates were immunoblotted using anti-Shp2 antibody, anti-pSrc(Y416) antibody, anti-Src antibody, anti-pPZR(Y241 or Y263) antibody, and anti-total PZR antibody. ERK1 / 2 was used as a loading control. [Figure 7A]This blot shows that Src kinase mediated PZR hypertyrosyl phosphorylation induced by NS-Shp2 or NSML-Shp2. SYF cells (Src- / -Fyn- / -Yes- / -MEF) and Src+ / + cells (SYF cells expressing wild-type Src) were infected with adenovirus expressing either GFP or Shp2E76A. Cell lysates were immunoblotted using anti-Shp2 antibody, anti-pSrc(Y416) antibody, anti-pPZR(Y241 or Y263) antibody, anti-PZR antibody, and anti-Src antibody. ERK1 / 2 was used as a loading control. [Figure 7B] This blot shows that Src kinase mediated PZR hypertyrosyl phosphorylation induced by NS-Shp2 or NSML-Shp2. SYF cells were transiently transfected with wild-type c-Src or kinase-dead c-SrcK295R / Y527F(KR / YF) and infected with adenovirus expressing either GFP, wild-type Shp2, or Shp2E76A. Cell lysates were immunoblotted using anti-Shp2 antibody, anti-pSrc(Y416) antibody, anti-pPZR(Y241 or Y263) antibody, anti-PZR antibody, and anti-Src antibody. ERK1 / 2 was used as a loading control. [Figure 7C] This blot shows that Src kinase induced hypertyrosyl phosphorylation of PZR. HEK-293 cells were transfected with Flag-tagged human PZR. Cell lysates were immunoprecipitated using an anti-Flag antibody. These immunoprecipitates were subjected to an in vitro Src kinase assay using recombinant Src protein. The reaction products were immunoblotted using an anti-pPZR (Y241 or Y263) antibody. [Figure 7D]This blot shows that Src family kinases induce hypertyrosyl phosphorylation of PZR. HEK-293 cells were constitutively active Src mutants and one of the following: HA-tagged wild-type zebrafish PZR (WT), PZR with a tyrosine 236 mutation (Y241F), PZR with a tyrosine 258 mutation (Y263F), or PZR with mutations in both (2YF). Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibodies and anti-HA antibodies. [Figure 8] Figure 8A shows blots and graphs illustrating increased Src complex formation with Shp2 mutants associated with NS / NSML. HEK-293 cells were transiently transfected with either Shp2 WT or one of the Shp2 mutants shown in the figure. Cell lysates were immunoprecipitated with anti-c-Src antibody (IP), and immune complexes were immunoblotted with anti-Shp2 antibody and anti-Src antibody (IB). Graphs represent mean ± SEM of concentration measurement analysis obtained from three independent experiments. Statistical significance was derived using Dunnett's test comparing Shp2 mutants to WT. *, P<0.05; **, P<0.01. Figure 8B shows blot images illustrating increased Src complex formation with Shp2 mutants and PZR associated with NS / NSML. HEK-293 cells were co-transfected with either Shp2 WT, E76A mutant, or Y279C mutant, along with either an empty vector, WT human PZR, or PZR 2YF mutant. Cell lysates were immunoblotted using anti-pPZR (Y241 or Y263) antibody or anti-Shp2 antibody. ERK1 / 2 was used as a loading control. Immunoblotted immune complexes using anti-Src antibody, anti-Shp2 antibody, and anti-PZR antibody. [Figure 9] This is a diagram illustrating a model showing the effects of NS and NSML mutants on PZR tyrosyl phosphorylation. [Figure 10]This is a panel of images showing the administration of dasatinib. Male Ptpn11D61G / + mice were intraperitoneally (ip) injected with the doses of dasatinib or DMSO control shown in the figures. After 24 hours, the mice were euthanized, cardiac tissue was collected, and immunoblotting was performed using the specified antibodies. [Figure 11] Figure 11A shows the prenatal administration plan for dasatinib in an NS mouse model. Pregnant mother mice were administered dasatinib daily from the time the animals were in utero at E7.5 until postnatal day 9 (P9). From postnatal day 10 (P10), dasatinib was administered directly to the mice by daily injection for 46 days (P56). The cardiac function of the mice was evaluated at P42 and P56. Figure 11B shows the postnatal administration plan for dasatinib in an NS mouse model. Starting at P10, dasatinib was administered to the mice daily for 32 days (P42), followed by a 14-day interruption (P56). The cardiac function of the mice was evaluated at P42 and P56. [Figure 12A] This is a panel of graphs showing that prenatal treatment with dasatinib improves cardiac function in NS mice. Pregnant mice were intraperitoneally injected with dasatinib (0.1 mg / kg) according to the protocol described herein. The number of mice in each group is shown in the figure. Statistical significance is indicated by *;P<0.05, **;P<0.01, ***;P<0.001, based on a two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 12B]This is a panel of graphs showing that treatment of postnatal NS mice with dasatinib improves cardiac function. Following the protocol described herein, NS mice were intraperitoneally injected with dasatinib (0.1 mg / kg), starting at P10. After 32 days of treatment, the mice's cardiac function was evaluated. The number of mice in each group is shown in the figure. Statistical significance is indicated by *; P<0.05, **; P<0.01, ***; P<0.001, based on a two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 12C] This is a panel of graphs showing that improved cardiac function was maintained after discontinuation of dasatinib treatment. Dasatinib (0.1 mg / kg) was administered intraperitoneally to NS mice, starting at P10, according to the protocol shown in Figure 1B. Cardiac function of mice given dasatinib for 32 days was re-evaluated after 2 weeks. The number of mice in each group is shown in the figure. Statistical significance is indicated by *;P<0.05, **;P<0.01, ***;P<0.001, based on a two-way ANOVA test. LV vol;s - Left ventricular volume in systole, LV vol;d - Left ventricular volume in diastole, FS - Fractional shortening, and EF - Ejection fraction. [Figure 13-1]This is a panel of images showing that c-Src kinase is a putative target for Noonan syndrome. Figure 13a is a schematic diagram of the wild-type full-length construct, N+C-SH2 construct, and PTP domain construct of human Shp2. Figure 13b is an image showing the detection of Myc-Src full-length co-transfected with Flag-Shp2 full-length, N+C, or PTP domain in HEK-293T cells. Protein-protein interactions were elucidated by immunoprecipitation. Figure 13c is an image showing the detection of the GST-SH3 domain of purified c-Src incubated overnight at 4°C with the His-tagged PTP domain of purified Shp2. Proteins were immobilized using GST Sepharose beads and separated by SDS-PAGE. The His-PTP domain in the GST complex was detected by immunoblotting using an anti-His antibody. WCL: Whole cell lysate, IP: Immunoprecipitation, IB: Immunoblotting. [Figure 13-2]This is a panel of images showing that c-Src kinase is a putative target for Noonan syndrome. Figure 13d is an image showing the phosphorylation level of Src(Y416). Mouse embryonic fibroblasts (MEFs) from Ptpn11D61G / + mice incubated with dasatinib at the concentrations shown in the figure for 18 hours. Whole cell lysates were immunoblotted with anti-p-Src(Y416) antibody, anti-Src antibody, anti-p-ERK1 / 2 antibody, and anti-ERK1 / 2 antibody. Tyrosyl phosphorylation of PZR was measured using a phospho-specific PZR antibody, and the molecular interaction between PZR and Shp2 was elucidated by immunoprecipitation. Figure 13e is a graph showing the phosphorylation level of Src(Y416). Figure 13f is a graph showing the phosphorylation level of ERK1 / 2. Figure 13g is a graph showing the amount of PZR phosphorylation at tyrosine 241. Figure 13h is a graph showing the amount of tyrosine-263 analyzed by concentration measurement. All data are shown as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to MEF of Ptpn11D61G / + treated with a vehicle (n=3 for each condition; one-way ANOVA test). [Figure 13-3]This is a panel of images showing that c-Src kinase is a putative target for Noonan syndrome. Figure 13i shows images of cardiac tissue immunoblotted with anti-Shp2 antibody, anti-p-ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-p-Src(Y416) antibody, anti-Src antibody, anti-p-PZR(Y263) antibody, and anti-PZR antibody. Three-week-old WT mice and Ptpn11D61G / + mice were intraperitoneally injected with vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). Cardiac tissue was isolated 18 hours later, and tissue lysates were immunoblotted. Figure 13j is a graph showing tyrosyl phosphorylation of ERK1 / 2. Figure 13k is a graph showing tyrosyl phosphorylation of Src. Figure 13l is a graph showing tyrosyl phosphorylation of PZR. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to cardiac tissue from Ptpn11D61G / + treated with a vehicle (n=3 for each condition; one-way ANOVA test). [Figure 14-1] This is a panel of images showing that dasatinib improves cardiac function in Ptpn11D61G / + mice. Figure 14a is a schematic diagram of prenatal administration of dasatinib to Ptpn11D61G / + mice. Figure 14b is a panel of images showing representative echocardiographic images at time P42 of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 14c is a graph showing ejection fraction (%) (EF) measured from the echocardiogram at time P42. Figure 14d is a panel of images showing representative echocardiographic images at time P56 of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 14e is a graph showing ejection fraction (%) (EF) measured from the echocardiogram at time P56. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 14-2]This is a panel of images showing that dasatinib improves cardiac function in Ptpn11D61G / + mice. Figure 14f is a schematic diagram of prenatal administration of dasatinib to Ptpn11D61G / + mice. Figure 14g is a panel of images showing representative echocardiographic images at time P42 of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 14h is a graph showing ejection fraction (%) (EF) measured from the echocardiogram at time P42. Figure 14i is a panel of images showing representative echocardiographic images at time P56 of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 14j is a graph showing ejection fraction (%) (EF) measured from the echocardiogram at time P56. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 14-3] This is a panel of images showing that dasatinib improves cardiac function in Ptpn11D61G / + mice. Figure 14k is a graph showing arterial systolic pressure at time P56, obtained from invasive hemodynamic studies of WT mice and Ptpn11D61G / + mice treated with a vehicle or dasatinib after birth. Figure 14l is a graph showing diastolic pressure at time P56 in WT mice and Ptpn11D61G / + mice treated with a vehicle or dasatinib after birth. Figure 14m is a graph showing mean arterial pressure (MAP) at time P56 in WT mice and Ptpn11D61G / + mice treated with a vehicle or dasatinib after birth. Figure 14n is a graph showing left ventricular blood pressure (LV pressure) at time P56 in WT mice and Ptpn11D61G / + mice treated with a vehicle or dasatinib after birth. All data show the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 15-1]This is a panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice saved by dasatinib. Figure 15a is a graph showing the heart weight at P56 measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth (n=8-9 for each group). Figure 15b is a graph showing the ratio of heart weight (HW) to body weight (BW) at P56 measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth (n=8-9 for each group). Figure 15c is a panel of images showing representative longitudinal sections of the heart at P56, stained using Masson's tricolor method, obtained from WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib after birth (scale bar = 2 mm). All data show the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 15-2] Figure 15f is a panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice saved by dasatinib. Figure 15d is a panel of images showing Masson's tricolor staining of the left ventricle at time P56 obtained from WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally (scale bar = 200 μm). Figure 15e is a graph showing the relative expression of the fibrosis marker gene Col1a2. Total cardiac RNA was isolated from WT mice and Ptpn11D61G / + mice (P56) treated with vehicle or dasatinib postnatally. Figure 15f is a graph showing the relative expression of the fibrosis marker gene Col3a1. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 15-3]This is a panel of images showing cardiomyopathy and cardiac fibrosis in Ptpn11D61G / + mice saved by dasatinib. Figure 15g is a graph showing the relative expression of Myh6(αMHC), a fetal cardiac gene. Figure 15h is a graph showing the relative expression of Myh7(βMHC), a fetal cardiac gene. Figure 15i is a graph showing the relative expression of Anf, a fetal cardiac gene. Figure 15j is a graph showing the relative expression of Bnp, a fetal cardiac gene. These genes were measured by quantitative RT-PCR (n=6 for each group). All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Figure 16-1] This is a panel of images showing that cardiomyocytes obtained from dasatinib-treated Ptpn11D61G / + mice exhibited normal Ca2+ signaling during excitation-contraction coupling. Figure 16a is an image showing Ca2+ excitation-contraction coupling measured in cardiomyocytes isolated from WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally. Representative traces of cardiomyocyte dynamics for calcium transient traces (top) and corresponding sarcomere length (leng) shortening traces (bottom). Figure 16b is a graph summarizing the data for relative calcium release (Rmag Ca2+). Figure 16c is a graph showing the percentage of sarcomere shortening (n = 111-162 cells obtained from 3 mice in each group). All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (two-way ANOVA test). [Figure 16-2]This panel of images shows cardiomyocytes obtained from dasatinib-treated Ptpn11D61G / + mice exhibiting normal Ca2+ signaling during excitation-contraction coupling. Figure 16d shows images of cardiac tissue immunoblotted with anti-SERCA2A antibody, anti-troponin I (tTnI) antibody, anti-troponin T (tTnT) antibody, and anti-tubulin antibody. Cardiac tissue was isolated from WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally, and tissue lysates were immunoblotted. Figure 16e is a graph showing SERCA2A expression. Figure 16f is a graph showing troponin I expression. Figure 16g is a graph showing troponin T expression. Expression was statistically evaluated after standardization using tubulin (n=6 for each group). All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (two-way ANOVA test). [Figure 17-1] This is a panel of images showing the effect of dasatinib on NS signaling in vitro. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with dasatinib for 18 hours. Whole cell lysates were immunoblotted using anti-Shp2 antibody, anti-p-Src(Y416) antibody, anti-Src antibody, anti-p-Raf1(Y341) antibody, anti-Raf1 antibody, anti-p-MEK1 / 2 antibody, anti-MEK1 / 2 antibody, anti-p-ERK1 / 2 antibody, anti-ERK1 / 2 antibody, anti-p-JNK antibody, anti-JNK antibody, anti-p-p38 antibody, anti-p38 antibody, anti-p-Akt antibody, and anti-Akt antibody (Figure 17a). Phosphorylation levels of Raf1(Y341) (Figure 17b) and MEK1 / 2 (Figure 17c) were statistically evaluated. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to Ptpn11D61G / +MEF treated with a vehicle (n=3 for each condition; one-way ANOVA test). [Figure 17-2]This is a panel of images showing the effect of dasatinib on NS signaling in vitro. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with dasatinib for 18 hours. Phosphorylation levels of JNK (Figure 17d) and Akt (S473) (Figure 17e) were statistically evaluated. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to vehicle-treated Ptpn11D61G / + MEFs (n=3 for each condition; one-way ANOVA test). Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with STI-571 (Figure 17f) or a Shp2 inhibitor (Figure 17g) for 18 hours. Tyrosyl phosphorylation of PZR was measured using a phospho-specific PZR antibody. [Figure 18-1] This is a panel of images showing the effect of dasatinib on NS signaling in vivo. Three-week-old WT mice and Ptpn11D61G / + mice were intraperitoneally injected with either vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). Figure 18a shows images of cardiac tissue immunoblotted with p-Raf1(Y341) antibody, Raf1 antibody, p-MEK1 / 2 antibody, MEK1 / 2 antibody, p-JNK antibody, JNK antibody, p-p38 antibody, p38 antibody, p-Akt(S473) antibody, and Akt antibody. Cardiac tissue was isolated after 18 hours, and tissue lysates were immunoblotted. [Figure 18-2]This is a panel of images showing the effects of dasatinib on NS signaling in vivo. Three-week-old wild-type mice and Ptpn11D61G / + mice were intraperitoneally injected with either a vehicle or dasatinib (0.1, 0.5, or 1.0 mg / kg). Figure 18b is a graph showing the phosphorylation level of Raf1(Y341). Figure 18c is a graph showing the phosphorylation level of MEK1 / 2. Figure 18d is a graph showing the phosphorylation level of JNK. Figure 18e is a graph showing the phosphorylation level of p38. Figure 18f is a graph showing the phosphorylation level of Akt(S473). All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001 indicate significance compared to Ptpn11D61G / + mice treated with the vehicle (n=3 for each condition; one-way ANOVA test). [Figure 19] This panel of images shows that postnatal dasatinib administration did not improve overall growth in Ptpn11D61G / + mice. Figure 19a is a graph showing the growth curves of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally. The difference between the treatment groups was significant from 3 weeks to 8 weeks of age (p<0.001, two-way ANOVA test). Figure 19b is a graph showing body weight measured at P42. Figure 19c is a graph showing body length measured at P42. Figure 19d is a graph showing body weight measured at P56. Figure 19c is a graph showing body length measured at P42. Figure 19e is a graph showing body length measured at P56. All data are mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 20-1] This panel of images shows that the features of facial dysplasia did not change in Ptpn11D61G / + mice treated with dasatinib. Figure 20a is a panel of representative images of the skull and mandible at P56 of WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally. [Figure 20-2]This is a panel of images showing that the features of facial dysplasia did not change in Ptpn11D61G / + mice treated with dasatinib. Figure 20b is a graph showing measurements obtained from skull length at P42 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20c is a graph showing measurements obtained from skull width at P42 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20d is a graph showing measurements obtained from the ratio of skull length to skull width at P42 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20e is a graph showing measurements of intercanthal distance (ICD) at time P42 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20f is a graph showing measurements of mandibular length at time P42 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20g is a graph showing measurements of skull length at time P56 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20h is a graph showing measurements of skull width at time P56 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20i is a graph showing the measured values ​​of the ratio of skull length to skull width at P56 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20j is a graph showing the measured values ​​of interocular distance (ICD) at P56 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 20k is a graph showing the measured values ​​of mandibular length at P56 in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. All data are shown as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group; two-way ANOVA test). [Figure 21-1]This panel of images shows that the splenomegaly phenotype in Ptpn11D61G / + mice was not eliminated by postnatal dasatinib treatment. Figure 21a is a panel of representative H&E stained histological images of the spleen at time P56 from 8-week-old WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally (scale bar = 200 μm). [Figure 21-2] This is a panel of images showing that the splenomegaly phenotype in Ptpn11D61G / + mice was not eliminated by postnatal dasatinib treatment. Figure 21b is a graph showing spleen weight at P42, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 21c is a graph showing the ratio of spleen weight to body weight at P42, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 21d is a graph showing spleen weight at P56, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 21e is a graph showing the ratio of spleen weight to body weight at P56, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. All data represent the mean ± SEM. *, p<0.05; ***, p<0.001. (n=7-10 for each group; two-way ANOVA test). [Figure 22]This is a panel of images demonstrating that dasatinib does not induce liver damage. Figure 22a is a panel of representative H&E stained liver images at time P56 from 8-week-old WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib postnatally (scale bar = 200 μm). Figure 22b is a graph showing the enzyme activity of alanine aminotransferase (ALT) in serum at time P42, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. Figure 22c is a graph showing the enzyme activity of alanine aminotransferase (ALT) in serum at time P56, measured in WT mice and Ptpn11D61G / + mice treated with vehicle or dasatinib. All data are mean ± SEM. *, p<0.05; ***, p<0.001. (n=7-10 for each group; two-way ANOVA test). [Figure 23] This image shows the molecular interaction between the SH3 domain of Src and the PTP domain of Shp2. The GST-tagged SH3 domain of purified c-Src was incubated overnight at 4°C with the His-tagged PTP domain of purified Shp2. The proteins were immobilized using GST Sepharose beads and separated by SDS-PAGE. The His-PTP domain in the GST complex was detected by immunoblotting using an anti-His antibody. [Figure 24] This panel of images demonstrates that NS-Shp2-induced PZR hyperphosphorylation is independent of both Shp2 phosphatase activity and c-Abl kinase activity. Mouse embryonic fibroblasts (MEFs) derived from Ptpn11D61G / + mice were incubated with STI-571 (Figure 24a) or a Shp2 inhibitor (Figure 24b) for 18 hours. PZR tyrosyl phosphorylation was measured using a phospho-specific PZR antibody. [Figure 25]This panel shows graphs illustrating the improvement of molecular markers for cardiomyopathy and fibrosis in NSML (Y279C / +) mice after dasatinib treatment. Total cardiac RNA was isolated from WT mice and Ptpn11Y279C / + mice (P42) treated with vehicle or dasatinib (0.1 mg / kg / day) postnatally. Fibrosis marker genes Col1a2 (Figure 25a) and Col3a1 (Figure 25b), cardiomyopathy marker ANP (Figure 25c), and fetal cardiac genes Myh6 (aMHC) (Figure 25d), Myh7 (bMHC) (Figure 25e), as well as the Myh6 / Myh7 ratio (Figure 25f), were measured by quantitative RT-PCR (n=6 for each group). All data represent mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (Two-way ANOVA test). [Modes for carrying out the invention]

[0023] Detailed description of the invention definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention relates. Any methods and materials similar to or equivalent to those described herein may be used in practice to verify the invention, but preferred materials and methods are described herein. The following technical terms are used in describing and claiming the invention:

[0024] Furthermore, it should be understood that the technical terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0025] As used herein, the articles “a” and “an” are used to refer to one or more (i.e., at least one) grammatical objects. For example, “an element” means one or more elements.

[0026] Where used herein, the term “about” when referring to measurable values ​​such as quantities and durations of time is intended to include variations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, such variations being appropriate for carrying out the disclosed method. Unless otherwise clearly indicated in the context, all numerical values ​​provided herein are qualified with the term “about.”

[0027] The phrase "abnormal protein tyrosine phosphorylation" refers to hyperphosphorylation or hypophosphorylation and / or abnormal protein kinase activity of one or more target proteins. In one embodiment, abnormal protein tyrosine phosphorylation is compared to a control.

[0028] The term “cardiovascular disease or condition” means any disease or condition affecting the cardiovascular system, including, but is not limited to, nerve conduction disorders, thrombosis, atherosclerosis, angina pectoris, hypertension, arteriosclerosis, myocardial infarction, congestive heart failure, cardiomyopathy, hypertension, arterial and venous stenosis, valvular heart disease, myocarditis, and arrhythmias. Cardiovascular disease conditions also include, but are not limited to, any clinical manifestations of disease conditions relating to the heart and the central or peripheral arterial and venous vascular system. For example, such clinical manifestations include, but are not limited to, pain, weakness, hypertension, elevated plasma cholesterol, elevated plasma fatty acids, tachycardia, bradycardia, abnormal electrocardiogram, external or internal bleeding, headache, dizziness, nausea, and vomiting.

[0029] The term “cardiac function” refers to the activity of the heart, or the interaction of the heart’s cells or tissues to perform that activity. Examples of cardiac function include, but are not limited to, the organization of myofibrils, the contractility of cardiomyocytes, and the proper delivery of blood and nutrients to the tissues where they are needed. Abnormal cardiac function (improper delivery of blood and nutrients to tissues) can lead to, but are not limited to, problems such as changes in blood pressure, thrombosis, changes in electrocardiogram, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, hypertrophy, stunted heart, heart failure (ventricular failure (left or right)), congestive heart failure, and cardiac arrest. Improvement of cardiac function may include, but are not limited to, the improvement, elimination, or prevention of at least one abnormal cardiac function, such as, myofibril tissue breakdown, abnormal cardiomyocyte contractility, cardiac fibrosis, abnormal blood pressure, excessive changes in blood pressure, thrombosis, changes in electrocardiogram, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure.

[0030] In this disclosure, “comprises,” “comprising,” “containing,” and “having,” etc., may have the meanings given to them in U.S. patent law, and may mean “includes,” “including,” etc. Similarly, “consisting essentially of” or “consists essentially” may have the meanings given to them in U.S. patent law, and this term is non-restrictive, allowing for more than that to exist, as long as the basic or novel features of that are not altered by the existence of more than that, except for aspects of the prior art.

[0031] "Congenital heart disease" refers to a category of cardiovascular disorders, including abnormalities in the cardiovascular structure that occur before birth.

[0032] "Effective dose" refers to the amount required to alleviate or improve at least one symptom of a disease compared to an untreated patient. The effective dose of an active compound used in the therapeutic treatment of a disease varies depending on the mode of administration, the age, weight, and overall health status of the subject.

[0033] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0034] A “fragment” means a portion of a polynucleotide or nucleic acid molecule. Preferably, this portion contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total length of the reference nucleic acid. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, or 2500 nucleotides (and any intermediate integer value). When applied to nucleic acid molecules, a fragment means a partial sequence of a large nucleic acid. A “fragment” of a nucleic acid molecule may be at least about 15 nucleotides in length; for example, at least about 50 to about 100 nucleotides; at least about 100 to about 500 nucleotides; at least about 500 to about 1000 nucleotides; at least about 1000 to about 1500 nucleotides; or about 1500 to about 2500 nucleotides; or about 2500 nucleotides (and any intermediate integer value).

[0035] "Low dose" refers to a therapeutically effective dose that is less than the dose typically prescribed for indications other than heart disease, congenital heart disease, heart failure, or similar conditions. In one embodiment, a low dose is less than a chemotherapy dose. In another embodiment, a low dose is less than a chemotherapy dose of a tyrosine kinase inhibitor, ranging from about 1 / 200th of that dose. In another embodiment, a low dose of a tyrosine kinase inhibitor improves at least one cardiac function. Dasatinib has been shown to be effective in preventing tumor development in mice at a dose of approximately 20 mg / kg (Kantarjian, H. et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 362, 2260-2270 (2010)). The therapeutic effect of dasatinib in humans has been reported to be approximately 2 mg / kg, which is equivalent to approximately 24 mg / kg in mice (Yu, EY et al. Phase II study of dasatinib in patients with metastatic castration-resistant prostate cancer. Clinical cancer research: an official journal of the American Association for Cancer Research 15, 7421-7428 (2009) and Apperley, JF et al. Dasatinib in the treatment of chronic myeloid leukemia in accelerated phase after imatinib failure: the START a trial. J Clin Oncol 27, 3472-3479, doi:10.1200 / JCO.2007.14.3339 (2009)). A low dose of dasatinib, 0.1 mg / kg (about 1 / 200th of the therapeutic dose), was sufficient to treat CHD-associated cardiac disease.

[0036] The terms “isolated,” “purified,” or “biologically pure” refer to substances that contain, to varying degrees, components that would normally accompany the substance in its natural state. “Isolate” means the degree of separation from the original source or environment. “Purify” means a higher degree of separation than isolation. A “purified” or “biologically pure” protein is one from which other substances have been sufficiently removed so that any impurities do not specifically affect the biological properties of the protein or cause other harmful consequences. That is, a nucleic acid or peptide is purified if it substantially contains no cellular material, viral material, or culture medium (if produced by recombinant DNA technology), as well as chemical precursors and other chemicals (if chemically synthesized). Purity and homogeneity are typically measured using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may indicate that a nucleic acid or protein produces essentially a single band on an electrophoretic gel. For proteins that can be modified, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

[0037] "Pharmacologically acceptable" refers to properties and / or substances that are acceptable to patients from a pharmacological / toxicological standpoint, and acceptable to pharmaceutical pharmacists from a physical / chemical standpoint regarding composition, formulation, stability, patient tolerance, and bioavailability. "Pharmacologically acceptable carrier" means a medium that does not interfere with the efficacy of the biological activity of the active ingredient and is not toxic to the host to which it is administered.

[0038] As used herein, the terms “pharmaceutical composition” or “pharmaceutically acceptable composition” mean a mixture of at least one compound or molecule useful within the scope of the present invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds or molecules to a patient. There are numerous techniques in the art for administering compounds or molecules, including, but not limited to, intravenous administration, oral administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, and topical administration.

[0039] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulant, that is involved in transporting a compound or molecule useful within the scope of the invention into or within the patient’s body so that it can perform its intended function. Typically, such a construct is transported or delivered from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable” in the sense that it can coexist with other components of the formulation, including compounds useful within the scope of the invention, and does not harm the patient. Some examples of substances that can function as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository waxes; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyhydric alcohols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, coexisting substances used in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antimicrobial and antifungal agents, as well as absorption retarders, etc., that are coexistent with the activity of compounds useful within the scope of the present invention and are physiologically acceptable to patients. Auxiliary active compounds may also be mixed into the composition. The “pharmaceutically acceptable carrier” may further include pharmaceutically acceptable salts of compounds or molecules useful within the scope of the present invention.Other additional components that may be included in the pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0040] "Protein Zero-like protein 1" or "MPZL1," also known as "Protein Zero-related" or "PZR," refers to a protein that is a cell surface protein of the immunoglobulin superfamily. PZR contains two immunoreceptor suppressive tyrosine motifs (ITIMs) responsible for binding to Shp2. When phosphorylated, PZR can specifically bind to Shp2, thereby activating Shp2's tyrosine phosphatase activity. Once activated, Shp2's tyrosine phosphatase activity works to dephosphorylate downstream substrates that transmit cellular signals. Shp2 can also transmit signals by acting as a scaffold or adapter protein that recruits other molecules / activities to specific complexes. Shp2 can regulate signal transduction in both catalyst-dependent and catalyst-independent ways.

[0041] A type of PZR isoform called PZR1b lacks ITIM and has a dominant-negative effect on full-length PZR and its Shp2 recruitment. Exemplary PZR sequences include human PZR or fragments found in GenBank accession numbers NM_001146191 and NP_001139663, as well as mouse PZR sequences or fragments found in NM_001001880 or NP_001001880. Much of the publicly known information about PZR relates to its role in cell signaling and cell migration via adhesion. However, it remains unclear whether PZR is involved in pathophysiological cell signaling, and consequently, the validity of PZR as a target for any human disease is still not understood.

[0042] "RAS opathy" refers to a group of genetic syndromes caused by germline mutations in genes encoding components or regulators of the Ras / mitogen-activated protein kinase (MAPK) pathway. These syndromes include neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with lentigo polycarcinoma, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiac-facial-skin syndrome, and Regius syndrome. The Ras / MAPK pathway plays a crucial role in regulating the cell cycle, as well as cell proliferation, differentiation, and aging, all of which are essential for normal development. Due to common potential Ras / MAPK pathway dysregulation, RAS opathy exhibits numerous overlapping phenotypic features. In some cases, these overlapping phenotypes may be present or caused by mechanisms operating independently of MAPK itself. The PZR / Shp2 complex, described herein, is located upstream of Ras.

[0043] Noonan syndrome (NS) is an autosomal dominant disorder that occurs in the United States at an incidence rate of approximately 1 in 1,000 to 2,500 live births. The most common cardiac defects in NS are pulmonary valve stenosis, atrial septal defect, and hypertrophic cardiomyopathy, each ranging in severity from mild to life-threatening. Noonan syndrome with lentigo (NSML) is a rare autosomal dominant disorder that has a "Noonan-like" appearance and a phenotype similar to NS, including lentigo, electrical conduction abnormalities, eccentric eyes, pulmonary valve stenosis, genital abnormalities, developmental delay, and hearing loss. Mutations associated with NS lead to increased phosphatase activity. Mutations associated with NSML lead to decreased phosphatase activity.

[0044] "Reference" means a standard substance or control. A "reference" is a designated standard substance or control used as a criterion for comparison.

[0045] As used herein, “sample” or “biological sample” means any sample that may contain cells of interest (e.g., cancer cells or tumor cells) of which a screening method or treatment is desired. A sample may be a biological sample, such as a biological fluid or biological tissue. In one embodiment, a biological sample is a tissue sample containing pulmonary artery endothelial cells. Such a sample may contain a variety of cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cells. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, or amniotic fluid.

[0046] The "Src family tyrosine kinases," or "SFKs," are a family of enzymes that catalyze the addition of phosphate groups to tyrosine residues in protein substrates. c-Src is a member of the SFK family.

[0047] A "Src family tyrosine kinase inhibitor" refers to a molecule that reduces or inhibits the phosphorylation of tyrosine residues on Src family protein substrates. Src family tyrosine kinase inhibitors can reduce or inhibit phosphorylation by interfering with tyrosyl phosphorylation, possibly by binding to tyrosine kinases or tyrosine residues with higher binding efficiency than tyrosine kinases or phosphate groups, and / or by preventing the effective binding of phosphate groups to tyrosine residues. Src family tyrosine kinase inhibitors include, but are not limited to, small molecule Src family tyrosine kinase inhibitors, Src family tyrosine kinase antagonists, neutralizing antibodies, and inhibitory peptides and / or oligonucleotides. Examples of small molecule Src family tyrosine kinase inhibitors include A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, and XL. This includes, but is not limited to, 228, artenusin, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and their analogues.

[0048] "Src homology 2 (SH2) domain-containing (SH2) protein tyrosine phosphatase-2" or "Shp2" is a member of the tyrosine-specific family of protein tyrosine phosphatases (PTPs). Shp2 is a tyrosine phosphatase that catalyzes the tyrosine dephosphorylation of proteins. Mutations in the human gene PTPN11 have been found to cause approximately half of Noonan syndrome cases and approximately one-tenth of NSML cases.

[0049] The terms "subject" or "patient," as used herein, may be human or non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and mouse mammals. Preferably, the subject is human.

[0050] The term "transmembrane glycoprotein" refers to membrane proteins that span both the inside and outside of the cell membrane. In one embodiment, transmembrane glycoproteins include immunoglobulin superfamily cell surface proteins such as PZRs.

[0051] As used herein, terms such as “treat,” “treating,” and “treatment” mean to alleviate or improve a disorder and / or its associated symptoms. It is understood that treating a disorder or condition does not necessarily, but does not exclude, that the disorder, condition, or its associated symptoms be completely cured or eliminated.

[0052] A "tyrosine kinase inhibitor" refers to a molecule that reduces or inhibits the phosphorylation of tyrosine residues on a protein substrate. Tyrosine kinase inhibitors can reduce or inhibit phosphorylation by interfering with tyrosyl phosphorylation, possibly by binding to tyrosine kinases or tyrosine residues with higher binding efficiency than tyrosine kinases or phosphate groups, and / or by preventing the effective binding of phosphate groups to tyrosine residues. Tyrosine kinase inhibitors include, but are not limited to, small molecule tyrosine kinase inhibitors, tyrosine kinase antagonists, neutralizing antibodies, and inhibitory peptides and / or oligonucleotides. Examples of small molecule tyrosine kinase inhibitors include, but are not limited to, afatinib, axitinib, bosutinib, cabozantinib, cejilanib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.

[0053] The ranges provided herein are understood to be abbreviated representations of all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange derived from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0054] A description of an aspect of a variable or aspect in this specification includes that aspect as any single aspect, or as a combination of that aspect with any other aspect or a part thereof.

[0055] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0056] composition Abnormal protein tyrosine phosphorylation, such as that of Src family tyrosine kinases and their substrates, has been found to be altered in subjects with cardiovascular disease. Abnormal protein tyrosine phosphorylation has also been found to be altered in subjects with certain diseases, such as RAS opathy. Inhibiting tyrosine kinase activity treats heart disease and improves at least one cardiac function. Inhibition also improves cardiovascular function in subjects with congenital heart defects associated with RAS opathy. The present invention includes compositions that inhibit tyrosine kinases, such as Src family tyrosine kinases, to improve at least one cardiac function, thereby preventing or reducing tyrosine phosphorylation. In one aspect, the present invention includes compositions comprising low doses of tyrosine kinase inhibitors that reduce tyrosine phosphorylation and improve at least one cardiac function in subjects that require it.

[0057] In one embodiment, low doses of tyrosine kinase inhibitors reduce abnormal tyrosine phosphorylation of transmembrane glycoproteins such as Src family tyrosine kinases and protein zero-related (PZRs).

[0058] In one embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 25 to 1 / 500 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 25 to 1 / 400 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 25 to 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 35 to 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 50 to 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of a low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 100 to 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 150 to 1 / 250 of the chemotherapy dose of tyrosine kinase inhibitor. In yet another embodiment, the low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 175 to 1 / 250 of the chemotherapy dose of tyrosine kinase inhibitor.Low doses of tyrosine kinase inhibitors are approximately 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 105, 1 / 110, 1 / 115, 1 / 120, 1 / 125, 1 / 130, 1 / 135, 1 / 140, 1 / 145, 1 / 150, 1 / 155, and 1 / 160 of the chemotherapy dose of tyrosine kinase inhibitors. The doses may be 1, 1 / 165, 1 / 170, 1 / 175, 1 / 180, 1 / 185, 1 / 190, 1 / 195, 1 / 200, 1 / 205, 1 / 210, 1 / 215, 1 / 220, 1 / 225, 1 / 230, 1 / 235, 1 / 240, 1 / 245, 1 / 250, 1 / 255, 1 / 260, 1 / 265, 1 / 270, 1 / 275, 1 / 280, 1 / 285, 1 / 290, 1 / 295, 1 / 300, and any fold change between them. In some embodiments, the chemotherapy dose of a tyrosine kinase inhibitor ranges from about 75 to about 170 mg / day or about 1.1 to about 2.4 mg for a 70 kg adult.

[0059] In another embodiment, the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cejilanib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In another embodiment, the composition comprises several of the tyrosine kinase inhibitors disclosed herein.

[0060] In yet another embodiment, low doses of tyrosine kinase inhibitors include Src family tyrosine kinase inhibitors, for example, but not limited to, A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, an inhibitor selected from the group consisting of artenusine, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and their analogs. In another embodiment, the composition comprises at least one Src family tyrosine kinase inhibitor.

[0061] In another embodiment, low doses of tyrosine kinase inhibitors improve at least one cardiac function. Cardiac function may include, but is not limited to, myofibrils, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. Abnormal cardiac function can lead to problems such as, but is not limited to, changes in blood pressure, thrombosis, electrocardiogram changes, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, heart failure (ventricular failure), congestive heart failure, and cardiac arrest. Improvement of cardiac function may include, but is not limited to, improvement, elimination, or prevention of at least one abnormal cardiac function such as, myofibrils, abnormal cardiomyocyte contractility, unregulated SERCA2A expression, cardiac fibrosis, abnormal blood pressure, excessive changes in blood pressure, thrombosis, electrocardiogram changes, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure.

[0062] In yet another embodiment, low doses of tyrosine kinase inhibitors produce an anti-fibrotic effect. Increased levels of fibrous components in the myocardium are associated with the progression of heart failure. Inhibiting tyrosine kinases at low doses reduces the accumulation of fibrous components in the myocardium.

[0063] Compositions that reduce abnormal protein tyrosine phosphorylation are also included in the present invention. Certain diseases, such as cardiovascular diseases or conditions like congenital heart disease, are characterized by abnormal protein tyrosine phosphorylation. Therefore, treatments that inhibit or reduce tyrosine phosphorylation of one or more transmembrane glycoproteins, such as protein zero-related or PZR, are included in the present invention. In another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with cardiovascular diseases or conditions. In yet another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with congenital heart disease. In yet another aspect, the present invention includes compositions that can reduce abnormal protein tyrosine phosphorylation associated with cardiovascular diseases or conditions related to RAS opathy.

[0064] method The present invention also includes methods for preventing or treating cardiovascular disease or conditions in subjects where it is needed. As described herein, inhibiting abnormal tyrosine phosphorylation prevents and / or treats cardiovascular disease or conditions. A composition comprising a low dose of a tyrosine kinase inhibitor is administered to subjects where it is needed, such as pediatric subjects, to reduce abnormal levels of tyrosine phosphorylation in order to prevent or treat cardiovascular disease or conditions.

[0065] In one aspect, the present invention provides a method for treating a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in a subject, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces the abnormal level of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0066] In another aspect, the present invention includes a method for treating a congenital heart disease, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0067] In yet another aspect, the present invention provides a method for treating a cardiovascular disease or condition associated with RAS opathy having abnormal protein tyrosine phosphorylation, comprising the step of administering a low dose of a tyrosine kinase inhibitor to a subject in need thereof, wherein the tyrosine kinase inhibitor reduces abnormal levels of protein zero-related (PZR) tyrosine phosphorylation and improves at least one cardiac function of the subject.

[0068] In one embodiment, the cardiovascular disease or condition in the method described herein is a congenital heart disease or cardiovascular disease or condition associated with RAS opathy, for example, selected from the group consisting of, but not limited to, neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with lentigo polycarcinoma (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiac-facial-cutaneous syndrome, and Regius syndrome.

[0069] In another embodiment, the method includes the step of administering a tyrosine kinase inhibitor to a subject who is a pediatric patient. The pediatric subject may be under 18 years of age. The pediatric subject may be under 12 years of age. The pediatric subject may be under 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 year of age. In another embodiment, the subject is a pediatric patient under 12 years of age. In an alternative embodiment, the method includes the step of administering a tyrosine kinase inhibitor to a subject who is older than 18 years of age.

[0070] In one embodiment, the method comprises administering a low dose of a tyrosine kinase inhibitor that reduces abnormal tyrosine phosphorylation of transmembrane glycoproteins such as Src family tyrosine kinases and protein zero-related (PZRs). In one embodiment, the abnormal levels of tyrosine phosphorylation include abnormal levels of tyrosine-phosphorylated protein zero-related (PZRs).

[0071] In another embodiment, the step of administering a low dose of a tyrosine kinase inhibitor improves cardiac function, including but not limited to myofibrils organizing, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis. In yet another embodiment, improvement of cardiac function may include, but not limited to, improvement, elimination, or prevention of at least one abnormal cardiac function, such as myofibrils breaking down, abnormal cardiomyocyte contractility, abnormally regulated SERCA2A expression, cardiac fibrosis, abnormal blood pressure, excessive blood pressure changes, thrombosis, electrocardiogram changes, arrhythmias, myocarditis, pericarditis, myocardial infarction, cardiomyopathy, and congestive heart failure. In yet another embodiment, the step of administering a low dose of a tyrosine kinase inhibitor brings about an antifibrotic effect.

[0072] In one embodiment, the dose of the low-dose tyrosine kinase inhibitor used in the method described herein is in the range of about 1 / 25 to about 1 / 500 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 25 to about 1 / 400 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 25 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 35 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 50 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the amount of the low-dose tyrosine kinase inhibitor is in the range of about 1 / 100 to about 1 / 300 of the chemotherapy dose of the tyrosine kinase inhibitor. In another embodiment, the low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 150 to 1 / 250 of the chemotherapy dose of tyrosine kinase inhibitor. In yet another embodiment, the low dose of tyrosine kinase inhibitor is in the range of approximately 1 / 175 to 1 / 250 of the chemotherapy dose of tyrosine kinase inhibitor.Low doses of tyrosine kinase inhibitors are approximately 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 100, 1 / 105, 1 / 110, 1 / 115, 1 / 120, 1 / 125, 1 / 130, 1 / 135, 1 / 140, 1 / 145, 1 / 150, 1 / 155, and 1 / 160 of the chemotherapy dose of tyrosine kinase inhibitors. The doses may be 1 / 165, 1 / 170, 1 / 175, 1 / 180, 1 / 185, 1 / 190, 1 / 195, 1 / 200, 1 / 205, 1 / 210, 1 / 215, 1 / 220, 1 / 225, 1 / 230, 1 / 235, 1 / 240, 1 / 245, 1 / 250, 1 / 255, 1 / 260, 1 / 265, 1 / 270, 1 / 275, 1 / 280, 1 / 285, 1 / 290, 1 / 295, 1 / 300, and any multiplier changes between them. In some embodiments, the chemotherapy dose of a tyrosine kinase inhibitor ranges from about 75 to about 170 mg / day or about 1.1 to about 2.4 mg for a 70 kg adult.

[0073] In yet another embodiment, the low-dose tyrosine kinase inhibitor is selected from the group consisting of afatinib, axitinib, bosutinib, cabozantinib, cejilanib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, restaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In another embodiment, tyrosine kinase inhibitors include Src family tyrosine kinase inhibitors, for example, but not limited to, A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL These include artenusine, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatannol, salakatinib, SrcI1, and their analogues. In another embodiment, the composition comprises several of the tyrosine kinase inhibitors disclosed herein. In yet another embodiment, the composition comprises at least one Src family tyrosine kinase inhibitor. In such embodiments, the tyrosine kinase inhibitors may be administered together or sequentially, by different routes of administration, or in the same or different pharmaceutical compositions.

[0074] The methods and compositions disclosed herein are also useful as treatments for cardiovascular diseases or conditions characterized by abnormal protein tyrosine phosphorylation in subjects with cardiovascular diseases or conditions.

[0075] Pharmaceutical composition The present invention also encompasses the use of the pharmaceutical compositions of the present invention for practicing the methods of the present invention. In one aspect, the present invention includes a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable carrier. In another aspect, the composition described herein is used in the manufacture of a medicament for the treatment of a cardiovascular disease or condition in a subject requiring it. In yet another aspect, the present invention includes a pharmaceutical composition comprising the composition described herein in combination with another therapeutic substance used in the treatment of a cardiovascular disease or condition. Such a pharmaceutical composition may be provided in a form suitable for administration to a subject and may comprise one or more pharmaceutically acceptable carriers, one or more additional components, or several combinations thereof. The composition described herein may comprise the compound intended in the present invention in combination with a physiologically acceptable salt, e.g., a physiologically acceptable cation or anion, as is well known in the art.

[0076] Pharmaceutical compositions useful in the methods of the present invention can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, oral, ocular, subarachnoid, intravenous, or other routes of administration. Other intended formulations include projected nanoparticles, liposomal formulations, encapsulated red blood cells containing active ingredients, and immunological formulations. The routes of administration are expected to be readily apparent to those skilled in the art and will vary depending on any number of factors, including the type and severity of the disease being treated, as well as the type and age of the animal or human patient being treated.

[0077] Formulations of pharmaceutical compositions described herein may be prepared by any method known or to be developed in the art of pharmacology. Typically, such preparatory methods include mixing the active ingredient with a carrier or one or more other auxiliary components, and then, if necessary or desirable, forming or packaging the product into desired single-dose or multi-dose units.

[0078] In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of at least one compound of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphoric acid and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0079] The practice of this invention will, unless otherwise specified, utilize conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which will be well within the comprehension of those skilled in the art. Such techniques are well described in literature such as “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); and “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention and may therefore be considered when conceiving and practicing the present invention. Techniques that are particularly useful for specific embodiments will be discussed in the following sections. [Examples]

[0080] The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should not be construed as being limited to the following examples, but rather as encompassing any and all modifications that become apparent as a result of the teachings provided herein.

[0081] Without further explanation, those skilled in the art will likely be able to prepare and utilize the compounds of the present invention and practice the methods of the claims using the foregoing description and the following exemplary examples. Accordingly, the following examples illustrate specific aspects of the present invention and should not be construed as limiting.

[0082] Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, manifesting as congenital heart defects, short stature, and facial dysmorphism. The complexity of Shp2 signaling is illustrated by the observation that patients with Noonan syndrome with lentigo polycarpa (NSML) exhibit symptoms similar to NS despite having PTPN11 inactivating mutations. Protein Zero-Related (PZR), a transmembrane glycoprotein that promotes cell migration in conjunction with the extracellular matrix, was identified as the major hyper-tyrosyl-phosphorylated protein in mouse models of NS and NSML. PZR hypertyrosyl phosphorylation was promoted in a phosphatase-independent manner by enhancing Src recruitment to Shp2 in NS and NSML. Therefore, PZR was identified as a target for NS and NSML. Enhanced recruitment of Shp2 to the membrane via PZR acted as a common mechanism to directly manage some of the overlapping pathophysiological features of these PTPN11 mutations.

[0083] The materials and methods used in conducting the experiments disclosed herein are described below.

[0084] Antibodies, chemicals, cell lines, and expression reagents. Rabbit monoclonal phospho-PZR (Y241) antibody and rabbit monoclonal phospho-PZR (Y263) antibody were produced in collaboration with Cell Signaling. Mouse monoclonal Src antibody, rabbit polyclonal Src antibody, rabbit polyclonal phospho-ERK1 / 2 (T202 Y204), mouse monoclonal ERK1 / 2 antibody, rabbit polyclonal phospho-Akt (S473) antibody, and mouse monoclonal Akt antibody were purchased from Cell Signaling. Rabbit polyclonal Shp2 antibody and rabbit polyclonal ERK1 / 2 antibody were purchased from Santa Cruz Biotechnology. Mouse monoclonal Shp2 antibody was purchased from BD Bioscience. Mouse anti-phosphotyrosine antibody 4G10 (05-321) was obtained from Merck Millipore, rabbit anti-GFP (TP401) from Acris, and mouse anti-HA.11 clone 16B12 from Covance. Rabbit polyclonal PZR (105-6) was generously provided by ZJ Zhao. Src family kinase inhibitors PP2 and SU6656 were purchased from Calbiochem. HEK-293 cells, NIH 3T3 cells, SYF (Src - / - Yes - / - Fyn - / - Mouse embryonic fibroblast (MEF) cells, and Src ++(SYF) cells overexpressing Src were purchased from ATCC and grown in growth medium (Dulbecco's modified Eagle medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum) at 37°C in a 5% CO2 incubator. Replication-deficient adenovirus (Ad) constructs containing wild-type Shp2 (Ad-Shp2 WT), replication-deficient adenovirus (Ad) constructs containing the E76A gain-of-function Shp2 mutant (Ad-Shp2E76A), and replication-deficient adenovirus (Ad) constructs containing green fluorescent protein (GFP) (Ad-GFP) were prepared as previously described (Eminaga, S., et al., J. Biol. Chem., 283:15328-15338). NIH 3T3 cells and SYF cells were infected with adenovirus at doses that resulted in a multiple of infection (MOI) of 50. pJ3Ω vectors containing SrcWT and pJ3Ω vectors containing the K295R / Y527F dominant-negative Src variant (SrcK295R / Y527F) have been previously described (Fornaro, M., et al., J. Cell Biol., 175:87-97). pIRES-GFP plasmids encoding Shp2 WT, gain-of-function / Noonan syndrome Shp2 variants (Shp2E76A and Shp2N308D), and Noonan syndrome with multiple lentigo Shp2 variants (Shp2Y279C and Shp2T468M) have been previously described (Kontaridis, MI, et al., J. Biol. Chem., 281:6785-6792). The zebrafish Shp2 mutant has been previously cloned (Jopling, C., et al., PLoS Genet., 3:e225). Zebrafish PZRs (zPZRs) were cloned from zebrafish embryo cDNA (from the tail bud stage to 48 hours post-fertilization (hpf)) by nested PCR. The Y236F, Y258F, and Y236F Y258F mutants of zPZR ITIM were generated using site-directed mutagenesis. The RPTPa signaling sequence and hemagglutinin (HA) tag were incorporated into the N-terminus of the zPZR.DNA transfection into HEK-293 and SYF cells was performed using lipofectamine 2000 according to the manufacturer's protocol.

[0085] MS analysis. The PhosphoScan method was performed as previously described (Rikova, K., et al., Cell, 131:1190-1203). Wild-type mouse hearts and Shp2 mutant (Noonan syndrome) mouse hearts were homogenized, sonicated, and centrifuged to remove cell fragments. The total protein content of each tissue was standardized using ProteinPlus Coomasie reagent (Pierce), proteins were reduced, alkylated, and digested overnight with trypsin (Worthington). Sep-Pak Classic C 18 The obtained peptides were separated from non-peptide substances by solid-phase extraction using a cartridge (Waters). The lyophilized peptides were redissolved, and the phosphopeptides were concentrated by immunoaffinity purification using pY-100 phosphotyrosine antibody (9411; Cell Signaling Signaling Technology). The peptides were eluted with 0.15% trifluoroacetic acid (TFA), and immediately before liquid chromatography-mass spectrometry (LC-MS) analysis, C 18 The samples were concentrated using a spin tip. For each sample, two injections were performed to create analytical replicas, increasing the number of tandem MS (MS / MS) identifications from each sample. Magic C 18Peptides were added directly onto a 10 cm × 75 μm PicoFrit capillary column packed with AQ reversed-phase resin. The column was developed using a 45-minute linear gradient of acetonitrile in 0.125% formic acid, delivered at 280 nl / min. Tandem mass spectra were collected using an LTQ-Orbitrap XL mass spectrometer running XCalibur with the Top 10 method, dynamic exclusion repeat count of 1, and repeat period of 30 seconds. MS spectra were collected in the Orbitrap component of the mass spectrometer, and MS / MS spectra were collected in the LTQ component. MS / MS spectra were processed using the SEQUEST and Core platforms (Gygi Lab, Harvard University). Searches were performed against the mouse NCBI database, including a reverse decoy database for all searches to estimate the false positive rate. Peptide assignments were obtained using the Core linear discriminant analysis module with a precision cutoff of 0.98. Cysteine ​​carboxamide methylation was designated as a static modification, while methionine oxidation and phosphorylation of serine, threonine, and tyrosine were permitted. Results were further refined using a mass-accuracy (5 ppm) filter and the presence of phosphotyrosine in the peptides. Unlabeled quantification was performed using Progenesis v4.1 (Nonlinear Dynamics). To ensure accuracy, peptide abundance data were manually reviewed in Progenesis for all peptides that changed by at least 2.0 times.

[0086] Animal handling - Ptpn11 D61G / +The mice were provided by Dr. Benjamin Neel (University of Toronto, Toronto) and genotyped as previously described (Araki T, Mohi MG, Ismat FA, Bronson RT, Williams IR, Kutok JL, Yang W, Pao LI, Gilliland DG, Epstein JA, Neel BG. 2004. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation. Nat Med 10:849-857). Briefly, Ptpn11 D61G / + Male mice were crossed with WT C75BL / 6 x SV129 female mice, and their offspring were genotyped by PCR and digestion with AgeI for the D61G allele.

[0087] Dasatinib treatment - N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate (dasatinib, BMS-354825) was purchased from Biovision. Dasatinib was dissolved in DMSO at a concentration of 10 mg / ml and then resuspended in vehicle (1× Dulbecco PBS) at a concentration of 200 μg / ml. Starting from day 10 after birth until week 6 of age (P42), WT male mice and Ptpn11 D61G / + Male mice were injected daily with dasatinib (0.1 mg / kg, intraperitoneally). Then, the injections were continued for 2 weeks or discontinued. Mice injected with vehicle served as controls. Body weight was measured weekly, and echocardiograms were performed at P42 (6 weeks of age) and P56 (8 weeks of age). Animal handling was approved by The Yale University Institutional Animal Care and Use Committee.

[0088] Echocardiographic Investigation - Cardiac size and function were analyzed by echocardiography using a Vevo770 console. Mice were lightly anesthetized by inhaling isoflurane (0.2% in O2). All measurements were obtained from 3 to 6 consecutive cardiac cycles, and their mean values ​​were used for analysis. Interventricular septum (IVS), left ventricular diameter (LVID), and left ventricular posterior wall thickness (LVPW) were measured from T-mode and M-mode tracing at both end-diastolic (d) and end-systolic (s) states. Diastolic measurements were performed using state-of-the-art methods from the American Society of Echocardiography. Left ventricular end-diastolic volume (LV vol,d) and left ventricular end-systolic volume (LV vol,s) were calculated from the TM-mode measurements. Ejection fraction (EF) (%) was calculated using the formula [(LVvol,d-LVvol,s) / LVvol,d]×100, and shortening rate (%) was calculated using the formula [(LVID,d-LVID,s) / LVID,d]×100.

[0089] Statistical Analysis - Statistical values ​​are presented as mean ± sem. P-values ​​were calculated using a two-way ANOVA (Tukey's multiple comparison) test. All statistical analyses were performed using GraphPad Prism 5. For all studies, a P-value less than 0.05 was considered statistically significant.

[0090] The materials and methods used in conducting the experiment of Example 2 disclosed herein are described below.

[0091] Antibodies, chemicals, cell lines, and plasmids - The following antibodies were used for either immunoblotting (IB) or immunoprecipitation (IP) as described. Mouse monoclonal Flag antibody (F1804, IP-1:100, IB-1:1,000) and mouse monoclonal biotin-labeled Flag antibody (F9291, IB-1:1,000) were obtained from Sigma. Mouse monoclonal Myc antibody (sc-40, IP-1:100, IB-1:1,000), mouse monoclonal biotin-labeled Myc antibody (sc-40B, IB-1:1,000), rabbit polyclonal Shp2 antibody (sc-280, IB-1:1,000), mouse monoclonal p38 antibody (sc-535, IB-1:1,000), and mouse monoclonal GST antibody (sc-138, IB-1:1,000) were obtained from Santa Cruz Biotechnology.Rabbit monoclonal phospho-PZR antibody (Y241; 8181, IB-1:1,000), rabbit monoclonal phospho-PZR antibody (Y263; 8088, IB-1:1,000), rabbit polyclonal phospho-Src antibody (Y416; 2101, IB-1:1,000), mouse monoclonal Src antibody (2110, IB-1:1,000), ma Rabbit monoclonal Raf1 antibody (No. 12552, IB-1:1,000), rabbit polyclonal phospho-MEK1 / 2 antibody (S217 / 221; No. 9154, IB-1:1,000), mouse monoclonal MEK1 / 2 antibody (No. 4694, IB-1:1,000), rabbit polyclonal phospho-ERK1 / 2 antibody (T202 / Y204; No. 9101, IB-1 :1,000), mouse monoclonal ERK antibody (No. 9107, IB-1:1,000), rabbit polyclonal phospho-p38 antibody (T180 / Y182; No. 9215, IB-1:1,000), rabbit polyclonal phospho-JNK antibody (T183 / Y185; No. 4668, IB-1:1,000), mouse monoclonal JNK antibody (No. 3708, IB-1 Rabbit polyclonal phospho-Akt antibody (S473; 9271, IB-1:1,000), mouse monoclonal Akt antibody (2967, IB-1:1,000), rabbit polyclonal SERCA2A antibody (9580, IB-1:1,000), and rabbit polyclonal troponin I antibody (4002, IB-1:1,000) were purchased from Cell Signaling. Rabbit polyclonal phospho-Raf1 antibody (Y341; ab192820, IB-1:1,000) and rabbit polyclonal α-tubulin antibody (ab4074, IB-1:1,000) were obtained from Abcam. Mouse monoclonal Shp2 antibody (610622, IB-1:1,000) was purchased from BD Bioscience. The mouse monoclonal His antibody (11922416, IB-1:1,000) was obtained from Roche. The rabbit polyclonal troponin T (MS-295, IB-1:1,000) was obtained from Thermo Scientific. The rabbit polyclonal PZR antibody (IB-1:1,000) was generously provided by ZJ Zhao.Dasatinib was purchased from Biovision, and STI-571 was obtained from LKT Laboratories. The Shp2 phosphatase inhibitor was generously provided by Z.-Y. Zhang (Indiana University). HEK-293T cells were purchased from ATCC, and mouse embryonic fibroblasts (MEFs) were obtained from WT mice and Ptpn11. D61G / + Cells were isolated from mice. Cells were grown in growth medium (Dulbecco's modified Eagle medium (DMEM) supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum) in a 37°C, 5% CO2 incubator. Human Src constructs, as well as Ptpn11 full-length constructs, N+C constructs, and PTP constructs, were prepared by PCR and cloned into pCMV-3Tag4a and pCMV-Tag2b vectors (Clontech laboratories). DNA transfection into HEK-293T cells was performed using lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol.

[0092] Immunoprecipitation and immunoblotting: Cells or cardiac tissue were dissolved on ice in lysis buffer (25 mM Tris-HCl, pH 7.4, 136 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM benzamidine, 1 mM PMSF, 1 μg / ml pepstatin A, 5 μg / ml aprotinin, and 5 μg / ml leupeptin). The cell or tissue lysate was incubated at 4°C for 30 minutes and clarified by centrifugation at 14,000 rpm at 4°C for 10 minutes. Protein concentration was measured using BCA reagent according to the manufacturer's instructions (Pierce). For immunoprecipitation, 500 μg of the lysate was incubated overnight at 4°C with 1 μg of the specified antibody. The immunocomplexes were collected on the surface of either Protein A Sepharose beads or Protein G Sepharose beads at 4°C for 4 hours, washed three times with the same lysis buffer, and then heated to 95°C for 5 minutes in sample buffer. The total lysate or immunocomplexes were subjected to SDS-PAGE and immunoblotting. Antibody binding sites were visualized using high-sensitivity chemiluminescence detection or the Odyssey Imaging System.

[0093] In vitro GST pull-down assay - Purified GST-Src SH3 and His-Shp2 PTPs derived from bacteria were provided by T. Boggon (Yale University). The pull-down assay was performed overnight at 4°C in 1 ml of lysis buffer containing GST-Src protein SH3 along with either His-Shp2 PTP or HEK-293 cell lysates overexpressing Flag-tagged Shp2. The SH3-bound Shp2 protein was affinity-purified for 1 hour at 4°C using BSA-coated GST-Sepharose beads. The interaction between the Src protein's SH3 and the Shp2 protein was examined by immunoblotting using anti-His antibody or anti-Flag antibody and anti-GST antibody.

[0094] Animal handling - Ptpn11 D61G / +The mice were provided by Dr. Benjamin Neel (University of Toronto, Toronto), and their genotypes were identified as previously described. 9 Simply put, Ptpn11 D61G / + Male mice were crossed with wild-type C75BL / 6 x SV129 female mice, and their offspring were genotyped by PCR and digestion using AgeI against the D61G allele. Dasatinib (Biovision) was suspended in a vehicle (1% DMSO dissolved in phosphate-buffered saline). For prenatal treatment, pregnant mice were intraperitoneally injected with dasatinib (0.1 mg / kg body weight) daily, starting at gestation day 7.5 (E7.5) and continuing until postnatal day 9 (in lactating females). Mice injected with the vehicle served as controls. Starting at P10 and continuing until postnatal week 8, either dasatinib or the vehicle alone was injected directly (intraperitoneally) daily into the pups. For postnatal treatment, dasatinib was injected (intraperitoneally) into the pups from P10 until postnatal week 6; injections were interrupted for 2 weeks. The handling of animals was approved by the Yale University Board of Animal Care and Use.

[0095] Histological examination - Hearts, livers, and spleens were isolated from wild-type and NS mice treated with vehicle or dasatinib. Tissues were fixed with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS), prepared for paraffin sectioning, and stained using hematoxylin and eosin (H&E) or Masson's tricolor staining method. Histological images were obtained under a bright-field microscope (Olympus BX51, Yale Liver Center).

[0096] Echocardiography - Mice were anesthetized in a sealed plastic container containing 1% isofurane in oxygen until immobile, and then moved to a warmed procedure board (37°C). Throughout the procedure, 1% isofurane was supplied via a nose cone connected to an isoflurane vaporizer to keep the animals anesthetized. A scan head was placed on the mouse's chest to acquire stable image signals (both B-mode and M-mode), and the data were analyzed using a Vevo 770 (VisualSonics). Using M-mode images, systolic and diastolic left ventricular peripheral wall thickness, chamber diameter, and interventricular wall thickness were measured. Ejection fraction (EF) (%) and shortening fraction (FS) (%) were calculated.

[0097] Hemodynamic Investigation - Anesthesia was induced by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). The animals were placed on a warm pad and an incision was made in the neck. The right carotid artery was exposed, and a 1.9 French tip transducer catheter (Millar Inc., Houston, TX) was inserted into the artery and then advanced into the left ventricle. Left ventricular pressure and heart rate, including high-fidelity positive and negative dp / dt, were measured under baseline conditions. Data were recorded and analyzed using LabChart software.

[0098] RNA Extraction and Quantitative Real-Time PCR Analysis - RNA was isolated from mouse hearts using the RNeasy kit (Qiagen, CA) according to the manufacturer's instructions. A total of 1 μg of RNA was reverse transcribed using a reverse transcriptase PCR kit (Applied Biosystems, CA) to produce cDNA. Real-time quantitative PCR was performed in sets of three using the Applied Biosystems 7500 Fast real-time PCR system and SYBR green gene expression master mix with the primer pairs listed below. TIFF0007840031000001.tif100144

[0099] Relative gene expression levels are all ΔC T The analysis was performed using the specified method and standardized for 18S rRNA expression.

[0100] Cardiomyocytes were isolated from 8-week-old mice using a modified Langendorff procedure, based on the method described in Xianghua Xu, et al. J Vis Exp. 2009; (28):1308, which involves enzymatic digestion of cardiac tissue for single-cell analysis. In short, the heart was rapidly extracted, cannulated, and mounted in a Langendorff apparatus. In the Langendorff apparatus, at 37°C, Ca 2+ The heart was perfused with a perfusion buffer (25 mM HEPES, 118 mM NaCl, 4.8 mM KCl, 2.0 mM KH2PO4, 2.55 mM MgSO4, 10 mM BDM, and 10 mM glucose). To digest the tissue, the heart was perfused with a buffer containing 0.5 mg / mL liberase TH (Roche Applied Science, Penzberg, Germany). After about 10 minutes, the heart was removed from the Langendorff apparatus and the right ventricle and atrium were removed. The left ventricle was isolated, cut into small pieces, and further digested in a digestion solution at 37°C for 5–10 minutes with mechanical agitation, then gradually ground to release individual cells. Large clumps of remaining tissue were transferred to fresh digestion buffer and the above process was repeated up to six times, or until all tissue was digested. Cells were removed from collagenase by gentle centrifugation, resuspended in FBS-containing buffer after several washing steps, and calcium (0.05–1.1 mM) was gradually reintroduced by stepwise addition of a high-concentration CaCl2 solution. The cells were allowed to stand for at least 1 hour before imaging.

[0101] Characterization of cardiomyocyte function - Cardiomyocytes were imaged in Tyrode's solution (150 mM: NaCl: 140, KCl: 5.4, CaCl2: 1.8, MgCl2: 1, HEPES: 25 mM, glucose: 10 mM). For calcium fluorescence imaging, 2.5 μM Fura-2 AM was added and pluronic acid (20% w / v) was added (complimented) to the Tyrode's solution, which was then loaded onto the cell pellet in the dark for 15 minutes. After 15 minutes, the cells were resuspended in fresh Tyrode's solution and stabilized until imaging. Ca of cardiomyocytes 2+ Transient and unloaded shortening contractions were measured using an inverted microscope (Nikon Eclipse, Chiyoda, Tokyo) equipped with a constant-temperature perfusion bath (Cell MicroControls, Norfolk, VA) under continuous perfusion of 37°C Tyrode's solution. Cells were stimulated with an electric field at 1 Hz. Contraction events were imaged in real time using a sarcomere length camera system (HVSL, Aurora Scientific, Ontario, Canada). Only rod-shaped cells with clearly defined sarcomere striations that contracted upon stimulation were selected for measurement. The length of the sarcomere was measured and recorded during 10 consecutive beatings, and then a single waveform was created by averaging all beatings. Calcium transient measurements were simultaneously recorded using alternating excitation wavelengths of 340 nm and 380 nm generated at an overall rate of 100 Hz by a RatioMaster fluorescence system (PTI, Birmingham, NJ). The fluorescence emission was filtered at a central wavelength of 510 nm and quantified to determine the response to alternating excitation wavelengths (F for each wavelength). 340 and F 380) Obtained. Ca 2+ The transient is the interpolated ratio (F) of the two fluorescence intensities at each time point. 340 / F 380The data was reported as follows: The data was recorded using the DAP5216a data acquisition system (Microstar Laboratories, Bellevue, WA) and processed using custom software written in MATLAB (MathWorks, Natick, MA). Peak sarcomere length shortening (Peak SL shortening), time to peak shortening (TTP), time to return to 50% length (RT50), and calcium transient magnitude (Ca 2+ R mag :Maximum F 340 / F 380 -Minimum F 340 / F 380 ), and the decay rate of calcium Tau(Tau Ca2+ ) was calculated by computer.

[0102] Statistical Analysis - All data represent the mean ± standard error of the mean (SEM). Differences between groups were evaluated using analysis of variance (ANOVA) along with Tukey's multiple comparisons, using the GraphPad Prism 6 statistical software program.

[0103] The results of the experiments disclosed herein are described below.

[0104] Example 1: Targeting cardiac disease via RAS opathy using tyrosine kinase therapeutic intervention. Figure 1A shows Ptpn11 D61G / + This figure shows the proteomics analysis of differentially tyrosyl-phosphorylated proteins in mouse hearts.

[0105] Figure 1B shows the heart of a wild-type mouse and Ptpn11 D61G / +This graph shows the log2-converted ratios of each phosphotyrosine-containing peptide in mouse heart. Figure 1C is a heatmap of differentially tyrosyl-phosphorylated peptides (phosphorylation sites are identified by MS and shown in parentheses). Figure 1D is a panel of extracted ion chromatograms and peptide sequences of PZRs containing tyrosine 242 (upper panel) and PZRs containing tyrosine 264 (lower panel) obtained by differential proteomics. Figure 1E shows the C-terminal amino acid sequences of PZRs in various vertebrates. Noonan syndrome mutant (Shp2 D61G / + Comprehensive phosphotyrosylproteomics of the hearts of mice carrying the knock-in mutation of ) reveals altered regulation of tyrosylphosphorylated proteins. MS analysis shows that the protein most highly tyrosylphosphorylated in these mice is PZR. Tyrosyl residues 264 and 242 are involved in Noonan syndrome (Shp2 D61G / + These were identified as increased PZR tyrosyl phosphorylation sites in the heart of mice. Given that PZR tyrosines 242 and 264 are highly conserved during evolution, they are likely important for PZR function. These results suggest that increased PZR tyrosyl phosphorylation may play a role in the development of the cardiac disease associated with Noonan syndrome. These results confirm that Y242 and Y264 are PZR hypertyrosyl phosphorylation sites in this NS mouse model.

[0106] Figures 2A-2E show immunoblots illustrating the characterization of PZR tyrosyl phosphorylation. The morphology (conformation) of the PZR hypertyrosyl phosphorylation site in NS and NSML mutants is also shown. When PZR phosphorylation-resistant mutants are expressed at the tyrosyl phosphorylation site present in mice, the ability to phosphorylate is impaired in cultured cells, as can be detected using phospho-specific anti-PZR(Y242) and anti-PZR(Y263). Shp2 mutants corresponding to those observed in either NS or NSML patients can induce PZR hypertyrosyl phosphorylation at Y241 and Y263. Similarly, zebrafish PZR exhibits the same characteristic of being tyrosyl phosphorylated at equivalent residues. These results suggest that Shp2 mutants associated with NS or leopard syndrome induce PZR hyperphosphorylation in various cell lines.

[0107] Figures 3A-3D are Ptpn11 D61G / + Mouse and Ptpn11 Y279C / + This shows PZR tyrosyl phosphorylation in the heart and cortex of mice. Using site-specific phospho-PZR antibodies, the inventors have demonstrated that Shp2 D61G / + We have shown that mice expressing allele knock-in mutations exhibit increased PZR tyrosyl phosphorylation in the heart and cortex. Similarly, Shp2 Y279C / + Mice expressing allele knock-in mutations also show increased PZR tyrosyl phosphorylation in the heart and cortex. These results demonstrate that both NS and NSML mutations, which enhance and reduce phosphatase catalytic activity respectively, can increase PZR tyrosyl phosphorylation. These results demonstrate that PZR is a target for both NS and NSML, suggesting that PZR corresponds to a novel common signaling component of these RAS opathies. These results demonstrate exemplary (that) PZR hypertyrosyl phosphorylation in the heart and cortex of NS and NSML model mice. These in vivo data confirmed the validity of computer-aided (Figure 1A-1E) and in vitro (Figure 2A-2E) experiments.

[0108] Figures 4A-4C are Ptpn11 D61G / + Images and graphs of PZR tyrosyl phosphorylation in mouse liver, kidney, and spleen are shown. PZR is Ptpn11 D61G / + High tyrosyl phosphorylation was observed in the liver, kidney, and spleen of mice. These results demonstrate high tyrosyl phosphorylation of PZR in various tissues of NS mice.

[0109] Figures 5A-5D are Ptpn11 D61G / + Mouse and Ptpn11 Y279C / + Images of ERK and Akt phosphorylation in mouse heart and cortex are shown. Ptpn11 D61G / + While the phosphorylation status of ERK and AKT in the heart and cortex of mice showed no substantial difference compared to wild-type mice, PZR was hypertyrosyl-phosphorylated under similar conditions (see Figures 3A-3D). These results implicitly suggest that there are no significant differences in the baseline levels of Shp2, phospho-ERK1 / 2, and phospho-Akt between NS mice and NSML mice. Furthermore, these results indicate that the effects of these RAS opathies on MAPK and AKT signaling in the heart and cortex are different from those that drive PZR hypertyrosyl phosphorylation.

[0110] Figures 6A–6B are blots showing the effect of Src family kinases on the Y241 and Y263 phosphorylation of PZR via NS / NMLS-Shp2. Mutants associated with NS and NMLS induce high tyrosyl PZR, which can be inhibited by cell pretreatment with the SFK inhibitor SU6656. These results suggest that SFK can phosphorylate both Y241 and Y263 of PZR. Furthermore, these results demonstrate that the high tyrosyl PZR phosphorylation induced by NS-Shp2 and NSML-Shp2 mutants is Src family kinase-dependent.

[0111] Figures 7A-7B are blots showing that Src kinases mediated PZR hypertyrosyl phosphorylation induced by NS / LS-Shp2. Figures 7C-7D are blots showing that Src kinases mediated PZR hypertyrosyl phosphorylation. These figures compare the effects of tyrosine kinase inhibitory activity on PZR tyrosyl phosphorylation. Cells expressing an activated Shp2 (Shp2-E76A) mutant were administered the tyrosine kinase inhibitors PP2 and SU6656. Both PP2 and SU6656 could inhibit PZR hypertyrosyl phosphorylation induced by Shp2-E76A, but SU6656 was more effective. PZR hypertyrosyl phosphorylation was completely inhibited by 1 μM SU6656, compared to 5 μM for PP2. These results support the idea that Src family kinases are responsible for PZR phosphorylation. Importantly, Src-mediated phosphorylation of PZR created a binding site (pY241 / pY263) for Shp2 to interact with PZR. These results implicitly suggest that c-Src directly phosphorylates the Shp2 binding site of PZR.

[0112] In Noonan syndrome, increased phosphorylation of Y241 and Y263 by Src resulted in harmful high levels of PZR / Shp2 complexes. The PZR / Shp2 complex has been proposed as a mechanism that promotes the development of congenerative heart disease in these patients.

[0113] Figures 8A-8B show increased Src complex formation with NS / NSML-related Shp2 mutants and PZRs. Figure 8A shows that mutant Shp2, known to cause either NS or NSML, can bind to c-Src with higher affinity compared to wild-type Shp2. Furthermore, these results suggest that c-Src directly phosphorylated the Shp2 binding site of the PZR.

[0114] Figure 9 illustrates a model illustrating the effects of NS-Shp2 and NSML-Shp2 mutants on PZR tyrosyl phosphorylation. This model is based on experimental data in which the Shp2 binding site of PZR was observed to be highly tyrosyl phosphorylated in the hearts of both NS and NSML mouse models. Increased PZR tyrosyl phosphorylation promoted increased Shp2 binding to PZR. The figure suggests that, as with other nearby potential Src substrates, increased PZR tyrosyl phosphorylation leads to increased Shp2 recruitment to PZR, further promoting PZR tyrosyl phosphorylation.

[0115] Furthermore, the NS and NSML mutants also interacted with the tyrosine kinase Src with high affinity. In summary, these indiscriminate interactions resulted in dysfunction of downstream signaling from PZR, which contributed to the development of congenital heart disease. It has been proposed that intervening Src tyrosine kinase activity reduces the PZR / Shp2 complex, correcting altered signaling from PZR and possibly other targets.

[0116] Figure 10 is a panel of images showing dasatinib administration. Male Ptpn11D61G / + mice were intraperitoneally injected with a specified dose of dasatinib or DMSO control. After 24 hours, the mice were sacrificially killed, cardiac tissue was collected, and immunoblotting was performed using total PZR antibody and pY(263)-PZR antibody. These results indicate that dasatinib injection into NS mice is effective in reducing PZR tyrosyl phosphorylation.

[0117] Figure 11A shows the prenatal administration plan for dasatinib in an NS mouse model. Pregnant mother mice were administered dasatinib daily from the time the animals were in utero at E7.5 until postnatal day 9 (P9). From postnatal day 10 (P10), NS mice were directly administered dasatinib by daily intraperitoneal injection for 6 weeks (P42) and 8 weeks (P56).

[0118] Figure 11B shows the postnatal administration plan for dasatinib in an NS mouse model. Dasatinib was administered daily to NS mice starting on postnatal day 10 (P10) for 6 weeks (P42). After 6 weeks, treatment was discontinued and cardiac function was measured. Subsequently, the same group of mice was evaluated after a 2-week cessation of dasatinib treatment.

[0119] In Figures 11A and 11B, cardiac function of these mice was evaluated at 6 and 8 weeks. These figures illustrate prenatal or postnatal dasatinb treatment strategies for NS mice. The dosing regimens described herein were designed to test three aspects of the efficacy of dasatinib for therapeutic intervention of NS-related cardiac disease. Because NS is a developmental disorder, the first dosing regimen, shown in Figure 11A, tested the efficacy of dasatinib in exerting a therapeutic effect when administered to developing embryos. The second evaluation revealed the efficacy of dasatinib in treating NS-related cardiac disease when administered postnatally. Since it is understood that therapeutic doses of dasatinib can be administered to patients postnatally, this strategy was more closely correlated with cardiac disease outcomes. This dosing strategy appears to reduce the risk of intrauterine complications. Finally, the third trial was intended to confirm whether dasatinib administration was necessary to continue treatment if cardiac function improved with treatment.

[0120] Figure 12A is a panel of graphs showing that prenatal treatment with dasatinib improved cardiac function in NS mice (as seen on page 42, Figure 11A). Figure 12B is a panel of graphs showing that postnatal treatment with dasatinib improved cardiac function in NS mice (as seen on page 42). Figure 12C is a panel of graphs showing that the improvement in cardiac function was maintained after discontinuation of dasatinib treatment (as seen on page 56, Figure 11B). These results provide evidence that Src signaling is involved in the pathogenesis of NS.

[0121] The results of these experiments demonstrated that low doses of dasatinib (defined herein as doses lower than those known to be effective in treating cancer) were effective in improving cardiac function in NS mice. In Figure 12A, these results, evaluated based on ejection fraction (EF) and fetal shortening (FS), demonstrated that when pregnant mice were injected with dasatinib, cardiac function was fully restored to wild-type parameters. In Figure 11B, dasatinib was shown to be effective even when administered post-development. This treatment still demonstrated the ability to completely correct cardiac function in NS mice. These results indicate that therapeutic administration of dasatinib to treat congenital heart disease in RAS opathy patients can be performed with substantially low risk by post-development administration. Finally, Figure 11C shows that cardiac function was maintained after discontinuing dasatinib for two weeks after effective cardiac function was achieved. These results demonstrate that once an effective treatment is achieved and cardiac function is restored, continuous exposure to dasatinib is unnecessary.

[0122] Example 2: Selective relief of cardiac defects in a mouse model of Noonan syndrome (NS) with dasatinib Shp2 consists of two Src homologous 2 (SH2) domains: a protein tyrosine phosphatase (PTP) domain and a carboxyl-terminal tail. NS-related Shp2 (NS-Shp2) mutations often occur in amino acid residues occupying the boundary between the amino-terminal SH2 domain and the PTP domain. The resulting mutations disrupt the self-repressive "closed" higher-order structure between the SH2 and PTP domains, favoring a more "open" configuration that promotes catalysis.

[0123] Protein zero-related proteins (PZRs), transmembrane glycoproteins containing two immunoreceptor tyrosine-based repression motifs (ITIMs) at their C-terminus, act as c-Src substrates and are major hypertyrosyl-phosphorylated protein and Shp2-binding targets in the heart of the NS mouse model. NS-Shp2 mutants interact with c-Src with high affinity, conferring the ability to indiscriminately target c-Src through PZR complex formation. Using zebrafish as a model for NS-mediated CHD, it has been proposed that PZR-Shp2-Src complex formation promotes abnormal signaling in NS-mediated CHD.

[0124] Crucial to this hypothesis is the ability of the NS-Shp2 mutant to exhibit enhanced interaction with c-Src, leading to increased c-Src-mediated signaling. This enhanced interaction between Shp2 and c-Src is likely to result from increased exposure of the binding surface within the PTP domain of Shp2, which would otherwise be unexposed in a "closed" higher-order structure. While Shp2 has been shown to form a complex with c-Src via its SH3 domain, the Shp2 region with which c-Src interacts has not yet been identified.

[0125] To address this, a series of Shp2 deletion mutants were designed (Figure 13a), co-transfected into HEK-293T cells, and complex formation was examined by co-immunoprecipitation (Figure 23). As expected, full-length Shp2 was detected in complex with c-Src, while Shp2 deletion mutants lacking the PTP domain were unable to interact (Figure 13b). Furthermore, in vitro binding assays confirmed that the PTP domain of Shp2 and the SH3 domain of c-Src directly interact (Figure 13c).

[0126] Because this interaction occurs within the PTP domain of Shp2, the "open" higher-order structure of NS-Shp2 mutants is thought to be more responsive to establishing a stable interaction with the SH3 domain of c-Src compared to wild-type Shp2. Therefore, it has been previously proposed that NS-Shp2 mutants form a more stable complex with c-Src at the membrane via PZR, and that this is a presumed mechanism for abnormal signaling mediated by c-Src. Importantly, these observations suggest that c-Src or members of the Src family kinase (SFK) are candidate partners for Shp2-mediated NS disease.

[0127] To investigate whether SFK is involved in NS pathogenesis, c-Src was pharmacologically inhibited to test whether inhibition of c-Src improves Shp2-NS signaling. To inhibit c-Src, dasatinib (Sprycel (copyright)), a dual Abl-Src kinase inhibitor approved for the treatment of chronic myeloid leukemia, was used. Treatment of mouse embryonic fibroblasts (MEFs) isolated from NS mice with dasatinib inhibited tyrosyl phosphorylation of c-Src, ERK1 / 2, and PZR (Figures 13d-13h and 24).

[0128] Inhibition of PZR tyrosyl phosphorylation by dasatinib also disrupted PZR / Shp2 complex formation (Figure 13d). Furthermore, Raf-1, MEK1, JNK, and Akt in NS-derived MEFs were also inhibited by dasatinib (Figure 17).

[0129] Since the BCR-Abl kinase inhibitor STI-571 (Gleevec (copyright)) did not reduce PZR tyrosyl phosphorylation, it was suggested that the inhibition of PZR tyrosyl phosphorylation and interference with the PZR / Shp2 complex were likely a result of the effect of dasatinib on c-Src rather than Abl (Figure 17). Furthermore, since the Shp2 inhibitor did not interfere with NS-Shp2-mediated PZR hypertyrosyl phosphorylation (Figure 17), it was suggested that NS-Shp2-mediated c-Src PZR tyrosyl phosphorylation occurs independently of the phosphatase activity of Shp2.

[0130] To investigate the in vivo effects of dasatinib on tyrosyl phosphorylation of c-Src and PZR by NS, mice containing a knock-in mutation (D61G) in which the Asp61 position of Shp2 is replaced with Gly61 were injected with dasatinib (Araki et al Nat Med 10, 849-857 (2004)). In this specification, these are referred to as "NS mice," and PtpN11 D61G / + Heterozygous mice reproduce many features of human diseases, including short stature, craniofacial abnormalities, myeloproliferative disorders, and CHD.

[0131] Dasatinib has been shown to be effective in preventing tumor development in mice at a dose of approximately 20 mg / kg (Shah et al., Science 305, 399-401 (2004)). The therapeutic effect of dasatinib in humans has been reported to be at a dose of approximately 2 mg / kg, which is equivalent to approximately 24 mg / kg in mice (Kantarjian et al., N Engl J Med 362, 2260-2270 (2010), Yu et al., Clinical Cancer Research 15, 7421-7428 (2009), Apperley J Clin Oncol 27, 3472-3479 (2009)). A low dose of dasatinib, 0.5 mg / kg, was sufficient to significantly inhibit both c-Src tyrosyl phosphorylation and PZR tyrosyl phosphorylation in the hearts of 3-week-old NS mice (Figures 13i-13l). In particular, these doses of dasatinib (0.1-0.5 mg / kg) did not affect ERK1 / 2 phosphorylation (Figures 13i-13l), Raf-1, MEK1, p38MAPK, or JNK in the hearts of 3-week-old NS mice (Figure 18). These results suggest that dasatinib at a dose as low as 1 / 250th of the effective chemotherapy dose (approximately 100-140 mg / day or approximately 1.4-2.0 mg / kg / day for adult patients with chronic myeloid leukemia) can inhibit PZR tyrosyl phosphorylation in the hearts of NS mice.

[0132] Furthermore, in the hearts of NS mice, dasatinib-induced inhibition of PZR tyrosyl phosphorylation was not linked to inhibition of ERK1 / 2 phosphorylation (Figure 13). Therefore, low doses of dasatinib interfered with NS-Shp2 signaling independently of the ERK1 / 2 pathway.

[0133] We evaluated the improvement of cardiac defects in NS mice by administering dasatinib to pregnant mother mice. Starting on embryonic day 7 and continuing until postnatal day 9 (in lactating females), wild-type pregnant mice crossed with NS mice were administered 0.1, 0.5, or 1.0 mg / kg of dasatinib daily interperitoneally. From postnatal day 10 (P10) onward, direct daily dasatinib injections to individual pups were resumed and continued until postnatal week 8 (P56) (Figure 14a). Dasatinib treatment at 0.5 mg / kg / day and 1.0 mg / kg / day resulted in embryonic lethality, but dasatinib treatment at 0.1 mg / kg / day showed no observable adverse effects (Table 1).

[0134] Cardiac function in NS mice was examined at weeks 6 and 8 based on echocardiography and invasive hemodynamics. Ejection fraction (EF) and fasting rate (FS) in untreated NS mice were significantly reduced by 35% compared to vehicle-treated wild-type mice (P<0.01). However, dasatinib-treated NS mice showed complete recovery of cardiac function at P42 compared to vehicle-treated NS mice (Figures 14b and 14c, and Table 2). However, continued administration of dasatinib for another two weeks induced heart failure in both wild-type and dasatinib-treated NS mice (Figures 14d-14e and Table 3). These data suggest that intrauterine dasatinib treatment can rescue the impaired cardiac function observed in NS mice. Therefore, c-Src activity contributes to the manifestation of symptoms in Shp2-NS CHD.

[0135] (Table 1) Ptpn11 treated with dasatinib prenatally D61G / + × Descendants from WT breeding animals (breeders) TIFF0007840031000002.tif24151

[0136] (Table 2) WT mice and Ptpn11D mice treated with vehicle or dasatinib before birth 61G / + Echocardiographic parameters at P42 in mice The data in TIFF0007840031000003.tif82150 represents the mean ± SEM. * p<0.05; ** , p<0.01 indicates significance compared to WT mice treated with the vehicle. ††, p<0.01 indicates Ptpn11 treated with the vehicle. D61G / + The study shows statistical significance compared to mice. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS: Intraventricular septum wall thickness; LVID: Left ventricular internal dimension; LVPW: Left ventricular posterior wall thickness; LV vol: Left ventricular volume; EF: Ejection fraction; FS: Fractional shortening; d: Diatole; s: Systole.

[0137] (Table 3) WT mice and Ptpn11D mice treated with vehicle or dasatinib before birth 61G / + Echocardiographic parameters of mice at P56 The data in TIFF0007840031000004.tif78151 represents the mean ± SEM. * p<0.05; ***A p<0.001 indicates statistical significance compared to vehicle-treated WT mice. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS: Intraventricular septum wall thickness; LVID: Left ventricular internal dimension; LVPW: Left ventricular posterior wall thickness; LV vol: Left ventricular volume; EF: Ejection fraction; FS: Fractional shortening; d: Diatole; s: Systole.

[0138] Dasatinib was effective in reducing the impact of NS-Shp2 on CHD after development. NS mice were treated with dasatinib (0.1 mg / kg / day) from P10 to P42 (Figure 14f). In addition to presenting with CHD, NS humans and NS mice exhibited stunted growth, facial dysmorphia, and splenomegaly similar to that of human disease. NS mice were found to exhibit stunted growth, facial dysmorphia, and splenomegaly, but dasatinib treatment did not improve any of these NS-related lesions (Figures 19-21). Furthermore, no evidence of liver injury was observed in either wild-type or NS mice treated with dasatinib (Figure 22).

[0139] However, when cardiac parameters were examined, dasatinib-treated NS mice (P42) exhibited fully restored cardiac function, as determined based on indicators such as EF and FS (Figures 14g and 14h and Tables 4-5). Unusually, when cardiac function was evaluated at a later point in time, i.e., two weeks after discontinuing dasatinib treatment, the same level of improvement in cardiac function was observed compared to wild-type controls treated with the vehicle (Figures 14i and 14j). Other cardiac parameters were also evaluated based on invasive hemodynamics, and these results showed significant recovery of aortic blood pressure and left ventricular pressure in dasatinib-treated NS mice (Figures 14k-14n and Table 6).

[0140] In summary, these data demonstrate that dasatinib, when administered post-developmentally to NS mice at doses below the therapeutic dose used for treating CML, provides selective efficacy in preventing heart failure in NS mice. Interestingly, the recovery of cardiac function in NS mice did not reverse even after dasatinib was removed, suggesting that the improvement in cardiac function is not temporary.

[0141] (Table 4) WT mice and Ptpn11D mice treated with vehicle or dasatinib after birth 61G / + Echocardiographic parameters at P42 in mice The data in TIFF0007840031000005.tif71149 represents the mean ± SEM. * p<0.05; ** †, p<0.01 indicates significant difference compared to WT mice treated with the vehicle. †, p<0.05 indicates significant difference compared to Ptpn11 mice treated with the vehicle. D61G / +The study shows statistical significance compared to mice. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS: Intraventricular septum wall thickness; LVID: Left ventricular internal dimension; LVPW: Left ventricular posterior wall thickness; LV vol: Left ventricular volume; EF: Ejection fraction; FS: Fractional shortening; d: Diatole; s: Systole.

[0142] (Table 5) WT mice and Ptpn11D mice treated with vehicle or dasatinib after birth 61G / + Echocardiographic parameters of mice at P56 The data in TIFF0007840031000006.tif70150 represents the mean ± SEM. *** , p<0.001 indicates significance compared to WT mice treated with the vehicle. †, p<0.05;†††, p<0.001 indicates Ptpn11 treated with the vehicle. D61G / + The study shows statistical significance compared to mice. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). IVS: Intraventricular septum wall thickness; LVID: Left ventricular internal dimension; LVPW: Left ventricular posterior wall thickness; LV vol: Left ventricular volume; EF: Ejection fraction; FS: Fractional shortening; d: Diatole; s: Systole.

[0143] (Table 6) WT mice and Ptpn11D mice treated with vehicle or dasatinib after birth61G / + Mouse hemodynamic analysis parameters at P56 time point The data in TIFF0007840031000007.tif57150 represents the mean ± SEM. ** p<0.01; *** p<0.001 indicates significance compared to WT mice treated with the vehicle. †, p<0.05;††, p<0.01 indicates Ptpn11 treated with the vehicle. D61G / + The results show statistical significance compared to mice. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparison).

[0144] To gain a more precise and deeper understanding of the cardiac phenotype observed in dasatinib-treated NS mice, macroscopic morphological and histological examinations of these hearts were performed. NS mice had smaller heart weights compared to wild-type mice (Figure 15a). The ratio of heart weight to body weight was significantly increased in NS mice (Figure 15b). Histological analysis also revealed that NS mice suffered from dilated cardiomyopathy (DCM), indicated by a significantly reduced systolic left ventricular septal thickness and an increased left ventricular chamber diameter (Figure 15c and Table 4).

[0145] As expected, histological examination of cardiac tissue revealed disordered myofibrils in the left ventricular wall of vehicle-treated NS mice (Figure 15d). In contrast, dasatinib-treated NS mice showed a significant recovery of all these pathological cardiac phenotypes to essentially the same level as vehicle-treated wild-type mice (Figures 15a-15d).

[0146] Another prominent feature of a dysfunctional heart is the occurrence of cardiac fibrosis. Consistent with the concept that dasatinib treatment prevents heart failure in NS hearts, fibrosis in dasatinib-treated NS mouse hearts was significantly reduced at the tissue level compared to vehicle-treated wild-type mice, and this was consistent with decreased mRNA expression levels of the fibrosis genes Col1a2 and Col3a1 (Figures 15d-15f). Deposition of fibrous components such as collagen, encoded by the Col1a2 and Col3a1 genes, is associated with heart failure. Therefore, the decrease in Col1a2 and Col3a1 expression is not inconsistent with the remission of heart failure by low-dose dasatinib treatment.

[0147] Re-expression of cardiac structural proteins, such as α-myosin heavy chain (MYH6) and β-myosin heavy chain (MYH7), is a sign of cardiomyopathy. In particular, inactivation of MYH6 and activation of MYH7 represent features of cardiac reprogramming that support the development of cardiomyopathy (Morita et al., J Clin Invest 115 (2005)). MYH6 expression in vehicle-treated NS mice was significantly downregulated compared to wild-type mice. Dasatinib treatment resulted in comparable levels of MYH6 expression in wild-type and NS mice (Figure 15g). MYH7 was significantly re-expressed in vehicle-treated NS mice, and this completely normalized after dasatinib treatment, returning to vehicle-treated wild-type levels (Figure 15h).

[0148] Evaluating atrial naturietic peptide (Anp) and brain naturiatic peptide (Bnp) further strengthened the efficacy of dasatinib in improving heart failure in NS mice. Both Anp and Bnp mRNA expression levels were significantly upregulated in NS mice compared to vehicle-treated wild-type controls (Figures 15i and 15j). In contrast, dasatinib-treated NS mice were completely rescued from the elevated mRNA expression levels of both Anp and Bnp (Figures 15i and 15j). In summary, these results support the conclusion that Src family kinase activity plays a crucial role in the development of NS-related CHD.

[0149] To determine whether the effect of dasatinib on NS cardiac function is specific to the cardiomyocyte, calcium (Ca) was measured in cardiomyocytes isolated from vehicle and dasatinib-treated wild-type and NS mice. 2+ The force dynamics were measured via ). Isolated cardiomyocytes were subjected to Ca under electrical pacing. 2+ Handling and contractile kinetics were characterized simultaneously. Relative calcium release rate (R) of NS cardiomyocytes. mag Ca 2+ The ) level was 55% higher compared to wild-type cardiomyocytes, and this difference was substantially improved in NS mice treated with dasatinib (Figures 16a and 16b).

[0150] Cardiomyocytes obtained from NS mice treated with the vehicle showed a lack of contractility, and peak shortening was 22% smaller than that of wild-type mice treated with the vehicle (Figures 16a and 16c). This result indicates that these same cardiomyocytes showed significantly greater Ca compared to wild-type mice. 2+ From the perspective of observing increased release, it is impressive, and in NS cells treated with the vehicle, myofilamentary Ca 2+This suggests a decrease in sensitivity. However, the rate of sarcomere shortening in NS cardiomyocytes was significantly lower, 22% lower than in wild-type cardiomyocytes (Figures 16a-16c and Table 7). Importantly, these differences were completely restored in cardiomyocytes isolated from dasatinib-treated NS mice (Figures 16a-16c and Table 7).

[0151] The molecular mechanisms of calcium handling and contractility changes are investigated in the sarcoplasmic reticulum (Sarco(endo)plasmic reticulum). 2+ - We investigated ATPase 2 (SERCA2A) by immunoblotting. In the myocardium, SERCA2A is the major isoform responsible for calcium delivery to the contractile mechanism. A prominent feature of heart failure is decreased SERCA2A expression, which leads to reduced Ca delivery to contractile proteins. 2+ A defect occurs in delivery, and therefore, the contractile force decreases.

[0152] Notably, the hearts of NS mice treated with the vehicle showed significantly reduced SERCA2A protein expression and increased TnI and TnT expression compared to wild-type mice (Figures 16d-16g). Consistent with the recovery of cardiac function in NS mice treated with dasatinib, cardiac tissue isolated from NS mice treated with dasatinib showed completely normalized SERCA2A expression levels (Figures 16d and 16e).

[0153] As a result of continuous remodeling that occurs during heart failure as a compensatory mechanism to maintain contractility, the contractile proteins troponin T (TnT) and troponin I (TnI) are upregulated. In vehicle-treated NS mice, both TnT and TnI were significantly increased compared to vehicle-treated wild-type mice (Figures 16d, 16f, and 16g). In NS mice treated with dasatinib, the expression levels of both TnT and TnI returned to levels comparable to those of vehicle-treated wild-type mice, indicating a complete recovery of the dysfunctional cardiac phenotype (Figures 16d, 16f, and 16g). In summary, these findings clearly demonstrate that post-developmental treatment with dasatinib in the NS mouse model alleviates myocardial contractile dysfunction.

[0154] (Table 7) WT mice and Ptpn11D mice treated with vehicle or dasatinib after birth 61G / + Ca2+ excitation-contraction coupling parameters of cardiomyocytes isolated from mouse hearts at time P56. The data TIFF0007840031000008.tif71149 represents the mean ± SEM. * p<0.05; *** , p<0.001 indicates significance compared to WT cardiomyocytes treated with the vehicle. †, p<0.05;†††, p<0.001 indicates Ptpn11 treated with the vehicle. D61G / + The study shows significant differences compared to cardiomyocytes. All p-values ​​were derived using two-way ANOVA (Tukey's multiple comparisons). TTP: Time to peak; RT50: Time from peak tension to 50% relaxation; RT90: Time from peak tension to 90% relaxation.

[0155] The transmembrane glycoprotein PZR was previously identified as the most abnormally hypertyrosyl-phosphorylated protein in NS mouse hearts (Eminaga et al., J Biol Chem 283, 15328-15338 (2008)). PZR is a Shp2-binding protein and an SFK substrate. High tyrosyl phosphorylation of PZR is a direct result of enhanced Src signaling via NS. These data suggest that c-Src plays a role in the propagation of CHD related to NS.

[0156] The data shown in Figures 25a-25f are from wild-type (WT) mice and NSML (Ptpn11) mice. Y279C / + This corresponds to a 6-week (P42) dasatinib treatment starting on day 10 postnatal (P10) in mice. Ptpn11 Y279C / + The mice were obtained from Dr. Kontaridis (Beth Israel Deaconess Hospital, Boston, MA) and bred as described (Marin, et al, J Clin. Invest., 121:1026-1043 (2011)). Mice were treated with either a vehicle or dasatinib at a dose of 0.1 mg / kg / day for 6 weeks, after which dasatinib treatment was discontinued and the mice were euthanized 2 weeks later. At the completion of the study, wild-type mice and Ptpn11 mice were used. Y279C / + Mice were sacrificially killed, and total RNA was isolated from their hearts. QPCR was then performed to detect mRNA expression of the genes Myh6 and Myh7, which are involved in the development of cardiomyopathy, and the genes col1a2 and Col3a1, which are involved in the development of cardiac fibrosis.

[0157] As shown in the figure, Ptpn11 treated with a vehicle Y279C / + The mice began to show signs of histological cardiomyopathy by 6 weeks of age, as evidenced by increased expression of ANP, Myh6, and Myh7 (Figures 25d and 25e). Furthermore, compared to wild-type mice treated with the vehicle, Ptpn11 Y279C / +In mice, cardiac fibrosis was incidentally increased (Figures 25a and 25b). However, Ptpn11 treated with dasatinib Y279C / + The mice showed complete recovery of ANP, Myh6, and Myh7 expression to wild-type levels. Significantly, Ptpn11 treated with dasatinib Y279C / + In mice, the Myh6 / Myh7 ratio, which represents the switching of fetal / adult myosin contractility genes, was also restored to wild-type levels (Figure 25f). In summary, these data demonstrate the effectiveness of low-dose dasatinib treatment in treating cardiomyopathy associated with Noonan syndrome with lentigo polycarpa (NSML) at the molecular level.

[0158] This is the first evidence that c-Src signaling is involved in the pathogenesis of NS. Other groups have reported that Shp2 is located upstream of SFK in a phosphatase-dependent manner (Zhang et al., Mol Cell 13, 341-355 (2004)). Here, we demonstrate a unique mechanism that leads to the “open” higher-order structure of NS-Shp2 mutants, which promotes c-Src signaling by achieving increased and localized PTP-Shp2 / Src-SH3 binding. Post-development administration of dasatinib at doses below the therapeutic dose required for CML treatment was sufficient to restore cardiac contractility and function. These data strongly suggest that c-Src is a central medium of pathogenesis by NS. With low doses of dasatinib, the Src pathway appears to be selectively affected, and ERK1 / 2 signaling was inhibited, but negligibly, at least in the cardiomyocyte. However, it is conceivable that ERK1 / 2 is present in a specific subset of cardiac cells affected by dasatinib treatment.

[0159] Importantly, analysis of calcium-mediated contractile coupling in cardiomyocytes isolated from dasatinib-treated NS mice clearly demonstrated that these cells are the site of action (through which dasatinib-induced c-Src inhibition exerts its effect on the contractile mechanism). Consistent with previous observations, the NS-Shp2 mutant exhibited increased Ca in the cardiomyocytes.2+ It increased signal transduction (Uhlen, et al. PNAS 103, 2160-2165 (2006)). Interestingly, the cardiomyocytes derived from NS mice showed decreased contractility, suggesting that Ca 2+ A decrease in sensitivity at the level of force contraction via this pathway was suggested. This decrease in sensitivity can be explained, at least to some extent, by a decrease in SERCA2A expression levels.

[0160] In summary, novel and unexpected therapeutic strategies for treating PTPN11-mediated CHD are described herein. These data identify the Src family kinases as a target class that leads to PTPN11-related CHD. Therapeutic strategies using "low-dose" dasatinib or other c-Src inhibitors may open new avenues for treating cardiovascular disease.

[0161] Other embodiments In this specification, any description of a list of elements in any definition of a variable element includes the definition of that variable element as any single element or as a combination (or partial combination) of the listed elements. Descriptions of embodiments in this specification include that embodiment as any single embodiment, or as a combination of any other embodiment or a part thereof.

[0162] The disclosures of each and all patents, patent applications, and publications referenced herein are hereby incorporated herein by reference in their entirety. While the present invention has been disclosed in relation to specific embodiments, it will be apparent that other embodiments and variations of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims shall be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition for treating cardiovascular diseases or conditions having abnormal protein tyrosine phosphorylation in a subject, comprising a low dose of a Src family tyrosine kinase inhibitor selected from the group consisting of A419259, AP23451, AP23464, AZD0424, AZM475271, CGP77675, ENMD2076, KB SRC 4, KX2-391, MNS, PD166285, PD180970, PKC-412, PP1, PP2, SU6656, TC-S7003, TG100435, TX-1123, WH-4-023, bosutinib, damnacanthal, salakatinib, and SrcI1. The Src family tyrosine kinase inhibitor reduces abnormal levels of tyrosine phosphorylation and improves at least one cardiac function in the subject, and the low dose is in the range of approximately 1 / 175 to 1 / 250 of the chemotherapy dose of the Src family tyrosine kinase inhibitor, A pharmaceutical composition comprising a cardiovascular disease or condition having abnormal protein tyrosine phosphorylation in the subject, which is associated with a RAS opathy selected from the group consisting of neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with lentigo polycarcinoma (Leopard syndrome), capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardiac-facial-cutaneous syndrome, and Regius syndrome.

2. The pharmaceutical composition according to claim 1, wherein the cardiovascular disease or pathological condition is a congenital heart disease.

3. The pharmaceutical composition according to claim 1, wherein cardiac function is selected from the group consisting of myofibrils organizing, cardiomyocyte contractility, SERCA2A expression, and cardiac fibrosis.

4. The pharmaceutical composition according to claim 1, wherein the Src family tyrosine kinase inhibitor is PP2 or SU6656.

5. The pharmaceutical composition according to claim 1, wherein the target is a pediatric patient.

6. The pharmaceutical composition according to claim 5, wherein the target population for pediatrics is under 12 years of age.

7. The pharmaceutical composition according to claim 1, wherein the subject is 18 years of age or older.

8. The pharmaceutical composition according to claim 1, wherein the abnormal level of tyrosine phosphorylation includes an abnormal level of tyrosine phosphorylated protein zero-related (PZR).

9. The pharmaceutical composition according to claim 8, wherein a low dose of a Src family tyrosine kinase inhibitor reduces PZR tyrosine phosphorylation.

10. The pharmaceutical composition according to claim 1, wherein a low dose of a Src family tyrosine kinase inhibitor provides an antifibrotic effect in cardiac tissue.

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