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"Mutation Mimicking Compounds That Bind to the Kinase Domain of Egfr" in Patent Application Approval Process

June 2, 2014

By a News Reporter-Staff News Editor at Clinical Trials Week -- A patent application by the inventors Berezov, Alan (West Hollywood, CA); Greene, Mark I. (Penn Valley, PA); Minkovsky, Natalie (Philadelphia, PA); Cai, Zheng (Wynnewood, PA); Zhang, Hongtao (Paoli, PA), filed on March 16, 2012, was made available online on May 22, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also The Trustees Of The University Of Pennsylvania).

This patent application is assigned to The Trustees Of The University Of Pennsylvania.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The erbB or EGFR family is a subclass of cell surface receptors with intrinsic tyrosine kinase activity known as receptor tyrosine kinases (RTKs). The EGFR family comprises four members: EGFR (also known as erbB1) itself, erbB2 (HER2/Neu), erbB3 and erbB4. EGFR plays a critical role in normal embryonic development and is also known to drive the growth of tumors.

"As for most RTKs the first step in the activation of EGFR is ligand induced receptor dimerization. The intracellular kinase domains in the ligand-induced EGFR dimer become activated by autophosphorylation in trans. Subsequent phosphorylation on tyrosines in the regulatory C-terminal tail creates binding sites for the recruitment of multiple downstream signaling molecules via interactions with their SH2 domains.

"Abberrant EGFR activation, resulting in EGFR overexpression (known as upregulation) or overactivity is strongly implicated in a cancers, including anal, breast, ovarian, head and neck, lung, pancreatic, and colorectal cancers and glioblastoma multiforme, and is already the target of several anti-cancer therapeutics. Mutations, amplifications or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers.

"The identification of EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR. Two major strategies have been used for suppressing aberrant EGFR signalling: antibody targeting of the receptor ectodomain and small molecule inhibition of the tyrosine kinase domain. The antibody approach provides high target specificity, but has limitations and challenges in drug development because of the protein nature of the therapeutic agent, including cost and delivery. Cetuximab and panitumumab are examples of monoclonal antibody inhibitors. Other monoclonals in clinical development are zalutumumab, nimotuzumab, and matuzumab. The monoclonal antibodies block the extracellular ligand binding domain. With the binding site blocked, signal molecules can no longer attach there and activate the tyrosine kinase.

"Another method of inhibiting abberant EGFR signalling is to use small molecules to inhibit the EGFR tyrosine kinase, which is on the cytoplasmic side of the receptor. Without kinase activity, EGFR is unable to activate itself, which is a prerequisite for binding of downstream adaptor proteins. Ostensibly by halting the signaling cascade in cells that rely on this pathway for growth, tumor proliferation and migration is diminished. Gefitinib, erlotinib, and lapatinib (mixed EGFR and ERBB2 inhibitor) are examples of small molecule kinase inhibitors which function as competitive TKIs by reversibly binding to the ATP site on the EGFR kinase domain. See e.g., Lynch, et al., 'Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib,' N. Engl. J. Med., 350: 2129-39 (2004); Paez, et al., 'EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy,' Science, 304: 1497-1500 (2004), each of which is incorporated by reference in its entirety.


"The advantages of small-molecule drugs over therapeutic proteins include the ease of manufacturing and administration, the potential for oral dosing, low immunogenity and applicability to a wider range of disease targets, including those inside the cell. Indeed, small molecule inhibitors of the tyrosine kinase domain of EGFR (i.e., Iressa.RTM. and Tarceva.RTM.) have been successfully developed as drugs, which directly target the EGFR. But not all patients can benefit from such drugs. Patients can be been divided into EGFR positive and negative, based upon whether a tissue test shows a mutation. One of the most common mutation that sensitizes tumors to small molecule tyrosine kinase inhibitors is the so-called L858R mutation, wherein Leu-858 in the EGFR peptide sequence is replaced by an Arg-858 (so-called 'L858R mutation'). EGFR positive patients have shown an impressive 60% response rate which exceeds the response rate for conventional chemotherapy. For example, see Jackman D M, et al., 'Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials'. Clin. Cancer Res. 15 (16): 5267-73 (August 2009). However, this mutation which allows for this success exists in only in a small sub-population (ca. 5%) of non-small cell lung cancer patients that harbor this particular mutation in the tyrosine kinase domain of EGFR. Minna, et al., 'Cancer. A bull's eye for targeting lung cancer therapy,' Science, 304: 1458-61 (2004), which is incorporated by reference herein in its entirety.

"In the metastatic setting, EGFR mutations are strong predictors of efficacy for the EGFR tyrosine kinase inhibitors (TKIs), erlotinib (Tarceva.RTM.) and gefitinib (Iressa.RTM.). Patients whose tumors harbor EGFR L858R mutations display a >70% radiographic response rate in prospective trials, including randomized phase III trials. Compared to those with EGFR wild-type tumors, patients with EGFR mutant tumors display a longer progression-free survival on EGFR TKI therapy than those who receive chemotherapy. Patients with metastatic EGFR mutant tumors treated with 'first-generation' EGFR TKIs have a median survival of more than two years. Prolonged survival may also be due to the fact that patients with EGFR mutant tumors have a better prognosis in general compared to those with EGFR wild-type tumors. Patients with EGFR mutant tumors treated with an EGFR TKI in the first-line setting may live longer than those treated in the second-line setting (30.5 months vs. 23.6 months, p=0.31).

"There is a need for small molecule pharmaceuticals which regulate the overexpression of EGFR so as to inhibit cell proliferative disorders characterized by over-activity and/or inappropriate activity of EGFR in a wider population of patients suffering from such disorders, including EGFR-related cancers."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "The present disclosure provides small molecule compounds capable of mimicking the effects produced by the drug-sensitizing kinase domain mutations known to occur in kinase domain of the epidermal growth factor receptor (EGFR). Without intending to be bound by any particular theory, it may be that these small molecules affect the kinase domain of EGFR so as to alter the signaling properties of the EGF receptor, make EGFR expressing tumor cells more EGFR dependent and thus sensitize these tumor cells to inhibition with the traditional TKI inhibitors. As such, the small molecules of the present invention can have the effect of improving the reach and efficacy of traditional EGFR and/or Src tyrosine kinase inhibitors (TKIs).

"Certain embodiments of the present invention provide pharmaceutical compositions comprising a compound capable of mimicking the L858R mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in an amount effective to mimic the mutation. Additional embodiments of these compositions characterized these compounds using exemplary structures, binding affinities, and using biological and other test data. Use of these compounds in the preparation of such compositions or medicaments is also considered.

"Other embodiments further provide that these pharmaceutical compositions comprise an EGFR tyrosine kinase inhibitor in an amount effective to inhibit a cell proliferative disorder characterized by over-activity and/or inappropriate activity of the EGFR, exemplary tyrosine kinase inhibitors being erlotinib (Tarceva.RTM.), gefitinib (Iressa.RTM.), or lapatinib (Tykerb.RTM.).

"Still other embodiments provide methods of inhibiting a cell proliferative disorder characterized by over-activity and/or inappropriate activity of a receptor comprising administering a pharmaceutically effective amount of a composition comprising a compound capable of mimicking the L858R mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR).

"Other embodiments provide method of treating a patient having a disease characterized by over-activity and/or inappropriate activity of an epidermal growth factor receptor (EGFR), comprising the step of administering to a patient in need of such treatment a pharmaceutical composition comprising a compound capable of mimicking the L858R mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in an amount in an amount effective to mimic the L858R mutation in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR).


"FIG. 1 provides an illustration of one possible representation of the binding of Tarceva.RTM. and one MMC (EEO3) in the cavities of the EGFR. The upper panel depicts a possible EGFR-Tarceva.RTM. complex, in which Tarceva.RTM. binds to the active site of the EGFR kinase close to the L858 residue. The lower panel depicts a possible binding conformation of EEO3 to the wild type (WT) EGFR-Tarceva.RTM. complex near the mutation site produces mutation-like effects by stabilizing the EGFR-Tarceva.RTM. complex and sensitizing EGFR to Tarceva.RTM.-induced inhibition.

"FIG. 2 provides a second illustration of one possible representation of the binding of Tarceva.RTM. and one MMC (EEO4) in the cavities of the EGFR.

"FIG. 3 shows curves of percent fluorescence as a function of MMC concentration for EEO3 and EEO4, and the associated binding constants derived therefrom.

"FIG. 4 illustrates the effect of the EGFR kinase inhibitors on inhibition of tumor cell proliferation by Tarceva.RTM.. The inhibitory effect of 20 .mu.M Tarceva.RTM. on proliferation of EGFR-expressing NE99 (FIG. 4A) and EGFR-negative T6-17 (FIG. 4B) cell lines has been tested in the absence (closed bars over 'Buffer') and presence (closed bars over MMCs) of 0.5 mg/ml concentrations of the designed EGFR kinase inhibitors. The inhibitory effects of each compound in the absence of Tarceva.RTM. (open bars) are shown as controls. The compounds enhance the inhibitory effect of Tarceva.RTM. on proliferation of the NE99 tumor cells that have high expression levels of EGFR. At the same concentrations, the compounds have no effect on non-EGFR-specific inhibition of T6-17 cell proliferation by Tarceva.RTM.. FIG. 4C shows results of a broader range of testing.

"FIG. 5 illustrates the effect of EEO3 and EEO4 on the enzymatic activity of a purified EGFR kinase (FIG. 5A) and on the inhibition of EGFR kinase by Tarceva.RTM. (FIG. 5B). The kinase activity was measured using a standard .sup.33P radioisotope assay that has been described in previous publications.

"FIG. 6 is a Western blot analysis illustrating the effect of EEO3 on the inhibition of EGFR phosphorylation by Tarceva.RTM. in NE91 cells. EGF-induced phosphorylation of EGFR was tested in the presence and absence of Tarceva.RTM. alone and Tarceva.RTM.+EEO3 combination. Total EGFR levels were measured as a control. FIG. 6B shows the effect of Tarceva.RTM. and EEO3 on the phosphorylation in mouse fibroblasts expressing L858R EGFR.

"FIG. 7 shows two Western Blot analyses showing the effect of EEO3 and Tarceva.RTM. on EGFR phosphorylation and downstream signaling.

"FIG. 8 illustrates the effects of EEO3 and Tarceva.RTM. at 0.25 .mu.M (FIG. 8A) and 1 nM (FIG. 8B) concentrations on anchorage independent growth of NE91 cells. Numbers of soft agar colony formations after three weeks with no treatment, with Tarceva.RTM. alone, EEO3 alone or Tarceva.RTM. plus EEO3 combination were counted using the AlphaImager 2000 imaging system.

"FIG. 9 illustrates the effect of MMCs on the apoptotic activity of Tarceva.RTM. in wild type (non-mutated) and mutant EGFR expressing cell lines. The cells were incubated in RPMI medium with 0.5% FBS in the presence of 1 .mu.M Tarceva.RTM., 1 .mu.M compound or Tarceva.RTM.+compound combination for 48 hrs, then stained with annexin V and analyzed by FACS.

"FIG. 10 graphically represents the results of in vivo testing of EEO3 (A) and EEO4 (B) in mice with AsPC1 tumors.

"FIG. 11 shows the effects of EEO3 and EEO4 on Tarceva.RTM.-induced inhibition of cell proliferation in human lung cancer cell line A549.

"FIG. 12 identifies wild-type amino acid (SEQ ID NO: 2) and corresponding nucleic acid sequences (SEQ ID NO: 1) for the human EGFR kinase domain. Residue number 1 of SEQ ID NOS: 1 and 2 corresponds to residue 695 of FIG. 12, and the remaining residues are numbered consecutively in a corresponding manner (for example, the 858 position in FIG. 12, corresponds to the 164 position of SEQ ID NOS: 1-3). Further, SEQ ID NO: 3 provides an amino acid sequence of human EGFR wherein the leucine residue at position 164 is substituted with arginine (L858R mutation)."

URL and more information on this patent application, see: Berezov, Alan; Greene, Mark I.; Minkovsky, Natalie; Cai, Zheng; Zhang, Hongtao. Mutation Mimicking Compounds That Bind to the Kinase Domain of Egfr. Filed March 16, 2012 and posted May 22, 2014. Patent URL:

Keywords for this news article include: Antibodies, Antineoplastics, Pharmaceuticals, Drugs, Genetics, Oncology, Gefitinib, Lapatinib, Immunology, Chemotherapy, Therapeutics, Blood Proteins, Lung Neoplasms, EGFR Inhibitors, HER2 Inhibitors, Immunoglobulins, Protein Kinases, Membrane Proteins, Peptide Receptors, Cell Proliferation, Phosphotransferases.

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Source: Clinical Trials Week

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