The very first Japanese Patent on PAK1-blockers (15K etc) for Cancer Therapy and Longevity

2017 Japanese Patent #JP6082488 (27/01, 2017)

Patentee:  Hiroshi Maruta, 14 Curtin Avenue, Brunswick West, Australia 3055.

Inventors:  Hideaki Takahashi, Yoshihiro Uto, Binh Cao Quan Nguyen, Pham Thi Be Tu, Shinkichi Tawata, Hiroshi Maruta

Representative: Ono International Patent Office, Tokyo, Japan.

Filing date :  16/03/2016 (Filing # 2016-052369)

Examined: 30/09/2016

Granted: 27/01/2017

The Invention Entitled:

Esters of acidic (COOH-bearing) PAK1-blockers, preparation, and their use in treatment of cancer and other PAK1-dependent diseases/disorders

The invention is described in the following statement:

*This invention is based on our finding in 2015 that 1,2,3-triazolyl esterization of COOH- bearing PAK1-blockers (ARC, CA and Ketorolac) via "Click Chemistry" boosts their anti-cancer activity by 100, >400 and >500 fold, respectively, mainly by increasing their cell-permeability without any loss of their water-solubility.

The invention claimed is: 

1.    PAK1-blocking esters of formula (1) or pharmaceutically acceptable salts thereof, for use in substantially blocking the abnormal activation of PAK1 in affected cells,

Formula (I)

wherein:

R₁ is selected from the “decarboxylated” forms of artepillin C (ARC), caffeic acid (CA), and R-ketorolac; and

R₂ is selected from a range of substituted groups including ortho-phenol, ortho-methoxy benzene, ortho-aniline, ortho-arginine phenyl ester, and ortho-aminohexyl benzene.

2.    A composition including a PAK-blocking ester of structural formula (1) or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically or cosmetically acceptable adjuvant, carrier or diluent.

3.    A method of treating PAK1 dependent diseases, that involves administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

4.    A method of suppressing PAK1 for treating diseases/disorders in which PAK1 is abnormally activated, that involves administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

5.    A method of treating cancer and other PAK1 dependent diseases according to claim 3, wherein PAK1 dependent diseases include any one or more of  solid tumors, Alzheimer’s disease, Parkinson’s disease, inflammatory diseases, multiple sclerosis and stomach ulcer, infectious diseases, diabetes (type 2), hypertension, epilepsy, schizophrenia, autism, depression, obesity, disorders such as hyper-pigmentation, alopecia (hair loss), pain and osteoporosis.

6.    A method of inhibiting formation and/or growth of a solid tumor according to claim 5, wherein the solid tumour include pancreatic cancer, colon cancer, breast cancer, prostate cancer, lung cancer, brain cancer (glioma), NF (types I and II), TSC, and multiple myeloma (MM).

7.    A method of treating a subject with cancers having an increased resistance to a conventional chemo-therapy or radio-therapy, including administering to the subject a therapeutically effective amount of a compound represented by structural formula (1) or a pharmaceutically acceptable salt thereof.

8.    A method of preventing either growth or metastasis of  cancers from a subject,

including administering to the subject a therapeutically effective amount of a compound represented by structural formula (1) or a pharmaceutically acceptable salt thereof.

9.    A method of preventing and/or treating a PAK1-dependent disease in a subject having an increased resistance to a conventional chemotherapy, the method including exposing the subject to an effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

10.  Cosmetics in general by administering a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof.

11.  A specific cosmetic method of improving skin-whitening according to claim 10.

12.  A specific cosmetic method of promoting hair growth according to claim 10.

Reference  concerning the above patent: 

Nguyen　BC,  Takahashi　H, Uto　Y,  Shahinozzaman MD,  Tawata　S, Maruta　H.

1,2,3-Triazolyl ester of Ketorolac: a “Click Chemistry”-based highly potent PAK1-blocking cancer-killer. Eur. J. Med. Chem, 2017, 126, 270-6. 

*We are currently seeking for a potential buyer (pharmaceutical partner etc ) of the exclusive license based on the above patent (Contact: maruta20420@yahoo.co.jp).

Innovative (Survivin-based) or (p21-based) cell culture screening system for highly cell-permeable PAK1-blockers

Several years ago, a unique adenoviral construct called “Ad-Surv-GFP” was developed originally in order to detect bladder cancers in urine of patients (1). This construct carries a fusion gene in which the promoter region (around 250 bp) of survivin gene is fused to cDNA of GFP, so that GFP is expressed under the control of survivin gene promoter. When bladder carcinoma cells are infected with this virus, GFP is expressed at a very high level, while the infected normal cells express GFP only at a very low level (1).  According to both our recent unpublished observation (2, the surviving-suppressor YM155 blocks PAK1 in cell culture) and the down-regulation of survivin expression in PAK1-deficient mice (3), it is now clear that PAK1 is essential for the activation of survivin gene promoter (Surv). Thus, in principle, this adenoviral construct or more preferably a mammalian expression vector harbouring Surv-LacZ fusion gene could be used for the GFP/LacZ-based screening of potent (highly cell-permeable) PAK1-blockers in cell culture of RAS-transformants such as A549 lung cancer cells and pancreatic cancer cells where PAK1 is abnormally activated. In general, "LacZ" signal is more readily detectable than "GFP" signal.

Interestingly, COX-2 gene promoter also requires PAK1 for its activation (4). Thus, there remains  another  possibility that COX-2-LacZ vector also could be used to screen for PAK1-blockers in cell culture. The most critical “technical” question is which promoter is more sensitive to PAK1 or PAK1-blockers. 

Alternatively, p21/WAF (CDK inhibitor) gene promoter could be used as a positive indicator for anti-PAK1 activity, because more than a decade ago we have shown that PAK1 down-regulates selectively the p21 expression (5). Unfortunately, however, "p21 promoter-LacZ " fusion construct has not been commercially available. However, recently I found that a few groups have used either "p21-promoter-LacZ (or -Luciferase) " constructs for cells or whole mice. That is very encouraging! 

In C. elegans (CL2070), we have successfully used “HSP16.2 gene promoter-GFP” fusion construct as a “positive” indicator for anti-PAK1 activity, and PAK1-blockers such as ARC and CAPE from propolis indeed boost the GFP expression in this worm (6).

References: 

 

1. Murali A, Kasman L, Voelkel-Johnson C. Adenoviral infectivity of exfoliated viable cells in urine: implications for the detection of bladder cancer. BMC Cancer. 2011; 11: 168.
2.  Nguyen BC, Ahn MR, Tawata S. Maruta H. YM155, the survivin suppressor, blocks the oncogenic/ageing kinase PAK1, and inhibits the migration of HUVECs in cell culture. manuscript in preparation.
3. Chen YC, Fueger PT, Wang Z. Depletion of PAK1 enhances ubiquitin-mediated survivin degradation in pancreatic beta-cells. Islets. 2013 ; 5: 22-8.
4. R Wu, AL. Abramson, MH. Symons, BM. Steinberg. Pak1 and Pak2 are activated in recurrent respiratory papillomas, contributing to one pathway of Rac1-mediated COX-2 expression. Int J Cancer. 2010, 127, 2230-7. 
5. Nheu T, He H, Hirokawa Y, Walker F, Wood J, Maruta H. PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition. Cell Cycle. 2004 ; 3: 71-4
6. Yanase, S； Luo Y；Maruta H. PAK1-deficiency/down-regulation reduces brood size, activates HSP16.2 gene and extends lifespan in C. elegans. Drug Discov. Ther. 2013, 7 (1), 29–35.