A Priori Activation of Apoptosis Pathways of Tumor (AAAPT) Technology 1: Sensitization of Tumor Cells Using Targeted and Cleavable Apoptosis Initiators in Gastric Cancer

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. The principle route of desensitization includes activation of survival pathways (e.g. NF-kB, PARP) and downregulation of cell death pathways (e.g. CD95, ASK1). As a result, it requires high dose of therapy to induce cell death which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing low-responsive and resistant tumor cells. Here we report a novel tumor sensitizer derived from the natural Vitamin E analogue (AMP-001). The drug design is based on a novel “A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate cell death pathways and inhibit survival pathways simultaneously. It involves an inbuilt targeting vector which targets tumor specific Cathepsin B, overexpressed by many cancers including gastric cancer. Our results indicate that AMP-001 sensitizes gastric cancer cells which resulted in expanding the therapeutic index of front-line chemotherapy doxorubicin both in vitro and in vivo nude mouse model. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AMP-001 as a neoadjuvant to chemotherapy to achieve a better efficacy and reduced off-target toxicity.


27
Tumor cells have a remarkable ability to circumvent endogenous and exogenous toxicities by deactivating 28 cell death pathways and thereby desensitizing themselves to interventions 1 . Defects in apoptosis pathways 29 (e.g. CD95, APO-1/Fas) would make tumor cells insensitive to chemotherapy 2 . For example, loss of CD95

45
The overall 5-year survival rates for the patient group with high AI (> 0.97) and low AI (< 0.97, p =0.001)

46
were 89.6 % and 69.2 % respectively 11 indicating that AI could be a potential biomarker of risk stratification 47 peroxidase-conjugated secondary antibody (ABclonal) and diaminobenzidine colorimetric reagent solution

245
The pro-apoptotic effect of AMP-001 was assessed using Annexin V/ staining assay. As shown in tumor weight in the combinational group was significantly lower than either doxorubicin or AMP-001. (Fig   332  7B, p < 0.001). Additionally, all mice did not undergo obvious body weight loss or abnormal symptoms in 333 drug treatments (Fig 7D). In alternate studies on AMP-001 in triple negative breast cancer model, we have 334 shown that high dose of AMP-001 (~200 mg/Kg) alone can have significant tumor regression with no 335 observable toxicity, despite higher dose 12 . Similarly, in the current studies also, there was no evident 336 histopathological abnormalities were observed in the vital organs such as heart, liver and kidney estimated 337 by HE staining (Fig 8). Ki67 (marker for cell proliferation) and CD31 (marker for micro vessel density)

338
were also examined by immunohistochemistry in the excised tumor sections. Although, doxorubicin also 339 downregulated the Ki67 expression, the combination group was most effective similar to the tumor 340 regression studies. The trend of CD31 expression was consistent with Ki67 (Fig 8). In order to determine 341 whether AMP-001/Dox combination treatment effectively promotes apoptosis in tumors, sections of excised tumors were also subjected to TUNEL assay. The results showed AMP-001 together with 343 doxorubicin treatment had more TUNEL positive cells compared to either doxorubicin or AMP-001 (Fig   344  8). In summary, the combination of doxorubicin and AMP-001 yielded a superior response compared to 345 either AMP-001 or doxorubicin in xenograft gastric cancer animal model.  (Fig 9C). This pattern of normal beating for cardiomyocytes was seen at all 357 concentrations including 250 µM AMP-001 ( Fig 9D). AMP-001 showed IC-50 greater than 250 µM, 358 compared to dox at 9.6 µM (25 times less, Fig 9F) which indicates a better safety profile for AMP-359 001. Even FDA approved drug (Sorafinib ~ 40 µM) has lower IC-50 compared to AMP-001 ( Fig 9E).

360
It is to be noted that IC-50 for cardiomyocytes has to be high (measure of safety) in contrast with IC-  intervention. Hence, our first approach was to sensitize the low or non-responsive tumor cells by activating 394 CD95 pathway, one of the major dysregulated cell death pathways. The second approach was to take 395 advantage of activation technology to ascertain if it entitles synergy with the standard chemotherapeutics.

396
The significant decrease of IC 50 for the combination of doxorubicin and AMP-001 for gastric cancer cells 397 (Fig 3-4)  in normal cells, for example in brain leads to neuro degenerative diseases 13 and hence, targeting is essential.

405
Our preclinical studies in vitro and in vivo revealed that AMP-001 has dual effects; at low dose it acts 406 as a tumor sensitizer inducting a reasonable cell death while, at higher doses it shows anti-tumorigenicity 407 on its own with little or no observable toxicity in non-target organs 12 . However, for our current studies we 408 used AMP-001 as a tumor sensitizer. The main objective is to enhance the efficacy of the currently used 409 chemotherapeutics using AMP-001 as a neoadjuvant to chemotherapy. The evidence showed that AMP- treatments but limited in its use due its cardiotoxicity, the combination of doxorubicin and AMP-001 may