COTI-2 is a small molecule discovered by Critical Outcome Technologies to reactivate mutant p53 through zinc chelation. It demonstrates strong efficacy against a wide range of p53 mutations in vitro and significant tumor growth inhibition in vivo without inducing resistance. Further studies confirmed COTI-2's p53-dependent mechanism of action and ability to modulate the PI3K/AKT pathway. An IND filing is planned in mid-2014 to study COTI-2 in gynecological cancers.
2. FORWARD LOOKING STATEMENTS
When used anywhere in this presentation, the words expects, believes, anticipates,
estimates, and similar expressions are intended to identify forward-looking
statements. Forward-looking statements herein may include statements addressing
future financial and operating results for Critical Outcome Technologies Inc., (COTI).
COTI has based these forward-looking statements on its current expectations about
future events. Such statements are subject to risks and uncertainties including, but
not limited to, the successful implementation of COTI’s strategic plans, the
acceptance of new products, the obsolescence of existing products, the resolution
of existing and potential future patent issues, additional competition, changes in
economic conditions, and other risks described in documents COTI has filed with
the Toronto Stock Exchange and Ontario Securities Commission.
All forward-looking statements in this document are qualified entirely by the
cautionary statements included in this document and such filings. These risks and
uncertainties could cause actual results to differ materially from results expressed
or implied by forward-looking statements contained in this document. These
forward-looking statements speak only as of the date of this document.
2
3. 3
COTI-2 INTRODUCTION
• COTI-2 is a small molecule with a novel mechanism of action, discovered by our
CHEMSAS process
– Originally thought to be an AKT modulator/inhibitor
• It is a 3rd generation thiosemicarbazone engineered for low toxicity and easily
synthesized in 3 steps
• It demonstrates strong in vitro and in vivo efficacy
• COTI-2 is effective in cancer cells/tumors with many common p53 mutations
N
N
N
H
S
N
N
N
3rd Generation
Thiosemicarbazone
4. 4
COTI-2 IC50 STRONGLY CORRELATED WITH p53 STATUS
• TP53 is the most frequently mutated gene in human cancer with mutation
frequencies ranging from 38% to 96%1
– Most frequent mutations occur in 6 “hotspot” codons2
• 23 cell line were evaluated for the relationship between their COTI-2 IC50 and p53
“hotspot” mutational status (amino acid residues 175, 248, and 273)
– COTI-2’s efficacy was strongly associated with the presence of p53 “hotspot” mutations
(Fisher exact, p = 0.0045)
1Freed-Pastor WA & Prives C (2012) Genes Dev. 26: 1268-1286.
2Yu X et al (2012) Cancer Cell, 21: 614-625..
LOW IC50
(< 250 nM)
HIGH IC50
(≥ 250 nM)
TOTAL
Hotspot p53 Mutations
(175, 248, 273)
13 0 13
WT p53 5* 5 10
TOTAL 18 5 23
Common p53 Gene Mutations
COTI-2 Efficacy Associated with p53 Hotspot Mutations
~28% of human cancers
* These cell lines also have mutations in p73 or the
PI3K/AKT/mTOR pathway
5. -10.00
-9.00
-8.00
-7.00
-6.00
-5.00
-4.00
-3.00
5
COTI-2 IC50 DISTRIBUTION IN HUMAN CANCER CELL LINES
• COTI-2 IC50 distribution in various human cancer cell lines as illustrated by a
waterfall chart shows 3 major groups of cell lines
Normal Human WBCs
MCF7 and NCI-H460:
WT p53 + PIK3CA mutation
U87MG:
WT p53 + PTEN mutation
NCI-H292:
WT p53 + p73 mutation
HCT116:
WT p53 + PIK3CA mutation
PANC-1:
p53 (R273H)
OVCAR-3 PK/PD xenograft
p53 (R248G)
GROUP 1 GROUP 2 GROUP 3
p53 = Wild-type
Mutants = p53 Wild-type AND p73, PI3K, or PTEN Mutant
p53 = Null Mutants
p53 = Hotspot & Non-Hotspot Mutants
6. 6
COTI-2 INDUCES A CONFORMATIONAL CHANGE IN MUTANT P53
• COTI-2 induces a ‘wild-type-like’ conformational change in mutant p53R175H in TOV-
112D ovarian cancer cell line
• Data also suggests a mutant p53 conformational change also occurs in PANC-1
(p53R273H) pancreatic and OVCAR-3 (p53R248Q) ovarian cancer cell lines
Experimental Design
• Fluorescently labeled antibodies specific for wild-type & mutant p53 used to assess cells grown in the presence/absence of COTI-2
• PAB1620-stained cells show wild-type p53 and PAB240-stained cells show mutant p53
• Cy3 refers to the fluorescent signal emitted by the fluor attached to the antibodies
• DAPI-staining indicates intact nuclei and provides the assurance that the cells are intact
7. 7
COTI-2 HAS NO AFFECT ON P53 WILD-TYPE CONFORMATIONAL STATUS
• COTI-2 has no affect on the wild-type p53 conformation as demonstrated by the
H460 NSCLC cell line, which was used as a control
Experimental Design
• Fluorescently labeled antibodies specific for wild-type & mutant p53 used to assess cells grown in the presence/absence of COTI-2
• PAB1620-stained cells show wild-type p53 and PAB240-stained cells show mutant p53
• Cy3 refers to the fluorescent signal emitted by the fluor attached to the antibodies
• DAPI-staining indicates intact nuclei and provides the assurance that the cells are intact
8. 8
COTI-2 TARGETS P53 MUTANT PROTEIN
• COTI-2 significantly reduces p53 mutant protein levels and significantly increases
wild-type p53 protein levels in TOV-112D cells likely by inducing a conformational
change
• COTI-2 has no significant effect on p53 protein levels in the H460 cell line, which
do not carry the mutant p53 protein
• (*) Significant difference in p53 protein levels between COTI-2 treated and untreated cells (control)
Mutant p53 Levels in the Presence/Absence of COTI-2 Wild-type p53 Levels in the Presence/Absence of COTI-2
0
20
40
60
80
100
120
TOV-112D H460
MeanFluorescenceIntensity
(ArbitraryUnits)
Cell Line
Control
COTI-2
*
0
20
40
60
80
100
TOV-112D H460
MeanFluorescenceIntensity
(ArbitraryUnits)
Cell Line
Control
COTI-2
*
9. 9
COTI-2 AFFECTS A WIDE RANGE OF P53 MUTATIONS
• COTI-2 inhibited a wide range of p53 mutations at concentrations 1.0 M
– Fifteen p53 mutations were inhibited by COTI-2 (shown below in red)
– The p53 mutations correspond to those most frequently occurring in various human
cancers
Experimental Design
• HCT116 p53 (-/-)
constructs were
transfected with
plasmids bearing various
p53 mutant genes
• Each transfectant was
exposed to 1.0 M of
COTI-2 for 72 hrs and
viability assessed
• The cell viability was
determined using the
CellTiter-Blue® Assay
Unlike other p53 targeted drugs in development COTI-2 targets a wider range of p53 mutations
N C
S
G C
H L
S C R W G Q C G S R Y
V D A L P W L H P S F
R175 C275R273R248G245Y220
10. 10
COTI-2 REACTIVATES MUTANT P53
• COTI-2 reactivates the tumor suppressor properties of p53 as demonstrated by the
induction of p21, which is a p53-dependent gene
– COTI-2 induces p21 at 3 and 12 h post-exposure in both the HCT116 p53 (R175H) and
(G245C) mutant constructs
– No difference in p21 protein levels were observed in the HCT116 p53 (-/-) construct
Experimental Design
• Cells were incubated with/without COTI-2 and protein extractions performed at 2 and 12 h post-incubation
• Protein levels were detected via western blots using p53, p21, and Erk2 specific antibodies
1 2 3 4
HCT116 p53 (R175H) HCT116 p53 (G245C)HCT116 p53 (-/-)
3h
- +
12h
- +
1 2 3 4
p53
p21
Erk2
COTI-2
3h
- +
12h
- +
3h
- +
12h
- +
1 2 3 4
11. COTI-2 REACTIVATES MUTANT P53 THROUGH ZINC CHELATION
• 8 genes involved in metal ion chelation up-
regulated by COTI-2 (p<0.01)
• Iron chelation unlikely to be significant effect
• Pre-treatment of p53 mutant cell lines with TPEN
markedly reduces COTI-2 effectiveness
• COTI-2 increases intracellular zinc concentrations
• Experiments are ongoing to better understand
the role of zinc in COTI-2 effectiveness
11
12. 12
CONFIRMATION OF P53-DEPENDENT MOA
• Gordon Mills, MD., PhD., Chair of the Department of Systems Biology and the Co-
Director of the Khalifa Institute for Personalized Cancer Therapy at The University
of Texas MD Anderson Cancer Center in Houston, Texas conducted a number of
cell-based experiments to further elucidate the activity of COTI-2 on various p53
mutations
• Dr. Mills’ research confirmed
– COTI-2 is most active in mutant p53 tumors
– COTI-2’s activity in many specific p53 mutations is striking
– COTI-2’s activity can be augmented by the presence of mutations in the AKT signaling
pathway
– Results of in vitro/in vivo studies in combination with the potential MOA warrant further
clinical development
“These results are encouraging given the central importance of p53 gene mutations in many cancers”
Dr. Gordon Mills, MD Anderson Cancer Center
13. 13
COTI-2 INDUCED NO SIGNIFICANT RESISTANCE
• COTI-2 induced no significant resistance through 5 generations in both cell lines
• Both Cisplatin and Placlitaxel induce significant increases in IC50 after first
generation of selection compared with COTI-2
• COTI-2 induced a statistically significant decrease in IC50’s (i.e., Collateral
Sensitivity)
0
0.5
1
1.5
2
2.5
3
Parental
cells
Round1
selection
Round2
selection
Round3
selection
Round4
selection
COTI-2
COTI-219
Cisplatin
Paclitaxel
0
1
2
3
4
5
6
7
8
Parental
cells
Round1
selection
Round2
selection
Round3
selection
Round4
selection
COTI-2
COTI-219
Cisplatin
Paclitaxel
IC50Ratio
DMS153 SCLC SHP77 SCLC
IC50Ratio
14. 14
COTI-2 PRODUCED SIGNIFICANT TGI AS A SINGLE AGENT
• COTI-2 administered PO produced a significant tumor growth inhibition as a single
agent in the OVCAR-3 ovarian cancer tumor model
Experimental Design
• OVCAR3 human tumor cells
inoculated subcutaneously in
the right and left flanks of
female athymic mice (NIH III
nu/nu), allowed to grow up to
75 to 100 mm3
• Groups of 12 mice each were
dosed PO with COTI-2 in
phosphate citrate buffer (75 or
100 mg/kg) with a schedule of
5X per week until study end
• Tumor volumes were graphed
as means (SE) and significant
difference between groups was
determined using Student’s T-
test (p<0.05)
• (*) Tumors significantly reduced by COTI-2 in all treatment groups relative to vehicle control
0
50
100
150
200
250
0 5 9 16 23 30 37 44 51 61
TumorVolume(mm3)
Study Day
Group 4 = Vehicle PO
Group 5 = COTI-2 75mg/kg PO
Group 6 = COTI-2 100mg/kg PO
Effect of Treatment on Tumor Volume
*
*
*
* *
*
* *
* *
* *
15. 15
SUMMARY
• COTI-2 is a small molecule with a novel mechanism of action, discovered by our
CHEMSAS process
• MOA
– COTI-2 appears to restore wild-type like functional activity to a wide range of p53
mutants through zinc chelation
• In vitro efficacy
– COTI-2 demonstrates nanomolar activity in multiple human cancer cell lines
– COTI-2 did not induce cross-resistance compared to cisplatin and paclitaxel
• In vivo efficacy
– When administered PO, COTI-2 induced significant tumor growth inhibition (85.5%) in an
ovarian tumor xenograft model without any overt toxicity in mice
• Clinical development strategy
– IND filing is planned for mid-2014
– Gynecological (ovarian, endometrial, and cervical) cancer patients failing 1st line therapy
• FDA Orphan status granted June, 2014 “for the treatment of ovarian cancer”
16. 16
COTI-2 NEGATIVELY MODULATES THE PI3K/AKT/MTOR PATHWAY
1Hennessy BT et al (2005) Nature Rev Drug Discov, 4: 988-1004.
• This pathway has been implicated in cancer and is involved in cell proliferation,
survival, motility and morphology1
• COTI-2 negatively modulates the PI3K/AKT/mTOR pathway
– COTI-2 directly inhibits PI3K via the upregulation of PIK3IP1
– Increased/restored p53 function stimulates PTEN activity
– Increased/restored p53 function leads to AKT protein degradation
PI3K
AKT
MDM2
p53
FOXO1 BAD GSK3B mTOR S6KCASP-9
RTK
Apoptosis
Cell cycle regulation
DNA repair
Apoptosis Cell cycle regulation Growth
Translation
PIK3IP1
PTEN
COTI-2 Upregulates PIK3IP1
Study # COTI-07-101