activity of 4-demethyl-4 ...5964adcf6c97e3ebad92-2d059e2ef73e5cbb778fc7d34841d99d.r25.… · b-16...
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Activity of 4-Demethyl-4-cholesteryloxycarbonylpenclomedine
(DM-CHOC-PEN) in Melanoma
P. Friedlander L.R. Morgan, E. Benes, A.H. Rodgers, R. Weiner, M.
Ware
Mount Sinai Medical Center, New York, NY; DEKK-TEC, Inc.,
New Orleans, LA; Tulane University Medical Center, Ochsner
Medical Center, New Orleans, LA
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Fast Facts
4-Demethyl-4-cholesteryloxy-carbonylpenclomedine
(DM-CHOC-PEN)
(SBIR/NCI Funded CA132,257)
Penclomedine analogs – PEN (R=CH3); DM-PEN (R=H);
DM-CHOC-PEN (R= - CO2 -cholesteryl)
DM-CHOC-PEN is a lipophilic polychlorinated neutral pyridine &
penetrates the BBB
N
OR
Cl
CCl3CH3O
Cl
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DM-CHOC-PEN Anti-CA Activity
• In Mice with -
Intracerebrally Implanted (IC) Human Xenografts
- U251 Glioblastoma – 20% CR (+LTS)
- D54 Glioblastoma – 20% CR (+LTS)
- MX-1 Breast Cancer – 17% (+LTS)
• In vitro
- Human – breast, NSCLC/SCLC, endometrial,
ovarian, GBM, pineal body tumor, schwannomas,
meningiomas.
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SPECIFIC AIMS
• Evaluate DM-CHOC-PEN’s anticancer activities in
a mouse melanoma model
• Study mechanism(s) of action for DM- CHOC-
PEN in a mouse melanoma model
• Compare anti-cancer activity with standard
chemotherapy in the above model
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METHODS
• Drugs – Dissolved in DMSO/tissue culture media,
saline or a egg yolk, soy bean oil, glycerin, water
emulsion.
• In vitro – cells grown in complete RPMI media @
36 C & 5% CO2.
• In vivo – B-16 cells implanted SC and animals
treated IP and PO 5-7 days after inoculation.
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RESULTS
B-16 mouse melanoma cells grow well in culture.
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RESULTS
B-16 mouse melanoma cells treated in vitro with DM-
CHOC-PEN
DM-CHOC-PEN induced intra-cellular melanin
production and cell death day-3.
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RESULTS
B-16 mouse melanoma cells treated with DM-CHOC-PEN
plus a free radical trapping agent (DCF-acetate)
B-16 cells plus DCF acetate B-16 cells plus DM-CHOC-
PEN + DCF-acetate
[Florescence is due to the release of DCF and oxidation
by free radical species (FRS)]
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Other Drugs and B-16 Melanoma
Drug IC50 (µg/mL)
DM-CHOC-PEN 0.5 +/- 0.01
Actinomycin D (Act-D) 0.5 +/- 0.02
cis-Platinum 1.5 +/- 0.1
4-Hydroperoxyifosfamide
(HOOI)
0.75 +/- 0.3
Doxorubicin (DOX) 0.7 +/- 0.1
Temozolamide (TMZ) >3.0
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RESULTS
Cell death patterns seen for DOX, TMZ, Act-D
and other agents - no melanin generated only cell
ghosts.
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DM-CHOC-PEN vs. TMZ and B-16 Mouse Melanoma
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HISTOLOGY
B-16 mouse melanoma model treated with DM-CHOC-
PEN
Histology of B-16 melanoma SC tumors - saline controls vs.
DM-CHOC-PEN treated mice.
[Notice the pigment laden cells w/vacuoles similar to what was seen
in vitro (arrows) ]
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Cross Section of a Melanocyte & its ‘Aurora’
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MELANIN – Chemistry
• Melanin - polymer of indole-5,6-quinone (Rapier)
• DOPA → DOPA-5,6-quinone + free radical species
• DOPA → 14 electrons & -19.8 kcal/mole on
oxidation
• Rich source of energy
• A one-dimensional semi-conductor for energy.
• High conjugated electronic resonance that transmits
electrons (mini-electron beam generator)
********
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DM-CHOC-PEN – Primary MOA
• Pseudo-alkylating via trichloromethyl moiety
• Trichloromethyl → dichloromethylene carbonium ion
• Adducts w/ guanine-N7 of DNA
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DM-CHOC-PEN – Toxicities
• Hepatic – hyperbilirubinemia (animals w/liver mets)
• No hematological
• No renal or cardiac
• No GI or neurotoxicity
• No pulmonary
**************************************
The above is pre-clinical mouse, rat, dog support data.
Presented – FDA, 2010 [IND 68,876].
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FACTS & FEATURES
(DM-CHOC-PEN)
FEATURE ADVANTAGE BENEFIT
Does not require
hepatic activation
Single IV dose
Long half life – +48 hrs
Crosses the BBB
Accumulates in CNS tumors; not normal brain tissue & not a substrate for Pgp transport.
Can use with other agents – binds guanine @ N7 vs. O6 (TMZ)
Non-neurotoxic
No behavioral or neurotoxicity noted in animal models
Cytotoxic to CNS cancers – both mets/primary lesions
Transient changes
Transient hepatic/lipid Δs; no bone marrow, renal, pulmonary, CV, neuro- toxicities noted.
Hepatic alterations & hyperlipidemia was transient ~ 36 - 48 hrs.
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CONCLUSION
In vitro, DM-CHOC-PEN had an improved IC50
vs. TMZ, cis-platinum, doxorubicin and other
experimental agents.
Evidence is presented that a mouse melanoma
model has improved survival with DM-CHOC-
PEN vs. TMZ and saline controls.
Cell death from DM-CHOC-PEN treatments
correlated with accumulation of intracellular
melanin and free radical specie generation.
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CONCLUSION (continued)
• Melanin cells, as seen in Fig. 2. represent classical
cancer cells with a resting low free energy (ΔF) and
high entropy (ΔS) – the alpha state.
• The interactions of DM-CHOC-PEN and DOPA
induced the formation of melanin – a high energy
component/storage that resulted in an increase in
cellular ΔF and a decrease in ΔS and the
development of highly pigmented cells in a new
resting high energy state – the beta state.
• The latter cells are in a well differentiated state and
die.
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ACKNOWLEDGEMENT
Grant support from the NCI/SBIR – R43/44
CA85021 and R43/44 CA132257 is
appreciated.