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

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

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.

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

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.

RESULTS

B-16 mouse melanoma cells grow well in culture.

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.

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)]

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

RESULTS

Cell death patterns seen for DOX, TMZ, Act-D

and other agents - no melanin generated only cell

ghosts.

DM-CHOC-PEN vs. TMZ and B-16 Mouse Melanoma

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) ]

Cross Section of a Melanocyte & its ‘Aurora’

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)

********

DM-CHOC-PEN – Primary MOA

• Pseudo-alkylating via trichloromethyl moiety

• Trichloromethyl → dichloromethylene carbonium ion

• Adducts w/ guanine-N7 of DNA

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].

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.

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.

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.

ACKNOWLEDGEMENT

Grant support from the NCI/SBIR – R43/44

CA85021 and R43/44 CA132257 is

appreciated.