DM-CHOC-PEN

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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

Fast Facts

4-Demethyl-4-cholesteryloxy-carbonylpenclomedine (DM-CHOC-PEN) (SBIR/NCI Funded CA132,257) OR Cl CH3O

Cl N

CCl3

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

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- CHOCPEN 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 DMCHOC-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 0.75 +/- 0.3 (HOOI) 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-CHOCPEN

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)

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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 Does not require hepatic activation Crosses the BBB

Non-neurotoxic

Transient changes

ADVANTAGE Single IV dose

BENEFIT Long half life – +48 hrs

Accumulates in CNS tumors; not normal brain tissue & not a substrate for Pgp transport. No behavioral or neurotoxicity noted in animal models

Can use with other agents – binds guanine @ N7 vs. O6 (TMZ)

Transient hepatic/lipid Δs; no bone marrow, renal, pulmonary, CV, neuro- toxicities noted.

Hepatic alterations & hyperlipidemia was transient ~ 36 - 48 hrs.

Cytotoxic to CNS cancers – both mets/primary lesions

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-CHOCPEN 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.