Conserved molecular mechanisms

underlying the effects of

small molecule chemotherapeutics

on cellsHEMANT SARIN, MD,

MScPH, ISTP

Why is it necessary to interrogate the conserved

biophysical properties of small molecule chemotherapies?

•Clinical efficacy remains questionable for the treatment of solid and hematopoietic malignancies

•Solid tumors in particular, for purposes of improving the tumor tissue selectiveness of enhanced permeation and retention (EPR)-based chemotherapy attached to optimally-sized nanoparticles

•Optimally-sized imageable dendrimer nanoparticle-based small molecule chemotherapy at ~9 nanometers (Hydrodynamic diameter = Dehydrated Diameters for such NPs)

•Sarin H: Effective transvascular delivery of chemotherapy into cancer cells with imageable nanoparticles in the 7 to 10 nanometer size range (Chapter 2). In: Current Advances in the Medical Application of Nanotechnology. Bentham Science Publishers Ltd.; 2012: 10-24

•Sarin, H. On the future development of optimally-sized lipid-insoluble systemic therapies for CNS solid tumors and other neuropathologies. Recent Patents on CNS Drug Discovery, 5 (3): 239-252, 2010

Traditional mechanisms of small molecule

chemotherapeutics based on:•Naked Target

–On protein receptor/enzyme in isolation, which provides information on relative binding affinities for intra-cellular proteins

•Cellular level in vitro–Provides information on the inhibitory concentrations needed to achieve tumor cell death and the overexpression status of induced pro- or anti-apoptotic protein forms

–But does not take into consideration cell membrane (CM) phospholipid or CM protein receptor interactions

Traditional mechanisms of small molecule

chemotherapeutics based on:•Systemic level in vivo

–Gives an idea of the dosing range necessary to achieve chemotherapeutic effect and tumor regression

INCOMPLETE INFORMATION:

On the modes, the levels and the character of interactions of

small biomolecules with cell membrane (CM) constituents

and at the intra-cellular levels

For the proper determination of the apoptotic potential of chemoxenobiotics at the

cellular level , important to know whether:•With and across cell membrane (CM) protein aqueous channels

•With CM surface protein receptors and endocytic

•With CM surface protein receptors and non-endocytic

•Directly with CM phospholipids themselves (if not across pores AND if not with CM surface protein receptors

For the proper determination of the apoptotic potential of

chemoxenobiotics at the sub-cellular level ,

important to know whether:•Nuclear, mitochondrial or mictroubular

•Mitochondrial

•Microtubular

•Non-microtubular (ie FKBP12/Inter-Domain (I-II/III) of FKBP52)

The conserved biophysical properties

of small molecule chemotherapeutics that predict the modes, the

levels and the character of interactions of small

biomolecules with CM constituents & at the intra-cellular levels

•Predicted chemoxenobiotic structure (2-D)

•Predicted molecular size (vdWD; nm)•Predicted internal (Core) lipophilicity (nm-1)

•Predicted external/peripheral hydrophilicity (nm-1) AND distribution over molecular space –Charge (Cationic or Anionic), Hydroxylation, Carbonylation, Etheroylation & Carboxylation

Small Molecule Chemoxenobiotic

Classification I•Pure Hydrophiles

Defined as xenobiotics with: (1) Insufficient (IS) intervening lipophilicity

(2) A hydrophilic (-) Log OWPC-to-vdWD ratio (nm-1 ) at physiologic of pH 7.4

-- Non-charged pure hydrophiles being CM aqueous channel pore permeable at vdWDs <0.78 nm and intra-cellularly localizing

Small Molecule Chemoxenobiotic Classification IIA•Hydro-lipophiles

Defined as xenobiotics with: (1) Sufficient (S) intervening lipophilicity (Log incorpOWPC-to-vdWD (nm-1 )

(2) A hydrophilic (-) Log OWPC-to-vdWD ratio (nm-1 ) at physiologic of pH 7.4

-- CM aqueous channel pore impermeable, and interaction, instead, with CM non-channel receptor, or CM receptor interaction

Small Molecule Chemoxenobiotic Classification IIB• Hydro-lipophiles

(A) Di-carboxylated (IS 1+ 1- S 0) Non-Channel Folic Acid Receptor-Mediated CM Endocytosis

(B) Non-Cationic (0), Mono-Cationic (S 1+ 0) or Di-Cationic (1+ S 1+)

Polyhydroxylated / Carbonylated / Etheroylated Channel Receptor -Mediated CM Endocytosis

(ie Na+/K+ ATPase; Ca2+ Channel)

(C) Di-Cationic (1+ IS 1+: 2+) Non-Channel Receptor -Mediated CM

Endocytosis

Small Molecule Chemoxenobiotic Classification IIIA•Lipophiles

Defined as xenobiotics with:

(1) Non-charged less lipophilic Toxicants with vdWDs <0.78 nm (CM channel pore permeable sub-CM interactors): Overall lipophilicity [(+) Log OWPC-to-vdWD ratio (nm-1 )]

AND (2) Xenobiotics with vdWDs >0.78 nm: Overall

lipophilicity [(+) Log OWPC-to-vdWD ratio (nm-1 )] with sufficient (S) intervening lipophilicity (Log incorpOWPC-to-vdWD; nm-1 ) in the presence of molecular hydrophilicity (Monohydroxylation / Monocarbonylated / Monoetheroylated / Monocarboxylated; Polyhydroxylated / Polycarbonylated / Polyetheroylated; Charge)

-- > (+) Overall Log OWPC-to-vdWD ratio (nm-1 )

Small Molecule Chemoxenobiotic Classification IIIA•Lipophiles

Sub-classes of Xenobiotics with vdWDs >0.78 nm and Overall lipophilicity [(+) Log OWPC-to-vdWD ratio (nm-1 )] with sufficient (S) intervening lipophilicity (Log incorpOWPC-to-vdWD; nm-1 ) in the presence of molecular hydrophilicity (Monohydroxylation / Monocarbonylated / Monoetheroylated / Monocarboxylated; Polyhydroxylated / Polycarbonylated / Polyetheroylated; Charge)

(1) Present isotropically [CM receptor hydrophobic core interactors

with vdWDs >0.78 nm (Receptor-Mediated CM Endocytosis)]

OR (2) Present anisotropically [CM Cholesterol /

phospholipid Glycerol-to-fatty acid-ester or CM receptor

hydrophobic core interactors with vdWDs >0.78 nm (potential for CM perturbomodulation)]

Small Molecule Chemoxenobiotic Classification IIIC• Lipophiles

(A) Anionic (S 1- 0) with Linear Structure, Cataniononeutral (IS 1+ 1- S 0) with Linear Structure, Anisotropic Polyneutral (S 0 0) with Linear Structure, Isotrophic Polyneurral with Linear or Compact Structure: CM Cholesterol or CM phospholipid Glycerol-to-fatty acid- ester Interaction or Disruption

(B) Cationic Isotrophic Polyneutral (0 S 1+ 0) with Flexible Linear Structure: Channel Receptor -Mediated CM Endocytosis (Ca2+ Channel: ie Verapamil)

(C) Cationic Less Isotrophically Polyneutral (0 S 1+ 0) with Compact Structure: Channel Receptor Obstruction without Endocytosis (Ca2+ Channel: ie Quinidine, a P-glycoprotein potential inducer) (D) Anisotrophic Polyneutral (S 0 0) with Compact Linear Structure: (Non-Channel) P-glycoprotein Receptor Binding without Endocytosis (ie Artemisinin: Sin Qua Non P-gp Inducer)

(E) Isotrophic Polyneurral (S 0 0) with Non-Linear Non-Compact Structure: Non-Channel Receptor-Mediated CM Endocytosis

(F) Di-neutral/Polyneutral Sterol with Non-Linear Compact Structure: Non-Channel Receptor-Mediated Pressuromodulation Antagonism (ie Sex Steroid Receptor Antagonists)

Nitroso-N-methylurea (MNU)

Temozolamide (TMZ)

Nitroso-N-ethylurea (ENU)

Cell Membrane (CM) Channel Aqueous Pore Permeation and DNA/RNA Adduction

Cell Membrane (CM) Channel Aqueous Pore Permeation and Nucleoside Substitution

Decitabine

5-Fluorouracil (5-FU)

3-Methyladenine

Gemcitabine

Cell Membrane (CM) Channel Aqueous Pore Permeation with the Potential to Bind to Cytochrome P450s: DNA Adduction and/or Crosslinking

Procarbazine

Carmustine (BCNU)

Cyclophosphamide

Cell Membrane (CM) Insertoassociation and Phospholipid Interspace Widening with Potential for 1ary Indirect Pressuromodulation

Cyclosporine A

Cell Membrane (CM) Cholesteroloassociation-to-CM Phospholipidoassociation: Cholesterol Removal-to-CM Phospholipid Pertubation and Potential for 1ary Indirect Pressuromodulation

Amphotericin B

Chlorambucil

Melphalan

Ketoconazole

Capecitabine

Fluconazole

Divalent Cationicity-Mediated Cell Membrane (CM) Receptor Vesiculo-Vacuolization Endocytosis, Sub-cellular Vacuolization along with Exosome Formation with Potential for CM Receptor-Mediated 3ary Indirect Shift Pressuromodulation

AMD3100 (Plerixafor)

Paraquat

Bleomycin

Carbonylation/Hydroxylation cum Cationicity-Facilitated Cell Membrane (CM) Channel Endocytosis and Mitochondrial VDAC Association: Non-association of Microtubule Tubulin to Mitochondrial Membrane (MM)

and Mitochondrial Anchorage Non-Mobility-Mediated MM Disruption/Mitochondria-Mediated Apoptosis

Vincristine

Doxorubicin

Dual Carboxylation-Facilitated Cell Membrane (CM) Receptor Endocytosis: Mitochondrial Membrane (MM) and Rough Endoplasmic Reticulum (RER) Membrane (RERM) Vesiculization

Methotrexate

Raltitrexed

Hydroxylation/Carbonylation/Dual Carboxylation-Facilitated Cell Membrane (CM) Receptor Endocytosis: Tubulin Polymerization Re-Polymerization Inhibition and Mitochondria-Mediated Apoptosis

to Rapamycin-Associated Protein Binding Tubulin Non-Binding

Etoposide (VP16)

Teniposide (VM26)

Colchicine

Paclitaxel (Taxol)

Ixabepilone

(+/-) Spiro-oxanthromicin A

Tacrolimus

Cell Membrane (CM) Receptor-Mediated Pressuromodulation Antagonism/Partial Antagonism: Antagonism/Partial Antagonism of Direct CM Receptor-Mediated Pressuromodulation

Hydroxytamoxifen (Afimoxitene)

Abiraterone

Cell Membrane (CM) Receptor-Mediated Antagonism/Partial Antagonism of Pressuromodulation Extracellulomodulation with Concomitant Receptor Kinase Inhibition:

Antagonism/Partial Antagonsim of Direct CM Receptor-Mediated Pressuromodulation +/- External Cationomodulation (>= 3+ -> 1+)

Gefitinib (Iressa)

Ceritinib

Erlotinib (Tarceva)

Lapatinib

MK-2206

Staurosporine

Afatinib

Imatinib

Crizotinib

Hydroxycamptothecin

AMD070

Topotecan

GM

-CS

F R

PD

GF

R

TR

AIL

RT

RA

IL R

Conclusions - I•Based on the observations herein, on the

modes, levels and character of interactions of xenobiotics and chemoxenobiotics with cells, analyzed in terms of the predicted conserved biophysical properties, insight has been gained into the specific mechanisms by which chemoxenobiotics enter cells and the organelles with which they interact to induce cytotoxcity

Conclusions - II•This knowledge is applicable towards

improving the effectiveness of combinationatory small chemotherapy regimens in current clinical use, for the treatment of solid and hematopoietic malignancies, including the order in which chemoxenobiotics are administered in combinationatory treatment regimens, in temporal proximity

Conclusions - III• It is anticipated that by the application of

this study's findings on the modes and character of cellular interactions, existing combinationatory chemotherapy regimens can be designed to be more efficacious, and furthermore, that by the incorporation of this knowledge into the algorithms for the design of personalized cancer treatments, the predictive accuracy of such algorithms can be further optimized

Conclusions - IV•For the curative treatment of solid

malignancies, small molecule chemoxenobiotics must be made to selectively accumulate within the uM in the tumor milieu, where they must remain for prolonged duration in order for uniform tumorocidal cytotoxcity to tumor and tumor-associated cells

Conclusions - IV• For the further translational development of novel

chemotherapeutic regimens employing optimally-sized and -designed biocompatible imageable dendrimer-based nanoparticles bearing labilely-attached small molecule chemoxenobiotics– Optimally-sized imageable dendrimer nanoparticle-

based small molecule chemotherapy at ~9 nanometers (Hydrodynamic diameter = Dehydrated Diameters for such NPs)•Sarin, H. Translational Theranostic Methodology for Diagnostic Imaging and the Concomitant Treatment of Malignant Solid Tumors. Inaugural Issue Neurovascular Imaging, 1(3), 2015