SYNTHESIS AND ANTIPROLIFERATIVE EFFECT OF NOVEL 13-(DI)ARYLALKYL BERBERINES IN

MALIGNANT MESOTHELIOMA CELLS

Paolo Lombardi,a,c Franco Buzzetti,a Ana Campillo,b Gaetano Fiorillo,a Paula Garcia,b Cristina Geroni,c Carmen Plasencia,b,c Anna Radresa,b Carmela Salvatore,c Tanjia Monir Syedaa

aNaxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, ItalybAromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain cAesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi, Ancona, Italy Email: p.lombardi@naxospharma.eu

Introduction: Malignant Mesotheliomas (MT)Introduction: Malignant Mesotheliomas (MT)

MTs are fatal asbestos-exposure-associated cancers MTs are fatal asbestos-exposure-associated cancers Sites of MT are the pleural cavity (90%) and the peritoneal area (7%)Sites of MT are the pleural cavity (90%) and the peritoneal area (7%) One-year survival time is < 40%One-year survival time is < 40%

Increasing incidence wordlwideIncreasing incidence wordlwide 14,200 MT cases/year are diagnosed worldwide (1994-2008 data)14,200 MT cases/year are diagnosed worldwide (1994-2008 data) The incidence has not yet peaked, but is predicted to reach the max in 2030 decadeThe incidence has not yet peaked, but is predicted to reach the max in 2030 decade

High levels of thymidylate synthase protein expression inHigh levels of thymidylate synthase protein expression in MT patients are the marker of lack MT patients are the marker of lack of efficacy of currrent treatments of efficacy of currrent treatments (pemetrexed, cisplatin)(pemetrexed, cisplatin)

Project Background: Thymidylate SynthaseProject Background: Thymidylate Synthase

Thymidylate Synthase (TS) is a key enzyme in the DNA synthesis andThymidylate Synthase (TS) is a key enzyme in the DNA synthesis andcell proliferation and represents one of the best-known drug targetscell proliferation and represents one of the best-known drug targets

in the anticancer area.in the anticancer area.

Current drugs are only TS inhibitors which rapidly induce 2-5fold increase of TSCurrent drugs are only TS inhibitors which rapidly induce 2-5fold increase of TSlevels in tumour cells. levels in tumour cells.

There is an obvious need for new compounds ableThere is an obvious need for new compounds ableto interrupt the abnormal production of TS in cancer cellsto interrupt the abnormal production of TS in cancer cells

TS

Actin

Control 5FU 5uM 5FU 20 uM

Expression of TS predicts clinical outcomes of TS inhibitors-containing Expression of TS predicts clinical outcomes of TS inhibitors-containing chemotherapy and increased TS levels are responsible for resistance. chemotherapy and increased TS levels are responsible for resistance.

Project Background: Thymidylate SynthaseProject Background: Thymidylate Synthase

In cells, TS is present as an enzymatically active dimeric form in equilibrium with a catalytically inactive monomeric form. In its turn, TS monomer is in equilibrium with a TS-mRNA complex by binding to its own mRNA.

The cellular levels of TS depends on its ability to interact with its own RNA

When clinically useful TS-directed inhibitors bind to dimeric TS, the enzyme discontinues its interaction with mRNA resulting in the synthesis of new TS that lowers the responsiveness of tumour cells

The level of expression of TS in cancer cells can be down-regulated with antisense oligos targeting TSmRNA specific sites, restoring the sensitivity to TS inhibitors.

An innovative mechanism of action: targeting TS translational autoregulationtargeting TS translational autoregulation

Project Background: Thymidylate SynthaseProject Background: Thymidylate Synthase

Western blot analysis of TS protein concentration in 2008 and C13* cells

EFFECT OF BERBERINE ON TS PROTEIN LEVEL AND ACTIVITY

Berberine: Background History Berberine: Background History Bitter-tasting isoquinoline quaternary alkaloid extracted from plants of the genus Berberis, Coptis and others

In use in the Ayurvedic and Chinese medicines since hundreds of years

It shows diverse pharmacological activities:

anti-microbial/parasitic, anti-diarrheal, anti-inflammatory,

anti-arryhthmic, cholesterol-lowering and anticancerA definite medical A definite medical potential has been potential has been established in a wide established in a wide spectrum of clinical spectrum of clinical applications including:applications including:

hyperlipidemia, metabolic syndrome, polycistic ovary syndrome, obesity, fatty liver disease, coronary artery disease

Berberine: Background History Berberine: Background History The precise molecular basis of its many

biological activities are still debated

modulation of protein expression by interaction with nucleic acids is postulated

The interactions between berberine and The interactions between berberine and nucleic acidsnucleic acids, reported since 1962, could , reported since 1962, could

lead to its anticancer effect lead to its anticancer effect

Mazzini, S. et al, Bioorg Med Chem, 2003, 505–514

Ferraroni, M. et al, Chem. Commun. 2011, 4917-4919.

intercalation

minor groove binding

Berberine represents an interesting Berberine represents an interesting and attractive natural lead compoundand attractive natural lead compound

Chemical modifications might select more specific medical indications

Chemical ProgrammeChemical Programme

Performing rational chemical Performing rational chemical modifications of berberine structure led modifications of berberine structure led

to a new class of derivatives with to a new class of derivatives with antitumour propertiesantitumour properties

resulting in derivatives with better (or different) biological effects with respect to the parent berberine

1 Waters ML, Curr Opin Chem Biol. 2002, 6, 736

Aromatic interactions are ubiquitous in nature, and their Aromatic interactions are ubiquitous in nature, and their geometry is relevant for the molecular recognition in geometry is relevant for the molecular recognition in

biological systemsbiological systems11

(Hetero)aromatic groups pending from a suitable position (Hetero)aromatic groups pending from a suitable position of the parent alkaloid skeletonof the parent alkaloid skeleton

linkers of variable length and functionality

geometric propensity for additional stacking-type, non-covalent aromatic interactions

Chemical ProgrammeChemical Programme

Chemical ProgrammeChemical ProgrammeAlkylation of enamine (7,8-dihydroberberine)

Uncommon aldehyde-enamine condensation1,2

generally from good to very good yields

1 Cook, AG, Enamines Synthesis, Structure and reaction, 1988, pag 200-201; 2 Iwasa, K, et al., Planta Medica, 1997, 196

from very low to low yields - better with activated halides or iodides - berberine back from loss of acetone major by-product

from low to moderate yields - berberine and tetrahydroberberine from disproportionation of enamine as major by-products

In vitroIn vitro Studies StudiesAntiproliferative effect in pleural mesothelioma Antiproliferative effect in pleural mesothelioma

MSTO-211H cellsMSTO-211H cells

R=4-OMe, NAX38

R=3-,4-,5-(OMe)3, NAX54

In vitroIn vitro Studies StudiesTS protein expression levels: time-course in mesothelioma MSTO-TS protein expression levels: time-course in mesothelioma MSTO-

211H cells at the IC50 dose at 72 h211H cells at the IC50 dose at 72 h

NAX038

Berberine NAX014 NAX012

NAX035

Solubility: Turbidimetric in vitro method

Passive permeability: Gentest™ Pre-coated PAMPA Plate System

In vitroIn vitro Studies Studies

Preliminary in vitro ADMEPreliminary in vitro ADME

all compound show good solubility (>20ug/mL) and better than

berberine

in general NAX compounds present an acceptable passive permebility (better than berberine)

suggesting a high intestinal absorption and good bioavailability

In vitroIn vitro Studies StudiesNAX035: antiproliferative activity on chemo-restistant human MT NAX035: antiproliferative activity on chemo-restistant human MT

cell lines in comparison with standardscell lines in comparison with standards

NAX035 NAX035 overcomes pemetrexed and cisplatin overcomes pemetrexed and cisplatin resistance in MT cells (collateral sensitivity)resistance in MT cells (collateral sensitivity)

Ex vivoEx vivo Studies StudiesEvaluation of TS levels in human mesothelioma tissue Evaluation of TS levels in human mesothelioma tissue

samples from nude mice treated with NAX035samples from nude mice treated with NAX035

NAX035 showed antitumour efficacy at tolerated oral and i.p. doses on human experimental MT model

A correlation between the efficacy of NAX035 and TS protein levels, was observed in vivo in tumour tissue samples examined at the end of the p.o. treatment

period

.

Conclusions Conclusions

Aknowledgements: Financial supports were provided by Ministero dello Sviluppo Economico (Grant. 01705 to Naxospharma) and by Agència per a la competitivitat de l'empresa ACC1O (Grants RDNET11-1-0001 and RD14-1-0072 to Aromics) under the 6th call of the EuroTransBio initiative, transnational project BERTA (BERberine as antiTumour Agents).

Innovative proprietary compounds, structurally related to the plant Innovative proprietary compounds, structurally related to the plant isoquinoline alkaloid berberine, able to interrupt the abnormal isoquinoline alkaloid berberine, able to interrupt the abnormal production of thymidylate synthase in cancer cells by targeting production of thymidylate synthase in cancer cells by targeting

translational autoregulation translational autoregulation

Novel mechanism of action, targeting the expression ofTS protein, differently from previous TS inhibitors

Efficacy on chemoresistant tumour cells

Antitumour efficacy and tolerability at the effective dosesby oral and intraperitoneal administration in a humanmesothelioma xenografted in nude mice