SELECTION OF A NOVEL BERBERINE DERIVATIVE WITH ANTITUMOUR EFFICACY ON HER2 POSITIVE

MURINE BREAST CANCERPaolo Lombardi,a,c Franco Buzzetti,c Gaetano Fiorillo,c Cristina Geroni,a

Elisa Pierpaoli,a,b Carmen Plasencia,a,d Mauro Provinciali,b Carmela Salvatore,a Tanjia Monir

Syedac aAesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi, Ancona, ItalybCentro Tecnologie Avanzate dell'Invecchiamento, INRCA-IRCCS, Via Birarelli 8, 60121 Ancona, Italy cNaxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, ItalydAromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain Email: p.lombardi@naxospharma.eu

Introduction: HER2Introduction: HER2++ Breast Cancer Breast Cancer

New agents exhibiting aNew agents exhibiting a mechanism of actionmechanism of action different in respect to current therapies might different in respect to current therapies might

offer a new option for treating HER2offer a new option for treating HER2++ BC patients BC patients

HER2HER2++ BC: BC:represents 20–30% of invasive BC associated with more aggressive disease progression and a poorer prognosis

HER2-targeting gold standard drugs show modest efficacy as single agent and substantial toxicity in combination

therapy

HER2+: human epidermal growth factor receptor 2 positive

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 anticancer

A 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 applications including: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

debatedmodulation 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, , reported since 1962, could lead to its anticancer effect could 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: Anticancer Properties &Berberine: Anticancer Properties & Activity Activity inin HER2 HER2++ BCBC

Anticancer properties of Anticancer properties of berberine in several berberine in several preclinical studies have preclinical studies have been published since many been published since many yearsyears

Berberine could be a useful adjuvant therapeutic agent in the treatment of HER2-overexpressing BC

Berberine suppresses the growth of HER2+ BC cells and promotes apoptosis by down-regulating the HER2/PI3K/Akt signaling pathway

Berberine represents an Berberine represents an interesting and attractive natural interesting and attractive natural

lead compoundlead compoundChemical modifications might select more specific medical indications

Chemical Chemical ProgrammeProgramme

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

led to a new class of derivatives led to a new class of derivatives with antitumour propertieswith antitumour 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, Aromatic interactions are ubiquitous in nature, and their geometry is relevant for the molecular and their geometry is relevant for the molecular

recognition in biological systemsrecognition in biological systems11

(Hetero)aromatic groups pending from a suitable position of the parent alkaloid skeletonlinkers of variable length and functionalitygeometric propensity for additional stacking-type, non-covalent aromatic interactions

Chemical Chemical ProgrammeProgramme

Novel berberine derivatives Novel berberine derivatives

Suppression of nascent protein synthesis by berberine appreciation of the differences in mRNA translational control between normal and cancer cells

has been reported by us and other

Chemical Chemical ProgrammeProgramme

targeting post-transcriptional control targeting post-transcriptional control processesprocesses

suppressing nascent protein synthesissuppressing nascent protein synthesisable to correct/compensate aberrant able to correct/compensate aberrant genetic expressions of cancer cellsgenetic expressions of cancer cells

In vitroIn vitro Studies StudiesChemical structures of tested berberine derivativesChemical structures of tested berberine derivatives

Antiproliferative activity against HER2+ human(SK-BR-3) and murine (N202.1A) breast cancer cell lines; time and

dose-dependence (Alamar Blue assay)quantification of apoptosis (Flow

Cytometry)

modulation of p53, p21WAF1, p16INK4a, PAI-1 and heparanase expression (RT-

PCR, Quantitative Real-Time mRNA analysis)

modulation of HER-2/neu expression and phosphorylation (Western Blot Analysis)

HER2 + human BC cells (SK-BR-3

HER2+ murine BC cells (N202.1A)

NAX Compounds: NAX Compounds: In vitroIn vitro Activity Activity inin HER2 HER2++ BC Cells BC Cells NAX014 is the most effective compoundNAX014 is the most effective compound

Method - Alamar Blue assay. The number of viable cells after treatment is expressed as a % of the vehicle treated control

NAX Compounds: NAX Compounds: In vitro In vitro Activity is Time-Activity is Time-dependentdependent

SK-BR-3 cellsSK-BR-3 cells

NAX014 is the most effective compoundNAX014 is the most effective compound

SK-BR-3 cellsSK-BR-3 cells Time 72hTime 72h

ControlControl BerberineBerberine

NAX014NAX014NAX012NAX012

8.7%

71.6%68.4%

44.2%

NAX Compounds: Induction of Apoptosis on HER2NAX Compounds: Induction of Apoptosis on HER2++ BC cellsBC cells

Quantification of Apoptosis by Flow Cytometry: apoptosis was measured through sub-diploid DNA peak analysis after staining with propidium iodide

NAX014 shows time-dependent apoptotic NAX014 shows time-dependent apoptotic effecteffect

Ctrl Berberine NAX012 NAX014 NAX035

NAX Compounds: Effect on HER2/neu Expression & NAX Compounds: Effect on HER2/neu Expression & PhosphorylationPhosphorylation

Unique ability of Unique ability of NAX014 to block total NAX014 to block total

HER2 expressionHER2 expression

Lapa

tinib

+Tr

astu

zum

ab

Treatment: NAX012 and NAX014 and berberine (BBR) 50µM for 24h

Graphs B and C represent quantification of the blots by the GS-670 imaging densitometer after HER2 normalization respect to β-actin (B) and pospho-HER2/β-actin normalization respect to HER2/β-actin (C). Results shown are representative of 2 independent experiments.

most probably by most probably by forming complex(es) forming complex(es)

with DNA and/or with DNA and/or mRNA mRNA

NAX014: Recovery of the NAX014: Recovery of the Antiproliferative Activity Antiproliferative Activity

SK-BR-3 human HER2+ BC cells treated with NAX014 (20µM) for 48h and then maintained in drug-free medium for 96h and 120h

NAX014 shows irreversible antiproliferative NAX014 shows irreversible antiproliferative effecteffect

NAX014: NAX014: In vivoIn vivo Acute and Chronic Toxicity in Acute and Chronic Toxicity in MiceMice

NAX014 LDNAX014 LD50 50 30.9 mg/kg30.9 mg/kgBerberine LDBerberine LD5050 10.9 mg/kg 10.9 mg/kg

Survival curves of FVB mice injected i.p. with 2.5, 5.0, 10 and 20 mg/kg of berberine

(BBR) or NAX014

Body weight changes in subchronic toxicity study (as percent of the day 0

weight)

NAX014

BERBERINE

Tumour Model: FVB Mice Her2/neuTumour Model: FVB Mice Her2/neu

Breast whole mount1 Week 12

Breast whole mount1 Week 25

Female mice develop Female mice develop spontaneous malignant, fatal, spontaneous malignant, fatal, breast tumours into the breast tumours into the mammary gland and metastases mammary gland and metastases (Muller et al. 1988)(Muller et al. 1988)

BC is palpable starting on Week BC is palpable starting on Week 2525

Mice bearing breast tumours (Week 25)

1. 3D techniqe.

TUM

TUM

Tumor expression

FVB-N 233 transgenic mouse model expresses the FVB-N 233 transgenic mouse model expresses the HER2/neu oncogene HER2/neu oncogene

NAX014: Antitumour Efficacy in HER2/neu Transgenic NAX014: Antitumour Efficacy in HER2/neu Transgenic MiceMice

FVB-N 233 Her2/neu mice FVB-N 233 Her2/neu mice treated intraperitoneally treated intraperitoneally with 2.5mg/kg of compounds (2xweek)x12with 2.5mg/kg of compounds (2xweek)x12

In mice treated with In mice treated with NAX014, but not in NAX014, but not in those which received those which received berberine (BBR), the berberine (BBR), the kinetics of appearance of kinetics of appearance of mammary tumours was mammary tumours was significantly different when significantly different when compared with control compared with control group (p=0.027) group (p=0.027)

NAX014: NAX014: In vivoIn vivo Antitumour Efficacy in Antitumour Efficacy in HER2HER2++ BC BC

FVB-N 233 Her2/neu mice treated FVB-N 233 Her2/neu mice treated intraperitoneally intraperitoneally with 2.5mg/kg of compounds with 2.5mg/kg of compounds (2xweek)x12(2xweek)x12

Tumour Number Tumour Growth Inhibition

Significant reduction of tumour volume in mice treated Significant reduction of tumour volume in mice treated intraperitoneallyintraperitoneally with NAX014 compared to control group with NAX014 compared to control group

(p<0.05 )(p<0.05 )

FVB-N 233 Her2/neu mice treated FVB-N 233 Her2/neu mice treated per per os with 20 mg/kg os with 20 mg/kg of NAX014 of NAX014 (2xweek)x8(2xweek)x8

Mea

n Tu

mor

Num

ber

Age (Weeks)

Tumour NumberAge (Weeks)

Tumour Growth Inhibition

Tum

or V

olum

e (%

vs C

ontr

ol)

Antimetastatic effectLung metastases NAX014 Control

% Mice with metastases

12.5 55.5

Cumulative no. 1 7

Mean size (mm) 6 ±0 5.7 ±1.8Maximum size (mm)

6 8

NAX014: NAX014: In vivoIn vivo Antitumour Efficacy in HER2 Antitumour Efficacy in HER2++ BC BC NAX014 is effective by NAX014 is effective by oral oral routeroute in delaying the onset in delaying the onset and the progression of HER2and the progression of HER2++ BC and shows antimetastatic BC and shows antimetastatic efficacyefficacy

IIn vivon vivo Evaluation of Vascularization of Mammary Tumours in HER2/neu Evaluation of Vascularization of Mammary Tumours in HER2/neu transgenictransgenic micemice

Microvessels density in tumor masses from controls, berberine and NAX014 treated mice. The vascular architecture was evaluated in vivo by using SDF videomicroscopy.

Tumour masses from control group Tumour masses from control group showed higher vessel density 17.2 showed higher vessel density 17.2 mm/mmmm/mm22 as compared to the as compared to the berberine 12.07 mm/mmberberine 12.07 mm/mm22 and the and the NAX014 groups 9.9 mm/mmNAX014 groups 9.9 mm/mm22 Statistical significance between Statistical significance between NAX014 versus control group NAX014 versus control group (p<0.01)(p<0.01)

NAX014: Conclusions NAX014: 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).

anticancer and anti-metastatic efficacy on HER2+ tumoursanticancer and anti-metastatic efficacy on HER2+ tumoursin vitro activity at µM concentrationsin vitro activity at µM concentrations

Unique ability to reduce cellular HER2 expression via a Unique ability to reduce cellular HER2 expression via a postulated postulated novel mechanismnovel mechanism

in vivoin vivo tolerability by i.p.and oral administration tolerability by i.p.and oral administration at the effective doseat the effective dose