SYNTHESIS AND ANTIPROLIFERATIVE EFFECT OF NOVEL 13-HALOPHENYLALKYL BERBERINES

IN HER-2+ BREAST CANCER CELLSPaolo 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, AnconabCentro Tecnologie Avanzate dell'Invecchiamento, INRCA-IRCCS, Via Birarelli 8, 60121 Ancona cNaxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, MilanodAromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain Email: p.lombardi@naxospharma.eu

BerberineBerberine

BerberineA definite medical potential has been established in a wide

spectrum of clinical applications in therapeutic areas such as hyperlipidemia, metabolic syndrome, polycistic ovary syndrome, obesity, fatty liver disease, coronary artery disease, where berberine has drawn most extensive attention as come out of scientific and patent literature and ongoing clinical trials (9, 2011 18, 2014)

Isoquinoline quaternary plant alkaloid used in the Ayurvedic and Chinese medicines since hundreds of years shows diverse pharmacological properties and activities:

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

anti-arrythmic, cholesterol-lowering, and anti-tumour

The precise molecular basis of its many biological activities are still debated.

Berberine Berberine Modulation of protein expression by interaction with nucleic acids is postulatedInteractions between berberine and nucleic acids have been reported since 19621

However, is the alkaloid a minor groove binder..... ...or an intercalator?

1 Yamagishi H, Interaction between nuclei acid and berberine sulfate, 1962, J Cell Biol, 15,589

Berberine Berberine

Berberine represents an interesting and attractive natural lead compound.

Rational chemical modifications might lead to more specific and selective medical indications

Berberine

Chemistry programme

(Hetero)aromatic groups pending from a suitable position of the parent alkaloid skeleton through linkers of variable length and functionality

possibly creating a geometric propensity for additional stacking-type, non-covalent aromatic interactions (intramolecular and/or molecule-cellular target).

N

O

O

OCH3

OCH3

X

(H2C) 1-6

N

O

O

OCH3

OCH3

X

(H2C) 0-5

13-phenylalkyl derivatives 13-diphenylalkyl derivatives

X= Cl, I, Br

Since aromatic interactions are ubiquitous in nature, and their geometry is relevant for the molecular recognition in biological systems1 that could result in better (or different) biological effects with respect to the parent berberine

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

Chemistry programmePatent US8188109B2

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

Alkylation of enamine (7,8-dihydroberberine)

Chemistry programme Prior art

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

generally from good to very good yields

Uncommon aldehyde-enamine condensation1,2

Chemistry programme Instant art

2 Iwasa, K, et al., Planta Medica, 1997, 196

1 Cook, AG, Enamines Synthesis, Structure and reaction, 1988, pag 200-201

Berberine and tetrahydroberberine are the major by-products from disproportionation reaction of dihydroberberine

Chemistry programme

Chemistry programme Even with glyoxylic acid 1

1 Fiorillo, G et al, An uncommon aldehyde-enamine condensation. Synthesis and antiproliferative activity of new berberine-derived (hetero)aryl amides. XXXIV Convegno Nazionale SCI, Divisione di Chimica Organica. Pavia (I), 10 September 2012.

Aldehyde sources Chemistry programme

Commercially available aldehydes

Commercially available corresponding alcohols followed by oxidation

Commercially available corresponding acids/esters followed by reduction

Homologation procedures from the above and others intermediates

DNA binding Chemistry programme

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

Derivatives possibly exhibiting ameliorated antitumour properties

Antiproliferative effects in human malignant mesothelioma cell lines

Antiproliferative effects in human malignant mesothelioma cell lines

Antiproliferative effects in different human cancer cell

lines

Advanced Technology Center for Aging ResearchScientific Technological Area IRCCS - INRCA

Mauro ProvincialiElisa Pierpaoli

Project Background Project Background Breast CancerBreast Cancer

Berberine inhibits cellular growth of breast cancer cells and promotes apoptosis by down-regulating the HER2/PI3K/Akt signaling pathway1

Breast Cancer (BC) is: the second most common cancer worldwide,the fifth most common cause of cancer death,the leading cause of cancer death in women (>1.4 million new cases and > 450,000 deaths annually)

BC rates are rising around the world The heterogeneity of BCs makes them both a fascinating and challenging solid tumour to diagnose and to treat.

Project Background Project Background Breast CancerBreast Cancer

BC characterized by overexpression of human epidermal growth factor receptor 2 (HER2) has been associated with more aggressive disease progression and a poorer prognosis.

Project Background Project Background Breast CancerBreast Cancer

HER2 is overexpressed in 20–30% of invasive BC thus, new therapies are desperately needed.HER2-targeting gold standard drugs show modest efficacy as single agent and substantial toxicity in combination therapy.

HER2 + human BC cells

(SK-BR-3)

Today BC HER2Today BC HER2+ + therapiestherapies

DRUGDRUG CompanCompanyy MOAMOA

Use - Cost x Use - Cost x month & FDA month & FDA Approval (yr)Approval (yr)

TrastuzumTrastuzumab ab Herceptin®

Genentech (US)Roche (EU)

Monoclonal Antibody against HER2 receptor

Combo with Chemio$4,500

2006

PertuzumaPertuzumabbPerieta®

Genentech Monoclonal Antibodyagainst HER2 receptor

Combo with Chemio & Herceptin

$6,000

2012

TDM-1TDM-1 Kadcycla®

Genentech Antibody-drug conjugate Ado-Trastuzumab emtansine

Single agent$9,800

2013

LapatinibLapatinibTykerb®

GSK EGFR & HER-2 tyrosine kinases inhibitor

Combo with Chemio$3,625

2007

Competitive landscapeCompetitive landscapeHER2 pathway and targets

Phase III drugs MOABKM120, LEE011 (Novartis); Palbociclib (Pfizer)

HER2 dowstream pathway

Me-too's of currently used antibodies HER2 transmembrane receptor domain

NAX compoundsNAX compounds

Chemical structures of Berberine chloride, NAX012, NAX013, NAX014, and NAX035.

Antiproliferative activity of NAX compoundsAntiproliferative activity of NAX compoundsagainst HER2+ breast cancer cellsagainst HER2+ breast cancer cells

SK-BR-3 cells24h treatment

N202.1A cells (murine)24h treatment

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

SK-BR-3 cells (human) 24h treatment

CellsIC50 µM

NAX014 NAX012 NAX013 NAX035 Berberine (BRB)

SK-BR-3 52.3 94.2 >100 >100 91.8N202.1A 20 40 >50 >50 NT

Pirpaoli E. BioFactors, 2013, 39, 672.

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

Time-dependent activity of NAX compoundsTime-dependent activity of NAX compoundsagainst HER2+ breast cancer cellsagainst HER2+ breast cancer cells

50 mM

0

20

40

60

80

100

120

0 24 48 72

Time

% o

f con

trol

NAX12B

NAX13B

NAX14C

NAX35B

BRB

10 mM

0

20

40

60

80

100

120

0 24 48 72

Time

% o

f con

trol

NAX12B

NAX13B

NAX14C

NAX35B

BRB

SK-BR-3 cellsSK-BR-3 cells

TimeIC50 µM

NAX014 NAX012 NAX013 NAX035 Berberine (BRB)

24 h 52.3 ±3.2 94.2 ±1.2 >100 >100 91.8±2.848h 30.7 ±2.1 46.6 ±2.5 >100 >100 58.4 ±1.972 h 26.5 ±6.7 31.9 ±2.9 >100 48.6 ±6.7 36.0 ±1.8

Pirpaoli E. BioFactors, 2013, 39, 672.

PI assay (SKBR3 + [50 uM] BRB analogs)

0

10

20

30

40

50

60

70

80

Apop

totic

nuc

lei (

%)

24h

48h

72h

24h 2,97 18,00 15,70 15,20 52,60

48h 7,88 27,70 54,20 44,40 65,10

72h 8,10 44,20 71,60 68,40 45,20

Ctrl BRB NAX12B NAX14C NAX35B

SK-BR-3 cellsSK-BR-3 cells

Induction of apoptosis of NAX compoundsInduction of apoptosis of NAX compoundsagainst HER2+ breast cancer cellsagainst HER2+ breast cancer cells

Time-dependent apoptotic effectTime-dependent apoptotic effect

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

Time 72hTime 72h

ControlControl BerberineBerberine

NAX014NAX014NAX012NAX012

8.7%

71.6%68.4%

44.2%

Pirpaoli E. BioFactors, 2013, 39, 672.

Breast whole mount1 Week 12

Breast whole mount1 Week 25

FVB-N 233 transgenic mouse model expresses the HER2/neu oncogene Female mice develop spontaneous malignant, fatal, breast tumours into the mammary gland and metastases (Muller et al. 1988).BC is palpable starting on Week 25.

Tumour model: FVB mice Her2/neu Tumour model: FVB mice Her2/neu

Mice bearing breast tumors (Week 25)1. 3D technique.

TUM

TUM

Tumor expression

EXP 1: Antitumour efficacy EXP 1: Antitumour efficacy in in HER-2/neu transgenic female HER-2/neu transgenic female

micemice

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

Tumour Number Tumour Growth Inhibition

NAX014 is effective NAX014 is effective in delaying the onset in delaying the onset and and the progression of HER2+ BC at well tolerated the progression of HER2+ BC at well tolerated

dosesdoses

High % of High % of tumour tumour free micefree mice after after NAX014 NAX014 treatment treatment

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

EXP 1: Antitumour efficacy in EXP 1: Antitumour efficacy in HER-2/neu HER-2/neu transgenic female mice transgenic female mice (2)(2)

EXP 2: Antitumour efficacy in EXP 2: Antitumour efficacy in HER-2/neu HER-2/neu transgenic female micetransgenic female mice

FVB-N 233 FVB-N 233 Her2/neu mice treated Her2/neu mice treated per os with 20 mg/kg per os with 20 mg/kg of NAX014 (2xweek)x8of NAX014 (2xweek)x8

NAX014 is effective by NAX014 is effective by oral route oral route in delaying the onset and the progression of HER2+ BCin delaying the onset and the progression of HER2+ BC

Age (Weeks)

Tumour Number

Age (Weeks)

Tumour Growth Inhibition

Antimetastatic efficacy in Antimetastatic efficacy in HER-2/neu transgenic female mice HER-2/neu transgenic female mice

NAX014 oral administration (20 mg/kg) Lung metastases NAX014 ControlMice with metastases (%) 12.5 55.5Cumulative no. of metastases

1 7

Mean size of metaststases (mm)

6 ±0 5.7 ±1.8

Maximum size of metastases (mm)

6 8

NAX014 shows antimetastatic efficacy by oral route NAX014 shows antimetastatic efficacy by oral route

FVB-N 233 FVB-N 233 Her2/neu mice treated per os with 20 mg/kg of NAX014 (2xweek)x8Her2/neu mice treated per os with 20 mg/kg of NAX014 (2xweek)x8

Tolerability in miceTolerability in mice (repeated IP administration) (repeated IP administration)

NAX012

0

25

50

75

100

125

0 5 10 15 20 25 30

Day

Body

Wei

ght (

% in

itial w

eigh

t)

0 mg/kg2.5 mg/kg5 mg/kg10 mg/kg20 mg/kg

NAX014

0

25

50

75

100

125

0 5 10 15 20 25 30

Day

Body

Wei

ght (

% in

itial

wei

ght)

0 mg/kg2.5 mg/kg5 mg/kg10 mg/kg20 mg/kg

NAX012(Body weight reduction) BBR

0

25

50

75

100

125

0 5 10 15 20 25 30

Day

Body

Wei

ght (

% in

itial

w

eigh

t)

0 mg/kg2.5 mg/kg5 mg/kg10 mg/kg

Berberine (Body weight reduction)

NAX014(Body weight reduction)Dose

(total dose)(mg/kg)

% Letality

NAX014 NAX012 Berberin

e

2.5 (20) 0 0 0

5 (40) 0 0 0

10 (80) 0 0 85.7

20 (160) 25 50 100

FVB mice; intraperitoneal (IP)treatment (2xweek)x4

NAX014NAX014Recovery of the antiproliferative activityRecovery of the antiproliferative activity

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

Irreversible Irreversible antiproliferantiproliferative effectative effect

HER2

p-HER2

β-actin

NAX014

Effect of NAX compounds on HER2/neu Effect of NAX compounds on HER2/neu expression and phosphorylation expression and phosphorylation

in HER2+ SK-BR-3 cellsin HER2+ SK-BR-3 cells

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

Treatment: NAX012 and NAX014 and berberine

50µM for 24h

Unique ability of Unique ability of NAX014 to block total NAX014 to block total HER2 expression, most HER2 expression, most probably by forming probably by forming complex(es) with DNA complex(es) with DNA and/or mRNA). and/or mRNA). Pirpaoli E. BioFactors, 2013, 39, 672.

Effect of NAX compounds on Effect of NAX compounds on expression of thymidylate synthase expression of thymidylate synthase

(TS) in MSTO cell line(TS) in MSTO cell line

Postulated mechanismPostulated mechanism

HER2 pathway and targets

NAX014 NAX014 is a novel anti-is a novel anti-HER2 agent, HER2 agent, targeting HER2 targeting HER2 expressionexpression

Dual Hispano-Italian spin Dual Hispano-Italian spin out newcoout newco

Cristina Geroni

Carmela Salvatore

Carmen Plasencia Narcis Clavell

ConclusionsConclusionsUnique ability to reduce cellular HER2 expression

NAX014

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 (Grant RDNET11-1-0001 to Aromics) under the 6th call of the EuroTransBio initiative, transnational project BERTA (BERberine as antiTumour Agents).

In vitro activity at µM concentrationsAntitumour and anti-metastatic efficacy on HER2+ tumours and tolerability at the effective doses in vivo by oral administrationCompound leading to a new series of 13- halophenylalkyl berberine derivatives with activity on HER2 overexpressing cancersFirst candidate for further development into Phase I clinical studies