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RECENT ADVANCES RECENT ADVANCES IN IN

ANTITUMOUR BERBERINEANTITUMOUR BERBERINEPaolo Lombardi,a,c Cristina Geroni,c Carmen Plasencia,b,c Carmela Salvatorec

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: [email protected]

mailto:[email protected]

Background history

Thymidylate Synthase: Thymidylate Synthase: a key enzyme for a key enzyme for cancer treatmentcancer treatment

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

targets in the anticancer area.targets in the anticancer area. TS is present as an enzymatically active dimeric form in TS is present as an enzymatically active dimeric form in

equilibrium with a catalytically inactive monomeric form. equilibrium with a catalytically inactive monomeric form.

TS monomer is in equilibrium with a TS-mRNA complex, by TS monomer is in equilibrium with a TS-mRNA complex, by binding binding

to its own mRNA. to its own mRNA.

The cellular levels (and activity) of TS depends on an The cellular levels (and activity) of TS depends on an autoregulatory loop which allows TS to control its own autoregulatory loop which allows TS to control its own

translation from its own mRNA. translation from its own mRNA.

Current drugs are only TS inhibitors which rapidly induce 2-5fold Current drugs are only TS inhibitors which rapidly induce 2-5fold increaseincrease

of TS levels in tumour cells. of TS levels in tumour cells.

There is an obvious need for new compounds There is an obvious need for new compounds able to interrupt the abnormal production of TS able to interrupt the abnormal production of TS

in cancer cellsin cancer cells

Expression of TS predicts clinical outcomes of TS inhibitors-Expression of TS predicts clinical outcomes of TS inhibitors-containing containing

chemotherapy and increased TS levels are responsible for chemotherapy and increased TS levels are responsible for resistance. resistance.

Thymidylate Synthase: Thymidylate Synthase: a key enzyme for a key enzyme for cancer treatmentcancer treatment

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

Antisense oligonucleotides Antisense oligonucleotides targeting TSmRNA targeting TSmRNA in cancer cellsin cancer cells

DNA minor groove DNA minor groove binders able to bind binders able to bind

to to TSmRNA TSmRNA We investigated We investigated distamycin, distamycin, DAPI and DAPI and Hoeschst 33258Hoeschst 33258

20082008 (sensible) and (sensible) and C13* C13* (resistant) (resistant) human human “ovarian” “ovarian” cancer cell cancer cell lineslines

DNA minor groove DNA minor groove binders able to bind binders able to bind

to to TSmRNA:TSmRNA:in vitro activity in vitro activity

TS expression after 72h-treatment with DNA minor groove TS expression after 72h-treatment with DNA minor groove binder compoundsbinder compounds

TS p

rote

in le

vel

(%of

Con

trol

)TS

Act

ivity

(%of

Con

trol

)

DAPI and HoechstDAPI and HoechstDistamycinDistamycin 2008 cells 2008 cells C13*cellsC13*cells

Berberine effect Berberine effect on TS expression on TS expression

20082008 (sensible) and (sensible) and C13* C13* (resistant) human (resistant) human “ovarian” cancer cell lines“ovarian” cancer cell lines

BerberineBerberineCell growth inhibitionCell growth inhibition

Sur

viva

l (%

of C

ontro

l)

TS p

rote

in le

vel(%

of c

ontro

l)

TS a

ctiv

ity(%

of c

ontro

l)

Effect of berberine on TS espressionEffect of berberine on TS espressionTS protein levels and activity were determined after a 72-h treatment with TS protein levels and activity were determined after a 72-h treatment with

berberineberberine

2008 cells2008 cells C13*cellsC13*cells

Berberine 0 5 10 0 5 Berberine 0 5 10 0 5 10 µM10 µM

TS protein level

BerberineBerberineBitter-tasting isoquinoline quaternary alkaloid extracted from plants of the genus Berberis, Coptis and others.

Berberine

In use in the Ayurvedic, Chinese and Native American medicines since hundreds of years. Pleiotropic substance with diverse pharmacological properties and activities:

anti-microbial/parasitic, anti-diarrheal, anti-inflammatory,anti-arryhthmic, cholesterol-lowering and anti-tumour

Berberine Berberine mechanism of mechanism of

action action The precise molecular basis of its many biological The precise molecular basis of its many biological

activities are still debated.activities are still debated.

Modulation of protein expression Modulation of protein expression by interaction with by interaction with nucleic acids is postulated.nucleic acids is postulated.

The interactions between The interactions between berberine and nucleic acids berberine and nucleic acids has been reportedhas been reported since 1962since 1962

J Cell Biol, 1962, 15, 589

However, is the alkaloid a DNA minor groove binder....However, is the alkaloid a DNA minor groove binder....

...or a DNA intercalator?...or a DNA intercalator?

Berberine: Berberine: DNA interaction DNA interaction mechanism mechanism

BerberineBerberine

A definite medical potential has been established in a wide spectrum of clinical applications including: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 studies 2011 23 studies 2015).

BerberineBerberine

Anticancer properties of berberine in Anticancer properties of berberine in preclinical studies preclinical studies

have been widely published since many years have been widely published since many years

however, there are no clinical trials in the area presently however, there are no clinical trials in the area presently Y. Feng, et al., Journal of Ethnopharmacology, on line accepted manuscript

Our findings

Berberine Berberine Berberine represents an interesting Berberine represents an interesting and attractive natural lead and attractive natural lead compoundcompoundChemical modifications might provide derivatives with better, different or specific biological effects and medical indications with respect to the parent berberine

By performing unprecedentedBy performing unprecedentedchemical modifications of thechemical modifications of theberberine structure, we obtained berberine structure, we obtained a new class of derivatives witha new class of derivatives withspecific antitumour propertiesspecific antitumour properties

Chemistry programmeChemistry programme

Aromatic interactions are ubiquitous in nature, and their geometry Aromatic interactions are ubiquitous in nature, and their geometry is relevant for the molecular recognition in biological systemsis relevant for the molecular recognition in biological systems11

linkers of variable length and functionality

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

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

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

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 programmeChemistry programme

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

Uncommon aldehyde-enamine condensation1,2

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

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

generally from good to very good yields

Berberine Derivatives Binding to DNA

N

O

O

OCH3

OCH3

Cl

(H2C)n

n = 1 NAX 044n = 2 NAX 043n = 3 NAX 039n = 4 NAX 042n = 5 NAX 056n = 6 NAX 057

N

O

O

OCH3

OCH3

Cl

(H2C)n

n = 0 NAX 045n = 1 NAX 046n = 2 NAX 035 n = 3 NAX 053n = 4 NAX 080n = 5 NAX 081

1D. Bhowmik, M. Hossain, F. Buzzetti, R. D’Auria, P. Lombardi, G.S.Kumar, J. Phys. Chem. B, 2012, 116, 2314−24.2D. Bhowmik, F. Buzzetti, G. Fiorillo, F. Orzi, T. Syeda Monir, P. Lombardi, G.S. Kumar, Med. Chem. Comm., 2014, 5, 226-31.

Berberine Derivatives Binding to DNA

N

O

O

OCH3

OCH3

Cl

(H2C)n

Nn = 1 NAX 071n = 2 NAX 120n = 3 NAX 075n = 4 NAX 077n = 5 NAX 079

S. Chatterjee, S. Mallick, F. Buzzetti, G. Fiorillo, T. M. Syeda, Paolo Lombardi, K. Das Saha, G. S. Kumar, RCS Adv., 2015, 5, 90632

Malignant Mesotheliomas Malignant Mesotheliomas (MT)(MT)

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

Increasing incidence wordlwide – rare tumourIncreasing incidence wordlwide – rare tumour 14,200 MT cases/year are diagnosed worldwide (1994-2008 data)14,200 MT cases/year are diagnosed worldwide (1994-2008 data) The incidence is predicted to reach the max in 2030 decadeThe incidence is predicted to reach the max in 2030 decade

High levels of High levels of thymidylate synthase thymidylate synthase protein expression in MT protein expression in MT patients are the marker of lack of efficacy of currrent patients are the marker of lack of efficacy of currrent

treatments treatments (pemetrexed, cisplatin(pemetrexed, cisplatin))

Berberine NAX Berberine NAX derivatives: derivatives:

antiproliferative effects antiproliferative effects in human mesothelioma in human mesothelioma

cell lines cell lines (high TS)(high TS)

N

O

O

OCH3

OCH3

Cl

(H2C)n

n = 0 NAX 045n = 1 NAX 046n = 2 NAX 035 n = 3 NAX 053n = 4 NAX 080n = 5 NAX 081

N

O

O

OCH3

OCH3

Cl

(H2C)n

n = 1 NAX 044n = 2 NAX 043n = 3 NAX 039n = 4 NAX 042n = 5 NAX 056n = 6 NAX 057

STO, MESOII = peritoneal mesothelioma cell lines MSTO = pleural mesothelioma cell line

TS protein expression levels: time-course in mesothelioma MSTO-211H cells at the IC50 dose at

72 h

Berberine NAX derivatives: Berberine NAX derivatives: effect on TS expressioneffect on TS expression

Berberine

NAX035NAX012

NAX038

NAX035NAX035

In vitro In vitro antiproliferative activity on chemo-restistant human MT antiproliferative activity on chemo-restistant human MT cell lines in comparison with standardscell lines in comparison with standards

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

Antitumour activity of i.p. and oral Antitumour activity of i.p. and oral NAX035, qdxw/wx5w on the NAX035, qdxw/wx5w on the peritoneal STO human peritoneal STO human mesothelioma s.c. xenografted in mesothelioma s.c. xenografted in nude micenude mice

Route Dose mg/kg

TVI% (+32) (PvsControls)

Max

BWL% TOX

i.p. 1 52 (0.1181) 8 0/9p.o. 10 72 (0.0434) 10 0/9p.o. 15 74 (0.0373) 5 0/8

Istituto Nazionale Tumori, Milano, ItalyIstituto Nazionale Tumori, Milano, Italy

Correlation between TS protein levels in vitro and in vivo in tumour tissue samples examined at the end of the p.o. treatment period

NAX035NAX035

Innovative proprietary compound, structurally Innovative proprietary compound, structurally related related

to the plant isoquinoline alkaloid berberine. to the plant isoquinoline alkaloid berberine. Novel mechanism of action, targeting the expression of TS protein, differently from previous TS inhibitors

Efficacy on chemoresistant tumour cells Antitumour efficacy and tolerability at the effective doses by oral and ip administrationin a human mesothelioma xenografted in nude mice

HER2HER2++ Breast Breast CancerCancer

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

treating HER2treating HER2++ BC patients BC patients

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

Berberine in Berberine in breast cancerbreast cancer

Antiproliferative activities of NAX Antiproliferative activities of NAX compds against HER2+ human compds against HER2+ human (SK-BR-3) and(SK-BR-3) and murine (N202.1A) breast cancer murine (N202.1A) breast cancer cellscellsChemical structures of tested berberine Chemical structures of tested berberine

derivativesderivatives

Centro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona, Italy

Time-dependent antiproliferative Time-dependent antiproliferative activities of NAX compounds activities of NAX compounds against HER2+ breast cancer SK-against HER2+ breast cancer SK-BR-3 cellsBR-3 cells

Time IC50 µM

NAX014 NAX012 NAX013 NAX035 Berberine

(BRB)24 h 52.3 ±3.2 94.2

±1.2 >100 >100 91.8±2.8

48h 30.7 ±2.1 46.6 ±2.5 >100 >100 58.4 ±1.9

72 h 26.5 ±6.7 31.9 ±2.9 >100 48.6 ±6.7 36.0 ±1.8

SK-BR-3 cellsSK-BR-3 cells


Effects of NAX compounds Effects of NAX compounds on HER2/neu expression on HER2/neu expression

and phosphorylationand phosphorylation

Treatment: NAX012 and NAX014 and berberine 50µM for 24h

HER2

p-HER2

β-actin

BioFactors, 2013, 39, 672.


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. BC is palpable starting on Week 25.

Tumour expression

NAX014: Antitumour NAX014: Antitumour efficacy efficacy in in

HER-2/neu transgenic HER-2/neu transgenic female micefemale mice

Tumour Number Tumour Growth Inhibition

NAX014 is effective NAX014 is effective in delaying the onset in delaying the onset and the progression of and the progression of HER2+ BC at well tolerated dosesHER2+ BC at well tolerated doses

FVB-N 233 FVB-N 233 Her2/neu mice Her2/neu mice treated IPtreated IP,, 2.5mg/kg of cmpds (2xweek)x122.5mg/kg of cmpds (2xweek)x12

High % Tumour Free Mice


NAX014: NAX014: In vivo In vivo acute and acute and chronic toxicity in mice chronic toxicity in mice

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 Growth Inhibition

Tumour Number

NAX014 is effective by NAX014 is effective by oral routeoral route in in delaying the onset and the delaying the onset and the progression progression of HER2of HER2++ BC and shows BC and shows antimetastatic efficacyantimetastatic efficacy

Antimetastatic effectLung metastases NAX014 Control% Mice with metastases

12.5 55.5

Cumulative no. 1 7Mean size (mm) 6 ±0 5.7 ±1.8Maximum size (mm)

6 8

NAX014: FVB-N 233 NAX014: FVB-N 233 Her2/neu Her2/neu mice treated mice treated per os with per os with 20 mg/kg20 mg/kg (2xweek)x8(2xweek)x8

Tum

our V

olum

e (%

Con

trol

)M

ean

Tum

our N

umbe

r

weeks

weeksCentro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona, Italy

NAX014: NAX014: in vivo in vivo evaluation of evaluation of vascularization of mammary tumoursvascularization of mammary tumours

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

Microvessels density in tumor masses from controls, berberine and NAX014 treated mice. The vascular architecture was evaluated in vivo by using SDF videomicroscopy.Centro Tecnologie Avanzate dell'Invecchiamento, INRCA,

Ancona, Italy

NAX014NAX014

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

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

Innovative proprietary compound, structurally Innovative proprietary compound, structurally related related

to the plant isoquinoline alkaloid berberine to the plant isoquinoline alkaloid berberine Unique ability to reduce cellular HER2 expression Unique ability to reduce cellular HER2 expression via via a postulated a postulated novel mechanismnovel mechanism

Conclusions

ConclusionsConclusionsBerberine exhibits diverse pharmacological Berberine exhibits diverse pharmacological properties in a wide spectrum of clinical properties in a wide spectrum of clinical applications applications that might prevent its use as a drug for a that might prevent its use as a drug for a definite therapy definite therapy the structure of berberine the structure of berberine represents a biologically represents a biologically interesting skeleton interesting skeleton berberine is an attractive natural berberine is an attractive natural lead compound lead compound rational chemical manipulation might lead rational chemical manipulation might lead to select the therapeutic areato select the therapeutic areas chemical modifications in appropriate positions chemical modifications in appropriate positions might discriminate might discriminate and narrow selective and narrow selective medical indicationsmedical indications

Berberine

Int J Mol Med, 2014, 34 409

Berberine unspecifically suppresses nascent protein synthesisby appreciation of the differences in mRNA translational control between normal

and cancer cells.

This has been reported by us and by others

ConclusionsConclusions

ConclusionsConclusionsHowever:

NAX012

NAX014

NAX035

Future Future perspectivesperspectives

BioFactors, 2013, 39, 652.

Future Future perspectivesperspectives

Tel24 5’-(TTAGGG)4-3’

NAX053

Financial supports were provided by

Ministero dello Sviluppo Economico (Grant. 01705 to Naxospharma)

andAgència 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).

AknowledgeAknowledgementsments

Science

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