Recent advances in antitumour berberine

Gaetano Fiorillo, Tanjia Monir Syeda , Paolo Lombardi

via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy

Email: staff@naxospharma.eu

Siena, 15-19 May 2016

Berberine

Extracted from plants of the genus Berberis,

Coptis and others.

In use in the Ayurvedic and Chinese medicines.

Anti-microbial/parasitic,

Anti-diarrheal, anti-inflammatory,

Anti-arryhthmic,

Cholesterol-lowering

Anticancer1,2

Berberine Chloride

1L. M. Guamán Ortiz, P.Lombardi, M. Tillhon, A. I. Scovassi, Molecules ,2014, 19, 12349-123672 L. M. Guamán Ortiz, P.Lombardi, M. Tillhon, A. I. Scovassi, Biochemical Pharmacology, 2012, 84, 1260–1267

2011: 7 clinical trials

2016: 25 clinical trials

Mondello, R. et al.,Bioorg Med Chem, 2003,

505–514 (NMR Studies)

Gratteri, P et al.Chem. Commun. 2011, 4917-

4919 (RX studies)

minor groove binding

BerberineDNA Interaction Mechanism

“ Interaction between nucleic acids and berberine sulfate “

Journal of Cellular Biology, 15, 1962, 589

…has been reported since decades…

Minor groove binding or Intercalation ????

Berberine represents an interesting and

attractive natural lead compound

Chemical modifications might select more

specific medical indications resulting in

derivatives with better (or different)

biological effects compared to the parent

berberine

Performing rational chemical modifications of

berberine structure led to a new class of

derivatives with antitumour properties

Chemical

Programme

Berberine

Aromatic interactions are ubiquitous in nature,

their geometry is relevant for the molecular recognition

in biological systems 1

Chemical

Programme

L = Linker with different functionalities

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)

Berberine derivativesSynthetic Methods_1

1)

from low to moderate yields -berberine and tetrahydroberberinefrom disproportionation of enamineas major by-products

2)

generally from good to very good yields

Uncommon aldehyde-enamine condensation1,2

1 Cook, AG, Enamines Synthesis, Structure and reaction, 1988, 2002 Iwasa, K, et al., Planta Medica, 1997, 196

Berberine derivativesSynthetic Methods_2

1) Commercially available aldehyde

2) Commercially available alcohol followed by oxidation

(e.g. : PCC or TEMPO)

3) Homologation starting from the above

Berberine DerivativesAldehyde Intermediates_1

Commercially

available

Berberine DerivativesAldehyde Intermediates_2

Commercially

available

Commercially

available

Commercially

available

Improved DNA intereaction

of berberine derivatives_1

1.770.35

2.11

11.01

7.6 7.586.8

0

2

4

6

8

10

12

Kix

10

-5(M

-1)

Interaction costants of NAXs 1

1.770.48 0.51

7.07

10.048.90

7.48

0

2

4

6

8

10

12

Kix

10

-5(M

-1)

Interaction costants of NAXs2

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.

n = 3

n = 4

Monophenyl Derivatives

Diphenyl Derivatives

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

n = 5

0

2

4

6

8

10

12

Ki x

10

-5 (

M-1

)

Interaction costants of NAXsPyridylalkyl Derivatives

Improved DNA intereaction

of berberine derivatives_2

Binding to human telomeric

G quadruplex

P. Gratteri, M. Ferraroni, C. Bazzicalupi, F. Papi, G. Fiorillo, L. M. Guamán Ortiz, A. Nocentini, A. I. Scovassi, P. LombardiChemistry An Asian Journal, 2016, 11(7),1107-15

NAX 053 – d[TAG3(T2AG3)T]

adduct crystal structure

Our1 and others’ studies identified

berberine as a novel, non specific

inhibitor of the nascent synthesis

of some proteins, supposedly

acting as a RNA silencing agent

Berberineeffect on TS expression

1G. Marverti, A. Ligabue, P. Lombardi, S. Ferrari, M. G. Monti, C. Frassinetti, M.P. Costi,Int. Journal of Oncology, 2013, 43, 129

2008 cells = cisplatin sensitive

C13 cells = cisplatin resistant

Berberine

NAX035NAX012

Berberine derivatives:

effect on TS over-expression

in mesothelioma cell lines

TS levels: time-course in mesothelioma MSTO-211H cells at the

IC50 dose at 72 h

NAX038

Berberine DerivativesAntiproliferactive effects against human

mesothelioma cell lines

STO, MESOII = peritoneal mesothelima cell linesMSTO = pleural mesothelioma cell lines

0123456789

IC50

[mM

]

Mesothelioma cell lines

STO

MESOII

MSTO

0

1

2

3

4

5

6

7

8

9

IC5

0[m

M]

Mesothelioma cell lines

STO

MESOII

MSTO

Antitumour activity of i.p. and oral

NAX035, on the peritoneal STO human

mesothelioma s.c. xenografted in nude

mice

Route Dose

mg/kgTVI% (+32)

(PvsControls)

Max

BWL% TOX

i.p. 1 52 (0.1181) 8 0/9

p.o. 10 72 (0.0434) 10 0/9

p.o. 15 74 (0.0373) 5 0/8

Correlation between TS protein levels in vitro and in vivo in tumour tissue

samples examined at the end of the p.o. treatment period

NAX 012 NAX 013 NAX 014 NAX 035 Berberine

24 h 94.2±1.2 >100 52.3±3.2 >100 91.8±2.8

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

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

Antiproliferative effect (IC50 mM)

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

M. Provinciali, P. Lombardi, et al, Biofactors, 39, 2013, 672-679

Berberine derivativesAntiproliferactive effects on HER2+

human Breast Cancer cells (SK-BR-3)

(SK-BR-3)

Tumour Number Tumour Growth Inhibition High % Tumour Free Mice

NAX014: antitumour efficacy in

HER-2/neu spontaneous tumours transgenic

female mice

Both i.p. [2.5mg/kg-(2xweek)x12]

and oral [20mg/kg-(2xweek)x8]

administrations of NAX014reduced tumour volume andnumber, delayed the onsetand progression of HER2+ BCcompared to control group

Tumour Growth Inhibition Tumour Number

i.p.

p. o.

Provinciali, M., Lombardi, P. et al., Carcinogenesis, 2015, 1–11

HER2

p-HER2

β-actin

Ctr

l

Different NAX compounds specifically

inhibit the expression of different

proteins

La

pa

tin

ib+

Tra

stu

zu

ma

b

NAX012NAX014 NAX035

Innovative proprietary compound, structurally related

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 i.p. administration

in a human mesothelioma xenografted nude

mice

Conclusions:

NAX 035

Conclusions:

NAX 014

anticancer and anti-metastatic efficacy on HER2+ tumours

in vitro activity at µM concentrations

in vivo tolerability by i.p.and oral administration

at the effective dose

Innovative proprietary compound, structurally related to the plant isoquinoline alkaloid berberine

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

Financial supports were provided by

Ministero dello Sviluppo Economico (Grant 01705) to Naxospharma srl (Project coordinator) and to Istituto Tumori Milano

and byAgència per a la competitivitat de l'empresa ACC1O (Grant RDNET11-1-0001) to

Aromics SL, Barcelonaunder the 6th call of the EuroTransBio initiative, Transnational joint project BERTA

(BERberine as antiTumour Agents).

Aknowledgeme

nts

Regione Lombardia (Grant 13810040) to Naxospharma srl and to Istituto di Genetica Molecolare – CNR Pavia, Project PLANT CELL

Ente Cassa di Risparmio di Firenze (Grant 2014.0309) to Department NEUROFARBA, University of Firenze