RECENT ADVANCES IN

ANTITUMOUR BERBERINE

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

Email: p.lombardi@naxospharma.eu

Paolo Lombardi

Bitter-tasting isoquinoline quaternary alkaloid

extracted from plants of the genus Berberis, Coptis and others.

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

+

crespino

bladder cancer

brain cancer

breast cancer

cervical cancers

chondrosarcoma

endometrial cancer

esophageal cancer

neuroblastoma

oral cancer

osteosarcoma

ovarian cancer

pancreatic cancer

prostate cancer

thyroid carcinoma

In vitro and/or in vivo and/or as traditional therapies

gastric cancer

hepatocellular cancer(China, high incidence, aflatoxin)

leukemia

lung cancer

multiple myeloma

melanoma

Berberine

Anticancer properties

Berberine

Ongoing clinical trials 9 studies 2011 43 studies 2017

Alzheimer cardiovascular diseasesdiabetes defective endothelial function glucose metabolism/metabolic syndrome

H. pylori infectionhyperglycemia/glycemic control hyperlipemia insulin sensitivity/secretioninflammation

non-alcoholic fatty liver disease obesity platelet aggregation polycystic ovary syndrome schizophrenia

Prevention of Colorectal Adenomas in Patients With Previous

Colorectal Cancer

2 Chinese studies

Xijing Hospital of Digestive Disease, Xi'an, Shaanxi Shanghai Institute of Digestive Disease

Berberine: our

reviews

Berberine: how did we step

into?

2008 (sensible) and C13* (resistant) human ovarian cancer cell

lines

0%

200%

400%

600%

800%

1000%

1200%

1400%

1600%

1800%

2000%

ctr

l

1µ

M c

DD

P

3µ

M c

DD

P

2µ

M 5

-FU

1µ

M c

DD

P+

2µ

M

5-F

U

3µ

M c

DD

P+

2µ

M

5-F

U

24

h 1µ

M c

DD

P

24

h 3µ

M c

DD

P

24

h 2µ

M 5

-FU

1µ

M c

DD

P 2

4h

2µ

M 5

-FU

3µ

M c

DD

P 2

4h

2µ

M 5

-FU

2µ

M 5

-FU

24

h

1µ

M c

DD

P

TS

pro

tein

le

vel (%

of

co

ntr

ol)

0%

50%

100%

150%

200%

250%

300%

350%

ctr

l

4µ

M c

DD

P

12µ

M c

DD

P

5µ

M 5

-FU

4µ

M c

DD

P+

5µ

M

5-F

U

12µ

M c

DD

P+

5µ

M

5-F

U

24h 4µ

M c

DD

P

24h 1

2µ

M c

DD

P

24h 5µ

M 5

-FU

4µ

M c

DD

P 2

4h

5µ

M 5

-FU

12µ

M c

DD

P 2

4h

5µ

M 5

-FU

5µ

M 5

-FU

24h

4µ

M c

DD

P

TS

pro

tein

le

vel (%

of

co

ntr

ol)

Thymidylate Synthase: a key enzyme for cancer

treatment

TS inhibitors (5-FU) and DNA-alkylators (cisplatin) rapidly induce 2-5fold

increase of TS activity and protein level in tumour cells.

Berberine effect on TS level/activity

and TSmRNA expression

2008 (sensible) and C13* (resistant) human ovarian cancer cell

lines

Berberine

Cell growth inhibition

Surv

ival (%

of C

ontr

ol)

0%

25%

50%

75%

100%

ctrl Berb 5µM Berb 10µM

TS

pro

tein

le

ve

l (%

of

co

ntr

ol)

TS

pro

tein

level(%

of

contr

ol)

TS

activity(%

of

contr

ol)

TS protein levels/activity and TSmRNA expression were

determined after a 72-h treatment with berberine

2008 cells C13*cells

Berberine 0 5 10 0 5 10 µM

TS protein level

In collaboration with

Berberine effect on (onco)proteins

does berberine disable the synthesis of protein at a nascent stage or degrade protein?

possible new MoA of berberine suppressing nascent protein synthesis?

might imply targeting (post)-transcriptional control processes at mRNA level?

depletion of level of protein (and enzymatic activity)

Int J Mol Med, 2014, 34 409

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

J Cell Biol, 1962, 15, 589

Berberine & Nucleic Acids

DNA minor groove binder....

...or a DNA intercalator?

Berberine & Nucleic

Acids

Berberine modifications

Berberine represents an interesting

and attractive natural lead compound

Chemical modifications might provide derivatives with

better, different or specific biological effects and medical

indications with respect to the parent berberine

By performing unprecedented

chemical modifications of the

berberine structure, we obtained

a new class of derivatives with

specific antitumour properties1

1) US Patent 8,188,109B2

Aromatic interactions are ubiquitous in nature, their geometry

is relevant for the molecular recognition in biological systems1

L = Linker of variable length

and different functionalities

(Hetero)aromatic groups pending from a

suitable position of the parent alkaloid

skeleton

geometric propensity for additional

stacking-type, non-covalent aromatic

interactions with targets

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

Chemistry programme

Berberine NAX DerivativesBinding to DNA

1.770.35

2.11

11.01

7.6 7.586.8

0

2

4

6

8

10

12

Kix

10

-5(M

-1)

Binding 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)

Binding 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.

In collaboration with

0

2

4

6

8

10

12

Ki x

10

-5 (

M-1

)

Pyridylalkyl 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

In collaboration with

Berberine NAX DerivativesBinding to DNA

Berberine NAX DerivativesBinding to Nucleic Acids

Malignant Mesotheliomas (MT)

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

Fatal asbestos-exposure-associated cancers

Sites of MT are the pleural cavity (90%) and the peritoneal area (7%)

One-year survival time is < 40%

Increasing incidence wordlwide – rare tumour

>15,000 MT cases/year are diagnosed worldwide

The incidence is predicted to reach the max in 2030 decade

Berberine NAX derivatives: antiproliferative

effects in human mesothelioma cell lines (high

TS)

0

1

2

3

4

5

6

7

8

9

IC50 [m

M]

STO

MESOII

MSTO

0

1

2

3

4

5

6

7

8

9

IC5

0 [m

M]

STO

MESOII

MSTO

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

In collaboration with

TS protein expression levels: time-course in mesothelioma

MSTO-211H cells at the IC50 dose at 72 h

Berberine NAX derivatives:

effect on TS expression levels

Berberine

NAX035NAX012

NAX038

In collaboration with

Berberine NAX derivatives:

effect on TSmRNA expression levels

In collaboration with

C NAX012 NAX035 NAX038 5-FU

TSmRNA expression in MSTO-211H cells after treatment with compounds for different times at the EC50 dose (72h)

NAX035: autophagic process

NAX03510uM

Control

HCT116

C

NAX035

HMA

Appearance of vesicles in treated cells

Typical hallmark: conversion of the proteinLC3I into its active form LC3II

In collaboration with

NAX035

In vitro antiproliferative activity on chemo-resistant human MT

cell lines in comparison with standards

NAX035 overcomes pemetrexed and cisplatin resistance

in MT cells (collateral sensitivity)

In collaboration with

Antitumour activity of i.p. and oral NAX035,

qdxw/wx5w on the 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

In collaboration with

In vitro in vivo

NAX035 – drug candidate

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

Novel mechanism of action, targeting the synthesis

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 administration in a human mesothelioma

xenografted in nude mice

Licensed-out (Barcelona) IND

Berberine inhibits cellular growth of breast cancer cells and promotes

apoptosis by down-regulating the HER2/PI3K/Akt signaling pathway

Berberine in breast cancer

HER2+ Breast Cancer

represents 20–30% of invasive BC associated with more aggressive

disease progression and a poorer prognosis

HER2+: human

epidermal growth

factor receptor 2

positive

HER2+ Breast Cancer: problems to

solveHER2-targeting gold standard drugs show modest efficacy as single agent

and substantial toxicity in combination therapy

Trastuzumab has been associated with significant adverse effects and, in

particular, with an increased risk of severe cardiac dysfunction

both de novo and acquired Trastuzumab resistance has been observed

price of therapy deemed too high for routine NHS in relation to benefits it gives

New agents:

- exhibiting a different mechanism of action

- tangibly lowering the out-of-the pocket cost burden by

patients

in respect to current targeted therapies might offer a

new option for treating HER2+ BC patients

Chemical structures of tested berberine derivatives

In collaboration with

Time-dependent antiproliferative activities

of NAX compounds against HER2+ breast

cancer SK-BR-3 cells

TimeIC50 µM

NAX014 NAX012 NAX013 NAX035 Berberine

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 ±å2.9 >100 48.6 ±6.7 36.0 ±1.8

Effects of NAX compounds on

HER2/neu expression and

phosphorylation

Treatment: berberine, NAX012 and NAX014 (50µM-

24h)

HER2

p-HER2

β-actin

Ctr

l

La

pa

tin

ib+

Tra

stu

zu

ma

b

In collaboration withSK-BR-3 cells

MSTO-211H cells

NAX014: apoptotic process

In collaboration with

BioFactors, 2013, 39, 672.

SK-BR-3 cells

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: efficacy in HER-2/neu transgenic

female mice treated i.p. 2.5 mg/kg (2xweek)x12

Tumour Number Tumour Growth Inhibition

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

doses

High % Tumour Free Mice

NAX014: in vivo evaluation of

vascularization of mammary tumours

Tumour mass vessel density

Microvessels density in tumour masses from

controls, berberine and NAX014 treated

mice. The vascular architecture was

evaluated in vivo by using SDF

videomicroscopy.

Ctrl

BBR

NAX014

17.2 mm/mm2

12.07 mm/mm2

9.9 mm/mm2

In collaboration with

NAX014: In vivo acute and

chronic toxicity in mice

NAX014 LD50 30.9 mg/kg Berberine LD50 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 NAX014Body weight changes in subchronic

toxicity study

(as percent of the day 0 weight)

NAX014

BERBERINE

Tumour Growth

Inhibition

Tumour Number

NAX014 is effective by oral route in delaying

the onset and the progression of HER2+ BC

and shows antimetastatic efficacy

Antimetastatic effect

Lung metastases NAX014 Control

% Mice with

metastases

12.5 55.5

Cumulative no. 1 7

Mean size (mm) 6 ±0 5.7 ±1.8

Maximum size (mm) 6 8

NAX014: FVB-N 233 Her2/neu mice

treated per os with 20 mg/kg

(2xweek)x8

Tum

ou

r V

olu

me

(% C

on

tro

l)M

ean

Tu

mo

ur

Nu

mb

er

weeks

weeks

NAX014 – drug candidate

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

NAX060 – follow-on

N

O

O

OCH3

OCH3

I

(H2C)2

Cl

NAX014

N

O

O

OCH3

OCH3

Cl

(H2C)3

Cl

NAX060

Cl

cell-cycle checkpoint molecules involved in cell senescence

Triple negative BC

NAX060 – follow-on

HER2+ ER+

TNBC

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

Alkylation of enamine (7,8-dihydroberberine) derivatives

from low to moderate yields - better with iodides or activated halides berberine and tetrahydroberberine from disproportionation of enamine as major by-products

2)

1)

Chemistry programme

berberine chloride(purchased from T&W Group- Shanghai)

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

Chemistry programme

generally from good to very good yields

Uncommon aldehyde-enamine condensation1,2

Esters, Amides, retro-Amides

even with glyoxylic acid

7,8-dihydroberberine

Conclusions

berberine exhibits diverse pharmacological properties in a

wide spectrum of clinical applications

that might prevent its use as a drug for a definite therapy

structure of berberine represents a biologically interesting

skeleton chemical manipulation might lead to select the

therapeutic areas

- easily available, cheap raw material

- trivial, standard chemical synthetic

processes - finished products with

low cost of goods

and manufacture