This document summarizes recent advances in the anticancer properties of berberine. Berberine is an alkaloid extracted from plants that has been used in traditional Chinese and Ayurvedic medicines. It has diverse pharmacological activities including antimicrobial, anti-inflammatory, and anticancer effects. Berberine shows anticancer activity against various cancer types in vitro and in vivo. Ongoing clinical trials are investigating berberine's effects on cancer as well as other diseases. Chemical modifications of berberine have produced derivatives with improved binding to DNA and anticancer activity. In particular, NAX035 has shown efficacy against mesothelioma in vitro and in vivo models at well-tolerated doses.

1. RECENT ADVANCES
IN
ANTITUMOUR BERBERINE
Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy
Email: p.lombardi@naxospharma.eu
Paolo Lombardi

2. 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

3. 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

4. Berberine
Ongoing clinical trials
9 studies 2011  43 studies 2017
Alzheimer
cardiovascular diseases
diabetes
defective endothelial function
glucose metabolism/
metabolic syndrome
H. pylori infection
hyperglycemia/
glycemic control
hyperlipemia
insulin sensitivity/secretion
inflammation
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

5. Berberine: our
reviews

6. 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%
ctrl
1µMcDDP
3µMcDDP
2µM5-FU
1µMcDDP+2µM
5-FU
3µMcDDP+2µM
5-FU
24h1µMcDDP
24h3µMcDDP
24h2µM5-FU
1µMcDDP24h
2µM5-FU
3µMcDDP24h
2µM5-FU
2µM5-FU24h
1µMcDDP
TSproteinlevel(%ofcontrol)
0%
50%
100%
150%
200%
250%
300%
350%
ctrl
4µMcDDP
12µMcDDP
5µM5-FU
4µMcDDP+5µM
5-FU
12µMcDDP+5µM
5-FU
24h4µMcDDP
24h12µMcDDP
24h5µM5-FU
4µMcDDP24h
5µM5-FU
12µMcDDP24h
5µM5-FU
5µM5-FU24h
4µMcDDP
TSproteinlevel(%ofcontrol)
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.

7. Berberine effect on TS level/activity
and TSmRNA expression
2008 (sensible) and C13* (resistant) human ovarian cancer cell
lines
Berberine
Cell growth inhibition
Survival(%ofControl)
0%
25%
50%
75%
100%
ctrl Berb 5µM Berb 10µM
TSproteinlevel(%ofcontrol)
TSproteinlevel(%of
control)
TSactivity(%ofcontrol)
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

8. 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

9. 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?

10. Berberine & Nucleic
Acids

11. 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

12. 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

13. Berberine NAX Derivatives
Binding to DNA
1.77
0.35
2.11
11.01
7.6 7.58
6.8
0
2
4
6
8
10
12
Kix10-5(M-1)
Binding costants of NAXs 1
1.77
0.48 0.51
7.07
10.04
8.90
7.48
0
2
4
6
8
10
12
Kix10-5(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

14. 0
2
4
6
8
10
12
Kix10-5(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 Derivatives
Binding to DNA

15. Berberine NAX Derivatives
Binding to Nucleic Acids

16. 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

17. Berberine NAX derivatives: antiproliferative
effects in human mesothelioma cell lines (high
TS)
0
1
2
3
4
5
6
7
8
9
IC50[mM]
STO
MESOII
MSTO
0
1
2
3
4
5
6
7
8
9
IC50[mM]
STO
MESOII
MSTO
STO, MESOII = peritoneal mesothelioma cell lines MSTO = pleural mesothelioma cell line
In collaboration with

18. 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
NAX035
NAX012
NAX038
In collaboration with

19. 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)

20. NAX035: autophagic process
NAX035
10uM
Control
HCT 116
C
NAX035
HMA
Appearance of vesicles
in treated cells
Typical hallmark: conversion of the protein
LC3I into its active form LC3II
In collaboration with

21. 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

22. Antitumour activity of i.p. and oral NAX035,
qdxw/wx5w on the STO human
mesothelioma s.c. xenografted in nude
mice
Route Dose
mg/kg
TVI% (+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

23. 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 mesotheliom
xenografted in nude mice
Licensed-out (Barcelona) IND

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

25. 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

26. HER2+ Breast Cancer: problems to
solve
HER2-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

27. Chemical structures of tested berberine derivatives
In collaboration with
Time-dependent antiproliferative activities
of NAX compounds against HER2+ breast
cancer SK-BR-3 cells
Time
IC50 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

28. Effects of NAX compounds on
HER2/neu expression and
phosphorylation
Treatment: berberine, NAX012 and NAX014 (50µM-
24h)
HER2
p-HER2
β-actin
Ctrl
Lapatinib+
Trastuzumab
In collaboration with
SK-BR-3 cells
MSTO-211H cells

29. NAX014: apoptotic process
In collaboration with
BioFactors, 2013, 39, 672.
SK-BR-3 cells

30. 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

31. NAX014: in vivo evaluation of
vascularization of mammary tumour
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

32. 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 NAX014
Body weight changes in subchronic
toxicity study
(as percent of the day 0 weight)
NAX014
BERBERINE

33. 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
TumourVolume(%Control)MeanTumourNumber
weeks
weeks

34. 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

35. NAX060 – follow-on
N
O
O
OCH3
OCH3
I
(H2C)2
Cl
NAX014
N
O
O
OCH3
OCH3
Cl
(H2C)3
Cl
NAX060
Cl

36. cell-cycle checkpoint molecules
involved in cell senescence
Triple negative BC
NAX060 – follow-on
HER2+ ER+
TNBC

37. 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)

38. 2 Iwasa, K, et al., Planta Medica, 1997, 1961 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

39. 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