lombardi iddst xi'an
TRANSCRIPT
RECENT ADVANCES IN
ANTITUMOUR BERBERINE
Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy
Email: [email protected]
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