8th Euro Global Summit on Cancer Therapy- Nov 03-05 2015 - Valencia - Spain

1. RECENT ADVANCESRECENT ADVANCES
ININ
ANTITUMOUR BERBERINEANTITUMOUR BERBERINE
Paolo Lombardi,a,c
Cristina Geroni,c
Carmen Plasencia,b,c
Carmela Salvatorec
a
Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy
b
Aromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain
c
Aesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi, Ancona, Italy
Email: p.lombardi@naxospharma.eu

2. Background history

3. Thymidylate Synthase:Thymidylate Synthase: a key enzyme for cancera key enzyme for cancer
treatmenttreatment
Thymidylate Synthase (TS) is a key enzyme in the DNA synthesis and cellThymidylate Synthase (TS) is a key enzyme in the DNA synthesis and cell
proliferation and represents one of the best-known drug targets in theproliferation and represents one of the best-known drug targets in the
anticancer area.anticancer area.
TS is present as an enzymatically active dimeric form in equilibrium with aTS is present as an enzymatically active dimeric form in equilibrium with a
catalytically inactive monomeric form.catalytically inactive monomeric form.
TS monomer is in equilibrium with a TS-mRNA complex, by bindingTS monomer is in equilibrium with a TS-mRNA complex, by binding
to its own mRNA.to its own mRNA.
The cellular levels (and activity) of TS depends on an autoregulatoryThe cellular levels (and activity) of TS depends on an autoregulatory
loop which allows TS to control its own translation from its own mRNA.loop which allows TS to control its own translation from its own mRNA.

4. Current drugs are only TS inhibitors which rapidly induce 2-5fold increaseCurrent drugs are only TS inhibitors which rapidly induce 2-5fold increase
of TS levels in tumour cells.of TS levels in tumour cells.
There is an obvious need for new compounds able toThere is an obvious need for new compounds able to
interrupt the abnormal production of TS in cancer cellsinterrupt the abnormal production of TS in cancer cells
Expression of TS predicts clinical outcomes of TS inhibitors-containingExpression of TS predicts clinical outcomes of TS inhibitors-containing
chemotherapy and increased TS levels are responsible for resistance.chemotherapy and increased TS levels are responsible for resistance.
Thymidylate Synthase:Thymidylate Synthase: a key enzyme for cancera key enzyme for cancer
treatmenttreatment

5. An innovative mechanism of action:
targeting TS translational autoregulationtargeting TS translational autoregulation
Antisense oligonucleotidesAntisense oligonucleotides targeting TSmRNAtargeting TSmRNA in canin can
cellscells

6. DNA minor grooveDNA minor groove
binders able to bind tobinders able to bind to
TSmRNATSmRNA
[D A P I µ M
0 1 0 2 0 3 0 4 0 5 0
SURVIVAL(%ofcontrol)
0
2 5
5 0
7 5
1 0 0
2 0 0 8 c e lls
C 1 3 * c e lls
[H O E C H S T ] µ M
0 5 1 0 1 5 2 0
SURVIVAL(%ofcontrol)
0
2 5
5 0
7 5
1 0 0
2 0 0 8 c e lls
C 1 3 * c e lls
We investigatedWe investigated
distamycin, DAPIdistamycin, DAPI
and Hoeschstand Hoeschst
3325833258
20082008 (sensible)(sensible)
andand C13*C13*
(resistant) human(resistant) human
““ovarianovarian”” cancercancer
cell linescell lines

7. DNA minor grooveDNA minor groove
binders able to bind tobinders able to bind to
TSmRNA:TSmRNA:
in vitro activityin vitro activity
TS expression after 72h-treatment with DNA minor groove binderTS expression after 72h-treatment with DNA minor groove binder
compoundscompounds
TS expression after 72h-treatment with DNA minor groove binderTS expression after 72h-treatment with DNA minor groove binder
compoundscompounds
TSproteinlevel
(%ofControl)
TSActivity
(%ofControl)
DAPI and HoechstDAPI and HoechstDistamycinDistamycin  2008 cells2008 cells
 C13*cellsC13*cells

8. Berberine effectBerberine effect
on TS expressionon TS expression
20082008 (sensible) and(sensible) and C13*C13* (resistant) human(resistant) human ““ovarianovarian””
cancer cell linescancer cell lines
20082008 (sensible) and(sensible) and C13*C13* (resistant) human(resistant) human ““ovarianovarian””
cancer cell linescancer cell lines
BerberineBerberine
Cell growth inhibitionCell growth inhibition
Survival(%ofControl)
0%
25%
50%
75%
100%
ctrl Berb 5µM Berb 10µM
TSproteinlevel(%ofcontrol)TSproteinlevel(%ofcontrol)
TSactivity(%ofcontrol)
Effect of berberine on TS espressionEffect of berberine on TS espression
TS protein levels and activity were determined after a 72-h treatment with berberineTS protein levels and activity were determined after a 72-h treatment with berberine
 2008 cells2008 cells  C13*cellsC13*cells
Berberine 0 5 10 0 5 10 µMBerberine 0 5 10 0 5 10 µM
TS protein level

9. BerberineBerberine
Bitter-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

10. BerberineBerberine mechanism ofmechanism of
actionaction
The precise molecular basis of its many biological activities are stillThe precise molecular basis of its many biological activities are still
debated.debated.
Modulation of protein expressionModulation of protein expression by interaction with nucleic acids isby interaction with nucleic acids is
postulated.postulated.
The interactions betweenThe interactions between berberine and nucleic acidsberberine and nucleic acids
has been reportedhas been reported since 1962since 1962
J Cell Biol, 1962, 15, 589

11. 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 interactionDNA interaction
mechanismmechanism

12. 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).

13. BerberineBerberine
Anticancer properties of berberine in preclinical studiesAnticancer properties of berberine in preclinical studies
have been widely published since many yearshave been widely published since many years
however, there are no clinical trials in the area presentlyhowever, there are no clinical trials in the area presently
Y. Feng, et al., Journal of Ethnopharmacology, on line accepted manuscript

14. Our findings

15. BerberineBerberine
Berberine represents an interestingBerberine represents an interesting
and attractive natural lead compoundand 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 unprecedentedBy performing unprecedented
chemical modifications of thechemical modifications of the
berberine structure, we obtainedberberine structure, we obtained
a new class of derivatives witha new class of derivatives with
specific antitumour propertiesspecific antitumour properties

16. Chemistry programmeChemistry programme
Aromatic interactions are ubiquitous in nature, and their geometryAromatic 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

17. 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, 1961
Cook, AG, Enamines Synthesis, Structure and reaction, 1988, pag 200-201
generally from good
to very good yields

18. Berberine Derivatives
Binding to DNA
1
D. Bhowmik, M. Hossain, F. Buzzetti, R. D’Auria, P. Lombardi, G.S.Kumar, J. Phys. Chem. B, 2012, 116, 2314−24.
2
D. Bhowmik, F. Buzzetti, G. Fiorillo, F. Orzi, T. Syeda Monir, P. Lombardi, G.S. Kumar, Med. Chem. Comm., 2014, 5, 226-31.

19. Berberine Derivatives
Binding to DNA
S. Chatterjee, S. Mallick, F. Buzzetti, G. Fiorillo, T. M. Syeda, Paolo Lombardi, K. Das Saha, G. S. Kumar,
RCS Adv., 2015, 5, 90632

20. Malignant Mesotheliomas (MT)Malignant Mesotheliomas (MT)
Fatal asbestos-exposure-associated cancersFatal asbestos-exposure-associated cancers
Sites of MT areSites of MT are the pleural cavitythe pleural cavity (90%) and the(90%) and the peritoneal areaperitoneal 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 ofHigh levels of thymidylate synthasethymidylate synthase protein expression in MTprotein expression in MT
patients are the marker of lack of efficacy of currrent treatmentspatients are the marker of lack of efficacy of currrent treatments
(pemetrexed, cisplatin(pemetrexed, cisplatin))

21. Berberine NAX derivatives:Berberine NAX derivatives:
antiproliferative effects inantiproliferative effects in
human mesothelioma cellhuman mesothelioma cell
lineslines (high TS)(high TS)
STO, MESOII = peritoneal mesothelioma cell lines MSTO = pleural mesothelioma cell line

22. 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

23. NAX035NAX035
In vitroIn vitro antiproliferative activity on chemo-restistant human MTantiproliferative activity on chemo-restistant human MT
cell lines in comparison with standardscell lines in comparison with standards
NAX035NAX035 overcomes pemetrexed and cisplatinovercomes pemetrexed and cisplatin
resistance in MT cells (collateral sensitivity)resistance in MT cells (collateral sensitivity)

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

25. NAX035NAX035
Innovative proprietary compound, structurally relatedInnovative proprietary compound, structurally 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 administration
in a human mesothelioma xenografted
in nude mice

26. HER2HER2++
Breast CancerBreast Cancer
New agents exhibiting aNew agents exhibiting a mechanism of actionmechanism of action different in respectdifferent in respect
to current therapies might offer a new option for treating HER2to current therapies might offer a new option for treating HER2++
BC patientsBC 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

27. Berberine inhibits cellular growth of breast cancer cells and
promotes apoptosis by down-regulating the HER2/PI3K/Akt
signaling pathway1
Berberine inhibits cellular growth of breast cancer cells and
promotes apoptosis by down-regulating the HER2/PI3K/Akt
signaling pathway1
Berberine inBerberine in
breast cancerbreast cancer

28. Antiproliferative activities of NAX compdsAntiproliferative activities of NAX compds
against HER2+ human (SK-BR-3) andagainst HER2+ human (SK-BR-3) and
murine (N202.1A) breast cancer cellsmurine (N202.1A) breast cancer cells
Chemical structures of tested berberine derivativesChemical structures of tested berberine derivatives
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona,

29. Time-dependent antiproliferative activities ofTime-dependent antiproliferative activities of
NAX compounds against HER2+ breastNAX compounds against HER2+ breast
cancer SK-BR-3 cellscancer SK-BR-3 cells
Time
IC50 µM
NAX014 NAX012
NAX01
3
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
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA,

30. Effects of NAX compounds onEffects of NAX compounds on
HER2/neu expression andHER2/neu expression and
phosphorylationphosphorylation
Treatment: NAX012 and NAX014 and berberine 50µM for
24h
HER2
p-HER2
β-actin
BioFactors, 2013, 39, 672.
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA,

31. 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.
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 efficacy inNAX014: Antitumour efficacy in
HER-2/neu transgenic female miceHER-2/neu transgenic female mice
Tumour Number Tumour Growth Inhibition
NAX014 is effectiveNAX014 is effective in delaying the onsetin delaying the onset and the progression of HER2+ BC at welland the progression of HER2+ BC at well
tolerated dosestolerated doses
NAX014 is effectiveNAX014 is effective in delaying the onsetin delaying the onset and the progression of HER2+ BC at welland the progression of HER2+ BC at well
tolerated dosestolerated doses
FVB-N 233FVB-N 233 Her2/neu miceHer2/neu mice treated IPtreated IP,, 2.5mg/kg of cmpds (2xweek)x122.5mg/kg of cmpds (2xweek)x12
High % Tumour Free Mice
ro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona, Italy

32. NAX014:NAX014: In vivoIn vivo acute andacute and
chronic toxicity in micechronic toxicity in mice
NAX014 LDNAX014 LD5050 30.9 mg/kg30.9 mg/kg
Berberine LDBerberine LD5050 10.9 mg/kg10.9 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
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona,

33. Tumour Growth
Inhibition
Tumour Number
NAX014 is effective byNAX014 is effective by oral routeoral route inin
delaying the onset and the progressiondelaying the onset and the progression
of HER2of HER2++
BC and shows antimetastaticBC and shows antimetastatic
efficacyefficacy
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 233NAX014: FVB-N 233
Her2/neuHer2/neu
mice treatedmice treated per os with 20per os with 20
mg/kgmg/kg
(2xweek)x8(2xweek)x8
TumourVolume(%Control)MeanTumourNumber
weeks
weeks
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA, Ancona,

34. NAX014:NAX014: in vivoin vivo evaluation ofevaluation of
vascularization of mammary tumouvascularization of mammary tumou
Tumour masses from control group showedTumour masses from control group showed
higher vessel density 17.2 mm/mmhigher vessel density 17.2 mm/mm22
asas
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 NAX014Statistical 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,

35. NAX014NAX014
anticancer and anti-metastatic efficacy on HER2+ tumoursanticancer and anti-metastatic efficacy on HER2+ tumours
in vitro activity at µM concentrationsin vitro activity at µM concentrations
in vivoin vivo tolerability by i.p.and oral administrationtolerability by i.p.and oral administration
at the effective doseat the effective dose
Innovative proprietary compound, structurally relatedInnovative proprietary compound, structurally related
to the plant isoquinoline alkaloid berberineto the plant isoquinoline alkaloid berberine
Unique ability to reduce cellular HER2 expression viaUnique ability to reduce cellular HER2 expression via
a postulateda postulated novel mechanismnovel mechanism

36. Conclusions

37. ConclusionsConclusions
Berberine exhibits diverse pharmacologicalBerberine exhibits diverse pharmacological
properties in a wide spectrum of clinical applicationsproperties in a wide spectrum of clinical applications
that might prevent its use as a drug for a definitethat might prevent its use as a drug for a definite
therapytherapy
the structure of berberine represents athe structure of berberine represents a
biologically interesting skeletonbiologically interesting skeleton
berberine is an attractive natural leadberberine is an attractive natural lead
compoundcompound
rational chemical manipulation might lead to selectrational chemical manipulation might lead to select
the therapeutic areathe therapeutic areas
chemical modifications in appropriate positions mightchemical modifications in appropriate positions might
discriminatediscriminate and narrow selective medical indicationsand narrow selective medical indications
Berberine

38. Int J Mol Med, 2014, 34 409
Berberine unspecifically suppresses nascent protein synthesis
by appreciation of the differences in mRNA translational
control between normal
and cancer cells.
This has been reported by us
and by others
ConclusionsConclusions

39. ConclusionsConclusions
However:
NAX012
NAX014
NAX035

40. FutureFuture
perspectivesperspectives
BioFactors, 2013, 39, 652.

41. FutureFuture
perspectivesperspectives
Tel24 5’-(TTAGGG)4-3’
NAX053

42. Financial supports were provided by
Ministero dello Sviluppo Economico (Grant. 01705 to
Naxospharma)
and
Agència 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).
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