ACTIVATION OF THE CYTOTOXIC EFFECT OF SOME CYTOSTATICS THROUGH EXPOSURE AT OPTICAL RADIATION

ACTIVATION OF THE CYTOTOXIC EFFECT OF SOME CYTOSTATICS THROUGH

EXPOSURE AT OPTICAL RADIATION

CEEX: nr.2-Cex-06-D11-32

Mihail Lucian Pascu1*, Alexandru Pascu1, Angela Staicu1, Madalina Dicu1, Doina Gazdaru2, Marieta Costache3, Jacques Barbe4, Abdallah Mahamoud4, Michelle Ferrari5, Silvia Rao5, Ligio Liggieri5, Benone Carstocea6

ACTIVATION OF THE CYTOTOXIC EFFECT OF SOME CYTOSTATICS

THROUGH EXPOSURE AT OPTICAL RADIATION

1National Institute for Laser, Plasma and Radiation Physics, Bucharest; 2 Physics Faculty, University of Bucharest; 3 Faculty of Biology, University of Bucharest;4Faculty of Pharmacy, Universite Mediterrannee of Marseille; 5 CNR- Institute for Energetics and Interphases, Genova 6 Central Military Hospital, Ophthalmology Clinic, Bucharest

APPLICATIONAPPLICATIONFighting resistance to treatment (microbes, Fighting resistance to treatment (microbes, malignant tumours, pseudotumours) by: malignant tumours, pseudotumours) by: - modifications induced in molecular - modifications induced in molecular structures of medicines following exposure to structures of medicines following exposure to optical (un-coherent, coherent) radiation.optical (un-coherent, coherent) radiation.- developing new ways to deliver the medicine - developing new ways to deliver the medicine to targetto target

Medicines: cytostatics, either commercially Medicines: cytostatics, either commercially available or specifically designedavailable or specifically designed

APPLICATIONAPPLICATION Levels/steps of research development:Levels/steps of research development:- exposure of the medicines solutions to - exposure of the medicines solutions to optical radiation and study the molecular optical radiation and study the molecular modifications, if any;modifications, if any;- evidence the modifications induced in cell - evidence the modifications induced in cell cultures at interaction with solutions cultures at interaction with solutions containing modified medicines; containing modified medicines; -evidence action of the modified medicine on -evidence action of the modified medicine on pseudotumour tissues produced on eyes;pseudotumour tissues produced on eyes;- generation and study of microdroplets - generation and study of microdroplets which contain medicines in solutionwhich contain medicines in solution

Spectral studies of some alkylphenyl- pyridinium compounds under light Spectral studies of some alkylphenyl- pyridinium compounds under light irradiationirradiation

Samples supplied by University of Marseille, Faculty of Pharmacy, Prof. Jacques Barbe.

Code number Series Bp (°C) BG 186 Acridine 64

BG204BG204 Acridine 128128

BG 558BG 558 Naphthyridine 130130

BG1120BG1120 PyridoquinolinePyridoquinoline 115-117115-117

Spectral studies of some alkylphenyl- pyridinium compounds Spectral studies of some alkylphenyl- pyridinium compounds under light irradiationunder light irradiation

N

SCH2 CH2 N(C2H5)2

NH2

N

SCH2 CH2

OCH3

N(C2H5)2

NN

O

H2C CH2 N(C2H5)2

BG204 BG558

(3-amino,9-thio [2-N-N-diethyl- (9-[2-N-N-Diethylaminoethyl]amino-ethyl] acridine) 1,9-diazantracen-10-one]

BG186 BG1120

(4-methoxy,9-thio [2-N-N-diethyl- (1,8 bis [thio(2-N-N amino-ethyl] acridine) acridine) diethylaminoethyl)], 9 methyl 4,5-diazantracene)

COMPOUND ABSORPTION BANDS

(nm)

EXCITATION BANDS

(nm)

EMISSION BANDS (nm)

OBSERVATIONS

BG 186 262, 347, 365, 405

280, 410 515 Exposure to optical radiation does not induce significant modifications of the spectra.

BG204 240, 273, 351, 369, 388, 480

310, 382, 440, 478

570 Absorption, fluorescence excitation and emission bands decrease with the irradiation time.

BG 585 256, 394 280, 410 450 Exposure to optical radiation does not induce significant modifications of the absorption spectra. Slight change in the fluorescence intensity with the increase of the exposure time.

BG1120 259, 379 300, 380, 410 476 The intensity of the absorption, fluorescence excitation and emission bands decrease with the increase of the exposure time.

Spectral studies of some alkylphenyl- pyridinium compounds Spectral studies of some alkylphenyl- pyridinium compounds under light irradiationunder light irradiation

Samples: Water solutions of BG204 (2x10-5 M ) , BG 558 and BG 1120 (5x10-5 M) - concentration currently used for clinical applications; Water solutions of BG 186 at 5x10-5M in 2% methanol

Irradiation: Xe lamp, power density 11mW/cm2, spectral range 200 -1100 nm, irradiation time: 4 min ÷ 180 min

Spectra: Absorption, fluorescence and FTIR spectra were made; for FTIR, solutions exposed to radiation up to 3 hours, then poured on KBr crystal and let one day to dry. The resulted solid samples were grounded and pressed at 1.575 x 105 kg cm-2 which allowed to

obtain pellets used further to be measured by FTIR.

BG 1120 ABSORPTION SPECTRA

200 300 400 500 600 700 8000.0

0.5

1.0

1.5

2.0

2.5

3.0

BG 1120 (5x10-5 M in distilled water)

Ab

sorb

an

ce

wavelength (nm)

unirrad. 7' 15' 30'

0 3 6 9 12 15

0.04

0.06

0.08

0.10

0.12

0.14

Ab

s. (

25

8.6

nm

)

t (min)

BG 1120

BG 1120 FLUORESCENCE EMISSION SPECTRUMBG 1120 FLUORESCENCE EMISSION SPECTRUM

300 400 500 600 700 800

0.000

0.005

0.010

0.015

0.020

Emission spectrum (ex

= 300 nm)

BG1120 5x10-5 M

F

luor

esce

nce

int.

(arb

.u.)

wavelength (nm)

unirrad. 7' 15' 30'

BG 1120 FLUORESCENCE EMISSION SPECTRA AT BG 1120 FLUORESCENCE EMISSION SPECTRA AT λλexex=270 nm=270 nm

300 350 400 450 500 550 600 6500

50

100

150

200

250

300

350

400

450

500

550

600

650

ex

=270 nm 0' 3' 6' 9' 12' 15'

Flu

ores

cenc

e in

tens

ity (

rel.u

nit.

)

wavelength(nm)

BG1120 conc= 5 *10-6M

0 3 6 9 12 15

100

200

300

400

500

600

Int.

(46

0 n

m)

time (min)

BG 1120 FLUORESCENCE EXCITATION SPECTRUMBG 1120 FLUORESCENCE EXCITATION SPECTRUM

200 250 300 350 4000.00

0.01

0.02

0.03

0.04

0.05

0.06

Excitation spectrum (em

= 460 nm)

BG1120 5x10-5 M

F

luo

resc

en

ce in

t. (

arb

.u.)

wavelength (nm)

unirrad. 7' 15' 30'

BG 1120 FTIR SPECTRUMBG 1120 FTIR SPECTRUM

4000 3500 3000 2500 2000 1500 1000 5000.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

BG1120 unirrad. BG1120 irrad.3 h

A

bsor

banc

e

(a.

u.)

wavenumbers (cm-1)

1400 cm-1, ring stretching mode in thiols680 cm-1, in-plane C – S - H bending

1098 cm-1, 1638 cm-1 and 1579 cm-1, C=S bond in lactam form

1400 cm-1 bending vibration in NH 43500 – 3100 cm-1, N–H stretching Vibration in NH4+

BG 1120: POSSIBLE PHOTOCHEMICAL PROCESSES

NN

CH3

NN

+ hv2CH2 CH2 N(CH3)2 +

+2H+2H

S S

SH SH2CH3-CH2-N(CH3)2

dithiol

aliphatic tertiary amine

S

NN

S

CH2-CH2-N(CH3)2

CH2-CH2-N(CH3)2

CH3

CH3

12 days after procedure, the eyes were injected BG 1120 under the conjunctiva, near the affected area.

“IN VIVO” STUDIES FOR PSEUDOTUMOUR TREATMENT ON RABBIT EYES

BEFORE INJECTING BG 1120

The Schmidt - Erfurth rabbit eye experimental model on conjunctive tissue inflammation and rich neovascularization. Pseudo-tumours with new vascularisations were induced by sewing a catgut stitch 2.0 at the sclero - corneal limbus and by injecting a prostaglandinic-like substance (Travatan 0.1 ml), with proinflammatory action nearby, under the conjunctiva.

NEOVASCULAR TISSUE

1st eye (not- treated) presents surrounding the necrotic stitch a large quantity of inflammatory cells as well as neovascularisations and a small amount of fibrosis.80x

The 2nd eye (in which not irradiated substance was introduced) has the same tissular modifications, however much reduced.100x

3rd eye ( irradiated substance) the quantity of the inflammatory infiltrate and oedema is decreased comparing to the 2nd eye. 200x

4th eye –irradiated, the neovascularisations and the inflammatory cells are diminished100x

CONCLUSIONS

The photo-transformation of the BG 1120 analysed samples shows that the molecules are photo-reactive and their therapeutic effect may be improved by exposure to UV-VIS radiation.

BG 1120 decreased inflammation and neovascularisations in the conjunctival tissue, especially after being irradiated.

Two mechanisms could be involved in the process: • the cytostatic mechanism: interference with the DNA synthesis and the cellular replication in the tissues with active proliferation.• the photosensitising mechanism that leads to the singlet oxygen and free radicals and finally to necrosis of the “treated” tissue.

Direct exposure of the eye injected not recommended.

THE CHEMICAL STRUCTURE OF: DAUNORUBICIN, DOXORUBICIN, IDARUBICIN (ANTRACICLINES)

Doxorubicin - ADRIABLASTIN Daunorubicin - RUBOMICIN

10-(4-amino-5-hydroxy-6-methyl-oxan-2-yl) oxy-6,8,11-trihydroxy-8-(2-hydroxyacetyl)- 1-methoxy-9,10-dihydro-7H-tetracene-5,12-dione

9-acetyl-7-( 4-amino-5-hydroxy-6-methyl- tetrahydropyran-2-yl) oxy- 6,9, 11-trihydroxy- 7,8,9,10-tetrahydrotetracene-5,12-dione

Epirubicin/FARMORUBICIN Idarubicin/ZAVEDOS

(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione

(8S,10S)-8-acetyl-10-[(2S,4S,5S,6S)-4-amino-5-hydroxy-6-methyl-oxan-2-yl]oxy-6,8,11 trihydroxy-1-methoxy-9,10-dihydro-7H-tetracene-5,12-dione

DAUNORUBICIN (DNR), DOXORUBICIN (DOX), IDARUBICIN (ANTRACICLINES)

-2000 analogues of DOX and DNR synthesised to reduce the negative effects on organisms;

- Only some of them, exhibiting a reduced degree of toxivity were clinically studied: Idarubicin, Epirubicin

-Antibiotics in the antraciclin class which act as “intercalator” and may generate free radicals.

ABSORPTION SPECTRA: DAUNORUBICIN, DOXORUBICIN, IDARUBICIN (ANTRACICLINES)

200 250 300 350 400 450 500 550 6000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Ab

sorb

an

ce

wavelenght(nm)

Daunorubicin c=10-4 M

Doxorubicin c=10-4 M

Idarubicin c=10-4 M

DAUNORUBICIN EMISSION FLUORESCENCE SPECTRUM (FOLLOWING EXPOSURE TO LASER BEAM)

200 400 600 800 1000

100

200

300

400

500

0' 3'

excit

=480 nm

daunorubicin

c=10-5 M

(nm)

inte

nsi

ty (

arb

.un

it)

Nd:YAG LASER WITH PARAMETRIC OSCILLATOR: PULSED REGIME, PULS REPRATE 10Hz, PULSE TIME WIDTH 5ns, PULSE AVERAGE ENERGY 25mJ

MICRODROPLETS STUDIES OBJECTIVESThe characterization of wetting properties of generated droplets, on hydrophobicand superhydrophobic surfaces, considering the specificity of the droplets content and their potential use in malignant tumours treatment. MEASUREMENTS-Surface Tensions (ST) -Contact AngleThe studies of the drug molecules adsorption performed at liquid-air and liquid-solid interface (surfaces with water contact angle above 90° - hydrophobic and above 150° - superhydrophobic).MATERIALSSolutions containing: * Anthracene derivative - 4,5 bis[thio(2NNdimethylaminoethyl)], 9methyl1,8 diazaanthracene, code BG1120; * Doxorubicin – pure substance commercially available; at 10 -3 M, 10 -4 M and 10 -5 M concentrations in waterMETHODDrop Shape AnalysisMEASUREMENTS SYSTEMS: - Profile Analysis Tensiometer (Sinterface, PAT 1)- Automatic Surface Tension Real-time Acquisition – ASTRA

Surface tension measurements

Air - water /medicines water solutions - solid interfaceContact angle water drop/solid surface θ < 90º: hydrophilic surface -

good wettingContact angle water drop/solid surface θ > 90º : hydrophobic surface -

poor wettingContact angle water drop/solid surface θ > 150º : super hydrophobic or ultraphobic surface – even poorer wetting

The drop shape is governed by the action of forces pulling at the three-phase contact line of the drop in the plane of solid.

σAS = σSW + σAW cos θ (Young equation, 1807)

Surface tension (N/m) = Surface energy (J/m2 )

Capillary flow

σAS

σAW

σSW

ConcentrationConcentration BG1120BG1120

γ (mN/m)γ (mN/m)

DoxorubicinDoxorubicin

γ (mN/m)γ (mN/m)

WaterWater

γ (mN/m)γ (mN/m)

SolventSolvent

C = 1x10C = 1x10-5-5M M 72.3 (72.3 (±0.8)±0.8) 72.5 (72.5 (±0.6)±0.6)

72.4 (72.4 (±0.6)±0.6)

Ultrapure waterUltrapure waterC = 1x10C = 1x10-4-4M M 71.1 (71.1 (±0.6)±0.6) 72.3 (72.3 (±0±0.9).9)

C = 1x10C = 1x10-3-3M M 71.0 (71.0 (±0.9)±0.9) 72.3 (72.3 (±0.4)±0.4)

C = 1x10C = 1x10-3-3M - ROM - RO 69.6 (69.6 (±1.2)±1.2) -- Distilled waterDistilled water

Measurements concerning Dynamic Surface Tension (DST) showed that in BG1120 case, the obtained values for the solutions prepared in laboratory and for the solution prepared four months ago, are closed; within the limits of the errors of measurement are close to the pure water value .

The same holds for Doxorubicin.


Volume

time (s)

0 200 400 600 800 1000 1200 1400 1600

volu

me

( l

)

37.0

37.2

37.4

37.6

37.8

38.0

38.2

38.4

Surface tension (left) and the control parameter – volume (right) – Doxorubicin’s concentrations.

Doxorubicin

time (s)

0 200 400 600 800 1000 1200 1400 1600

ST

(mN

/m)

71.0

71.5

72.0

72.5

73.0

73.5

74.0

C=10-5MC=10-4MC=10-3M


Contact angle measurements – superhydrophobic surface

The values obtained at the contact angles measurements at liquid-solid interface for superhydrophobic surface

ConcentrationConcentration BG1120BG1120

αα (o)(o)

contact anglecontact angle


αα (o)(o)


WaterWater

αα (o)(o)


SolventSolvent

C = 1x10C = 1x10-5-5M M 157.9 (157.9 (±0.6)±0.6) 169.0 (169.0 (±1.4)±1.4)

170.1 (170.1 (±0.4)±0.4)

Ultrapure Ultrapure waterwater

C = 1x10C = 1x10-4-4M M 162.9 (162.9 (±0.5)±0.5) 169.4 (169.4 (±0.6)±0.6)

C = 1x10C = 1x10-3-3M M 156.6 (156.6 (±±1.4)1.4) 170.0 (170.0 (±1.4)±1.4)

C = 1x10C = 1x10-3-3M – 4 monthsM – 4 months 163.1 (163.1 (±1.3)±1.3) -- Distilled waterDistilled water

The values represent the mean values (of the left and right angle) obtained

into a set of data corresponding to a drug concentration

Droplet images on superhydrophobic

surface, left – water, right – BG1120

Temperature = 20oC

Capillary outer diameter=0.21 mm

Droplet volume = 5µl

The time evolution of the contact angles; the mean values are represented for the left and right angles of the droplets (generated on the same local superhydrophobic surface) for a set of data corresponding to BG1120 concentrations; the control parameter was represented by the drop size (h)

Drop size (h)

time (s)

100 200 300 400 500 600 700h.

drop

(cm

)

0.1

0.2

0.3

0.4

BG1120_superhydrophobic surface

time(s)

0 100 200 300 400 500 600 700

c.an

gle(

°)

146

148

150

152

154

156

158

160

162

164

166

C=10-4MBG1120 Ro



Doxorubicin_superhydrophobic surface

time (s)

0 100 200 300 400 500

c.a

ngl

e (

o )

150

155

160

165

170

175

C=10-5MC=10-4MC=10-3M

Drop size (h)

time (s)

0 100 200 300 400 500

h.d

rop

(cm

)

0.10

0.15

0.20

0.25

0.30

0.35

The time evolution of the contact angles; the mean values are represented for the left and right angles of the droplets

Contact angle measurements – hydrophobic surfaceConcentrationConcentration BG1120BG1120

αα (o)(o)



αα (o)(o)


WaterWater

αα (o)(o)


SolventSolvent

C = 1x10-5M 136.0 (136.0 (±1.3)±1.3) 134.6 (134.6 (±0.8)±0.8)

135.7 (135.7 (±0.8)±0.8)

Ultrapure Ultrapure waterwater

C = 1x10-4M 134.6 (134.6 (±1.2)±1.2) 133.5 (133.5 (±1.4)±1.4)

C = 1x10-3M 133.2 (133.2 (±1.1)±1.1) 128.1 (128.1 (±0.7)±0.7)

C = 1x10-3M – 4 months 133.2 (133.2 (±1.0)±1.0) -- Distilled Distilled waterwater

The contact angles time evolution. The mean values are represented for the left and right angles of the droplets for BG1120 and pure water; the control parameter is the drop size (h)

BG1120_hydrophobic surface

time (s)

0 200 400 600

c.a

ng

le (o)

120

122

124

126

128

130

132

134

136

138

140

C=10-5MC=10-4MC=10-3MC=10-5M RO Apa pura

Drop size (h)

time (s)

0 200 400 600

h.d

rop

(cm

)

0.02

0.04

0.06

0.08

0.10

H2O on superhydrofobic surface BG1120 on superhydrofobic surface

Droplet volume = 5 µl = ct.; Dcapillary = 0.21 mm

MOVIE

Conclusions

The measurements show that the equilibrium values of the ST are constant in time and equal for all drugs concentrations - the ST values do not depend on the drug concentrations in the solutions.

The values are close to pure water ones – i.e. at drug concentrations which were used the adsorption effects at water-air interface are absent or very low and the distribution of drug molecules in droplet is uniform and remains constant in time

The equilibrium values of contact angles of droplets on superhydrophobic surfaces do not depend on the solution concentrations - the droplets do not wet the surface; they have a good compatibility with it, from the point of view of the droplet instrumentation.

The equilibrium values of the contact angles of the droplets on hydrophobic surfaces do not depend on the solution concentrations and are nearly as high as for the pure water.

Conclusions

Staff: 12 people; two young scientists hired Equipment aquisition, such as : Nd:YAG/OPO laser, computing equipment, optics equipment/components to measure LIF, etc.

Oportunity to cooperate/continue cooperation in Romania and abroad.

-In Romania: * Continuing: +University Bucharest , Faculty of Physics

+ Central Military Hospital, Ophthalmology Clinic, Bucharest

* New: + University Bucharest, Faculty of Biology

-Abroad: * New: + Faculty of Pharmacy, Universite Mediterrannee of Marseille; + CNR- Institute for Energetics and Interphases, Genova

Conclusions

International cooperation:-European COST networks:

+Network P21 Physics of Droplets+ Networks B16, BM 0701 Resistance to treatment+ Romanians in foreign laboratories: 2

> STSM at Genova; in preparation at Berlin/Max Planck Institute> working stage at Faculty of Biology, University of Becancon

+ Foreign scientists 1: from Genova to Bucharest, end 2008 Published papers in ISI journals: 5, at the moment Communications: 22+ In Romania at National conferences: 8 (CNF, Annual Conference of the Physics Faculty, University of Bucharest, National Conferences of opthalmology, INDLAS)+ INVITED PAPERS at International conferences: 14 (4 LASER FLORENCE, Italy; 3 COST medicine/biology conferences – Istanbul, Copenhagen, Budapest; 1 AMAA - Bucharest; 1 SIS 2008 – Berlin; 1 COST physics conference – Erlangen; 3 International opthalmology conferences- Sinaia, Viena, Bucharest; 1 accepted at the Gordon Conference 2009, Galvestone USA )

ACTIVATION OF THE CYTOTOXIC EFFECT OF SOME CYTOSTATICS THROUGH EXPOSURE AT OPTICAL RADIATION

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