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58 ITN Annual Report – 2009

Radiopharmaceutical Sciences Isabel Rego Santos

The Radiopharmaceutical Sciences Group

developed and implemented expertise and facilities to

carry on basic/applied oriented research and

technology transfer on nuclear tools for SPECT and

PET molecular imaging and for targeted

radiotherapy. The group is multidisciplinary and its

expertise involves organic and coordination chemistry,

bioconjugation, radiochemistry, animal and cell

studies, and molecular biology.

Our expertise and facilities enables to perform

research in modern Radiopharmaceutical Sciences, an

important topic in Life Sciences, justifying also our

participation in National and International research

projects and the support of an International

Pharmaceutical Company. Our expertise and facilities

are also extensively used to provide education and

training at different levels.

The main achievements during 2009 are described in

this report. However, we would like to emphasize the

following:

Facilities:

Our animal housing facilities and our molecular

biology laboratory have been reinforced, respectively,

with the installation of equipment for nude mice and

equipment for DNA analysis (PCR and Gel

Documentation System). We have also fully

implemented the solid phase peptide synthesis

methodology using the recently acquired Microwave-

Peptide-Synthesizer.

Research:

1 – Novel 99mTc(I) receptor-targeted polymeric

nanoprobes, based on dextran-mannose conjugate

derivatives, were synthesized. One of these

nanoprobes showed favourable biological features for

sentinel lymph node (SLN) detection, an important

issue in the management of patients with newly

diagnosed breast cancer.

2- The potencial for Multi Drug Resistance (MDR)

imaging of some of our myocardial perfusion

compounds was evaluated. The results obtained

indicated that some of them act as Pgp substrates,

being promising for MDR imaging during anticancer

chemotherapies.

3 - In vitro enzymatic studies with iNOS have shown

that the affinity to the enzyme of the inhibitor-

containing conjugates was improved upon metallation

with “Re(CO)3”. Such affinity depends on the spacer

length between the inhibitor and the metal center.

Education and Training

1-Graduation:

Radiopharmacy teaching at ESTSeL and at Faculty of

Pharmacy/University of Lisbon. Under a protocol,

Nuclear Medicine students/ESTeSL are trained at ITN

(two weeks/year ).

2-Post-graduation:

a) Coordination of the Master Course Biomedical

Inorganic Chemistry: Diagnostic and Therapeutical

Applications (ITN/UL). Coordination and teaching of

Radiochemistry and Biomedical Inorganic Chemistry

in the same MSc course.

b) Coordination and teaching of Radiopharmaceutical

Chemistry in the Master Course Pharmaceutical and

Therapeutical Chemistry/Faculty of Pharmacy/UL.

c) Teaching of Chemical Systems and Reactivity in

the Master Course in Chemistry, Faculty of

Sciences/UL

d) Lectures in PhD Teaching Programs organized by

Universities/Associated Laboratories, namely

ITQB/UNL.

3-Young scientists:

Several young scientists are being trained in the group,

playing a major role in our projects.

Expertise Provided:

Nuclear Medicine Centers, Portuguese Medicines

Evaluation Agency, IAEA, Foreigner Science

Foundations and International Conferences.

Publications:

Peer-Review International Journals – 17; Reports - 5;

Communications – 21; Thesis: MSc – 1, PhD - 3.

Invited Lectures and Seminars: 4.

Research Team

Researchers

I. SANTOS, Princ., Group Leader

A. PAULO, Princ.

J. D. G. CORREIA, Princ.

M. P. C. CAMPELLO, Aux.

M. C. OLIVEIRA, Aux.

L.GANO, Aux.

F. MARQUES, Aux.

P. RAPOSINHO, Aux.

C.FERNANDES, Aux.

F. MENDES, Aux., Contract.

G. MORAIS, Aux., Contract. (Since Sep.)

R. GARCIA, Pos-Doc, FCT grant (until Sep.)

S. GAMA, Pos-Doctoral, FCT grant

P. S. ANTUNES, Pos-Doctoral, FCT grant

M. K. S. BATISTA, Pos-Doctoral, FCT grant

Students

S. LACERDA, Ph.D. student, FCT grant

(until March)

C. XAVIER, Ph.D. student, FCT grant

(until March)

E. PALMA, Ph.D. student, FCT grant

C. MOURA, Ph.D. student, FCT grant

T. ESTEVES, Ph.D. student, FCT grant

C. NETO, Ph.D. student, FCT grant

B. OLIVEIRA, Ph.D. student, FCT grant

S. CUNHA, Ph.D. student, FCT grant

F. SILVA, Ph.D. student, FCT grant

M. MORAIS, Ph.D. student, FCT grant

F. LUCENA, M.Sc. student

C. FRANCISCO, BIC grantee, POCI (until Sept.)

R. FRAGOSO, Undergraduate student

M. ARAÚJO, Undergraduate student

Technical Personnel

A. RODRIGUES

E. CORREIA

RADIOPHARMACEUTICAL SCIENCES

ITN Annual Report – 2009 59

99mTc(I)-labeled mannosyl-dextran for sentinel lymph node detection

M. Morais, J. D. G. Correia, C. Fernandes, M. Martins1, S. Pereira,

2 I. Santos

Objective

The main goal of this project is the design and

synthesis of novel 99mTc(I) receptor-targeted

polymeric nanoprobes for sentinel lymph node (SLN)

detection. These nanoprobes, which are based on

dextran-mannose conjugate derivatives, aim at the

improvement of accuracy in the identification and

characterization of SLN by nuclear imaging, having

important therapeutic and prognostic significance in

patients with newly diagnosed breast cancer.

Results

The polymeric conjugate dextran-(amine)7-(pyrazolyl-

diamine)8-(mannose)15 (1) was synthesized by

functionalization of dextran with mannose units and

pyrazolyl-diamine (pz) chelating units to recognize

mannose receptors present in macrophages and for

metal stabilization, respectively. The conjugate was

characterized by 1H/13C-NMR spectroscopy, and its

purity determined by size-exclusion HPLC. The

hydrodynamic radius of the polymeric particles in

solution was measured by Dynamic Light Scattering

(DLS) and the Zeta Potencial, at physiological pH,

was determined by electrophoretic mobility using

Laser Doppler Velocimetry (LDV) (Fig. 1).

A

Size (d nm)

Size Distribution by number

Number(%)

0

10

20

30

0.1 1 10 100 1000

A

Size (d nm)

Size Distribution by number

Number(%)

0

10

20

30

0.1 1 10 100 1000

0

10

20

30

0.1 1 10 100 1000

B

0

200000

400000

600000

800000

-200 -100 0 100 200

Tota

l C

ounts

Zeta Potential DistributionZeta Potencial Distribution

Zeta Potencial (mV)

Total Counts

B

0

200000

400000

600000

800000

-200 -100 0 100 200

Tota

l C

ounts

Zeta Potential DistributionZeta Potencial Distribution

Zeta Potencial (mV)

Total Counts

Size (nm) Zeta Pot. (mV)

Dextran 4,26 ± 0,42 -9,88 ± 0,48

1 7,04 ± 0,27 7,32 ± 0,62

Fig.1 Hydrodynamic radius (nm) by number of particles in solution

(A) and Zeta Potencial (B) of dextran and 1.

Reaction of 1 with fac-[99mTc(CO)3(H2O)3]+ gave, in

quantitative yield and high specific activity, the

polymeric conjugate fac-[99mTc(CO)3(k3-1)]+ (2),

whose chemical identity was established by

comparing its HPLC chromatogram (γ-detection) with

that of the rhenium (2a) surrogate (UV-detection)

(Fig. 2). Compound 2 is stable both in vitro and in

vivo.

2a (M = Re)

2 (M = 99mTc)

Fig.2 HPLC chromatograms of 2 and 2a.

Static imaging studies of Wistar rats injected

subcutaneously with 2 in the footpad have shown that

the compound retains strongly in the first lymph node

(LN), exhibiting an high 1st LN/2nd LN ratio (~10:1) at

3h post-injection (Fig. 3).

1st Lymph node

2nd Lymph node

1st Lymph node

2nd Lymph node

Fig.3 Gamma-camera static images of Wistar rats injected

subcutaneously with 2 in the footpad at 1 h and 3 hours p.i..3

The favorable biological features presented by 2,

namely the negligible accumulation in non-target

organs, make this compound very promising for

further evaluation in other animal models, which is

currently underway.

Published work:

M. Morais, J. D. G. Correia, I. Santos, Dextran-mannose conjugates bearing a pyrazolyl-diamine chelator for labelling with the fac-[99mTc(CO)3] unit, IAEA Meeting, 18-22th May, Athens, Greece.

M. Morais, J. D. G. Correia, I. Santos, 99mTc(I) – Nanocarrier as Versatile Platform for Sentinel Lymph Node Detection., 2nd European Summer School in Nanomedicine, 12-16th June, Cascais, Portugal.

1 Dep. Chemistry, Univ. Aveiro 2 CICECO., Univ. Aveiro 3 Performed by S. Subramanian, India, under the framework of a

“CRP, Development of 99mTc Radiopharmaceuticals for Cancer

Diagnosis and Sentinel Node Detection, IAEA.

3 h 1 h



99mTc(I) Tricarbonyl complexes functionalized with melanin-binders for in vivo targeting of melanoma

C. Moura, L. Gano, P. Raposinho, A. Paulo, I. Santos

An enlarged family of tricarbonyl 99mTc(I)

complexes, anchored by pyrazolyl-containing

chelators and bearing N-(diethylaminoalkyl) groups

as melanin-avid pharmacophores, have been studied

as new radioactive probes for in vivo detection of

metastatic melanoma. All evaluated complexes

showed an high in vitro affinity for melanin, but a

rather poor tumor uptake. Looking for compounds

with improved in vivo biological properties, related 99mTc(I) complexes have also been newly synthesized

and evaluated (Fig. 1). These complexes showed a

promising tumor uptake (1.90 - 2.17% ID/g at 1 h p.i.) in B16F1

melanoma-bearing mice (Fig. 2), which depends on the spacer length between the bioactive fragment and the

metal.

__________________________________________________________________________________________

Evaluation of Novel Pyrazolyl-alkylamine Pt(II) Complexes as Anti-Cancer Drugs

C. S. Francisco, S. Gama, F. Marques, A. Paulo, I. Santos, M. Ravera,1 E. Gabano,

1 D. Osella

1

Expecting to obtain a synergistic effect of DNA platination and

intercalation on cell cytotoxicity, we have studied Pt(II) complexes

anchored by pyrazolyl-diamine ligands bearing anthracene derivatives as

DNA-binding groups. These

complexes were characterized

by common analytical

techniques (e.g. 195Pt NMR,

Fig. 1) and their cytotoxicity

evaluated against cisplatin-

sensitive (A2780) and

cisplatin-resistant

(A2780cisR) human ovarian

cancer cell lines (Fig. 2). In

order to establish a structure-activity relationship, related complexes

with pyrazolyl-alkylamine ligands of different electronic and/or

steric properties are currently under study.

____________________ 1 DiSAV, Univ. del Piemonte Orientale “Amedeo Avogadro”, Alessandria, Italy

__________________________________________________________________________________________

Organometallic Complexes Bearing Bioactive and/or NLS Peptides for Targeted Auger Radiotherapy

T. Esteves, A. Paulo, I. Santos, P. Nanda,1 C. Smith

1

99mTc(I) tricarbonyl complexes anchored by pyrazolyl-diamine ligands bearing

anthracenyl or acridine orange fragments have the ability to accumulate in the

nucleus of tumor cells

with a significant DNA

intercalation (Fig. 1).

Therefore, this type of

complexes emerged as

promising building

blocks to develop site-

directed radioprobes for in vivo imaging and/or Auger

radiotherapy. To achieve such goal, multifunctional

complexes bearing bombesin analogs (Fig. 2) and/or NLS

(Nuclear Localization Signal) peptides have been isolated and fully characterized. The in vitro evaluation of

these radioconjugates is underway in order to assess their specificity for tumor cells, ability to accumulate in the

nucleus and suitability for Auger targeted radiotherapy.

_______________ 1 Research Division, Harry S TrumanMemorial Veterans’ Hospital, Columbia, MO 65201, Missouri, USA

Fig.2

( )n

( )n

n=2n=1

pKaMeasurement

k’

pH

Fig.1

- 2.6549 ppm

LPtCl

- 2.6549 ppm

LPtCl

Fig.1

Fig.2

Fig. 1

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

NH2

O

HN

CH3

H3C CH3

HN

N

H3C CH3

S

H3C

NH2

O

NH

ONH

HO

O

NHNH

HO

Re

COCO

N

OC

N NH2

N

HNO

N+ NN

2+

O

O

Fig.2



Estradiol-based metal complexes for breast cancer diagnosis.

C. Neto, M. C. Oliveira, L. Gano, F. Marques, I. Santos, T. Thiemann1.

Following our previous

work on the design and

evaluation of new probes

for targeting estrogen

receptor (ER), a novel

estradiol derivative labeled

with fac-[99mTc(CO)3] has

been synthesized and characterized by HPLC comparison with its

rhenium surrogate. The 99mTc(I)-complex, obtained in high

radiochemical yield and purity, is stable in total blood, human

serum and liver homogenate up to 4h incubation at 37ºC. Receptor binding affinity, cell studies and in vivo

evaluation of this 99mTc-complex as a biomarker for the molecular imaging of ER expressing tumors are in

progress.

____________________ 1 Dep. of Chemistry, Fac. of Sciences, United Arab Emirates Univ., United Arab Emirates

________________________________________________________________________________________

Novel radiolabeled receptor tyrosine kinase inhibitors for in vivo targeting of EGFR

C. Neto, M. C. Oliveira, L. Gano, C. Fernandes, I. Santos, M. Kuchar1, T. Kniess

1

Searching for SPECT/PET radiotracers for in vivo imaging of EGFR

tumors, novel

anilinoquinazoline

-based compounds

were synthesized,

characterized and

labeled with 125I

and 18F.

The radioiodinated compounds were obtained in high yield and

with high specific activity. In vitro cellular assays have shown

high uptake by A431 cells. For some of the iodinated

compounds, biodistribution studies revealed low accumulation

in most organs and a small thyroid uptake. The

radiofluorination using the [18F]KF/Kryptofix complex are still

under optimization.

____________________ 1 PET-Tracer Group, Institute of Radiophamacy, FZD, Germany

_________________________________________________________________________________________

Radiolabeled VEGFR tyrosine kinase inhibitors as potential SPECT tracers for angiogenesis imaging

M. C. Oliveira, L. Gano, I. Santos, M. Kuchar1, T. Kniess

1

Oxindole-based molecules, used as lead structures for VEGFR-TK inhibitors, were synthesized, characterized

and labeled with 125I. The radiocompounds were obtained in high radiochemical

yield and their purity (≥ 98%) and identity were confirmed by HPLC. High

radiochemical stability

in saline solution and in

human serum was

found up to 24h when

kept in the dark at

37ºC. The lipophilic

compounds (logP ≈ 2.25) present a low plasma protein

binding (5-10%), and biodistribution studies in healthy

CD-1 mice indicated a rapid clearance from most

organs, including blood, as well as a high in vivo

stability. Based on these lead compounds further

studies are underway.

___________________ 1 PET-Tracer Group, Institute of Radiophamacy, FZD, Germany

OH

OH

NN

N

NH2

Tc

CO

CO

CO

+

O N

N

NH

Cl

F

NH

X.

X=125I, 18F

N

O

H

I125

0

2

4

6

8

10

12

14

1 h 4 h

%I.D./g

Bone

Muscle

Blood

Kidney

Thyroid

Intestine

Liver

0

3

6

9

12

1 h 4 h

% I.D./ g

LiverKidneyLungMuscleBloodThyroid



Metabolic Studies of Ether-Containing Tris(pyrazolyl)methane 99m

Tc(I) Complexes

C. Fernandes, L. Gano, A. Paulo, I. Santos

Tris(pyrazolyl)methane 99mTc(I)-complexes with ether substituents

at the 3-, 4- and 5- positions presented a remarkable in vivo stability

(Fig. 1) and hold potential as myocardial imaging agents. By

contrast, complexes with tris(pyrazolyl)methane bearing ether

substituents uniquely at the 4-position did not accumulate in the

myocardium and showed

extensive metabolization

(Fig. 2). The in vivo

behavior of these

chelators, together with

the possibility of their

derivatization through

the central carbon atom,

make them promising as bifunctional chelators. As the observed

metabolization is expected to improve the biological performance

(e.g. blood clearance and overall excretion) bioconjugates with

adequate biomolecules are being explored.

Bifunctional DOTA-like chelators for biomolecules radiolabeling

R. Garcia, F.Silva, S. Lacerda, M Batista, L. Gano, R. Fragoso, M. P. C. Campello, P. Fousková1, E.Toth

1, A.

Paulo, I. Santos

Within our interest on

macrocyclic complexes of

trivalent radiometals for

molecular imaging, we have

evaluated new In/111In complexes

of a DOTA-like chelator bearing

a quinazoline pendant arm (Fig.s

1 and 2) and a 67Ga radiocomplex

of DO3ASH. 111InL, an hydrophilic neutral radiocomplex, exhibits high stability under physiological conditions,

presenting a very low cell internalization level. By contrast, the 67Ga-DO3ASH complex, also prepared in high

yield, presents a favourable biological profile, being in progress the functionalization of DO3ASH with clinically

relevant biomolecules.

_________ 1 Centre de Biophysique Moléculaire, CNRS, Rue Charles Sadron 45071 Orléans Cedex 2, France

Re(I)-complexes as potent inhibitors of iNOS

B. L. Oliveira, F. Mendes, F. Figueira, J. D. G. Correia, P. D. Raposinho, I. Santos, A. Ferreira1, C. Cordeiro,

1

A. P. Freire1

Aiming to find new 99mTc(I) compounds for

probing inducible Nitric Oxide Synthase (iNOS) in

vivo, we synthesized Re(I)/99mTc(I) complexes

containing pendant L-arginine analogs. In vitro

enzymatic studies with iNOS have shown that the

affinity to the enzyme of the inhibitor-containing

conjugates is improved upon metallation with

“Re(CO)3” (A). The most remarkable result was

obtained with the complex containing NO2-L-arg

(1) coupled to the chelator through a 6 carbon

linker. This complex presented a considerable

inhibitory action (Ki = 6 µM), comparable to that

of the free inhibitor (NO2-L-arg; Ki = 3 µM).

Complex 1 was also able to inhibit NO production (~46 %) in LPS-stimulated RAW 264.7

macrophages (B, C). ________________________ 1 Centro de Química e Bioquímica, Dep. de Química e Bioquímica, Fac. de Ciências da Univ. de Lisboa, Portugal.

A

C

B

L1 1

NN

N NH2

NO2NHNH

COOH

NH

NHO

( )3

NN

NNH2

NO2NHNH

COOH

NH

NHO

( )3

ReOC

COCO

[Re(CO)3]+

L1K i = 36 uM

1K i = 6 uM

Fig.1 Time (min) 99mTc-TMEOP Blood

KidneyUrineHeart IntestineLiver0 5 10 15 20 25Time (min) 99mTc-TMEOP Blood

KidneyUrineHeart IntestineLiver0 5 10 15 20 250 5 10 15 20 25

+

N

N

O

O

O

H

Tc

N

N

O

O

O

O C CO

CO

N

N

O

O

O

C

99m

Time (min) 99mTc-TMEOP BloodKidneyUrineHeart IntestineLiver

0 5 10 15 20 25Time (min) 99mTc-TMEOP BloodKidneyUrineHeart IntestineLiver

0 5 10 15 20 250 5 10 15 20 25

+

N

N

O

O

O

H

Tc

N

N

O

O

O

O C CO

CO

N

N

O

O

O

C

99m

Fig.2

0 5 10 1 5 20 2 5

1h p.i .

2h p.i .

B lood

+H

Tc

N

N

O

CO

CO

OC

N

NO

O N

N

C

99m

Time (min) 0 5 10 1 5 20 2 5

1h p.i .

2h p.i .

B lood

+H

Tc

N

N

O

CO

CO

OC

N

NO

O N

N

C

99m

0 5 10 1 5 20 2 5

1h p.i .

2h p.i .

B lood

+H

Tc

N

N

O

CO

CO

OC

N

NO

O N

N

C

99m

Time (min)

urine



0

5

10

15

20

25

Up

take/a

cti

vit

y (

%)

Internalization Cellular uptake

5 60 180

A B

Time (min)5 60 180

0

50

100

150

Bone/Muscle Ratio Bone/Blood Ratio

Fig.2

Novel multi-functional bone-seeking agents

E. Palma, J. D. G. Correia, L. Gano, S. Casimiro1, L. Costa,

1 I. Santos

The discovery of a synergistic cytotoxic effect of bisphosphonates

combined with antitumoral agents has been well demonstrated in both pre-

clinical and clinical setting. This fact prompted us to propose new

multifuntional compounds for simultaneous delivery of radiation,

chemotherapy and bisphosphonate

units to bone metastic lesions.

Therefore, we have introduced

novel complexes of the type fac-

[M(CO)3(k3-L)]+ (M = Re or 99mTc;

L = pz-pamidronate, 1; L = pz-

alendronate, 2; L = pz-Glu-

alendronate, 3) and evaluated the biodistribution profile of the 99mTc

complexes in mice (Fig. 1). The compounds were stable at physiological

conditions and presented high bone uptake and high target to non target

ratio, 4 h p.i. (Fig. 2). Their conjugation to cytotoxic units and evaluation

is in progress.

____________________ 1 Inst. de Medicina Molecular, Fac. de Medicina, Lisboa, Portugal

New 99m

Tc probes for monitoring multidrug resistance: From cardiology to oncology

F. Mendes, L. Gano, C. Fernandes, A. Paulo, I. Santos

Lipophilic cationic myocardial perfusion radiopharmaceuticals have been used for cancer detection and

monitoring of tumour Multi Drug Resistance (MDR). The complexes 99mTc-TMEOP and 99mTc-DMEOP,

promising myocardial SPECT imaging agents, were evaluated for MDR imaging. The uptake and efflux kinetics

of 99mTc-TMEOP were assessed in human cancer cells and in derivative drug-resistant lines, which overexpress

Pgp1 (responsible for

MDR) (Fig. 1). The

uptake of 99mTc-TMEOP

is comparable with 99mTc-

Sestamibi, being greatly

reduced in cells over-

expressing Pgp (Fig. 2). 99mTc-TMEOP was further

evaluated in vivo and it

was showed that it could

act as a substrate of Pgp.

Melanoma MC1R-targeting with 99m

Tc(CO)3-labeled cyclic αααα-MSH analogs. P. Raposinho, J. D. G. Correia, C. Oliveira, I. Santos

Following our work on MC1R-targeting, with cyclic radiolabeled α-

melanocyte-stimulating hormone (α-MSH) analogs for melanoma

imaging, two new cyclic compounds were synthesized and conjugated

to a pyrazolyl-based bifunctional chelator (pz). The binding affinity of the peptide-pz conjugates pz-βAla-Nle-c[Asp-His-D-Nal(2’)-Arg-Trp-

Lys]-NH2 (IC50 = 0.3 nM) and c[CO-p-C6H4-CO-His-D-Nal(2’)-Arg-

Trp-Lys]Lys(pz)-NH2 (IC50 = 11.9 µM) to the MC1R has been

determined by competitive binding assays using 125I-(Tyr2)-NDP, and a

significant loss of affinity for the second conjugate was found. Both

compounds were labeled with fac-[99mTc(CO)3]+ and the

radiocomplexes (A and B) evaluated in vitro and in vivo. For both, a

rapid and significant cellular uptake was found (Fig. 1), but their tumor

uptake was negligible in melanoma-bearing mice.

0

2

4

6

8

15 30 45 60 120 180

Time (min)

Up

tak

e (%

to

tal/m

illio

n c

ells)

MCF7

MCF7 neo

MCF7 pgp

Fig. 2 Fig. 1

0

5

10

15

%I.D/g organ 4 h p.i....

Blood Muscle Liver Kidney Bone

12

3

Fig.1

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