FIRST APPLICATION OF THE IART APPROACH

WITH A NEW Re-188 LABELED BIOTIN DERIVATIVE

M. Pasquali
1
, C. Trapella
2
, R. Guerrini
2
, A. Boschi
1
, L. Uccelli
1
, E. Janevik-Ivanovska
3
, A. Duatti*
1
1
University of Ferrara, Lab of Nuclear Medicine, Dept of Radiological Sciences, Ferrara, Italy
2
University of Ferrara, Dept of Pharmaceutical Sciences, Ferrara, Italy
3
University of Skopje, Inst of Pathophysiology and Nuclear Medicine, Skopje, Macedonia
Introduction
Breast conservative surgery followed by sentinel node biopsy (SNB) and postoperative regional radiotherapy represents the treatment
of choice in patients with early breast cancer.
1
Several modalities of accelerated partial breast irradiation (APBI) have recently been
proposed. These methods include external-beam radiation therapy (EBRT), three-dimensional Conformal External-Beam
Radiotherapy, interstitial brachytherapy (HDR-BT), the MammoSite breast brachytherapy, targeted intraoperative radiotherapy
(TARGIT) and single fraction intraoperative radiotherapy (IORT/ELIOT). As an alternative approach, the application of a radionuclide
therapy with the combined administration of avidin and radiolabeled-biotin could be able to deliver a radiation dose to the operated
breast. This new procedure, known as Intraoperative Avidination for Radionuclide Therapy (IARTs),
1
consists of two steps: (1)
avidination of the anatomical area of the tumour with native avidin injected by the surgeon into and around the tumour bed, and (2)
targeting the anatomical area of the tumour by intravenous (IV) injection of radiolabelled biotin, 1 day later. Avidin is a 66-kDa
glycosylated and positively charged protein, which shows extremely high affinity for the 244-Da vitamin H, biotin (k
d
= 1015 M).13
Since, it is well known that the inflammatory reaction displays cation-exchanging properties, after surgery, avidin is expected to be
retained at the tumour bed site for several days and to act as a new receptor for radiolabelled biotin.
188
Re is a high-energy -emitter
(E
max
= 2.1 MeV), with a relatively short physical half-life (T
1/2
= 17.1 h) and a long penetration range in tissue (R
max
= 11.2 mm). Biotin
can be easily labelled with Rhenium-188 (
188
Re). Thus,
188
Re-biotin is particularly suitable for radionuclide therapy and the probability of
killing the majority of neoplastic cells is related to the so-called cross-fire effect.
Experimental
Preparation of the
99m
TcN-Biotin complex. The product was prepared using a two-step procedure as follows. (1) Na[
99m
TcO
4
] (0.9 mL,
100 MBq4 GBq) were added to a vial containing 1.0 mg of SDH and 0.10 mg of SnCl
2
(suspended in 0.10 mL of saline). The mixture
was kept at room temperature for 15 min. (2) To the resulting solution 0.1 mg of the Cys-Cys-biotin ligand (dissolved in 0.5 mL of
saline) and 0.5 mg of PCN (dissolved in 0.5 mL of saline containing 2.0 mg of -hydroxypropylcyclodextrin) or, alternatively, 0.5 mg of
PTA (dissolved in 0.5 mL of saline) were added simultaneously. The reaction vial was heated at 80 C for 30 min. Yield 96%.
Preparation of the
188
ReN-Biotin complex. A similar procedure was employed for preparing the analogous Re-188 nitrido complex
starting from generator-eluted Na[
188
ReO
4
]. A key difference with the Tc-99m preparation, however, was that 15 mg of sodium oxalate
were added in Step (1) to ensure formation of the [
188
ReN]
2+
group. Yield 89%.
Stability studies. Stability of the resulting conjugates was investigated in physiological solution and in human serum as well as
traschelation by cysteine and glutathion following well-established procedures.
5
Both Tc-99m and Re-188 compounds were found to
remain unchanged after 4 hours of incubation in the above conditions.
Binding studies. Avidin/biotin affinity studies were performed using both HPLC analysis and a spectrophotometric method based on the
reagent 4-hydroxazobenzene-2-carboxylic acid (HABA). Both methods were aimed at determining the binding stoichiometry of the
Cys-Cys-Biotin ligand (L), and of the corresponding Tc-99m and Re-188 complexes, towards avidin (Av) in comparison with natural
biotin (Vitamin H = VH) at a 1:4 Av/L molar ratio, which is the saturation ratio for the interaction Av/VH.
HABA assay. The complex between HABA and Av (HAv) was obtained by adding 90.0 L of a Av solution (prepared by dissolving 10.4
mg of Av in 40 mL of a 0.05-M sodium phosphate buffer, pH = 6.0) to 1.0 mL of a filtered HABA solution (prepared by dissolving 24.2
mg of HABA in 10 mL of 0.01-M NaOH). Avidin affinity of L was tested by adding to the resulting HAv solution , increasing amount s of
L according to different Av/L molar ratios (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:16 and 1:32).The absorbance of the different solutions was
measured at 500 nm by a UV/Vis spectrophotometer, and HAv affinity at 1:4 molar ratio was determined by comparing OD
500
(1:4) with
the saturation OD
500
value. Compound L showed high binding capacity for avidin (approximately, 98% of natural VH).
HPLC assay. Affinity of the Tc-99m and Re-188 complexes for Av was assessed by HPLC (Beckman System) according to the
following procedure. Experimental conditions: column, Agilent Zorbax Bio Series, GF-250 (4.6 9.4 mm); detector, UV and
radiometric; mobile phase, A, phosphate buffer, 0.4 M, pH = 7.4, B, phosphate buffer, 0.4 M, pH=7.4, flow rate, 1 ml/min; isocratic,
030 min, %B = 50. An aliquot of Av was mixed with the appropriate complex in 1:4 molar ratio. After incubation for 5 min at RT, the
solution was injected into the HPLC system. Results showed that approximatelty 98.9% of the complex remains tightly bound to Av at
this molar ratio. The retention time of the radioconjugate complex with avidin (with both Tc-99m and Re-188) was 11.7 min whereas
that of the free metal-biotin radiocompound (with both Tc-99m and Re-188) was 18.5 min.
Biological studies. Preliminary biological studies have been conducted in rats according to the animal welfare regulations of the Italian
local authorities. Female SpragueDawley rats, weighing 200-250 g, were housed for 1 week with free access to food and water for
subsequent biodistribution studies. Animals were anesthetized with an intramuscular injection of a mixture of ketamine (80 mg kg-1)
and xilazine (19 mg kg-1). After surgical exposure of a subcutaneous small area in one posterior leg, avidin was locally administered by
puncturing the exposed muscle tissue with an insulin syringe and, then, the injured area was sutured. Tc-99m activity (1.8 MBq in 50
L) was injected in the jugular vein after surgical exposure, and the whole animal was imaged at 10 and 30 min post injection (A and B
in Figure 4, respectively). High localization of activity was found in the subcutaneous region locally administered with avidin (shown by
an arrow in Figure 4). Activity was also found in liver, kidneys and urine.
The labeling approach
The labeling of biotin was conducted using a novel approach
2
that is briefly outlined as follows. Generally, technetium--nitrido
complexes possess a square-pyramidal geometry where the terminal TcN group occupies an apical position. Accordingly, there
remain four residual positions on the basal plane left available for coordination by other ligands. Studies carried out on various classes
of technetium nitrido complexes showed that the combination of a tridentate -donor ligand, having S
-
, N, and S
-
as donor atoms, with a
monodentate -acceptor monophosphine ligand (PR
3
) affords a highly stable arrangement when coordinated around the TcN core . A
pictorial representation of this type of complexes (called 3+1 complexes) is reported in Figure 1. The essential point here is that the
tridentate SNS-type ligand should comprise a combination of three-donor atoms selected from the restricted set of S
-
and N atoms,
while the monodentate ligand, PR
3
, has to be taken from the category of -acceptor ligands.
Figure 1
The labeling of biotin was conducted using a novel approach
3
that is briefly outlined as follows. Generally, technetium--nitrido
complexes possess a square-pyramidal geometry where the terminal TcN group occupies an apical position. Accordingly, there
remain four residual positions on the basal plane left available for coordination by other ligands. Studies carried out on various classes
of technetium nitrido complexes showed that the combination of a tridentate -donor ligand, having S
-
, N, and S
-
as donor atoms, with a
monodentate -acceptor monophosphine ligand (PR
3
) affords a highly stable arrangement when coordinated around the TcN core . A
pictorial representation of this type of complexes (called 3+1 complexes) is reported in Figure 1. The essential point is that the
tridentate SNS-type ligand should comprise a combination of three-donor atoms selected from the restricted set of S
-
and N atoms,
while the monodentate ligand, PR
3
, has to be taken from the category of -acceptor ligands.
The 3+1 method could be potentially employed for the labeling of any bioactive molecule. It was found that a combination of two
aminoacids (or pseudoaminoacids) provides an excellent source of SNS-type ligands (Figure 2a, M = Tc, Re). Beside this,
monophosphine ligands having variable substituent groups(Figure 2b) can be used to obtain final conjugate complexes having different
lipophilic characters (PCN = tris-cyanoethylphosphine; PTA = 1,3,5-triaza-7-phosphaadamantane).
Figure 2
a
b
M = Tc, Re; X = monodentate co-ligand
IsoCys = isocysteine
ma = mercaptoacetic acid
Cam = cysteamine
= bioactive group
Synthesis and labeling of a bifunctional Cys-Cys-biotin ligand
The molecular structure of the biotin derivative employed in this work is reported in Figure 3. This ligand was obtained by linking a Cys-
Cys chelating system (Figure 2a) to a functionalized biotin through a Pauson-Khand
3
reaction involving the condensation of an allyl
group (on the biotin) with an alkyne group (on the Cys-Cys) in the presence of dicobalt octacarbonyl [Co
2
(CO)
8
]. The same Figure
illustrates also the coordination around the TcN core when PCN was used as ancillary monodentate ligand. A n identical result was
obtained with the water-soluble phosphine PTA. The analogous Re-188 conjugate complexes were obtained by simple replacement of
the [
99m
TcN]
2+
core with the [
188
ReN]
2+
core.
Figure 3
Figure 4
Conclusions
New biotin derivatives labeled with Tc-99m and Re-188 have been prepared using a novel labeling approach. The resulting Tc-99m
and Re-188 conjugate complexes form a perfect matching pair as a consequence of their identical molecular structure. Biological
characterization of the new radiolabeled biotin agents showed that they exhibit high in vitro affinity for avidin. This finding was
confirmed by in vivo experiments indicating that avidin, locally administered in a small subcutaneous area of rats leg, is capable of
accumulating radiolabeled biotin. Further studies are required to clearly establish whether the observed localization is fully regulated by
the binding interaction of avidin with labeled biotin.
References
1) Paganelli, G., Ferrari, M., Cremonesi, C., et al. IARTs: Intraoperative avidination for radionuclide treatment. A new way of partial
breast irradiation. The Breast (2007) 16, 1726.
2) Duatti; A., Cyr; J., Uccelli, L.; Boschi; A.,; Srinivasan; A.; Pasqualini; R. Radioactive metal complexes comprising a tridentate
complexing sequence. European Patent Number: EPI1913959; Publication date: 2008-04-23.
3) Crawford, J.L., Kerr, W. J., McLaughlin, M., et al. Use of a highly effective intramolecular PausonKhand cyclisation for the formal
total synthesis of ()-- and -cedrene by preparation of cedrone. Tetrahedron (2006) 62, 11360-11370.