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Alpha-Emitters for Immuno-Therapy: A Review of Recent Developments from Chemistry to Clinics

Sandrine Huclier-Markai1,*, Cyrille Alliot2,3, Nicolas Varmenot2,4, Cathy S. Cutler5 and Jacques Barbet2,3

1Laboratoire Subatech, UMR 6457, Ecole des Mines de Nantes / Université de Nantes / CNRS-IN2P3, 4 Rue A. Kastler,

BP 20722, F-44307 Nantes Cedex 3, France; 2ARRONAX GIP, 1 Rue Aronnax, F- 44817 Nantes Cedex, France;

3CRCNA- Inserm, Université de Nantes, U892, 8 Quai Moncousu 44007, Nantes Cedex 1, France;

4Institut de Cancéro-

logie de l’Ouest – René Gauducheau – Bd J. Monod – 44805 Saint Herblain Cedex, France; 5University of Missouri

Research Reactor Center, Columbia, MO 65211, USA

Abstract: Alpha-particles are of considerable growing interest for Targeted Alpha Therapy (TAT). TAT gains more atten-tion as new targets, chemical labeling techniques and -particle emitters are developed but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available -particle emitters. This article is an up-to-date overview of the literature concerning -emitters used for of cancer. It briefly describes the nuclear characteristics, the production parameters (targets, extraction and purification), the complexa-tion properties of these radionuclides to chelates and biological vectors and finally draws-upon the preclinical and clinical studies that have been performed over the past two decades. Radiobiology and dosimetry aspects are also presented in this paper.

Keywords: 212Pb, 212Bi, 213Bi, 211At, 223Ra, 225Ac.

INTRODUCTION

The use of radionuclides in medicine is based largely on the discoveries of two critical concepts; the “tracer principle” and the “Magic Bullet”. In 1913, George De Hevesy devel-oped the tracer approach and was the first to recognize that radionuclides could be used as tracers to follow how the na-tive element or compounds containing the element were dis-tributed either in plants or animals [1]. He based his discov-ery on the principle that radioactivity has the advantage of being easily detected at very low quantities, allowing for the introduction of minute quantities, nano- to picomoles, that will not perturb the system. Thus the radiolabelled tracer allows for noninvasive measurement of distribution and function in a biological system. Later, in 1927, C. Regnaud and A. Lacassagne predicted that the ideal agent for cancer therapy would be composed of heavy elements capable of emitting radiation at molecular levels which selectively bind in the protoplasm of cells one seeks to destroy [1-4]. Fur-thermore, the “Magic Bullet” concept was proposed from the experience of 19th century German chemists (principally Paul Ehrlich) by selectively staining tissues for histological examination, and in particular, selectively staining bacteria. Ehrlich reasoned that if a compound could be made that se-lectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity. Hence, a "Magic Bullet" would be created that killed only the targeted organism. A problem with the use of the Magic Bullet concept as it emerged from its histological *Address correspondence to this author at the Laboratoire Subatech, UMR 6457, Ecole des Mines de Nantes / Université de Nantes / CNRS-IN2P3, 4 Rue A. Kastler, BP 20722, F-44307 Nantes Cedex 3, France; Tel/Fax: ???????????????; E-mail: huclier@subatech.in2p3.fr

roots is that people confused the dye with the agent of tissue selectivity and antibiotic activity. The name "Magic Bullet" was used in the 1940 movie Dr. Ehrlich's Magic Bullet, which depicts his life and focuses on Salvarsan (arsphe namine, "compound 606"), his cure for syphilis. This “Magic Bullet” concept has been extended to biomolecules particu-larly antibodies utilized as targeting molecules to transport toxins such as radionuclides selectively to receptors that are over expressed on certain diseased cells such as tumour cells. This concept has been expanded to include a host of nano-carriers from small molecules such as folic acid to peptides and proteins, microspheres and most recently nanoparticles. Both tracer and Magic Bullet concepts have been utilized to develop radiopharmaceuticals.

Radiopharmaceuticals are drugs that consist of two parts: a radionuclide that imparts the mechanism of action through its decay, attached to a targeting biomolecule or organic ligand that carries or determines the localization of the ra-diopharmaceutical. They can be used either for diagnostics for the noninvasive imaging of disease or as therapeutics to deliver a toxic payload selectively to a tumour site (a radi-onuclide emitting non-penetrating radiations: electrons, al-pha-particles, for instance).

Among the radionuclides used in nuclear medicine, al-pha-particles are of considerable growing interest for Tar-geted Alpha Therapy (TAT). Due to their short range in tis-sue (a few cell diameters), and high linear-energy-transfer (LET), -particles are especially suited for targeting micro-metastases and single tumour cells present in leukaemia’s and other blood-borne diseases [4-10]. The development of

depends mainly on the availability of a radionuclide with a suitable linker moiety and a biological carrier (organic

2 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 Huclier-Markai et al.

ligand, peptide, protein and/or monoclonal antibody). But TAT gains more attention as new targets, chemical labeling techniques and -particle emitters are developed and made available as stated by Elgqvist in 2011 [11]. In radioimmu-notherapy (RIT), the primary goal is to improve delivery to cancer cells and therapeutic efficacy, whilst minimizing tox-icity to normal cells. Different approaches have been inves-tigated to achieve this, such as developing small molecular weight carriers, pretargeting methods, multidosing or frac-tionation, locoregional administration and using cocktails of radiolabelled monoclonal antibodies to simultaneous target-ing multiple antigens. Some of these approaches have been encouraging, but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available -particle emit-ters. Radionuclides originally investigated in this context are namely 211At, 212Bi, 213Bi, and 225Ac. Their commercial availability was previously discussed in detail by Fisher in 2008 [12] and is not emphasized in this paper. More re-cently, other radionuclides such as 149Tb, 227Th, have been considered for TAT and tested in vitro and or in animal models. (Table 1) summarizes the main characteristics of these elements, their advantages and drawbacks. The concept of TAT has moved thus to the reality with increasing clinical experiences, including gliomas, ovarian cancer, metastatic melanoma, metastatic prostate cancer, lymphoma and acute myeloid leukaemia [13]. The clinical studies carried out to date have been promising, although making TAT safe and economically feasible remains a challenge [14]. The differ-ent targeting constructs used hither to that may be promising carriers for TAT in the future were presented and discussed in an excellent review from Olafsen et al. [14] and will not be discussed again in this paper. The constructs include en-zymatically cleaved antibody fragments (Fab and F(ab˙)2 fragments); genetically engineered antibody fragments (scFv monomer, dimer (i.e. diabody) and tetramer, CH2 domain deleted antibody fragments); other targeting protein con-structs such as affibodies and peptides, as well as liposomes and nanoparticles.

This article is an up-to-date overview of the literature concerning -emitters used for of cancer. It briefly de-scribes the nuclear characteristics, the production parameters (targets, extraction and purification), the complexation prop-erties of these radionuclides to chelates and biological vec-tors and finally draws-upon the preclinical and clinical stud-ies that have been performed over the past two decades. Ra-diobiology and dosimetry aspects are also presented in this paper.

RADIOBIOLOGICAL AND DOSIMETRIC CONSID-ERATIONS

The interest of alpha-particle-emitting radionuclides for immunotherapy lies in the physical and radiobiological properties of alpha-particles as compared with those of pho-tons and electrons [16]. Compared to standard radioisotopes beta emitters used in therapy, the energy deposition along the path, or linear energy transfer (LET), of an alpha-particle can be 100 to 1000 times greater, ranging from 60 to more 110 keV/ m for typical alpha-emitter radionuclide energy rang-ing from 2 to 10 MeV. In addition, the LET along an indi-vidual particle’s path is not constant, even increasing at the

end of the track where, typically for heavy charged particles, the Bragg peak occurs. The resulting high ionization density along the track, increases the probability of yielding DNA double strand breaks (DSBs) and consequently, the lethality power. In other words, because of the high concentrated dose deposited along the track and short range in tissues on the order of cellular dimensions, alpha-particles have a high probability of inducing damage to DNA, making them quite cytotoxic. By contrast of radiation-induced cellular inactiva-tion for low-LET radiation, requires the accumulation of sublethal damage, achieved only at much higher doses.

This specific radiation quality of alpha-particles, charac-terized by localized spatial distribution of the imparted en-ergy and high density of ionization per unit path length, causes direct DNA damage rather than indirect free radical-mediated DNA damage. The major radiobiological conse-quences of this, representing advantages of high-LET versus low-LET radiation, are an independence from dose-rate and oxygen effect [17].

The spatial distribution of alpha-particle sources as well as the geometry of the cells (thickness, diameter of the cell nucleus, distribution of DNA, etc…), have to be considered as important parameters in order to correlate the absorbed dose distribution with the observed biological response for the tissue of interest (tumour and/or critical organs).

To achieve estimated dose distribution, accurate dosime-try calculations require knowledge of the activity distribution as a function of time at the cellular and subcellular levels as well as an accurate representation of the geometry [16, 18]. This is not reachable by clinical scintigraphic imaging be-cause the spatial resolution is of the order of several mm, 2 orders of magnitude greater than the microscopic scale at which one dose is deposited with alpha-particles [19]. In addition, because of the important stochastic variations in the energy deposited in cell nuclei, macroscopic dosimetry ap-proaches become less relevant and microscopic approaches are required. The fundamental quantities to be considered in microdosimetry are specific energy (energy per unit mass) and linear energy (energy per unit path length through the target), calculated using analytical methods (convolution via Fourier transforms) or Monte Carlo simulation methods [20]. Nevertheless, the use of macro or micro dosimetric tech-niques for the calculation or estimation of the absorbed dose has to be considered [21].

Finally, alpha-particle-emitters may produce alpha-particle-emitting daughters, some of which may have rather long half-lives. Thus, dosimetry calculations must take into account the distribution of both the parent and all daughters [16]. This could contribute to the absorbed dose and to bio-logical effects. Moreover, the daughters’ biological distribu-tion may be dominated by a high affinity for tissues such as bone or kidneys, leading to a risk in terms of toxicity.

LEAD-212

Production Route and Extraction / Purification from the

Target

Lead-212 is produced by the decay chain of 228Th and can be obtained from a generator of 224Ra (daughter of 228Th). In the current generator system, according to Narbutt

Alpha-Emitters for Immuno-Therapy Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 3

Table 1. Main Properties and Production Routes of Radioisotopes of Interest

RN Particles

Emitted T1/2

E

(MeV)

Mean

Tissue

Range

( m)

Production

Mode

Usual

Chelating

Agent

Application Advantages Drawbacks

149Tb 1 , e+ 4.1

hours 4 ~ 30

142Nd(12C,5n)149

Dy or by proton

bombardment of

Ta

DOTA Micrometastases Unspecific

irradiation Poor availability

211At

(Two decay

routes)

7.2

hours 6 / 7,4 67.5 209Bi ( , 2n)

Labeling

through acti-

vated stanny-

lated or di-

fluoroboronyl

synthons

Monoclonal

antibody at-

tachment, m-

astato-benzyl-

guanidine

Long half life

Poor availability

Radiolabeling

stability ques-

tioned

212Pb

2

Decaying to

Bi-212

10.6

hours 7.8 600

228Th decay

chain

DOTA

TCMC

liposomes

Radioactive

label for therapy

using antibod-

ies, cellular

dosimetry

Available as a 224Ra generator

Not an alpha-

emitter. Decays

into bismuth-

212

212Bi 1 , 1 60.55

min 6.09 40-100

228Th decay

chain

CHX-A”-DTPA

liposomes

Monoclonal

antibody at-

tachment used

for cancer

treatment, cellu-

lar dosimetry

studies

Available as a

generator

208Tl decay

product with a

E = 2.6MeV

+

Short half life

213Bi 1 , 2 45.7

min 5.87 40-100

225Ac decay

chain CHX-A”-DTPA

AML, prostate

cancer, multiple

myeloma, non-

Hodgkin’s-

lymphoma,

colon cancer

Available within

a generator form Short half life

223Ra 4 , 2 11.4

days 5.7 – 7.5 ~ 60

227Th decay

chain

Zoledronic acid,

EDTMP

liposomes

Bone seeking in

the prostate

cancer

Available as a

generator

No fully chelat-

ing agent avail-

able

225Ac 5 , 3 10

days 5.8 – 8.4 ~ 70

229Th decay

chain Cyclotron

HEHA

DOTA

EDTMP

liposomes

AML, ovarian

cancer, breast

cancer,

neuroblastoma

229Th/225Ac

generator avail-

ability

Stablility of

chelation +

controlling the

fate of daughters 227Th and 225Ac decay into a series of alpha-emitters that include 223Ra (11.4 days) and 219Rn (4 sec) for 227Th and 221Fr (4.8 min) and 213Bi (45.6 min) for 225Ac. 212Pb decays into the alpha-emitter 212Bi (60.6 min) after emitting an electron. [3, 4, 8-10; 15] TCMC is the abbreviation of 1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane.

and Bilewicz [22], the 228Th / 212Pb generator can be pre-pared using DOWEX–50x8 cation exchange resin. 228Th is adsorbed onto the resin in 0.01 M HCl, while 212Pb is eluted from the column using 1 M HCl or HI (0.5M) in approx. 70% yield [10]. Currently 10 mCi generators are available. This type of generator is produced by AREVA Med within the project Thorium AREVA for Oncology (TAO).

Sometimes, an additional purification step is necessary using Chelex–100 in order to remove the by-products in-duced by radiolysis of the resin itself [23]. 212Pb is loaded (~ 30 kBq) at pH 5 in an acetate buffer solution [24]. After

washing (about 25 mL), 212Pb is eluted from the column with 5 M HNO3 [25].

Unfortunately, radiolytic effects limit the scale of organic resin-based 224Ra-212Pb-212Bi generator to levels insufficient for clinical use; thus evaporation based generator systems have been developed to overcome this problem [10, 26].

212Pb can be used as an in situ generator of 212Bi [27]. However the destruction of radioimmunoconjugates is a ma-jor difficulty in radiolabeling a mAb with 212Pb due to the high charge. Studies with 212Pb are limited to approaches

4 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 Huclier-Markai et al.

using an in vivo generator as described by Horak et al. [28] in which 212Bi is the source of -particles.

Complexation Chemistry and/or Nanoparticles

Aqueous solution chemistry of lead(II) complexes was investigated by Pippin et al. [29] in the course of developing 203Pb radiolabelled monoclonal antibodies as imaging agents. The stability constant of the 1:1 complex Pb-DOTA was found to be 24.3 (0.2). The biodistribution performed in that study suggested that 203Pb-DOTA labelled monoclonal anti-bodies were stable in vivo and thus useful for tumour local-ization of Pb(II) radionuclides. A study in mice with radiola-belled mAb 212Pb-DOTA-103A showed severe bone marrow toxicity, which resulted in the death of all animals [30, 31]. Brechbiel and Gansow evaluated the catabolism of different chelators attached to monoclonal antibodies and found that for internalizing antibodies, CHX-A”-DTPA or DOTA con-jugates used for bismuth or lead isotopes respectively are adequate and result in long retention of the tracer in the tar-geted cells [32]. This may not be important for short-lived radionuclides such as 213Bi but is of importance for 212Pb as when 212Pb decays to its 212Bi daughter, the 212Bi is released [33] from the chelate and could possibly be released intracel-lularly. Although the chelating agent used (DOTA) is known to form strong complexes with both Bi and Pb, a significant part of Bi (~35 %) is released from the carrier molecule after the 212Pb/212Bi radioactive decay and results in the formation of highly ionized daughter atoms [33].

Although direct biomedical applications of 212Bi are lim-ited by its relatively short half-life of 60.6 min; interestingly, its radionuclide parent (212Pb) may be considered as a longer lived (T1/2=10.6h) in vivo indirect source for -emission. To avoid undesirable radiotoxicity from localization of un-chelated 212Bi or 212Pb in natural binding sites, quite stringent requirements are mandated for the complexes (i) both lead and bismuth complexes should be kinetically inert to disso-ciation in vivo, (ii) the bismuth complex should be suffi-ciently stable thermodynamically so that recoil of 212Bi formed from the 212Pb - decay does not destroy the bismuth complex and (iii) the bismuth complex formed should sur-vive valence shell electronic reorganizations induced by nu-clear conversion of the gamma rays associated with 212Pb decay. Interpretation of decay effects have been studied for 143,144Ce or 177Yb [35, 36]. Asano et al. [37] found that no decomposition of 171Tb-Cy-DTA (i.e. CDTA) formed from the parent 171Er complex when no free metal was present in solution since a recombination of dissociated metal ion and ligand could be possible. The 212Pb could also be encapsu-lated into liposomes as previously reported for copper, scan-dium or bismuth. The formation, the characterization, the stability and in-vivo distribution as a function of lipid bilayer membrane were first examined by Rosenow et al. [38]. These authors showed that liposome-associated 212Pb was rapidly taken up in large quantities by the liver and spleen. Additionally they showed liposomes could be stabilized re-maining at least partially intact in vivo and thus in circulation in the serum; 212Pb liposomes effectively suppressed an anti-body response at high doses of activity. The chemical fate of the 212Bi-DOTA complex formed by the - decay of 212Pb-(DOTA)2- has been examined by Mirzadeh et al. [33]. The authors developed a useful methodology for evaluating the

chemical integrity of coordination complexes in such sys-tems. Notably, they investigated whether the complex ion which resulted from decay of 212Pb(DOTA)2- was intact and stable by analysing solutions initially containing this ion for amount of DOTA-complexed and uncomplexed 212Bi after attaining transient equilibrium with 212Bi. The authors found that the fraction of 212Bi not complexed to DOTA repre-sented the fraction of breakup of 212Bi(DOTA)- formed from

- decay of the parent complex. The break-up of the 212Bi-DOTA complex was ascribed to the internal conversion of -rays emitted by the excited 212Bi nuclide. Another study has been recently performed on such systems showing that an effective retention of 212Bi after - decay of 212Pb is achiev-able with 100 nm liposomes [25].

Preclinical /Clinical Studies

A clinical trial is ongoing (AREVA Med LLC, Univer-sity of Alabama, Birmingham, USA, NCT01384253), in which Pb is being used for targeting malignant cells with the trastuzumab antibody, as a potential treatment for metas-tatic disease. This Phase I trial is designed to determine the safety profile of Pb-TCMC-Trastuzumab and its dose in patients with HER-2 positive intraperitoneal cancers (ovar-ian, peritoneal, pancreatic, stomach and breast). No data from this clinical trial has been published.

BISMUTH-212 AND 213

Bismuth compounds, as a chemical element, have been widely used in the clinic for centuries because of their high effectiveness and low toxicity in the treatment of a variety of microbial infections, including syphilis, diarrhoea, gastritis and colitis. The first account was reported in 1786 by Louis Odier for the treatment of dyspepsia. Apart from microbial activity, compounds with isotopes of bismuth exhibit anti-cancer activities. In particular, 212Bi and 213Bi compounds have also been used as targeted radio-therapeutic agents for cancer treatment, and their ability to reduce the side-effects of cisplatin in cancer therapy (chemotherapy). 212Bi has been tested in animal studies leading to RIT- [39].

Although the chemistry of lead and bismuth complexa-tion is largely described in the literature, the stability of the complexes obtained with the radioactive isotopes of these elements is not.

Production Route and Extraction / Purification from the Target

The bismuth radioisotopes are available from generators 224Ra and 225 Ac respectively. Both 212 Bi and 213 Bi decay via branched pathways that result in both and emission. 213 Bi lacks the high abundance of energetic and potentially haz-ardous gamma emissions of 212Bi and thus is a more attrac-tive candidate notably for RIT. 212Bi can be obtained from the decay chain of 228Th. It decays via a branched pathway by -emissions to stable 208Pb. However, the short physical half-life of 212Bi can be a problem in terms of the time required for radioimmunocon-congate (RIC) labeling processes and delivery to the tumour. This problem has been partially offset by the construction of generators of 224Ra for the production of 212Bi.

Alpha-Emitters for Immuno-Therapy Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 5

The radiometal 213Bi has the same physico-chemical properties as 212Bi. It decays via a branched pathway by and emissions to stable 209Bi. The emitted energy (90.3%) is by -emission (major energy of 8.4MeV). Due to a gamma-ray of 440keV (26%), imaging of tumour uptake is possible and can be used to calculate dosimetry. 213Bi is pro-duced from the decay of 225Ac. A generator has been devel-oped for clinical use [40, 41] and has been described else-where [42, 43].

Bismuth Complexes and Biocoordination Chemistry

Bi(III) forms stable complexes with aminopolycarboxy-late (APC) and polyaminopolycarboxylate (PAPC) ligands. According to Pearson hard and soft acid-base theory, Bi(III) is a borderline metal ion that has a high affinity for multiden-tate ligands containing O and N donor atoms [44], but also forms stable complexes with S and halogens. The chemistry and structure of Bi(III) complexes with APC and PAPC ligands was reviewed by Stavila et al. in 2006 [45]. From that paper, the main characteristics could be summarized as the following. The stability constants of Bi(III) complexes of APC and PAPC are usually very high and could be isolated even at low pH, as for instance log KML (ETDA-Bi) = 26.7 (0.5); log KML (DOTA-Bi) = 30.3 (0.5) or log KML (DTPA-Bi) =30.7 (0.5).

More precisely, the labeling of biological carrier mole-cules with Bi is generally performed using chelating agents based on derivatives of DTPA or DOTA. Although Bi(III) has a strong tendency to hydrolyze, in the presence of these strong chelating ligands it can be stabilized up to pH 10. Denticity of the ligands is a factor determining the stability of the complexes as well as charge of the ligand, the preor-ganization, and the steric efficiency in which the ligand sur-rounds the Bi(III) ion to form a cage-like structure. The larger Bi(III) ion (1.03Å for a coordination number of 6 and 1.18Å for a coordination number of 8) forms 1:1 or 1:2 com-plexes with APC and PAPC ligands and exhibits coordina-tion numbers between 7 and 10. However, the octa-coordination of Bi(III) is by far the most frequent. Hassfjell and Brechbiel [46] have provided data concerning the chela-tion of some PAPC ligands as carrier molecules for 212/213Bi in cancer therapy. Stavila et al. [45], state that greater re-search towards the formation of Bi(III) APC and PAPC complexes with higher stability in vivo and lower cytotoxic-ity is needed, as well as the optimization of biocompatibility through a better design of new ligands.

A review of bismuth with potential targeting molecules, including peptides, proteins and enzymes was published in 2007 by Yang and Sun [47]. To selectively deliver bismuth to the site of disease, a chelate ligand such as DOTA or DTPA are commonly used as they form stable complexes. More frequently, the chelate is conjugated to a monoclonal antibody (mAb) or a fusion protein, via modification of the ligand to produce a bismuth radiolabelled compound, and introduced into the host, to target specific cell types and sites of disease [47, 48]. Derivatives of DTPA are particularly suitable for 213Bi labeling of antibodies due to their fast complexation.

Antibody Labeling with Bi-212 or Bi-213

The conjugate of 213Bi CHX’’-A-DTPA complex with a humanized anti-CD33 antibody HuM195, an anti-CD45 mAb and an anti-prostate-specific membrane antibody have been used in preclinical models of leukaemia and prostate cancer [49, 50]. 213Bi-labelled HuM195 was in a phase I/II clinical trial for advanced myeloid leukaemia [51]. In 2005, a review on cancer RIT with alpha-emitting radionuclides [8] described in detail all the preclinical in vitro and animal studies that have been performed with 212Bi and 213Bi. For 213Bi, human studies were performed that demonstrated spe-cific and potent cell killing ability against leukaemia with no significant toxicity [52] and showed that targeted -particle therapy is feasible in humans [53]. 213Bi-C595 has been shown to inhibit growth of pancreatic cell clusters and pre-angiogenic lesions in vivo, indicating that 213Bi treatment may have a role as adjuvant therapy to prevent early recur-rence [54].

Promising preclinical and clinical results have been achieved, and many studies have demonstrated the superior-ity of pretargeted RIT (PRIT) compared with conventional RIT. PRIT with -emitters has been reviewed by Lindegren and Frost in 2011 [55]. Briefly, pretargeting consists in in-jecting unlabelled modified antibody (pretargeting agent) enabling maximum binding to the tumour cells, then fol-lowed by the administration of the radiolabelled ligand spe-cific to the pretargeting agent (usually a small carrier ligand). In that case, the target to background ratios can be enhanced compared with direct targeting using radiolabelled antibod-ies.

Kozak et al. in 1986 [39] were the first to demonstrate that 212Bi can be bound to a mAb (anti-TAc directed to hu-man interleukin 2 IL-2 receptor) conjugated via a bifunc-tional metal ligand DTPA. The stability of 212Bi-DTPA was demonstrated in two animal models of erythroid leukaemia [56] and leukaemia l [57]. A study in a murine model of hu-man colon carcinoma (LS174T) was performed [58] show-ing significant antitumoural effects. In mice bearing a human ovarian tumour (SK-OV-3), Horak et al. [28] proposed a nanogenerator approach (mAb AE1 anti HER2/neu) radiola-belled via bifunctional p-SCN-Bz-DOTA in which it was shown that a prolongation of survival was obtained while no toxic effect was observed. 213Bi-DOTA Biotin conjugate in tumours has been shown to lead to complete elimination of tumour xenografts in some animals [59]. In this study, the group receiving PRIT with 213Bi-DOTA-biotin treatment showed a significant therapeutic effect in which 7 out of 10 mice were cured. 213Bi-DOTATOC somatostatin analogue was evaluated in a preclinical animal model [60]. Results showed a significant decrease of tumour growth without acute or chronic hematologic toxicities, and only mild acute nephrotoxicity but without evidence of chronic toxicity. The efficiency of the same 213Bi-DOTATOC was compared to 177Lu-DOTATOC in human pancreatic adenocarcinoma cells and results indicated that 213Bi-DOTATOC was therapeuti-cally more effective, showing four times greater induction of apoptosis than 177Lu-DOTATOC [61]. Park et al. [58] per-fomed a preclinical trial in which conventional RIT was compared to PRIT using 213Bi for treatment of non-Hodgkin lymphoma. PRIT showed a high fraction of complete remis-sion. By contrast, due to the unfavourable pharmacokinetics

6 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 Huclier-Markai et al.

of the antibody and the short half- life of 213Bi, no significant uptake was seen in RIT.

Another approach has been developed using the in-vivo generator 225Ac/213Bi for disseminated metastatic cancer. Liposomes encapsulating 225Ac were formulated and shown to retain the potentially toxic daughters at the tumour site. Sofou et al. [63] have developed passive encapsulation of 225Ac and tested the retention of actinium and its daughters by stable pegylated phosphatidylcholine cholesterol lipo somes of different sizes and charge. These authors showed that multiple 225Ac radionuclides could be entrapped per liposome but due to the large size of the liposomal structures required to contain the daughters, the approach is better suited for locoregional therapy.

Nanoparticles have been recently evaluated as a possible mechanism to stably chelate 225Ac and its daughters.

Preclinical / Clinical Studies

The potential of targeted therapy with the alpha-emitter 213Bi has been successfully demonstrated in a number of pre-clinical studies and several clinical trials have provided evi-dence for its feasibility, safety and therapeutic efficacy. Ac-cording to Sgouros [64], clinical trials of alpha-particle emit-ters have demonstrated the expected hallmarks of targeted alpha particle-emitter therapy — anti-tumour efficacy with minimal toxicity. The 213Bi Phase I/II trials against acute myeloid leukaemia (AML) demonstrated complete responses in patients whose tumour burden had been previously re-duced by cytarabine. The responses in this very high risk population lasted up to 12 months. Myelosuppression was tolerable and no significant extramedullary toxicity was ob-served [65, 66]. In addition, there are also on-going trials in Europe and Australia. These trials are investigating targeted 213Bi against lymphoma, progressive glioma, and melanoma [66-68].

A recent phase I/II trial was conducted to determine the maximum tolerated dose (MTD) and antileukemic effects of 213Bi-lintuzumab, the first targeted -emitter, after partial cytoreductive chemotherapy [69]. Lintuzumab (HuM195), a humanized anti-CD33 antibody, targets myeloid leukaemia cells and has modest single-agent activity against acute mye-loid leukaemia (AML). Thirty-one patients with newly diag-nosed or relapsed/refractory were treated with cytarabine (200 mg/m2/d) for 5 days followed by 213Bi-lintuzumab (18.5-46.25 MBq/kg). The results of this trial showed that sequential administration of cytarabine and 213Bi-lintuzumab was tolerable and could produce remissions in patients with AML. Clinical experience with 213Bi has been established in Phase I and Phase II trials of leukaemia [53, 69], lymphoma [70], malignant melanoma [71-73], and glioma [67, 74]. Morgenstern et al., 2011 [75] have reviewed therapy with 213Bi, from preclinical studies to clinical experiences. The conclusions of these authors are that 122 patients received therapy with 213Bi-labelled radioconjugates in clinical trials, with excellent retention at the tumour site, no adverse effect, that clearly establish the therapeutic effectiveness. Nonethe-less, the main drawback is the availability of 225Ac/213Bi but should be implemented to maximize progress in clinical test-ing of 213Bi.

ASTATINE-211

Production Route and Extraction / Purification from the

Target

Astatine-211 is a short-lived -emitting radionuclide (T1/2=7.2 h). Its half-life is long enough to permit multi-step synthetic procedures and it is compatible with the pharma-cokinetics of a wide variety of potential cell-specific target-ing agents. It decays by two pathways: one by electron cap-ture (58%) to 211Po (T1/2=0.5 s) which decays by prompt al-pha-emission to stable 207Pb (E =7.45 MeV); and the second route is by emission (42%) to 207Bi (T1/2=31.55 y) (E =5.84 MeV) then by electron capture to stable 207Pb. The mean energy of both alpha-particles is about 6.78 MeV. Astatine-211 is produced via a cyclotron by the nuclear reaction 209Bi( ,2n)211At, but 210At may be simultaneously produced via 209Bi( ,3n)210At. Production of 210At must be avoided because it decays to 210Po, an alpha-emitter with a long half- life (T1/2=138.376 d), which is a bone seeker. The major im-pediment to 211At availability is the need for a medium en-ergy -particle beam for its production. A complete review of the cyclotron production, the recovery of 211At from the target has been recently published [76] together with aspects of astatine availability, and discussion for circumventing astatine supply. So to optimize the ratio between both radi-onuclides, the alpha energy was set to 28 MeV [77, 78]. There are about 30 cyclotrons in the world that have the beam characteristics capable for the 211At production. Only one other team [79, 80] uses an alternative production reac-tion via Li3+ projectiles, natPb(7Li, xn)211At , at 35 MeV. Other routes of production exist and have been summarized elsewhere [76].

Liquid-liquid extraction was the principle procedure to purify no-carrier-added astatine. It was extracted by using different organic solvents (CS2, diisopropylether, etc...). Some authors continue to purify astatine by liquid-liquid extraction using diisopropylether (DIPE) [81]. In addition, 211At, could be purified principally by dry distillation at 650°C in a tubular oven with a nitrogen flush. Dry distilla-tion of astatine results in its recovery in small volumes nec-essary for radiochemical synthesis in a fast time and a recov-ery yield of between 75 and 85%.

Astatine Complexes and Biocoordination Chemistry

The chemistry of astatine is diverse. At the contrary of different metals previously mentioned, astatine is principally conjugated to an antibody via a covalent linker. The major labeling reagent is MeATE (N-succinimidyl 3-(trimethyl stannyl)benzoate and its derivatives. In brief, astatine is added to a solution of MeATE and N-iodo or N-chloro suc-cinimide in organic solvent (CHCl3,) to give N-succinidimyl-astatobenzoate (SAB) by destannylation. The nuclide reacts with the precursor for about 10 minutes at room temperature under gentle agitation. Then different purification procedures are used to isolate the SAB compound by HPLC techniques.

A novel radioastatination procedure developed recently by the Wilbur group to increase in vivo stability and prevent the de-astatination of antibodies uses the metal behaviour of astatine confirmed by recent studies on basic astatine chem-istry [82, 83]. The mechanism is a complexation of astatine

Alpha-Emitters for Immuno-Therapy Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 7

by a boron cage (closo-decaborate). NaAt/chloramine T (to control the oxidation state of astatine) and boron cage are mixed in acetic acid/methanol/water at room temperature for 10 minutes. The yield is dependent on the boron cage, 84% for decaborate and 53% for dodecaborate. The advantage of this method is the ability to attach the antibodies to the boron cage first and then to radiolabel with astatine.

Pruszynski et al. [84] have developed the ability to attach astatide anions to soft metal cations (Rh(III) and Ir(III)), which are also complexed by a bifunctional ligand (macro-cyclic thioether ligand 16aneS4-diol). Astatide, soft metal cations and 16aneS4-diol are mixed in a water-ethanol solu-tion. The solution is brought to pH 4 and heated at 80°C for 2 hours. The labeling yields were higher than 80%. A more exhaustive review of 211At by Wilbur et al. has been recently published [85]. Nearly 50 reagents studied in the develop-ment of pendant groups for labeling with 211At were de-scribed in that paper.

Antibody Labeling

Numerous approaches have been developed to selectively deliver 211At to tumours [3, 8], the use of colloids [86], pharmaceutical molecules [87, 88], melanin precursors [89], substrate carriers [90], thymidine analogues [91], biotin ana-logues [92] and bisphosphonate complexes [93-95]. Asta-tine-211 has also been conjugated to MAbs, such as the anti-CD20 rituximab, or to antibody fragments [96-100]. With a number of these compounds, it has been demonstrated that it is possible to kill cells selectively with only one to five -particles [95, 99, 100]. But 211At-labelled monoclonal anti-bodies may not be suitable for targeted -particle therapy if they have to be administrated systemically due to the rela-tively short half- life of 211At and the long duration needed to achieve a maximum tumour-to-normal tissue adsorbed dose ratio [101]. Using a bifunctional chelating agent (N-succinimidyl 3-(trimethylstannyl)-benzoate), Zalutsky et al.'s group [96, 97] has developed a two-step method for radiola-beling intact human/mouse chimeric MAb 81C6 (which re-acts with an epitope of the extracellular matrix glycoprotein tenascin of glioma and other tumours) and fragments F(ab') 2 of human/mouse chimeric MAb Me1–14 (reactive with chondroitin sulphate proteoglycans of human glioma and melanoma).

A more recent study [98] has demonstrated the initial proof of principle for the MRT (modular recombinant trans-porter) approach for designing targeted alpha-particle-emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT (N-succinimidyl 3-[211At] astato-5-guani dinomethylbenzoate for cancer cells overexpressing EGFR suggests that this 211At-labelled conjugate has promise for the treatment of malignancies, such as glioma, which over-express this receptor.

In a recent review [55], Lindegren and Frost have sum-marized the recent developments in particle pretargeting approaches. A summarization is given in (Table 2).

Vaidyanathan and Zalutsky [103] discussed the recent developments in the application of 211At-labelled radiophar-maceuticals; whereas the various reagents used for radiola-beling have been reviewed by Wilbur [85]. Although not all

211At-labeling reagents are reviewed, nearly 50 reagents studied in the development of pendant groups for labeling with 211At were described in that paper.

Preclinical /Clinical Studies

Only 2 studies have been published for 211At-labelled molecules in humans. Median survival in recurrent malig-nant brain cancer patients following administration of 211At-labelled anti-tenascin antibody into the surgically created tumour resection cavity was increased from the historically expected 25 to 30 weeks to 54 weeks [104]. Two patients with recurrent glioblastoma were alive 151 and 153 weeks after 211At-labelled chimeric 81C6 therapy [105].

The other study dealt with 211At-MX35 F(ab’)2 for ovar-ian cancer patients in a phase I study. Intraperitoneal admini-stration of (211)At-MX35 F(ab')(2) showed it was possible to achieve therapeutic absorbed doses in microscopic tumour clusters without significant toxicity [106].

ACTINIUM-225

225Ac decays by -emission; there are successively 5 -emissions and 3 -emissions, most of them of high energy. 225Ac shares 2 advantages with 223Ra. It does not emit high energy -rays, thus facilitating transport, and it has a rela-tively long half-life allowing transport to distant sites as well as the study of slow biological processes.

Production Route and Extraction / Purification from the Target

225Ac can be obtained in several ways. It can be produced by natural decay of 233U [107]. Additionally it can be ob-tained by neutron irradiation of 232Th leading to 233U [15]. By multiple electron capture in 226Ra, depending on the neu-tron flux, harness and dose, mixtures of 227Ac, 228Th and 229Th can be obtained. The separation of the nuclides is achieved on a titanium phosphate column [15, 108]. This system is ideal for 227Ac production, the source for 223Ra. After production, 225Ac has to be purified on a Dowex-50 column before being used for antibody radiolabeling or loaded onto a resin for the production of 213Bi with an 225Ac / 213Bi generator [8, 109].

Proton induced reaction on 226Ra leads to 225Ac directly via a (p, 2n) reaction. This speedy production mode easily adapts to rising needs of 213Bi but requires repeated irradia-tion of 226Ra. The process seems cheaper and easier to main-tain than a 229/228Th generator, which requires more shielding and degrades due to radiolysis.

Because 225Ac radioimmunoconjugates act as atomic nanogenerators, delivering an -particle cascade to a cancer cell, they are approximately 1,000 times more potent than 213Bi-containing analogs [7, 110]. Although this increased potency could make 225Ac more effective than other -emitters, the possibility of free daughter radioisotopes in circulation after decay of 225Ac raises concerns about the potential toxicity of this radioisotope.

Actinium Complexes and Biocoordination Chemistry

Two related macrocyclic chelates were identified as po-tentially useful and explored to attach 225Ac to monoclonal

8 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 Huclier-Markai et al.

Table 2. Pretargeting Approaches Published With 211

At (From [55])

Compound Assay Results Reference

Biotin-3-[211At]astatoanilide

([211At]AtBA) In vitro with strepavidin Low stability in serum [100]

3-

([211At]astatobenzoyl)norbiotinamide

([211At]AtBB)

[90]

Nido-carboranyl with biotin deriva-

tives In vitro / biodistribution

Increased stability of the nido-

carboranyl biotin derivatives as

compared with the stannylated

biotin derivatives

[83]

Polylysine Biodistribution

Low normal tissue uptake with

clearance in the liver for the High

Molecular Weight compounds and

in the kidneys for the Low Molecu-

lar weight compounds

Proof-of-principle of avidin-

conjugated antibody-trastuzumab as

pertargeting agent on SKOV 3 cell-

line.

[53]

antibodies. The first was 1,4,7,10,13,16-hexaazacyclohexa decane-N,N’,N”,N”’,N””,N””’-hexaacetic acid (HEHA) and the second was 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA). The 225Ac-HEHA complex was demonstrated to be less stable than the 225Ac-DOTA complex in in vivo experiments [111]. In addition, the two monoclonal anti-body/antigen systems that were examined using HEHA-constructs were non-internalizing immune-complexes and the targeted constructs could still release the daughters sys-temically. Another chelating agent evaluated was [112]: 2-(4-isothiocyanatobenzyl-1,4,7,1,13,16-hexa-azacyclohexa-decane-1,4,7,10,13,16)-hexa-acetic-acid (HEHA-NCS). With this ligand it has been possible to successfully radiolabel 3 antibodies (CC49, T101, BL-3) with 225Ac.

The macrocyclic ligand DOTA and its derivatives have been used effectively for labeling antibodies [7]. 225Ac-DOTA has a history of unfavourable radiolabeling chemistry and poor metal-chelate stability. Thus, a 2-step procedure was developed in which 225Ac is first conjugated to DOTA-SCN followed by attachment of this construct to the anti-body [7, 110, 111, 113]. The [225Ac]DOTA-SCN has been coupled to several IgG systems [113] and lead to a im-munoreactivity ranging from 25% to 81% depending on the antibody. This increased potency of the 225Ac constructs compared with 213Bi can be explained by the longer half-life of 225Ac and by the ability of 225Ac conjugates to act as atomic nanogenerators, emitting 4 -particles within an indi-vidual tumour cell as it decays.

The potential usefulness of liposomes as carriers of 225Ac was studied in 2004 by Henriksen et al. [114]. They showed that sterically stabilized liposomes could be loaded with 225Ac with excellent stability in serum in vitro. Sofou et al., [115] have demonstrated that 225Ac was passively entrapped in multivesicular liposomes (MUVELs). PEGylated MU-

VELs yielded 98% 225Ac retention and 18% retention of the last daughter 213Bi for 30 days. MUVELs were then conju-gated to an anti-HER2/neu antibody trastuzumab and exhib-ited strong binding and significant internalization (83%) by ovarian carcinoma SKOV3 cells.

There is considerable interest in utilizing nanoparticle constructs to stably complex metals for diagnostic and thera-peutic applications. Nanoparticles are being developed as an alternative due to their size and ability to circumvent some of the hurdles encountered with traditional agents. These nanoparticles are further undergoing modification by at-tachment of biomolecules such as peptides and antibodies to act as targeted delivery systems. Part of what is driving the use of nanoparticles is that conventional agents have resulted in low therapeutic indices due to biodistribution where only a minute portion of the intravenously administered drug reaches the target but large doses are delivered to normal tissues. Nanoparticles have unique properties that can be optimized to allow for higher penetration and retention in tumour cells. Furthermore, unlike conventional agents where only one radionuclide can be delivered per carrier, nanopar-ticles offer the advantage of delivering several radionuclides per carrier, increasing the dose and thus effectiveness of the drug. This is extremely important when low numbers of re-ceptors are present and only a few targeting molecules can be delivered.

Recently researchers have been evaluating using nanoparticles to stably complex actinides to sequester the daughter radionuclides that result from decay such as in the case of 225Ac [116, 117]. These methods are often referred to as in vivo generators that start with the stable complexation of a parent radionuclide that then undergoes decay to radio-active daughters that upon decay result in significantly higher dose to the tumour site. Conventional chelating tech-

Alpha-Emitters for Immuno-Therapy Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 9

niques often result in the loss of the daughters upon decay of the parent and toxicity to normal tissues that lower the over-all therapeutic efficacy.

Decay of 225Ac to stable 209Bi results in the release of four -particles and greater than 27 MeV of energy. Attach-ment of 225Ac to standard targeting agents using standard chelating agents, upon decay results in the release of the daughters. For example, 225Ac can be incorporated into a LaPO4 nanoparticle matrix. Studies were undertaken to evaluate lanthanum phosphate nanoparticles to which 225Ac was encapsulated to determine if the parent and daughters would remain sequestered upon decay. To test if these agents could be successfully used in selective targeting the La(225Ac)PO4NPs were conjugated to the monoclonal anti-body mAB 201B. This antibody was chosen as the targeting occurs within minutes of injection to the thrombomodulin of lung endothelium. As mentioned previously decay of 225Ac results in stable 209Bi, with the release of four -particles. The studies showed a retention of ~50% of daughter nuclides within the La(225Ac)PO4NPs over a period of one month. Biodistribution and imaging studies in mice showed ~ 30% of the La(225Ac)PO4NPs accumulated in mouse lungs at 1 h post injection resulting in a greater than 200% ID/g. To fur-ther enhance the potential of sequestering multiple parti-cles from 225Ac, these investigators went on to design gold-coated lanthanum gadolinium phosphate nanoparticles (NPs) for the purpose of both retaining 225Ac and its daughters and providing a versatile platform for attaching targeting agents for various tumour types within the body. In order to retain 225Ac and its daughters, 225Ac was embedded into a binary mixture of LaGdPO4 nanoparticles via hydrolysis of sodium tripolyphosphate in the presence of La3+ and Gd3+ ions. Next, citrate reduction of gold created a shell of Au on the surface of the LaGdPO4 particles. Magnetic LaGdPO4-AuNPs are then separated from nonmagnetic AuNPs using a 0.5-T NdFeB magnet. TEM analysis of these LaGdPO4-AuNPs particles showed monodisperse particles with average diame-ters of 4–5 nm. Radiochemical analysis indicated that LaG-dPO4-AuNPs without additional layers sequestered 60.2 ± 3.0% of the first decay daughter of 225Ac, 221Fr. Subsequent epitaxial growth with additional LnPO4 layers increased daughter retention. The addition of two shells of LaGdPO4 and one shell of Au increased 221Fr retention to 69.2 ± 1.7%, while the addition of four shells of GdPO4 and one shell of Au increased retention to 92 ± 1.0%. Retention of the first decay daughter is essential to minimize normal tissue toxic-ity. Daughter sequestration in these first-generation particles was high, but retention was improved by additional layers of Au and/or LnPO4. The authors have gone onto attach bio-molecules such as peptides or antibodies such as mAb 201B and plan to evaluate this in the future. The properties of low toxicity and favorable biodistribution make the LaGdPO4-AuNP system a promising platform for targeted alpha ther-apy with 225Ac.

Antibody Labeling

225Ac has been developed into potent targeting drug con-structs and is in clinical use against acute myelogenous leu-kaemia. The paper from Scheinberg and McDevitt [118] reviewed notably some aspects of biological studies in ani-mal models with 225Ac, the targeting with monoclonal anti-

bodies as well as the targeting nanogenerator approach. The first development was reported in vitro with [225Ac]DTPA-antibody construct with IgG1. However, the DTPA chelate moiety did not stably bind the 225Ac. Another radiolabelled antibody DTPA-construct, [225Ac]-DTPA-201B was exam-ined [119]. The DTPA was replaced by HEHA and the [225Ac]-HEHA-201B antibody construct was evaluated [120]. Nevertheless, the HEHA chelate released 225Ac and the non-internalizing antibody-antigen complex compro-mised the use of this system for RIT. A fragment of the CC49 antibody was coupled to HEHA and radiolabelled with 225Ac which showed only a marginal therapeutic effect.

Preclinical / Clinical Studies

The marked increase in potency of 225Ac constructs over 213Bi-containing analogs in vitro led to studies in nude mice bearing human prostate carcinoma and lymphoma xeno grafts. Single nanocurie doses of 225Ac-labelled tumour spe-cific antibodies significantly improved survival controls and cured a substantial fraction of animals [7, 118]. An excellent review on 225Ac was written by Miederer et al., 2008 [111] in which all the biodistribution aspects, the monoclonal anti-bodies and other carriers have been described, together with pharmacokinetics, radiobiology and dosimetry aspects. Miederer et al. [111] additionally described the successful Phase I clinical trial with [225Ac]DOTA-HuM195 in USA.

Clinical investigations in humans, using 223Ra for therapy of painful skeletal metastases in prostate and breast cancer patients, showed a strong and consistent reduction in alkaline phosphatase levels [121, 122]. In a large fraction of prostate cancer patients, this was accompanied by reduced prostate-specific antigen relative to baseline. Myelosuppression was minimal and thrombocytopenia was not dose-limiting. The alpha-emitting radionuclide, 225Ac has a decay scheme that includes 3alpha-particle-emitting daughters. The last alpha-emitting daughter in the series is 213Bi. The cytotoxicity of this in vivo isotope generator or “nanogenerator” is 1000 times more potent than 213Bi, in vitro, and has demonstrated remarkable efficacy in preclinical studies [123]. In a first-in-human phase I dose escalation study of this nanogenerator, AML patients treated with a single infusion of 23 to 170 μCi (0.5 to 2 μCi/kg) have demonstrated dose-related reduction in peripheral blood and bone marrow blasts with no acute or delayed toxicity at 10 month follow-up [124]. Accrual to this trial continues.

RADIUM-223

223Ra can be obtained from a generator of 227Ac (21.8 years). It decays by 4 -emissions and 2 -emissions to sta-ble 207Pb. These 4 -emissions confer an advantage from a therapeutic point of view, but also represent a major draw-back for stable radiolabeling. Furthermore, 219Rn gas emitted during the decay of 223Ra can redistribute in the body and be responsible for toxicity to healthy normal tissues. For target-ing bone metastases, 223Ra is the ideal radionuclide because simple cationic radium (radium chloride form) can be used for this purpose. A phase I trial evaluated the safety and tol-erability of this radiotherapeutic in breast and prostate cancer patients with bone metastases [125]. Attempts have also been made to evaluate its use after incorporating into

10 Current Topics in Medicinal Chemistry, 2012, Vol. 12, No. 23 Huclier-Markai et al.

liposomes or mAbs. Notably, loading 223Ra into liposomes coated with folate-F(ab’)2 was developed by Yao et al. [31]. Vaidyanathan and Zalutsky [103] gave an overview of the current status of 223Ra for targeted -particle radiother-apy.Studies on chelating agents have been published and one of the most promising would be calix[4]-tetraacetic acid but requires more development [126].

THORIUM 227

Production Route and Extraction / Purification from the

Target

227Th is produced indirectly. The first step consists of neutron irradiation of 226Ra to produce 227Ra. By beta decay this radionuclide gives 227Ac then 227Th. A purification pro-cedure is necessary to obtain no-carrier-added 227Th. 227Ac and thorium’s daughters (223Ra, 219Rn, 215Po, 211Pb, 211Bi, 207Tl, 207Pb) are discarded using strong anion exchange resin (AG1X8) in nitric acid 7M. 227Th is then recovered with hy-drochloric acid 12M. Before radiolabeling this solution is evaporated to dryness and the 227Th resuspended in HCl 0.01M.

Thorium Complexes and Biocoordination Chemistry

Scarce studies have been performed with 227Th. From our knowledge, only one laboratory in Norway has worked re-cently with 227Th. As for 225Ac, the conjugation of 227Th with antibody occurs in two steps [127, 128]. First, 20 L of 10 mg/L solution of p-SCN-benzyl-DOTA was added to a solu-tion containing 20 L (150 mg/mL) of L-ascorbic acid and 20-150 L of 100-300 MBq/mL 227Th in a 2 mL glass vial. The pH was adjusted to 5.5 with NH4CH3COO. The reaction mixture was then incubated for 40 min at 55-60°C.

To promote antibody conjugation, 1-2 mg of antibody was added to 227Th-DOTA solution after cooling to 37°C. The pH was adjusted to pH 8-9 with carbonate buffer. After 45 min, DTPA was added to the solution to complex free 227Th and the mixture was purified by gel filtration using saline phosphate buffer (pH 7.4). The final specific activity of the radioimmunoconjugate was about 500-1000 Bq/ g.

Really scarce data exist on radiolabeling and to our knowledge no clinical trial have been started using 227Th.

CONCLUSIONS

This review documents the efforts made over the past 20 years to develop -emitting radio-immunoconjugates for TAT in cancer patients (from physics to clinics). Significant advances have been made in radioisotope production meth-ods, chelation chemistry and in the development of novel approaches to deliver metals stably and precisely in vivo. Current sources of alpha radiation were reviewed and some prospects for clinical application were discussed. These growing advances are highlighting the need for more specific and selective methods of attaching and stabilizing metals with properties of interest for therapy. The radiobiology and microdosimetry are well understood and are the keys in bio-logical targeting. Notably the dose to the normal tissues to the one received by the tumour is the limitation of their use. Pretargeting is one way to circumvent this limitation. Pro-

gress in these areas will lead to advances in the application of targeted alpha therapy (TAT) for cancer therapy. In the review made by Allen B.J. in 2011 [19] advantages and drawback of TAT were summarized. Nonetheless, alpha-particle therapy is still a work in progress and promising preclinical and clinical trials lead to changes in cancer ther-apy.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Declared none.

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