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4. STUDIES ON 105Rh AND LABELED MOLECULES

4.1. INTRODUCTION

The preparation of 105Rh as a NCA radionuclide was fi rst reported by Morris and Khan in 1966 [1] followed by Kobayashi in 1967 [2] and by Lessing et al. in 1975 [3]. The potential of 105Rh as a therapeutic radionuclide was identifi ed by Troutner in 1987 [4]. Production of NCA 105Rh by (n,γ) reaction followed by radiochemical processing was reported by Grazman and Troutner [5]. The author joined Prof. Troutner in the year 1987 to work on the development of 105Rh labeled radiopharmaceuticals under a project funded by the US Depart-ment of Energy (DOE). This chapter describes the author’s contributions in the production of 105Rh and the development of 105Rh labeled molecules.

4.2. INORGANIC CHEMISTRY OF RHODIUM

Rhodium (Z = 45) is second row, group 9 transition metal of the platinum group. Rhodium is found in nature as mononuclidic with 103Rh as the only stable iso-tope. The element has an electronic confi guration of [Kr]4d85s1 and the most common oxidation state of rhodium is +3 (d6), though 0 to +6 are also observed. Octahedral d6 complexes of rhodium favor a low-spin ligand fi eld arrangement where all d valence electrons occupy the three low-energy t2g orbitals, thus form-ing a closed-shell confi guration. While there are more than twenty radionuclides reported, the long-lived radioisotopes of rhodium are 99Rh (T1/2 = 16.1 d), 101mRh (T1/2 = 4.34 d), 101Rh (T1/2 = 3.3 y), 102mRh (T1/2 = 2.9 y), 102Rh (T1/2 = 207 d) and 105Rh (T1/2 = 35.36 h). Among the different radioisotopes, 105Rh has potential to be used as a therapeutic radionuclide whereas the uses of other isotopes are not documented.

4.3. RADIONUCLIDIC CHARACTERISTICS OF 105Rh105Rh decays with a half-life of 35.5 h and emitting three beta particles of en-ergies 560 keV (70%) and 260 keV (30%). There are two gamma emissions of 319 keV (19%) and 306 keV (6%). The low energy β– particles are suitable for certain specifi c applications in targeted therapy such for antibody labeling, bone pain palliation as well as for the development of radiopharmaceuticals targeting

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metastasis. The two low energy, low abundance gamma rays can be used for mapping the in vivo uptake of the administered radiotracer.

4.4. PRODUCTION OF 105Rh

Natural rhodium 103Rh, which is mononuclidic, on reactor irradiation results in the formation of short-lived 104Rh (T1/2 = 42 s) and 104mRh (T1/2 = 4.4 m), both decay to stable 104Pd (stable). Hence, the production of radioisotopes of rhodium by using rhodium target is ruled out. 105Rh is produced by the indirect method via the (n,γ) reaction of 104Ru followed by β– decay to 105Rh. The neutron acti-vation cross-section of this reaction, however, is low (0.5 barns). The nuclear reaction is given below:

β–, T1/2 = 4.44 h104Ru(n,γ)105Ru ► 105Rh

Natural ruthenium is found as a mixture of several isotopes: 96Ru (5.52%), 98Ru (1.88%), 99Ru (12.7%), 100Ru (12.6%), 101Ru (17.0%), 102Ru (31.6%) and 104Ru (18.7%). Irradiation of natural Ru will lead to the production of 97Ru (T1/2 = 2.9 d), 103Ru (T1/2 = 39.35 d) and 105Ru (T1/2 = 4.44 h). Being short-lived, 105Ru decays to 105Rh which can be separated by chemical methods.

The main drawback of this method is that 105Rh needs to be separated from other ruthenium radionuclides. Also natural ruthenium is often associated with iridium impurity which will also get activated during irradiation leading to the formation of 192Ir (T1/2 = 74.4 d) and 194Ir (T1/2 = 19 h). Hence, the iridium ra-dionuclides also need to be removed during the radiochemical processing.

Enriched 104Ru is preferred for the production of 105Rh. A radiochemical separation to isolate 105Rh from the unspent ruthenium target is needed. Enriched targets are expensive and hence recovery of the enriched target will be an ad-ditional task while using enriched 104Ru target.

Grazman and Troutner described the production of 10-100 mCi (0.37-3.7 GBq) quantities by irradiating natural ruthenium at the Missouri University Research Reactor (MURR) and the process chemistry was developed which yielded 105Rh suitable for radiolabeling ligands [5]. In this process, the ruthenium metal target was purifi ed to remove iridium impurity prior to irradiation. The radiochemical processing involved dissolution of the irradiated target, ruthenium metal powder, in KOH and converting the ruthenium to RuO4 by passing chlo-rine gas. The RuO4 is extracted in carbon tetrachloride and subsequently re-covered and purifi ed to get NCA 105Rh with specifi c activity > 700 mCi/μmol. The author used the 105Rh prepared by this method in all the radiolabeling studies reported in his papers [6-11] and described in the subsequent sec-tions.

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4.5. DEVELOPMENT OF 105Rh LABELED MONOCLONAL ANTIBODIES

The 105Rh program at the University of Missouri started by Prof. Troutner with the objective to radiolabel monoclonal antibodies for radioimmunotherapy and was funded by the DOE grant. Development of radiolabeled antibodies as diagnostic and therapeutic radiopharmaceuticals was a widely investigated topic in the eighties. Specifi c activity requirements

105Rh is available in NCA form and hence of very high specifi c activity (theoretical specifi c activity is 843 Ci/mg (30 TBq/mg)). The theoretical spe-cifi c activity corresponds to 88 500 mCi/μmol. However, the 105Rh practically obtained had specifi c activity > 700 mCi/μmol only as other complexing metals are expected to be present which will reduce the effective specifi c activity of 105Rh. The molecular weight of the monoclonal antibody is 150 000 D. Hence, by labeling monoclonal antibodies with 105Rh, a specifi c activity of ~5 mCi/mg could be obtained. For radioimmunotherapy, up to 100 mCi of 105Rh activity was expected to be given which could have been incorporated in about 20 mg of the antibody which was considered reasonable. However, if there is lesser metal contamination in the radionuclide preparation, the specifi c activity can be much higher and the amount of monoclonal antibody to be administered can be reduced to much lower levels.

Rhodium-105 cannot be directly incorporated to the antibody molecule. The bifunctional chelating agent (BFCA) approach for radiolabeling of bio-molecules was just evolving at the time when the author started working with 105Rh. Our interest was to develop good BFCAs for making stable rhodium complexes. Hence, studying the inorganic chemistry aspects of rhodium was important in order to design suitable ligands and bifunctional chelating agents that will form stable complexes with rhodium.

As the author started working on the development of 105Rh radiopharma-ceuticals very little prior knowledge existed on this topic. Hence, the work on the development of 105Rh labeled monoclonal antibodies involved:

design of ligands and bifunctional chelating agents suitable for complexing • rhodium,synthesis and characterization of rhodium complexes at macroscopic levels,• preparation and characterization of • 105Rh complexes,coupling of the complexes with the biomolecules,• purifi cation of the labeled biomolecules,• stability studies of the • 105Rh labeled biomolecules,immunoreactivity of the antibody molecules.•

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4.6. DEVELOPMENT OF BIFUNCTIONAL CHELATING AGENTS FOR RHODIUM

Rhodium(III) forms kinetically inert complexes with ligands having S, N, P donor atoms. The complexes formed are generally in octahedral geometry. Various amounts of substitution with the ligands leaving 1-3 chloride atoms in the coordination sphere are usual with the rhodium complexes. Complexes of rhodium with amines, thioethers, phosphines, pyridines, alkenes, carbon mon-oxide etc. are reported. Rhodium (III) chloride which also is referred to as hydrated rhodium trichloride (RhCl3 (H2O)3) is the reactive form and used for complexation studies. Rhodium chloride can exist in different forms with dif-ferent levels of hydration and consequently different charges such as [RhCl3(H2O)3]

0, [RhCl2(H2O)4]+, [RhCl(H2O)5]

2+ exists in solution. Rh(III) complexes are usually synthesized in aqueous solutions of RhCl3(H2O)3 and in the presence of alcohol. The alcohol present in the reaction mixture is supposed to reduce the Rh(III) to Rh(I) to assist the substitution reaction, and then atmos-pheric oxygen present in the reaction reoxidizes the Rh(I) to generate the ki-netically inert Rh(III) complex [12].

Fig. 4.1. Bifunctional chelating agents synthesized and used for radiolabeling 105Rh and conjugating with gamma globulin

I II

III IV

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4.6.1. DESIGN AND SYNTHESIS OF BIFUNCTIONAL CHELATING AGENTS FOR RHODIUMThe bifunctional ligands should be able to chelate rhodium strongly and also should have a functional group such as –NH2 or –COOH to link it to the bio-molecule. The proteins also have free –NH2 and –COOH groups and hence peptide bonds could be developed by coupling complimentary groups. Figure 4.1 shows the BFCAs synthesized as part of the author’s research work at the University of Missouri-Columbia. These included a bidentate (IV), two triden-tate (I & II), and a pentadentate ligands (III). Also several other chelating ligands were synthesized during the period for radiolabeling with 99mTc for the develop-ment of diagnostic radiopharmaceuticals. A tetradentate amine phenol ligand was used for the complexation of rhodium and studied at macroscopic level [13].

4.7. RHODIUM COMPLEXES

Two rhodium complexes with a bidentate and a tetradentate ligand were syn-thesized and its structure determined by single crystal X-ray diffraction studies which are described below.

4.7.1. SYNTHESIS AND X-RAY CRYSTAL STRUCTURE OF A RHODIUM (III) COMPLEX WITH AN AMINE PHENOL LIGAND [14]A new rhodium (III) complex of an amine phenol ligand N,N’-bis-(2-hydroxy-benzyl)-1,3-diaminopropane was synthesized and characterized by X-ray dif-fraction studies. The synthesis of the ligand was done by reacting two equiva-

Fig. 4.2. ORTEP diagram of Rh(III) complex of N,N’-bis-(2-hydroxybenzyl)-1,3-di-aminopropane

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lents of salicylaldehyde with one equivalent of 1,3-diaminopropane and reduc-ing the resultant Schiff base with sodium borohydride [13]. The rhodium complex was synthesized by refl uxing 0.36 mmol (100 mg) of the ligand in 20 ml of ethanol with 0.36 mmol (94.6 mg) of RhCl3·3H2O at pH 8. The solution was fi ltered and left in a refrigerator for crystallization. The orange yellow crystals obtained after several days were collected and used for X-ray diffrac-tion studies. Diffraction data were collected by using MoKα radiation on an Enraf-Nonius CAD4 automated diffractometer. The structure consists of a di-nuclear complex with two μ-hydroxy bridges between the two Rh atoms which is complexed to a double deprotonated ligand molecule (Fig. 4.2). The overall charge of the molecule is neutral due to deprotonation of all phenolic groups. The rhodium atoms were coordinated to two amine nitrogen atoms, two phe-nolic oxygen atoms and two μ-hydroxy oxygen atoms in a near octahedral fashion. The studies were useful to understand the complexation behavior of the amine phenol ligands with rhodium. However, the phenomenon of binu-clear complex formation was making the tetradentate backbone unsuitable for developing into a BFCA for complexing with rhodium. It was also not sure whether the binuclear formation will be taking place only at macroscopic level and at radiotracer level the complex formed might be mononuclear. 4.7.1.1. Rhodium complexes of a bidentate secondary amine oxime ligands [11]A new bidentate amine oxime ligand, 3-benzylamino-3-methyl-2-butanone oxime, was prepared by reacting 3-chloro-3-methyl-2-butanone oxime with

Fig. 4.3. ORTEP diagram of Rh(III) complex of 3-benzylamino-3-methyl-2-butanone oxime

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benzyl amine in methanol under refl uxing condition. The rhodium complex of the ligand was prepared by reacting the ligand with rhodium chloride at pH 7. The orange colored solution formed was fi ltered and the fi ltrate upon slow evaporation gave orange crystals of the rhodium complex. The crystals were isolated and characterized by 1H-NMR, IR spectroscopy and X-ray crystallo-graphy. The X-ray data were collected as described above. The crystal structure consisted of discrete mononuclear, neutral and slightly distorted octahedral complex containing two ligand molecules (Fig. 4.3). The octahedron around rhodium was completed with two trans-chlorine atoms and four nitrogen atoms from the two bidentate amine oxime ligands. The bifunctional analogue of this ligand, 3-[N-(4-aminobenzyl)]amino-3-methyl-2-butanone oxime, was also synthesized and used for radiolabeling with 105Rh and conjugation to human gamma globulin.

4.8. COUPLING OF BIOMOLECULES WITH PREFORMED 105Rh COMPLEXES

Rhodium (III) do not form complexes with ligands at room temperature and heating the ligands with 105Rh in chloride form to higher temperature is essential to achieve good complexation yields. The complexes once formed are kineti-cally inert and stable for extended period of time as well as during challenge studies with other ligands having good coordinating groups. Rhodium com-plexes of several BFCAs were prepared and conjugated with protein molecules. The stability of the 105Rh-BFCAs was established by challenge studies with cysteine and EDTA ligands in large excess. Cysteine has a thiol, amine and carboxylic acid groups and hence is a strong chelating molecule.

The studies done by the author were the fi rst complexation studies with 105Rh in the literature and hence detailed work was needed to optimize the conditions to get high complexation yields. The studies involved varying the pH of the reaction, use of different buffer solutions, varying reaction time, varying the ligand metal ratio etc. The ligands were always dissolved in ethanol which catalyzed the complexation reaction presumably through the reduction of Rh(III) to Rh(I) [15] the intermediate for complexation, however, the positive role played by ethanol in rhodium complexation was understood much later.

Complexation of the ligands with 105Rh was done by using carrier Rh(III)Cl3·3H2O together with 105Rh tracer. The complexation involved refl ux-ing the Rh(III) carrier with 105Rh tracer followed by the addition of calculated amount of ligand in ethanol : saline (1:1) solution. The reaction was monitored by TLC and the complexation generally took about 20 min to an hour. The complexes were characterized byTLC as well as by a novel method ‘magne-sium oxide adsorption’ developed during the course of the studies (described) later [16].

94

At the initial stage of the studies, either albumin or globulin was used as model proteins. Human gamma globulin has identical structure as that of the antibodies and could mimic the characteristics of the monoclonal antibodies.

4.8.1. RADIOLABELING OF PROTEINS WITH 105Rh USING 4-p-TOULIC ACID-DIETHYLENETRIAMINE (PTDET) [6] The bifunctional chelating agent, PTDET (ligand I, Fig. 4.1), has the carboxyl group (–COOH) as the coupling group and three amine nitrogen for complexa-tion with Rh(III). The complex formed was expected to have other monodentate ligands such as chloride anion or neutral water molecules to complete the octa-hedral complexation sphere. PTDET was synthesized by a three-step synthetic procedure. The fi rst step involved protection of primary amine groups of di-ethylenetriamine with phthalic anhydride followed by reaction of the diphthaloyl protected triamine with α-bromo-p-toulic acid. The diphthaloyldiethylene-triamine was subjected to acid hydrolysis to obtain the target ligand. The charac-terization of the ligand was done by NMR, IR and elemental analysis. Complexation with 105Rh and coupling with gamma globulin

The 105Rh complexes were prepared as follows: RhCl3·3H2O dissolved in 1:1 ethanol : saline was spiked with 105Rh and refl uxed for a few minutes. The ligand dissolved in 1:1 ethanol : saline is added to this solution and the mixture was heated for about another 20 min. The complexation yields were estimated by TLC in silica gel or by using the magnesium oxide powder adsorption tech-nique [16]. The complexation of 105Rh with the ligand gave complex yields ~90% at optimized conditions.

The BFCA contained –COOH group which was to be coupled to the free amino or carboxylic acid groups available in the protein. Coupling agents such as carbodiimide, diphenyl phosphoryl azide or isobutyl chloroformate were used for the coupling reaction. The conjugation yields were estimated by gel permeation chromatography using sephadex G 75. Precipitation of the protein with trichloroacetic acid was also done at times as a quick quality control tech-nique. Stability of the 105Rh complex coupled proteins was established by chal-lenging with 100-fold excess of EDTA as well by estimating the radiochemical purity at different time intervals.

The main problem encountered in these studies was poor conjugation yields irrespective of the coupling method and bond formed. The overall conjugation yields, decay corrected, were typically about 30% needing further work on both the preparation of new chelating agents as well as coupling chemistries. Qual-ity control techniques such as thin layer chromatography and adsorption by magnesium oxide powder was established during these studies.

95

4.8.2. RADIOLABELING OF PROTEINS WITH 105Rh USING 4-(p-AMINOBENZYL)-DIETHYLENETRIAMINE (ABDET) [7]Though the complexation yield of 105Rh with PTDET described above was found to be high ~90%, the coupling yield of 105Rh labeled PTDET was found to be low, ~50%. Hence, the overall yield of conjugation was 35-45% which together with the decay loss was unacceptable for making radiolabeled anti-bodies with suffi cient specifi c activity. Hence, the need for better BFCAs was felt. As the major problem in PTDET was the conjugation effi ciency, BFCA with an amine group as the coupling moiety was desired and hence the new BFCA, ABDET was synthesized.

The synthetic scheme for the preparation of ABDET was a bit more diffi cult needing a four-step procedure (Fig. 4.4). Diethylenetriamine was fi rst protected with diphthaloyl derivatization followed by condensation with 4-(p-nitro)ben-zyl bromide. The resultant product was deprotected by acid hydrolysis to get the nitro derivative of the BFCA. Hydrogenation on Pd activated charcoal was done to get the BFCA. Purifi cation by recrystallization and characterization of the product by NMR and elemental analysis were done at each synthetic stage to get the fi nal pure product. Complexation with 105Rh and coupling with gamma globulin

Complexation of the BFCA with 105Rh was done at pH 9 in 0.5 M bicarbo-nate buffer and the reaction mixture contained ethanol as before. The complexa-tion yields were about 90% as observed with PTDET. For coupling with gamma globulin, the complex was treated with thiophosgene (CSCl2) dissolved in CHCl3 to convert the –NH2 group to –NCS (isothiocyanate) group. The aqueous layer was separated and the CHCl3 layer was evaporated to remove excess thiophosgene. The activated complex in both aqueous and organic

Fig. 4.4. Synthetic scheme of 4-(p-aminobenzyl)-diethylenetriamine

96

layer were combined and used for coupling with protein dissolved in borate buffer. Some very interesting observations were made in these experiments. The complex in the aqueous layer was less reactive than the complex in the organic layer, presumably due to the fact that the activated complex was more soluble in organic layer than the unactivated complex. Extraction into chloro-form could be used for purifying the complex prior to conjugation with the protein.

Conjugation was studied using unactivated complex as well as trying to activate the complex formed from the nitro derivative. These were not ex-pected to show any binding with the gamma globulin. Both the blank studies showed insignifi cant conjugation with protein.

The 105Rh-ABDET-gamma globulin was purifi ed by sephadex gel permea-tion chromatography. One addition to this set of reaction was the estimation of the immunoreactivity of the 105Rh labeled gamma globulin by affi nity chromato-graphy. The author’s prior work on radioimmunoassay was useful for doing the affi nity chromatography studies. Sepharose coupled with anti-IgG was used for the studies. The 105Rh labeled antibody was bound to the sepharose column indicating that the there was no compromise on the immunoreactivity of the radiolabeled IgG molecules.

The results with this BFCA were more successful. The complexation yields were slightly better, however, the signifi cant advantage was that the conjugation effi ciency was very high (> 90%) and the immunoreactivity of IgG was retained post radiolabeling. The results of the studies indicated that coupling reaction after activation of the –NH2 group by using thiophosgene is a better method than the use of –COOH group and other conjugating reagents. Solubility of the activated complex in organic solvents was an advantage as it could be used for purifi cation of the activated complex. The experiments were successful as the results were suitable for extension to monoclonal antibody labeling. One im-portant lesson learned from these studies was that 105Rh complexes at high yields can be prepared by conducting the reaction at pH 9 in bicarbonate medium, a condition subsequently used by many researchers. The deprotonation of the amine nitrogens at pH 9 was expected contribute to the better complexation yields.

4.8.3. RADIOLABELING OF PROTEINS WITH 105Rh USING 1,7-BIS(2-HYDROXYBENZYL)-4-(p-AMINOBENZYL)- -DIETHYLENETRIAMINE (BHABDT) [8]The studies with the BFCA, ABDET was successful and the results were adapt-able for radiolabeling of antibodies with 105Rh. However, one of the objectives was to develop a BFCA which will not only radiolabel 105Rh, but can also be used for radiolabeling 99mTc to developed matched pair of radiolabeled mono-clonal antibodies to develop diagnostic and therapeutic radiopharmaceuti-cals.

97

In parallel to the 105Rh, the author was also involved in the development of novel 99mTc tracers. Amine phenol ligands were found to be good complexing ligands for 99mTc and capable of giving neutral and lipophilic complexes [13, 17-21]. Studies showed that these ligands also formed complexes with rhodium [14]. Hence, efforts were made to make a BFCA with the amine phenol backbone that will not only be suitable for 105Rh but also for 99mTc. A pentaden-tate amine phenol (BHABDT) ligand with benzyl amine group for conjugation was hence synthesized. The synthesis of this ligand was even more diffi cult and challenging as it involved a six-step synthetic procedure (Fig. 4.5) of which the fi rst three steps were same as that used for the synthesis of ABDET.

The 4-(p-nitrobenzyl)-diethylenetriamine derivative, the intermediate for the synthesis of ABDET was condensed with salicyladehyde to get the Schiff base which was subsequently reduced with sodium borohydride to get 1,7-bis(2-hy-droxybenzyl)-4-(p-nitrobenzyl)-diethylenetriamine. The purifi ed nitro deriva-tive was hydrogenated under Pd activated charcoal to get the fi nal product, BHABDT. The intermediates and fi nal product were characterized by elemental analysis and 1H- and 13C-NMR studies.

Fig. 4.5. Synthetic scheme for 1,7-bis(2-hydroxybenzyl)-4-(p-aminobenzyl)-diethylene-triamine

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The complexation and conjugation studies with this ligand gave results similar to the ligand ABDET. Overall conjugation yields, decay corrected, could be obtained up to 75%. 105Rh labeled IgG prepared by this method showed high immunoreactivity as estimated by affi nity chromatography.

99mTc labeling of the BFCA, BHABDT was also done and conjugated to gamma globulin with very high yields [22]. The studies were highly successful as the BFCA was capable of complexing a diagnostic and therapeutic radionu-clide could be synthesized and radiolabeling studies successfully demonstrated.

4.8.4. CYSTEINE AS A BFCA FOR RADIOLABELING PROTEINS WITH 105Rh [9]As the synthesis of the BFCAs was taking time, our attention turned to some of the naturally occurring ligands which have suitable coupling groups such as –NH2 or –COOH to be used as BFCAs to radiolabel 105Rh. The naturally oc-curring amino acid, cysteine was thought to be good for complexing with rhodium and conjugating to antibodies. 105Rh complexes were prepared with three equivalents of cysteine to metal at both acidic and basic pH and used for coupling with human serum albumin using carbodiimide as the coupling agent. A peptide linkage was expected to form in this case.

Complexation of cysteine ligand with 105Rh was done at both acidic as well as basic pH and conjugation with gamma globulin was attempted. The coupling yields were about 30% when the complex was prepared at acidic pH and no coupling was seen when the complex was prepared at basic pH in bicarbonate buffer, the most preferred condition for the preparation of 105Rh complexes as established in the previous studies. The poor coupling yield of the complex prepared at basic pH was thought to be due to the involvement of the –COOH group in coordination with the metal ion. The results were only partially suc-cessful indicating the need for well designed ligands for complexation and conjugation with proteins.

4.8.5. 105Rh-HEMATOPORPHYRIN [10]Porphyrins are avid chelators of metal ions and hence the possibility of using one of the naturally occurring porphyrin derivatives as a BFCA was tried. The studies gave some very interesting results which are discussed below.

8,13-bis(1-hydroxyethyl)-3,7,12,17-tetramethyl-21H,23H-porphine-2-18--dipropionic acid (Hematoporphyrin IX) (Fig. 4.6) is a commercially available porphyrin derivative with four nitrogen atoms in the ring and two carboxylic acid groups in the periphery. The four nitrogen atoms can be used for coordina-tion with metal ions and possibly one of the –COOH groups also might par-ticipate in the complexation reaction. As the two carboxylic acid groups were proximal one of it was guessed to be available for conjugation with an amino group in the proteins. Radiolabeling of porphyrins with 111In and 209Pd were al-ready reported prior to this work [23, 24].

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The radiolabeling of hematoprophyrin with 105Rh was achieved in high yields (~90%) at optimized conditions with 20% excess ligand at pH 9 bicar-bonate buffer at elevated temperature. The 105Rh labeled hematoprophyrin could be purifi ed by passing the reaction mixture over a silica gel chromatography. Elution with saline removed uncomplexed rhodium, whereas elution with 1:1 saline : acetone eluted the 105Rh-hematoporphyrin. Gentle heating of this fraction removed acetone.

The complex formed was highly stable. Coupling of the complex with proteins using carbodiimide as the conjugating agent was tried. One surprising observation revealed was that 105Rh-hematoporphyrin was bound to protein at very high yields almost quantitatively even without the use of any coupling agent. The percent activity incorporated with gamma globulin with and without the coupling agent was the same. We postulated that the protein binding mecha-nism of 105Rh-hematoporphyrin may be similar to the carrying of heme by hemoglobulin. In that case ion is coordinated to four nitrogens of the tetrapyr-role ring and one of the two remaining valencies is satisfi ed by nitrogen of histidine from the protein and the last valency by oxygen [25].

Radiolabeled hematoporphyrin derivatives are bound to tumor and it was likely that the 105Rh-hematoporphyrin or the 105Rh complex of other porphyrins also could have tumor affi nity. Hence, the possibility of using 105Rh labeled porphyrins as a tumor targeting agent useful for targeted therapy. The author was interested in pursuing this work, however, could not be continued as he returned to India at the end of the two-year postdoctoral associate ship. Studies in this direction are still worth pursuing.

Fig. 4.6. Hematoprophyrin IX

100

4.9. RADIOCHEMICAL PURITY EVALUATION OF 105Rh COMPLEXES BY MAGNESIUM OXIDE ADSORPTION [16]

Development of 105Rh based radiolabeled compounds started at Missouri under the guidance of Prof. Troutner. One of the problems faced was the non-avail-ability of appropriate quality control techniques to characterize the complexes. The starting material RhCl3 used is in the form [RhCl6–x·xH2O]x–3. Depending on the condition of the starting material and the intermediate steps, it can be in several species and this was confi rmed by paper electrophoresis in which the starting material was spread from the point of spotting to the anode upon paper electrophoresis. It was often essential to use buffer medium in order to do complexation and the buffers used were acetate, carbonate, citrate, borate etc. and all of which were weak chelating agents. On refl uxing the buffers with the starting material, the species of 105Rh changed. Upon complexation with the ligands, the Rh was expected to coordinate with the donating atoms in the ligand and displace the anions/monodentate ligands attached to the rhodium atom. Hence, the complexes formed were highly heterogeneous and their characterization at radiotracer level was a real challenge.

Like in all other tracer studies, the application of the conventional techniques such as paper chromatography, thin layer chromatography and paper electro-phoresis was tried to ascertain the complexation yields. However, there was problem in obtaining consistent results capable of accurately estimating com-plexation yields while using different ligands. Hence, several blank experiments were carried out to rule out the possibility of errors while interpreting the com-plexation results. Thin layer chromatography was found applicable to a certain extent.

It was a suggestion of Prof. Lo, who was working in the group as a visiting scientist from the National University of Taiwan to use magnesium oxide (MgO) adsorption as a quality control technique. Lo had personal experience of using MgO to adsorb a large number of metal ions. Hence, detailed studies were carried out to explore the possibility of using MgO adsorption as a quality control technique.

The technique developed by us for estimation of 105Rh complexes involved the addition of a speck (~50 mg) of MgO powder to the mixture containing rhodium complex solution and unreacted rhodium. It was observed that the rhodium which was not complexed to the ligands was adsorbed whereas rho-dium complexes remained in solution. The technique turned out to be practi-cally very useful and hence a validation study for this quality control technique was done and published.

The validation included making Rh complexes of several ligands, starting material, RhCl3 and intermediate prepared by refl uxing the starting material with carbonate buffer. TLC was used as a comparative a method, wherever

101

applicable. Based on the results obtained, the MgO adsorption technique became a standard QC technique for estimation of the complexation yields. Several scientists who subsequently worked with rhodium complexes at Missouri used this method as a quality control technique. The MgO adsorption technique was extended to the radiochemical processing of 105Rh [26].

Despite the success with several ligands to estimate the complexation yield, the method failed when it was used for characterization of 105Rh-hematopor-phyrin. It was observed that not only the uncomplexed Rh, 105Rh-hematopor-phyrin also got adsorbed in the MgO pellet. This failure prompted the author to search for other quality control methods. Solvent extraction technique using methyl isobutyl ketone (MIBK) was found to be suitable for the estimation of the complexation yield of 105Rh-hematoporphyrin. The complex had a high partition coeffi cient of about 20 in MIBK aqueous extraction.

4.10. PRODUCTION OF 105Rh USING NATURAL RUTHENIUM TARGET [27]

The author upon his return to Bhabha Atomic Research Centre started a program on the development of 105Rh radiopharmaceuticals. The author together with his PhD student developed the process chemistry for the preparation of 105Rh using natural ruthenium target. The process chemistry developed is different from the procedure reported by Grazman [5]. The natural ruthenium metal target used had a purity of 99.9% and was obtained from Light and Company, USA. No pre-purifi cation of the target was done as the radiochemical process developed was expected to purify the iridium impurities. The potential nuclear reactions while irradiating natural ruthenium target in the nuclear reactor is Table 4.1. Neutron capture reactions leading to the formation of different radionuclides when natural ruthenium is irradiated

Target % natural abundance

Cross-section (barns)

Radionuclide formed Half-life

96Ru 5.5 0.25 97Ru 69 d98Ru 1.86 8 99Ru Stable99Ru 12.7 4 100Ru Stable100Ru 12.6 6 101Ru Stable101Ru 17.0 5 102Ru Stable102Ru 31.6 1.3 103Ru 39.4 d104Ru 18.7 0.5 105Ru 4.04 h191Ir 37.3 400 192Ir 74.4 d193Ir 62.7 110 194Ir 19 h

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given in Table 4.1. The table also includes the iridium radionuclides that will be co-produced from the impurity in the target.

The standardized radiochemical processing included the separation of ru-thenium metal as well as ruthenium radionuclides (97Ru and 103Ru), stable 105Pd formed from the decay of 105Rh and stable 106Pd formed by the neutron capture of 105Rh followed by β– decay (16 000 barns). Process also included removal of iridium impurities,192Ir and 194Ir. The radiochemical processing developed also removes all metal contaminants and get 105Rh suitable for radiolabeling studies.

4.10.1. IRRADIATIONAbout 100 mg of natural ruthenium metal target (99.9% pure) sealed in an aluminium irradiation can was irradiated at a neutron fl ux of ~3x1013 n cm–2 s–1 in the Dhruva Reactor of the Bhabha Atomic Research Centre. The irradiation was done for 5-7 days depending on the operational cycle of the reactor. At the end of bombardment the target was cooled for 24 h in order to convert major part of 105Ru to 105Rh.

4.10.2. RADIOCHEMICAL PROCESSINGThe irradiated target was dissolved in KOH and KIO4 in double distilled water followed by heating to convert the ruthenium to RuO4

2–. This species is then converted to RuO4 by acidifi cation with 1 M sulfuric acid and extracted into carbon tetrachloride. The organic phase containing RuO4 was removed and discarded as radioactive waste. Multiple extraction steps were needed for the near complete removal of ruthenium radionuclides. The aqueous phase contain-ing 105Rh, traces of radiorutheniums and radioiridiums was further processed to get 105Rh.Removal of iridium impurities

The aqueous phase was evaporated to dryness, cooled and taken in 7 M hydrochloric acid followed by the addition of H2O2 to oxidize iridium impurities to Ir(IV). The Ir(IV) formed was removed by multiple extraction with tributyl phosphate (TBP). After removal of iridium impurities, the aqueous phase was evaporated to near dryness. The residue which is rich in KCl salt was leached with concentrated hydrochloric acid, followed by cooling to precipitate KCl which was removed by centrifugation. Purifi cation of 105Rh

The supernatant which contained 105Rh was purifi ed by cation exchange resin chromatography. The excess acid and salts were removed by washing the column with water and the 105Rh was eluted with 1 M HCl. The effl uent and washings were dried and reconstituted in water to get 105Rh for radiolabeling studies. The fi nal radiochemical form of 105Rh was expected to be in RhCl3·3H2O or as RhCl6-x·xH2O.

By following the procedure developed above, about 10-20 mCi of 105Rh per batch could be prepared consistently. The radionuclidic purity of 105Rh produced

103

as determined by gamma spectroscopy was high and suitable for radiolabeling studies. The 192Ir was the only radionuclidic impurity and was about 1.2-1.5 μCi in a batch of 15 mCi of 105Rh at the end of radiochemical processing. No further attempts were made to reduce the iridium impurities.

The 105Rh prepared by the above method was not used for any complexa-tion studies as the author’s interest shifted to other more easily producible radionuclides.

4.11. CURRENT STATUS OF 105Rh AS A THERAPEUTIC RADIONUCLIDE

There were some continued effort from the University of Missouri on the de-velopment of 105Rh complexes and a few more publications in 105Rh appeared in the literature post authors return from Missouri [28-34]. The author on his return to the Bhabha Atomic Research Centre started a program on the development of 105Rh radiopharmaceuticals, but abandoned it in favor of other radionuclides that can be more easily produced, as described in the subsequent chapters. There were other isolated reports such as the development of 105Rh-EDTMP as a potential bone pain palliation agent [35]. Other than that there is practically no interest shown in this radionuclide for the development of therapeutic radio-pharmaceuticals.

4.12. LESSONS LEARNED FROM 105Rh RADIOPHARMACEUTICALS DEVELOPMENT

Rhodium-105 was identifi ed by Prof. Troutner as a potential therapeutic radio-nuclide for antibody labeling due to its favorable radionuclidic characteristics. Iodine-131 was the preferred isotope for antibody labeling. However, the long half-life (T1/2 = 8.02 d) was thought to be too high and not matching with the biokinetics of the monoclonal antibody. High abundant and high energy gamma rays in 131I decay is also a major disadvantage. The patient undergoing therapy will need long hospitalization. Hence, the interest was to fi nd an isotope with 1-3–day half-life and having lower percentage of gamma emission and can be produced in NCA form.

The main reason for the selection of 105Rh was that the 35 h half-life was compatible with the biological half-life of the monoclonal antibody. The 35 h half-life was also comfortable for the radiochemical processing of the isotope and formulation of the radiopharmaceutical. Being produced by (n,γ) followed by β– decay the radionuclide offered very high specifi c activity needed for radiolabeling a macromolecule like monoclonal antibody (MW 150 000 D).

104

The scientifi c research done towards the above direction was highly suc-cessful. The radionuclide with high specifi c activity (> 700 mCi/μmol) could be produced. Suitable bifunctional chelating agents could be developed and complexed with 105Rh, the complexation yields were higher and fi nally the complexes could be coupled to human gamma globulin with high effi ciency without compromise of the immunoreactivity. Based on the data collected, 105Rh looked to be potentially useful therapeutic radionuclide. The prestige of the Missouri radiopharmaceuticals group was also very high thanks to the introduc-tion of the radiopharmaceuticals, 99mTc-HMPAO (Ceretec®) and 153Sm-EDTMP (Quadramet®) into the market around the same time with patent rights to Uni-versity of Missouri-Columbia. Several radiopharmaceuticals companies were interested in acquiring the patent rights for the new developments on 105Rh and fi nally the University granted the rights of 105Rh radiopharmaceuticals to the DOW Chemical Company which eventually took two US patents with Prof. Troutner, John, the organic chemist who started the synthesis work and the author as the co-inventors [36, 37]. In addition, the author and his colleagues were successful in getting several publications in high impact journals. Despite the high promise showed by 105Rh, it failed to live up to the expectation. No 105Rh based therapeutic agents eventually entered a human clinical trial. 105Rh still continues as a ‘potential’ radionuclide for radiotherapy and this status is not expected to change.

The reasons for the failure of 105Rh are worth analyzing. As per the author’s analysis, the most important cause of the failure of it to emerge as a useful radionuclide for therapy is the diffi culty in its production. The cross-section for neutron activation of 104Ru is only 0.5 barns, which means that at saturation irradiation, the amount of activity that can be produced at end of bombardment is only about ~7.8 Ci when one g of enriched target is irradiated at a neutron fl ux of 1014 n cm–2 s–1. At least half of it will be lost due to decay during radio-chemical processing and radiopharmaceuticals production. If radionuclide therapy has to become successful, large quantities of the radionuclide is need-ed to treat the large number of patients who might benefi t from the new modal-ity. A large scale successful therapy cannot be started with 105Rh due to the diffi cult production logistics. The radiochemical processing is highly compli-cated needing multiple steps to remove the ruthenium target from 105Rh. The incomplete removal of ruthenium will reduce the specifi c activity of 105Rh as ruthenium like rhodium will chelate with most of the ligands. Hence, the spe-cifi c activity needs to be redefi ned as activity per unit mass of total metal rather than activity per unit mass of rhodium. The production also needs highly enriched target otherwise several ruthenium impurities are co-produced and the radiochemical processing has to be done with very high amounts of radioactivity. Hence, production with natural target will result in large quantities of radioactive waste. The enriched target is expensive and the ‘per barn cost index’ as defi ned in chapter 3 is very high for 104Ru as the cross-section of the

105

nuclear reaction is only 0.5 barns. The most important lesson learned from the large experience gained with 105Rh is that the feasibility of production of the radionuclide should be the most important consideration while selecting a radionuclide for development of targeted radiopharmaceuticals.

Nevertheless, 105Rh has a special place in author’s scientifi c career as it was his entry to the fascinating science of therapeutic radiopharmaceuticals. The guidance received from Prof. Troutner helped the author to achieve scientifi c maturity. Identifying good scientifi c problems, planning experiments, interpret-ing data, writing papers and replying to the reviewer’s comments etc. were refi ned during the two-year stay at the University of Missouri. Prof. Troutner’s advice for new scientifi c ideas was to scan the journals which are outside ones field of specialization; the journals of own fi eld might not give much new ideas. Following this advice helped the author to design several new ligands by taking cue from papers appearing in inorganic chemistry and organometallic chemistry journals. Hematoporphyrin IX was identifi ed as a BFCA by a few days of search in the Aldrich catalogue.

The experience gained by the author in working with 105Rh was highly useful to start the therapeutic radiopharmaceuticals program at the Bhabha Atomic Research Centre upon his return from the University of Missouri in 1989 and the small research group started by the author has grown over the years and is one of the highly accomplished groups in the fi eld of therapeutic radiopharmaceuticals.

4.13. CONCLUSIONS

This chapter described the work done by the author on the development of 105Rh based radiopharmaceuticals. Production of the radionuclide, synthesis of new bifunctional chelating agents, complexation studies at macroscopic and micro-scopic levels, conjugation with protein and purifi cation of radiolabeled proteins as well as assessment of the immunoreactivty of the proteins were part of the work done. The work described in this chapter is the basis of eleven publica-tions co-authored by the author in addition to two US patents. Based on the author’s experience, the conclusion is that 105Rh do not have the possibility to be developed into successful therapeutic products.

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106

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