Radiochemical and biological evaluation of novel Sm-153/Ho-166-amino acid-chitosan complexes

Radiochemical and biological evaluation of novel 153 Sm/ 166 Ho-amino acid–chitosan complexes F. Marques, a L. Gano, a M. K. S. Batista, b,c C. A. R. Gomes, c P. Gomes, b and I. Santos a 153 Sm/ 166 Ho-chitosan complexes have been considered promising agents for internal radiation therapy. By direct administration, complexes solution converts into a gel, at physiological pH, allowing its retention for a long time. Herein, we report on the synthesis of 153 Sm/ 166 Ho complexes with the novel amino acid–chitosan polymers, N-(c-propanoylvalin)–chitosan (CHICO-val) and N-(c-propanoyl-aspartic acid)–chitosan (CHICO-asp). The main goal of this study was to obtain data on the radiochemical and biological behaviour of these complexes and information regarding their therapeutic potential when compared to 153 Sm/ 166 Ho-chitosan. Radiolabelling yield of 153 Sm/ 166 Ho-amino acid–chitosan complexes was dependent on polymer concentration but less dependent on pH. Radiochemical stability was shown to be higher for amino acid–chitosans than for chitosan, with 153 Sm/ 166 Ho-CHICO-val being stable up to 3 h, while 153 Sm/ 166 Ho-CHICO-asp is stable up to 24 h. In the presence of ascorbic acid radiochemical stability of 153 Sm/ 166 Ho-CHICO-val and 153 Sm/ 166 Ho-CHICO was improved, decreasing for 153 Sm/ 166 Ho-CHICO-asp. In vivo behaviour of 153 Sm complexes was studied in mice. The radioactive amino acid–chitosans can be directly injected into blood stream without significant retention on injection site, being trapped by liver. Biodistribution studies suggest that the radioactive amino acid–chitosans, due to its water solubility and stability may be considered potential candidates to be further explored for liver targeted nuclear therapy. Keywords: radiolanthanides; chitosan derivatives; amino acid; polymers; internal radiation therapy Introduction Internal radiation therapy (IRT) with unsealed b-emitting radionuclides has emerged for the treatment of cancer as an alternative to external radiotherapy. b-emitters have advantages for the treatment of tumours owing to their physical characteristics, in particular their short soft tissue penetration range, delivering high radiation dose to tumour without adverse radiation effects on the surrounding normal tissues. 1 Among the b-emitters, 166 Ho (t 1/2 26.8 h, b max 1.85 MeV (51%), g0.081 MeV (7.5%) and 153 Sm (t 1/2 46.8 h, b max 0.81 MeV (21%), g0.103 MeV (38%) appear to be good candidates for IRT. 2 Chitosan, a polymer of 2-deoxy-2-amino-D-glucose obtained by N-deacetylation of chitin has unique properties for a broad variety of applications. 3–5 Its chemical structure is characterized by the degree of acetylation (DA), which also determines its ability to chelate metal ions. 3 Chemical modifications to enhance chelating properties, selectivity and metal ion capacity of chitosan have been proposed and various chitosan derivatives have been designed to improve these features. 6 Keeping this in mind, N-(2-carboxyethyl)–chitosans have been prepared and their metal complexation properties and potential applications have been studied. 7–9 Owing to its capacity to complex b-emitter metal ions through its amino groups, chitosan appears to be a promising therapeutic alternative for local treatment of tumours. 10 The main features of these complexes are their solubility under acidic conditions and the easy conversion to gel at the local of administration (physiological pH), with the corresponding retention of the radionuclide. Based on this principle, 153 Sm/ 166 Ho-chitosan complexes have been prepared with relatively high radiochemical purity (490%) and their biological behaviour has been considered promising for local therapy of prostate and skin cancer, 11–13 brain glioma, 14 renal cysts 15 and rheumatoid arthritis. 16–17 Following these results, 166 Ho-chitosan (DW-166HC) has been approved in Korea as a radiopharmaceutical for the treatment of small hepatocellular carcinoma (HCC). 18,19 However, DW-166HC is sensitive to degradation induced by radiation and its use immediately after synthesis is highly required. We have been working on the chemical modification of chitosan amino groups in order to improve both the polymer water solubility over a broader pH range and its chelating 79 Research Article Received 24 January 2008, Revised 28 October 2008, Accepted 1 November 2008 Published online 16 December 2008 in Wiley Interscience (www.interscience.wiley.com) DOI: 10.1002/jlcr.1571 J. Label Compd. Radiopharm 2009, 52 79–83 Copyright r2008 John Wiley & Sons, Ltd. a Instituto Tecnolo ´gico e Nuclear, Estrada Nacional 10, 2686-953 Sacave ´m, Portugal b CIQUP, Departamento de Quı ´ mica, Faculdade de Cie ˆncias, Universidade do Porto, Rua do Campo Alegre 687, P-4169-007 Porto, Portugal c LAQUIPAI, Departamento de Quı ´ mica, Faculdade de Cie ˆncias, Universidade do Porto, Rua do Campo Alegre 687, P-4169-007 Porto, Portugal *Correspondence to: F. Marques, Instituto Tecnolo ´gico e Nuclear, Estrada Nacional 10, 2686-953 Sacave ´m, Portugal. E-mail: [email protected] properties, for potential biomedical applications. 7–9 Recently, we have prepared two novel amino acid–chitosan polymers, N-(g-propanoyl-valin)–chitosan and N-(g-propanoyl-aspartic acid)–chitosan, from low molecular weight chitosan and N a -(3-bromopropanoyl)–valine or N a -(3-bromopropanoyl)–aspartic acid, respectively. 20 Structures of chitosan (CHICO, 1), N-(g-propanoyl-valin)–chitosan (CHICO-val, 2a)andN-(g-propanoylaspartic acid)–chitosan (CHICO-asp, 2b) are presented in Figure 1. In view of the interesting properties of our novel amino acid–chitosan derivatives, 21 we decided to explore the coordination capability of these modified chitosan towards 153 Sm/ 166 Ho, two therapeutically relevant radionuclides. Herein, we describe the synthesis of 153 Sm/ 166 Ho-N-(g-propanoylvalin)–chitosan and 153 Sm/ 166 Ho-N-(g-propanoyl-aspartic acid)– chitosan as well as our data on their radiochemical and biological behaviour as compared with the respective chitosan analogous. Results and discussion Synthesis and stability of the radiolabelled complexes Irradiation of 152 Sm/Ho-nitrates yielded 140 MBq/mg ( 153 Sm) and 190 MBq/mg ( 166 Ho) with high radionuclidic purity as confirmed by the typical g-ray spectrum. 22 The complexation reaction conditions were optimized in order to obtain 153 Sm/ 166 Ho-complexes with high radiochemical purity. The labelling yield of 153 Sm/ 166 Ho-CHICO–amino acid complexes was not highly dependent on the pH, but depends on the polymer concentration. Complexes were obtained with yields higher than 98% at pH 3–7 with 1% (w/v) polymer solution, while the complexation yield drastically decreases at lower concentration (Table 1). In contrast, the labelling yield of 153 Sm/ 166 Ho-CHICO complexes was highly dependent on both parameters, reaching only 98% at pHC3 and using a 1% CHICO solution. 11,16 The stability of the CHICO–amino acid complexes was followed by ITLC, using the chromatographic systems indicated in the experimental section. Both complexes have in vitro radiochemical behaviour different than that of their CHICO analogous. While the latter complex remains in the origin, in accordance with its colloidal nature, the CHICO-asp and CHICOval complexes migrate with R f values of approximately 0.4 and 0.6, respectively (Table 2). Nonetheless, in both cases the presence of free radiolanthanide was never detected. After formation, 153 Sm/ 166 Ho-CHICO-asp/CHICO-val complexes showed relatively high radiochemical purity within a sufficient time period for eventual clinical applications ( 153 Sm/ 166 Ho-CHICO-val is stable up to 3 h and 153 Sm/ 166 HoCHICO-asp is stable up to 24 h), while 153 Sm/ 166 Ho-CHICO appeared to be more sensitive and degrading with storing. 23 As an example, we show in Figure 2 how fast 153 Sm-CHICO degrades leading to new radiochemical species with time. As shown in Figure 2, 153 Sm-CHICO leads to a new species with R f E0.22, 30 min after formation, and with time new species are formed progressively with higher R f values. The amino acid derivatives are stable for a longer period of time and the decomposition corresponds to the formation of a new radiochemical species along with the original ones. The different behaviour found for our complexes may be due to the presence of one or two carboxylate groups in the chitosan backbone, which may contribute for structural stabilization of the radioactive complexes. Considering that ascorbic acid (AA), an antioxidant radical scavenger, is often used as a food additive or as a stabilizer in radiopharmaceutical compositions, 24,25 we have decided to evaluate the effect of this compound on the radiochemical stability of 153 Sm/ 166 Ho-chitosan derivatives and at the same time on 153 Sm/ 166 Ho-chitosan. As can be seen in Figure 2, 80 Table 1. Labelling yield vs polymer concentration at the pH range 3–7 Polymer Conc. (% w/v) Labelling yield (%) CHICO-val 0.5 0 1100 CHICO-asp 0.5 0 1100 Table 2. Radiochemical behaviour of 153 Sm/ 166 Ho-amino acid–CHICO complexes R f 153 Sm/ 166 Ho-CHICO 153 Sm/ 166 Ho-CHICO-val 153 Sm/ 166 Ho-CHICO-asp 153 Sm/ 166 Ho(NO 3 ) 3 00.6 0.4 0.9–1.0 Figure 1. Molecular structures of chitosan (1), N-(g-propanoyl-valin)–chitosan (2a) and N-(g-propanoyl-aspartic acid)–chitosan (2b). F. Marques et al. www.jlcr.org Copyright r2008 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2009, 52 79–83 the radical scavenger has slightly improved the stability of 153 Sm-CHICO, in agreement with the results previously described by Zoldners et al. 24 The stability of this compound seems to be relatively dependent on the nature of the solution components. In acetic acid solutions, for example, Zoldners et al. described that chitosan degrades with the formation of more water-soluble fragments along the time, as a result of oxidative and hydrolytic splitting of the polymer chains. Moreover, it has also been referred that 166 Ho-chitosan is radiation sensitive, heading for decreasing in the complex viscosity. 23 The increase stability of 153 Sm-CHICO in the presence of AA may be due to the effect of AA as a scavenger of possible radicalar species and/ or to the additional coordination capability of AA. 23,24 Regarding the 153 Sm/ 166 Ho-CHICO–amino acid complexes, the addition of AA (2% AA solution) has significantly affected its radiochemical behaviour. For compound 153 Sm/ 166 Ho-CHICO-val its stability enhanced significantly up to 24 h. In contrast, the addition of AA to 153 Sm/ 166 Ho-CHICO-asp resulted in a high degree of degradation of the initial complexes, with formation of several species. Based on the thermo-stability and structural studies previously performed for CHICO and for CHICO-amino acid polymers, by differential scanning calorimetry and scanning electron microscopy, the CHICO-asp has a looser macromolecular structure than its CHICO-val counterpart and a highest thermo-sensitivity and water-holding capacity, which implies that its supramolecular structure is weaker than that of its counterpart (Figure 3). 20,21 Thus, while CHICO-val has a compact and continuous film-like structure, CHICO-asp has a highly porous morphology, probably due to electrostatic repulsions between its higher number of carboxylate groups. The porous morphology of CHICO-asp may allow AA, a wellknown chelating agent, 26,27 to efficiently penetrate into the polymeric matrix and compete for metal complexation with the polymer chelating groups. In CHICO-val the structure is more compact and stable so another oxygen donor co-ligand may lead to a mixed-ligand complex, which will be anchored on the donor groups of the amino acids as well as on the AA, leading to the stabilization of the radiometal by a synergic effect. 24,26 Biodistribution of the 153 Sm-labelled complexes in mice Preliminary biodistribution studies were only performed for 153 Sm-complexes. For comparison, 153 Sm-CHICO was also studied in the same animal model. As expected, the 153 Sm-CHICO was almost completely retained at the site of administration and no radioactivity excretion was found up to 24 h. On the contrary, the CHICO-amino acid complexes could be injected directly into the blood stream by the tail vein. For both complexes a high liver uptake was found, despite i.v. injection and the excretion was very slow or negligible. However, the two complexes have shown slightly different biodistribution patterns (Figure 4). While 153 Sm-CHICO-val was associated to high hepatic radioactivity retention that enhanced over time, the 153 Sm-CHICO-asp was slowly cleared from the liver into the intestine, which is a disadvantage if one wants to treat liver cancer. Additionally, the uptake by the liver should be speed up in order to reduce the radiation dose to the blood cells. The significant retention of activity in the liver found for 153 Sm-CHICO-val can probably be due to the in vivo formation of radiochemical species of colloidal/polymeric nature. Experimental Chemicals: Enriched samarium oxide (Sm 2 O 3 , 98.4% as 152 Sm) was purchased from Campro Scientific and natural Ho 2 O 3 (99.9%) from Strem. Chitosan (abbreviated CHICO) (MW150 000; DA = 0.10) was supplied by Sigma-Aldrich. 81 Figure 2. Radioactive distribution on the ITLC-SA strips of 153 Sm-CHICO complex obtained in the absence and presence of ascorbic acid over time. Figure 3. SEM micrographs (100 magnification) of CHICO (A), CHICO-val (B) and CHICO-asp (C). F. Marques et al. J. Label Compd. Radiopharm 2009, 52 79–83 Copyright r2008 John Wiley & Sons, Ltd. www.jlcr.org Instant thin layer chromatography (ITLC) strips were supplied by Polygram, Macherey-Nagel. N-(g-propanoyl-valin)–chitosan (abbreviated CHICO-val) and N-(g-propanoyl-aspartic acid)–chitosan (abbreviated CHICO-asp) were synthesized and purified according to methods previously reported. 21 All materials were reagent grade unless otherwise specified. Synthesis of the peptide chitosan polymers: The synthesis of the two novel CHICO-based polymers was reported in detail elsewhere. 21 Briefly, N a -(3-bromopropanoyl)-valine and N a -(3bromopropanoyl)-aspartic acid were prepared by reacting the relevant L-amino acid tert-butyl esters with 3-bromopropanoic acid in the presence of dicyclohexylcarbodiimide (DCCI) as coupling reagent. After being isolated by column chromatography on silica, the tert-butyl esters were successfully identified by 1 H and 13 C NMR. These esters were then cleaved by acidolysis with neat TFA, with quantitative formation of the corresponding free carboxylic acids N a -(3-bromopropanoyl)-valine and N a -(3bromopropanoyl)-aspartic acid, whose structures were also confirmed by NMR. The carboxylic acids obtained as above described were covalently attached to chitosan as follows: chitosan was dissolved in water containing 4 M equivalents of N a -(3-bromopropanoyl)-valine or N a -(3-bromopropanoyl)-aspartic acid. The reactions were allowed to proceed at 601C under magnetic stirring and with daily additions of NaHCO 3 to keep pH within the 6–8 range. The peptide–chitosans were purified by dialysis against deionized water for 5–7 days, and then freeze-dried. Production of 153 Sm and 166 Ho: 153 Sm and 166 Ho were produced by 152 Sm(n,g) 153 Sm and 165 Ho(n,g) 166 Ho reaction, respectively, in the ITN Nuclear Research Portuguese Reactor (1 MW). Irradiation was performed using nitrate targets (Sm/Ho, 10 mg) prepared from the correspondent isotopically enriched 152 Sm 2 O 3 or natural Ho 2 O 3 under a thermal neutron flux of 1.5 10 13 n/cm 2 s and epithermal neutron flux of 3.1 10 11 n/cm 2 s for 1–2 h. Following irradiation, the targets were reconstituted in H 2 O to yield 1% (w/v) 153 Sm/ 166 Ho-nitrate solution for complex preparation. The radionuclide purity was assessed by gspectrometry with a Ge(Li)-detector (Canberra) and activities were measured by a radioisotope calibrator (Aloka, Curiemeter IGC-3, Tokyo, Japan). Radiolabelling procedure: The preparation of 153 Sm/ 166 Hocomplexes with a high labelling yield (498%) was optimized. Typically, 40 mLof1% 153 Sm(NO 3 ) 3 solution or 20 mLof1% 166 Ho(NO 3 ) 3 solution was added to 700 mL of CHICO or CHICObased polymer solution in 1% (v/v) acetic acid, followed by thorough stirring for 20 min at room temperature. The radiolabelling pH of the resulting solutions was 3.0 for 153 Sm/ 166 Ho-CHICO complex and 7.0 (by adding an appropriate volume of 1 N NaOH solution) for 153 Sm/ 166 Ho-peptide-chitosan based complexes. The labelling efficiency was accomplished by ITLC, using ITLC-SA strips developed with MeOH:H 2 O:acetic acid (50:50:0.5) as the mobile phase. In this system, the radiochemical species migrate with the R f presented in Table 2. Radioactive distribution on the ITLC-SA strips was detected by using a Berthold LB 505 detector coupled to a radiochromatogram scanner. The stability of the 153 Sm/ 166 Ho-CHICO or 153 Sm/ 166 HoCHICO-based complexes was assessed by measuring the radiochemical purity by ITLC over time, up to 24 h. The effect of the radical scavenger AA (2% AA solution) on the stability of the complexes was also studied. In vivo studies: Biodistribution studies were carried out using normal CD-1 mice from Charles River, Spain, according to previously reported procedures. 22 Animals were intravenously injected, via the tail vein, or intraperitoneally injected with 100 ml (3.7 MBq) of the radiolanthanide complexes. After 1 and 24 h animals were killed by cervical dislocation, the main organs were dissected and the radioactivity counted in a gcounter (Berthold LB 2111, Germany). Biodistribution results were expressed as percent of injected dose per total organ (% I.D./organ). Concluding remarks Concerning the best strategy to deal with inoperable HCC, there is a general lack of consensus. Classical approaches include trans-arterial chemo-embolization (TACE) or IRT through administration of radionuclide–lipiodol complexes that have the advantage of being highly retained by hepatic tumours. 28–35 However, TACE is not suitable for advanced disease or patients with an obstructed (thrombotic) portal vein, whereas radiolabelled lipiodol (e.g. 131 I-lipiodol) is associated with pain on injection and occasional induction of severe pneumopathies. 30,31 Radioimunotherapy can be a way to circumvent the above problems. But treatments with the monoclonal 131 I-Hepama-1, though associated with low toxicity, usually requires hepatic artery ligation, whereas polyclonal 131 I rabbit antiferritin IgG has been related to thromobocytopenia. 31 Alternatively, radiolabelled glass or resin microspheres have been explored as potentially safer IRT agents, but their clinical efficacy has not been proved so far. Successful treatment of liver tumours in an animal model was achieved with 166 Ho-labelled poly-(L-lactic acid) microspheres, which opens new possibilities towards the use of microspheres built from biocompatible materials. 31 In this context, radiolabelled chitosan-based polymers appear as a most attractive and cost-effective choice, as chitosan is cheap, biodegradable, bioadhesive and biocompatible, and has low toxicity and low immunogenicity. Indeed, the approved 166 Ho-chitosan complex (DW-166HC), was found to be a highly effective and safe new radiopharmaceutical agent for IRT against liver cancer. 19 The CHICO-amino acid polymers, described herein, were found to be suitable ligands for the preparation of stable 153 Sm/ 166 Ho-complexes with high labelling yields (498%) and high radiochemical purity over the time. As compared with unmodified chitosan, the amino 82 Figure 4. Whole body excretion and biodistribution in the most relevant organs of 153 Sm-CHICO complexes at 1 and 24 h after administration in mice. F. Marques et al. www.jlcr.org Copyright r2008 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2009, 52 79–83 acid derivatives present several advantages for eventual clinical applications: water solubility over a wide pH range and quantitative formation of non-colloidal radiolanthanide complexes. Despite their higher fluidity, the radiolabelled CHICO-amino acid complexes have higher stability than their CHICO counterparts and may be potentially interesting to be explored for the treatment of HCC and liver metastasis, when treating the whole liver makes it more suitable as compared with resection or liver transplantation. 36 In addition, in a clustered metastasis or primary tumour, the cross-fire effect of b  energy particles might also be beneficial. 36,37 Preliminary biological results have shown that 153 Sm-CHICO-amino acid complexes present high liver uptake specially CHICO-val. This targeting of the liver may also be exploited for liver-specific targeting and with the appropriate radionuclide, for imaging. 38 Whether the biodistribution profile of this radioactive amino acid–chitosan could be improved in order to increase the blood clearance and then minimize the radiation dose to non-target organs the chance to administer it via i.v. injection may be a significant advantage relatively to the compounds advocated for the same purpose, which require local administration. The improved radiochemical and biological characteristics of 153 Sm/ 166 Ho-CHICO–amino acid complexes highlight the interest to further explore the chemical modification of chitosan in order to introduce functional groups to modulate its pharmacokinetics, coordination capability towards different radionuclides and specificity to design specific radiotracers. 39 Acknowledgement The authors wish to thank the ITN Portuguese Research Reactor for the production of 153 Sm and 166 Ho. P. 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