J Radioanal Nucl Chem (2012) 292:395–399 DOI 10.1007/s10967-011-1417-z
Technetium(I) tricarbonyl complexes: potential precursors of the radiopharmaceuticals. Part II: phenethylbiguanide (phenformin) L. Fuks • E. Gniazdowska • P. Koz´min´ski I. Herdzik-Koniecko
•
Received: 26 August 2011 / Published online: 13 September 2011 Akade´miai Kiado´, Budapest, Hungary 2011
Abstract The radiosynthesis of [99mTc]-phenformin ([99mTc]-1-phenethylbiguanide) complex and its suitability as precursor of the radiopharmaceuticals for the tumor imaging was assessed. Radiochemical purity of the [99mTc]complex was determined using radio-TLC and was studied by the HPLC with radiochemical detection, while its stability in challenge experiments. The results show, that due to the sufficient stability and lipophilicity of the complex, it fulfills our expectations for promising radiopharmaceutical precursor not only for the diagnostic agent, but also for the drug suitable for the oncological Auger electron therapy. Keywords Tricarbonyl technetium(I) Phenethyl biguanide Auger electron therapy Radiopharmaceuticals
Introduction Up-today technetium-99m was used predominantly in diagnostic nuclear medicine because, in addition to an appropriate half-life of ca. 6 h, it was supposed to be a pure gamma ray emitter (Ec = 140.5 keV). However, transition from its metastable to the ground state includes process of the internal conversion, which causes the release of an average of four Auger or Coster–Kronig electrons per decay [1]. In the past, toxic effects of low-energy electron emitter, and subsequently possibility to be applied in the therapy of tumors, had frequently been assumed to depend on the covalent binding of the Auger electron-emitting radionuclide to nuclear DNA [2]. Nevertheless, several L. Fuks (&) E. Gniazdowska P. Koz´min´ski I. Herdzik-Koniecko Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland e-mail:
[email protected]
investigators have shown that various agents (e.g., steroids, growth factors, and DNA intercalators) radiolabeled with such isotopes are also highly toxic to mammalian cells (exponential decrease in survival) [3, 4]. Recently published results of the preliminary studies on the possibility of applying [99mTc]-complexes as the Auger electron therapy (AET) radiopharmaceuticals have confirmed this hypothesis [5–9]. All these studies assume that the radionuclide should be conjugated to carriers that specifically facilitate internalization the drug. So these carriers should allow it reach the nuclear DNA and stop its replication. It has been recently demonstrated that [99mTc]-induces in vitro double-strand breaks in circular dsDNA when carried in its direct vicinity by DNA binding moieties, such as intercalators [10]. [99mTc]-labeled imaging agents used in nuclear medicine are traditionally based on the N,S- or N,O-ligand systems. To our knowledge, up-to-now N,N-ligands have not been described by others than Neves et al. [11–16]. Above mentioned studies concerned the oxotechnetium(V) and oxorhenium(V) complexes. Biguanides and their N-substituted derivatives have attracted considerable attention for treatment e.g. of the recognized hyperglycemy [17–19]. In particular, metformin (1,1-dimethylbiguanide hydrochloride) and phenformin (N0 -beta-phenethylformamidinyliminourea; 1-phenethylbiguanide hydrochloride) are known as the antidiabetic medicaments and have been used for over 30 years. Biguanides have been also proposed as antimalarial drugs [20], and more recently for therapeutic treatment of pain, anxiety or memory disorders [21]. As complexing agents, biguanides containing at least five nitrogen donor atoms are mainly the bidentate chelators. Nevertheless, in some cases they may act as the monodentate ligands. They are considered strong r- and p-donating ligands which form stable complexes with transition metal
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HCl
NH
NH NH
NH
NH2
Fig. 1 Molecule of the phenformin hydrochloride— potential DNA intercalating agent
ions by overlapping vacant d-orbitals of the metal with the filled p orbitals of the ligand [22, 23]. Transition metal complexes of recognized therapeutical properties are these of copper(II), nickel(II), cobalt(II) or platinum(II) and -(IV) [24–29]. It has been also shown that similarly to the ligands numerous complexes, beside the anticancer properties, exhibit also other therapeutic properties: antibacterial, antimalarial and/or anti-inflammatory [30]. Surprisingly, except studies of Neves et al. [11–16], radiolabeled biguanides have not been proposed neither as diagnostic agents nor as drugs applied in the nuclear medicine. Presented work is a part of a more general program carried out to find novel inorganic and/or organometallic therapeutic agents, which can be applied in the AET. In this paper we describe synthesis and characteristics of the monovalent tricarbonyl [99mTc]-technetium complex with the potential in binding the nuclear DNA. Namely, the complex of phenformin (phenethylbiguanide hydrochloride—Fig. 1) was of our interest. Molecule of the ligand contains the flat benzene ring, so it seems to be capable of intercalating the DNA major groove. Simultaneously carried out studies performed in our group on the platinum(II)-phenformin complex have shown, that the latter indeed intercalates dsDNA and exhibits significant cytotoxic properties. Results will be published soon.
Materials and methods Phenformin was purchased from Sigma and used as obtained. Solvents for synthesis, crystallization, and TLC
(p.a., POCh, Gliwice, Poland) were also used without further purification. Water was bi-distilled from quartz. [99mTc]-radionuclide was eluted from the commercially available 99Mo/99mTc generator (IAE Radioisotope Centre POLATOM, Swierk-Otwock, Poland) as a Na[99mTc]O4 in 0.9% saline (ca. 1 GBq/mL). Synthesis of the [99mTc(I)]-tricarbonyl complex Two step synthesis was accomplished according to procedure proposed by Alberto [31] improved by Zhang [32] (Scheme 1). A kit containing 5.5 mg NaBH4, 4 mg Na2CO3 and 10 mg Na–K tartrate was purged with CO gas prior to addition of the generator eluate: Na[99mTc]O4. The solution was heated to 85 C and kept hot for 30 min. Yield of the fac{[99mTc](CO)3(H2O)3}? intermediate complex (determined by RP-HPLC) was higher than 95%. Then, 1 mL of an aqueous solution containing the intermediate complex (*1 GBq/mL; pH * 9) was set to react with 0.1 mL of the phenformin solution (10-2 M) for 30 min at 65 C. The reaction mixture was purified by isolating the appropriate radioactive peak (Rt = 21.6 min) from the column (Fig. 2). The reaction yield was ca. 95% as measured by the RPHPLC. Radiochemical purity of the [99mTc]-complex was evidenced by two radio thin layer chromatography (RTLC) analyses [33]. The complex remains at the origin, when the RTLC chromatogram is developed in acetone and migrates with the eluent front in the aqueous saline solution. Chromatographic studies
Scheme 1 Tricarbonyl technetium(I) complexed by phenformin—potential precursor of the radiopharmaceuticals
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TM
RP-HPLC separations were performed with Supelcosil LC-C18 HPLC columns, analytical (4.6 9 250 mm; particle size 5 lm) and semipreparative (10 9 250 mm). Merck L7100 chromatograph equipped with the UV–Vis detector (Shimadzu SPD-10AVP) and the radiometric detector with a well-type NaI(Tl) crystal (home-made, INCT, Warsaw) was used. The solvents were: TEAP buffer, 0.05 M, pH 2.25 (solvent A) and MeOH (HPLC grade;
Technetium(I) tricarbonyl complexes
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20000
(C)
after the experiment, samples of the aqueous phase were analyzed by RP-HPLC to check whether the complex had not decomposed in the course of the experiments.
(B)
Ligand exchange experiments
10000 0
cpm
600 400 200 0 6000
(A)
4000 2000 0 0
10
20
30
R t (min)
Fig. 2 HPLC of the [99mTc]-species (A) reaction mixture, (B) pre99m Tc]O4 cursor—[99mTc](CO)3(H2O)? 3 , (C) Na[
solvent B). Elutions were made in the gradient: 0–3 min: 0% B, 3–6 min: 0–25% B, 6–9 min: 25–34% B, 9–20 min: 34–100% B, 20–25 min 100% B. The flow rate was 1 mL/ min or 2 mL/min when the analytical or semipreparative column, respectively, was used. UV detection was at 235 nm and/or c-detection were applied. RTLC was performed on the Kieselgel 60WF254 aluminum plates (Merck). Distribution of radioactivity on the plates was determined using an automatic TLC analyzer SC-05 (home-made, INCT, Warsaw). Paper electrophoresis experiments were performed with chromatography paper strips (Paper Chromedia GF 83, Whatman; 20 9 1 cm), pre-treated with phosphate buffer (0.1 M, pH 7.40). The analyses were carried out for 60 min at 200 V in the same phosphate buffer. A midi horizontal electrophoresis unit (Sigma-Aldrich) device was used. After drying, distribution of the radioactivity along the strips was determined with the same SC-05 TLC analyzer. Lipophilicity determination Lipophilicity determination of the [99mTc(I)]-phenformin complex was determined by standard procedure based on the extraction in the systems (a) n-octanol/PBS (saline phosphate buffer; pH 7.40) and (b) n-octanol/0.9% NaCl aqueous solution, which mimic the physiological conditions [34]. In details, starting solution containing [99mTc]complex was isolated by the RP-HPLC, evaporated under argon stream prior to re-dissolving in 500 lL of PBS or 500 lL of 0.9% NaCl aqueous solution respectively. Triplicate spiked aqueous phase was shaken with n-octanol (vol.:vol. 1:1) at ambient temperature for 15 min (the time being sufficient to reach equilibrium) and centrifuged (5,600 rpm; 5 min). Concentration of the [99mTc]-compound in both phases was determined by c-radiation counting using a well-type NaI(Tl) detector. Immediately
The [99mTc(I)]-phenformin complex was isolated from the reaction mixture using semipreparative RP-HPLC, then incubated at 37 C with 10 mM solutions of histidine or cysteine in the PBS buffer (pH 7.40). RP-HPLC analyses of the incubated solutions were performed in different time periods, up to 4 h, since starting the incubation. Because of short physical half-life of the technetium-99 m radionuclide (ca. 6 h) and short biological half-life of known [99mTc]species (ca. 4–6 h) we assumed, that longer experiments are not necessary. In the stability studies in rat serum–0.1 mL of the RPHPLC isolated complex was added to 0.9 mL of rat serum (Sigma-Aldrich), and the mixture was incubated at 37 C. At certain time intervals up to 4 h since beginning of the incubation, 0.1 mL samples were withdrawn, mixed in an Eppendorf tube with ethanol (0.3 mL) and shaken to precipitate proteins. Then, the samples were centrifuged (14,000 rpm; 5 min) and the supernatants were separated. The radioactivity concentrations of both supernatant and precipitate were measured using the well-type NaI(Tl) detector. To check that the complex had not converted into other water-soluble radioactive species, aliquots of the supernatant were analyzed by the RP-HPLC.
Results and discussion In order to have a better chance of success in application, apart from proper biological properties (e.g. biodistribution, pharmacokinetics or safety for the patients) prospect drugs must have also appropriate physicochemical properties. Such properties as solubility, lipophilicity or stability are among the fundamental properties of a drug candidate. Synthesis of the complex, as well as its time-dependent stability, was checked by RP-HPLC analysis carried out at different time intervals after the synthesis. Results of the analyses performed for the [99mTc(I)]-(phenformin) complex, as well as of its precursor fac-{[99mTc](CO)3(H2O)3}?, showed that even in mild conditions (the deaerated reaction mixture, pH 7–8, room temperature) the species decompose slowly by re-oxidation to the pertechnetate. The detectable amounts (ca. 5% of Na[99mTc]O4 was found already after 4 h (as presented in Fig. 2). Such result, however, seems to be acceptable because the aforementioned period is of the same range as the biological (the rate of excretion) and physical (the rate of decay) half-lives of [99mTc] drugs, i.e. ca. 4–6 hrs and 6 hrs, respectively [35].
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An important feature of drugs, which determines their bioavailability, biodistribution, CNS penetration, metabolism,1 etc. is lipophilicility/hydrophobicity. As a fundamental property of matter, lipophilicity is a descriptor that can help to predict the transport and impact of chemicals in physiological systems. So, drug candidates are obligatory screened according to their distribution in the standard system of n-octanol/PBS solution and n-octanol/0.9% NaCl aqueous solution, respectively [34]. A less lipophilic compound: •
•
•
is characterized by greater aqueous solubility accompanied by greater bioavailability. If the desired concentration of a species cannot be reached, even the most potent in vitro substance cannot be an effective drug. may be less sequestered by fatty tissue and therefore is easy to excrete. As a result, it leads to decreased accumulation and impacts the systemic toxicity by the substance. may not be ideal for penetration through certain barriers. For oral and intestinal absorption the ideal value of log P is less than 1.5. A drug targeting CNS should ideally have a log P value around 2, while a drug intended for sublingual absorption may have a log P value even greater.
In the presented work, partition coefficients have been found to be rather low: log PTc = 0.0025 ± 0.0004 [system (a)] and log PTc = 0.068 ± 0.014 [system (b)]. Range for these values does not differ significantly from this found in the literature for the phenformin (log Plig & 0.1 [38]). It can be, however, easy modified: simple addition of one benzene ring and formation of the 2-ethylnaphthalene biguanide increases lipophilicity of the potential ligand to log Plig & 1.5 (value predicted theoretically in our group). Stability of the isolated from the reaction mixture [99mTc(I)]-phenformin complex was examined in challenge experiments carried out by incubating it (temp. 37 C) in the presence of an excess of strongly competing natural ligands. Such ligands, commonly accepted in the radiopharmacy as standards, are histidine and cysteine. Obtained values of the percentage of intact [99mTc(I)]-complexes after the period up to 4 h, are presented in Table 1. It can be seen that no significant differences in the stability of both species have been observed. Within time period after the injection required for the nuclear medicine procedures with the [99mTc] containing compounds, the complex
Table 1 Percentage of intact [99mTc(I)]-complex in challenge experiments incubated in 37 C with an excess of histidine or cysteine [99mTc(I)]-complex Histidine (%)
Cysteine (%)
0h
100
100
1h
96
95
2h 4h
95 86
95 84
appeared to be sufficiently stable against exchange of each of the sulfur containing ligands. Stability of the isolated complex was also studied in the solution containing the rat serum. In the course of the experiment, a small part of the complex interacted with the serum components, and the reaction products (mainly, labeled proteins) precipitated. Measurements of radioactivity of both supernatant and precipitate (protein) fractions indicated that the fractions of the complex, which had been bound by the serum components, amounted to 11.9% after 30 min, 14.4% after 1.5 h and 27.5% after 4 h. Taking into account that content of the aggressive proteins in the human serum is smaller than that in the rat serum, we can expect that stability of the [99mTc(I)]-complex, in the previous medium will be significantly higher. Concluding, results of the stability experiments fulfill our expectations for the complex as promising radiopharmaceutical precursor. Finally, the isolated complex was examined by paper electrophoresis. The radioelectrophoretogram shows the peak, what does not shift from the starting point (Fig. 3). Simultaneously pertechnetate moves towards anode in the same experimental condition. This observation confirms our expectation that the complex is the uncharged species and should be of importance for the DNA intercalation— the sandwiching of a molecule between two adjacent pairs of bases in the DNA double helix. It is a textbook knowledge, that for a metal complex to be an intercalator it should either be planar or have an extended planar component which can slot between base pairs. It will be shown elsewhere, that because of the planar aromatic ring in the phenformin species—platinum complexed by this ligand may intercalate the DNA. So, we can expect that due to emission of the Auger electrons, [99mTc(I)]-phenformin complex may be proposed as precursor of the anticancer therapeutical drug based on this radionuclide.
1
Bioavailability is a measurement of the rate and extent of a therapeutically active drug that reaches the systemic circulation and is available at the site of action [36, 37]. By definition, when a medication is administered intravenously, its bioavailability is 100%. However, when a medication is administered via other routes (such as orally), its bioavailability decreases (due to incomplete absorption and first-pass metabolism). CNS—central nervous system.
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Conclusions The potential DNA intercalating complex containing the phenethylbiguanide ligand and the tricarbonyl technetium(I), has been synthesized with good yield in the mild
Technetium(I) tricarbonyl complexes
Fig. 3 Migration distance of the tricarbonyl technetium(I)phenformin complex determined by paper electrophoresis
conditions. Stability examination in the standard radiopharmaceutical experiments has shown that the compound may be proposed as the potential antitumor drugs precursor for the AET. Acknowledgment The work was carried out within the grant of Polish Ministry of Science and Higher Education No N N204 141437.
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