Synthesis and evaluation of a macrocyclic bifunctional chelating agent for use with bismuth radionuclides

Nuclear Medicine and Biology 28 (2001) 409 – 418

Synthesis and evaluation of a macrocyclic bifunctional chelating agent for use with bismuth radionuclides K. Garmestania, Z. Yaob, M. Zhangc, K. Wongb, C.W. Parkb, I. Pastand, J.A. Carrasquillob, M.W. Brechbiela,* a

Radioimmune & Inorganic Chemistry Section, ROB, DCS, National Cancer Institute, Building 10, Room B3B69, 10 Center Drive, Bethesda, MD 20892-1002, USA b Nuclear Medicine Department, CC c Metabolism Branch, DCS, NCI d Laboratory of Molecular Biology, DBS, NCI; NIH, Bethesda, MD, USA Received 19 August 2000; received in revised form 9 October 2000; accepted 26 October 2000

Abstract The detailed synthesis of the bifunctional chelating agent 2-(p-isothiocyanatobenzyl)-1,4,7,10,13-pentaazacyclopentadecaneN,N⬘,N⬙,N⵮,N⬙⬙-pentaacetic acid (BF_PEPA) is reported. This ligand was conjugated to monoclonal antibody B3 and the resultant immunoconjugate radiolabeled with 205,206Bi. The in vivo stability of the radiolabeled immunoconjugate, and targeting characteristics were determined by biodistribution studies in A431 xenograft tumor-bearing mice sacrificed at 0.5, 1, 2, 4, and 24 hr. Results indicate that BF_PEPA appears to not be a suitable bifunctional chelating agent for sequestering isotopes of Bi(III) for radioimmunotherapy applications. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Bismuth;

205; 206

Bi; Radioimmunotherapy; Bifunctional chelating agent; ␣-Therapy

1. Introduction The treatment of cancer by the use of radiolabeled monoclonal antibodies continues to be attractive and supported by recent clinical reports [19,21,36,41]. One variable that influences effectiveness of radioimmunotherapy (RIT) is the choice of the radionuclide and its associated emission characteristics [40] which is then related to the type of disease to be treated and thus directly related to the tissue penetration requirements and inherent cell killing ability of the isotope [33]. Therapy of solid tumors has been exacted with ␤⫺ emitters [1,12,44]. The range of the ␤⫺ particle is several millimeters and the majority of energy deposition does not occur immediately along the emission track [33]. The effective and optimal tissue range of ␤⫺ particles then may not be optimal for treatment of small clusters of cells or single cells, micrometastatic disease, leukemias and lymphomas [18,27,33]. Contrary, the energy from ␣-particles

* Corresponding author. E-mail address: [email protected] (M.W. Brechbiel).

(4 –9 MeV) is deposited over a very short distance (40 –100 ␮m) in tissue, resulting in a dense track of ionizing radiation and is cytotoxic at a dose rate as low as 1 cGy/hr [24]. Thus, treatment of these and related diseases may be more efficient with ␣-emitters which combine high cytotoxicity and a short tissue range [8]. Recently, considerable efforts have been invested in the development of the ␣-emitters 212Bi (t1/2 ⫽ 60 min) [17], 213Bi (t1/2 ⫽ 46 min) [20,28,32] and 211 At (t1/2 ⫽ 7.2 h) [45,46] resulting clinical trials [38,47], despite the potential limitation of 213Bi due to the short half-life. Alternately, 225Ac (t1/2 ⫽ 10 d) has been proposed as an alternate ␣-emitting radionuclide to obviate the short half-lives of 213Bi and 211At, and to take advantage of a 4 ␣-emission cascade [15,16]. Recently, efforts from this laboratory have involved the screening and development of a suitable bifunctional chelating agent for 225Ac [8,10,11]. While screening candidates for this application, the chelating agent 1,4,7,10,13-pentaazacyclopentadecane-N,N⬘,N⬙,N⵮,N⬙⬙-pentaacetic acid (PEPA) [22] was noted to possess significant selectivity for 213 Bi during the screening of this ligand for possible use with 225Ac. This then provided the impetus for the synthesis

0969-8051/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 9 6 9 - 8 0 5 1 ( 0 0 ) 0 0 2 0 3 - 1

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of a bifunctional derivative of PEPA, (2-(p-isothiocyanatobenzyl)1,4,7,10,13-pentaazacyclopentadecane-N,N⬘,N⬙,N⵮,N⬙⬙-pentaacetic acid (BF_PEPA). For RIT applications, 212,213Bi must be linked as the metal complex to a monoclonal antibody (mAb) or immunoprotein via a suitable bifunctional chelating agent wherein that complex must be adequately thermodynamically and kinetically stable to minimize release of the isotope to minimize toxicity in vivo [14]. Derivatives of diethylenetriamine pentaacetic acid (DTPA), CHX-DTPA’s conjugated to monoclonal antibodies, have been shown to form complexes with Bi(III) isotopes both rapidly and with suitable stability for in vivo applications [3,17,32]. The macrocyclic ligand DOTA also forms suitably stable complexes with Bi(III) isotopes [23], however, the formation kinetics associated with this chelating agent were found to be unacceptable in conjunction with the short half-lives of the therapeutic Bi(III) isotopes [37]. Thus, the study of an alternate macrocyclic ligand wherein increased denticity was proposed to counter a potentially overly large cavity. By virtue of the larger, less constrained ring the possibility of forming a suitably stable complex that permitted improved formation rates as compared to DOTA was considered attractive. Evaluation of BF_PEPA was performed then by conjugation to monoclonal antibody B3 that recognizes Lewisy and has been very well studied with several bifunctional chelating agents [5–7]. Additionally, as a control, the B3 conjugate with the chelating agent CHX-A⬙ DTPA radiolabeled with 205,206Bi was then employed as a standard for acceptable in vivo stability in that this chelating agent has previously been shown to meet the requirements for such applications with the Bi(III) isotopes [3,17,32]. Evaluation of the in vivo stability of the Bi(III) complex formed with the BF_PEPA immunoconjugate was performed by means of a biodistribution experiment. This method was chosen as opposed to a simple serum stability measurement on the basis that the result obtained would be authoritative and capable of detecting subtleties that may be overlooked by in vitro methods [42]. Herein, we report the detailed synthesis of BF_PEPA, preparation of the conjugate with monoclonal antibody B3, radiolabeling with 205,206Bi, and the in vivo stability of this radioimmunoconjugate as compared to that of the 205,206Bi labeled CHX-A⬙ DTPA-B3 immunoconjugate evaluated in mice bearing A431 xenograft tumors.

2. Materials and methods 2.1. Synthesis BOC-p-Nitro-phenylalanine and BOC-iminodiacetic acid disuccinimidyl ester were prepared as described in the literature [4,29]. The tert-butyl bromoacetate was purchased

from Fluka (Ronkonkoma, NY). All other reagents were purchased from Aldrich (Milwaukee, WI), Sigma (St. Louis, MO), or Fluka and used without further purification. Ion-exchange resins were obtained from Bio-Rad Laboratories (Richmond, CA). 1 H and 13C NMR were obtained using a Varian Gemini 300 instrument (Palo Alto, CA). Chemical shifts are reported in ppm on the scale relative to TMS, TSP, or solvent. Proton chemical shifts are annotated as follows: ppm (multiplicity, integral, coupling constant (Hz)). Chemical ionization mass spectra (CI-MS) were obtained on a Finnegan 3000 instrument (San Jose, CA). Fast atom bombardment mass spectra (FAB-MS) were acquired on a Extrel 400 (Pittsburgh, PA). Exact mass FAB-MS was obtained on a JOEL SX102 spectrophotometer (Peabody, MA). The exact mass measurements in FAB were obtained using an accelerating voltage of 10 kV with the samples being desorbed from a matrix using 6 keV xenon atoms. Mass measurements were performed at 10,000 resolution using electric field scans with the sample peak bracketed by two poly(ethylene glycol) reference ions. Elemental analyses were performed by Atlantic Microlabs (Atlanta, Georgia). The HPLC system components (Beckman Instruments, Fullerton, CA) were as follows: a pair of 114 M pumps, a 165 dual-wavelength variable uv detector, controlled through a 406 analogue interface module, running under System Gold software, and a Beckman 4.6 ⫻ 25 cm ultrasphere ODS 5␮ column. A 25 min gradient from 100% 0.05 M Et3N/HOAc to 100% MeOH at 1mL/min was employed for all HPLC chromatography.

2.2. BOC-p-Nitro-phenylalaninyl glycine ethyl ester BOC-p-Nitro-phenylalanine (10.23 g, 33.0 mmol,), glycine ethyl ester hydrochloride (4.64 g, 33.3 mmol,) and triethylamine (4.63 mL, 33.3 mmol) were dissolved in ethyl acetate (300 mL) and anhydrous DMF (200 mL). EDC (6.38 g, 33.3 mmol) was added and the solution stirred 18 hours at room temperature. The solution was diluted with in ethyl acetate (500 mL) and extracted with water (100 mL); 5% NaHCO3 (100 mL ⫻ 3); brine (100 mL); 1.5 M HCl (100 mL ⫻ 2) and brine (100 mL ⫻ 2). The organic layer was dried over Na2SO4, filtered and concentrated to yield a light tan solid (11.5 g, 88%). 1 H NMR (DMSO-d6) ␦ 8.460 (t, 1H, J ⫽ 4.8), 8.169 (d, 2H, J ⫽ 8.7), 7.573 (d, 2H, J ⫽ 8.7), 7.082 (d, 2H, J ⫽ 8.7), 4.284 (m, 1H), 4.101 (q, 2H, J ⫽ 7.8), 3.874 (m, 2H), 3.141 (dd, 1H, J ⫽ 13.8, 4.2), 2.866 (dd, 1H, J ⫽ 13.8, 10.8), 1.260 (s, 9H), 1.195 (t, 3H, J ⫽ 6.6); 13C NMR (DMSO-d6) ␦ 170.96, 169.53, 155.44, 147.19, 144.64, 130.43, 123.81, 78.23, 61.64, 54.96, 41.18, 38.08, 28.06, 13.98; CI-MS (M⫹⫹1) 396. Anal. Calc. for C18H25N3O7: C, 54.67; H, 6.38; N, 10.63. Found: C, 54.29; H, 6.39; N, 10.39.

K. Garmestani et al. / Nuclear Medicine and Biology 28 (2001) 409 – 418

2.3. BOC-p-nitro-phenylalaninyl glycine N-(2-aminoethyl) amide (1) Ester 10 (18.92 g, 47.9 mmol) was taken up as a slurry in methanol (50 mL) and added to ethylenediamine (400 mL) while vigorously stirring. The reaction solution was allowed to stir for 18 hours at room temperature. After thin layer chromatography (1:4, MeOH:CHCl3) indicated no starting material, the ethylenediamine was removed by vacuum rotary evaporation. The oily residue was dried under vacuum for 24 hours after which an orange solid was isolated (18.64 g, 95%). 1 H NMR (DMSO-d6) ␦ 8.288 (t, 1H ,J ⫽ 5.7), 8.176 (d, 2H, J ⫽ 7.8), 7.747 (t, 1H, J ⫽ 5.1), 7.564 (d, 2H, J ⫽ 8.7), 7.155 (d, 1H, J ⫽ 7.8), 4.255 (m, 1H), 3.704 (m, 2H), 3.155 (dd, 1H, J ⫽ 13.8,3.9), 3.105–3.032 (m, 2H), 2.875 (dd, 1H, J ⫽ 14.4,10.8), 2.568 (t, 2H, J ⫽ 6.0), 1.279 (s, 9H); 13C NMR (DMSO-d6) ␦ 171.67, 168.82, 155.65, 146.96, 146.42, 130.81, 123.29, 78.36, 55.29, 42.23(2C’s), 41.20, 37.13, 28.03(3C’s); CI-MS (M⫹ ⫹ 1) 410. Anal. Calc. for C18H27N5O6: C, 52.79; H, 6.66; N, 17.11. Found: C, 52.81; H, 6.61; N, 17.32. 2.4. p-Nitro-phenylalaninyl glycine N-(2-aminoethyl) amide dihydrochloride Amide 1 (17.6 g, 43 mmol) was added to anhydrous dioxane (800 mL) previously saturated with HCl(g) while cooled in an ice bath. The suspension was stirred for 18 hr after which the solvent was diluted with diethyl ether (200 mL) and cooled in the refrigerator for 6 hr. The precipitate was collected on a Buchner, washed with diethyl ether, and dried under vacuum to leave a light tan solid (16.9 g, 96%). 1 H NMR (D2O) ␦ 8.270 (d, 2H, J ⫽ 8.7), 7.547 (d, 2H, J ⫽ 9.0), 4.359 (t, 1H, J ⫽ 6.6), 3.986 (d, 1H, J ⫽ 16.8), 3.832 (d, 1H, J ⫽ 17.7), 3.532 (t, 2H, J ⫽ 5.7), 3.366 (d, 2H, J ⫽ 7.8), 3.157 (t, 2H, J ⫽ 6.0); 13C NMR (D2O) ␦ 174.68, 172.61, 150.51, 144.93, 133.81, 127.32, 57.01, 45.35, 42.14, 39.83, 39.46; CI-MS (M⫹ ⫹ 1) 310. Anal. Calc. for C13H21N5O4Cl2: C, 40.84; H, 5.55; N, 18.32. Found: C, 40.93; H, 5.76; N, 18.42. 2.5. 1-(tert)-Butyloxycarbonyl)-5-(p-nitrobenzyl)-3, 6, 9, 14,-tetraoxo-1, 4, 7, 10, 13-pentaazacyclopentadecane (2) In a 5 L 3-necked Morton flask, anhydrous dioxane (3.5 L) was heated to ⬃90°C. BOC-iminodiacetic acid disuccinimidyl ester (4.27 g, 0.01 mmol) in DMF (50 mL) was loaded into a 50 mL gas-tight syringe. The dihydrochloride prepared above (3.82 g, 0.01 mmol) was taken up in anhydrous DMSO (⬃40 mL), treated with triethylamine (3.06 mL, 0.022 mmol) and vigorously stirred. The triethylamine hydrochloride was filtered off on a Buchner and washed with additional DMSO to bring the total volume of the filtrate up to 50 mL. The solution was then loaded into a 50 mL gas-tight syringe. Both syringes were locked onto a

411

syringe pump and added to the dioxane such that the addition was complete after 24 h. Five additions were made such that the total amount of the reactants was 4 ⫻ 10 mmol and 2.4 mmol in each reactant, respectively. After the final addition, the reaction was heated for an additional 18 hr and then cooled to room temperature. The reaction was concentrated to a foam by rotary evaporation and the residue dissolved in chloroform (500 mL). The chloroform was washed with water (200 mL), 5% NaHCO3, (2 ⫻ 200 mL), brine (200 mL), 1 M HCl (2 ⫻ 200 mL), brine (200 mL) and water (100 mL). The CHCl3 layer was dried over Na2SO4 and reduced to dryness. The residue was chromatographed in two portions silica gel columns (3 ⫻ 40 cm) eluted with a 0%–15% MeOH in CHCl3 gradient to isolate the pure macrocycle as a tan solid (6.00 g, 27.9%). 1 H NMR (CDCl3) ␦ 8.525 (br.t, 1H, J ⫽ 7.8), 8.159 (d, 2H, J ⫽ 9.0), 7.887 (br.m, 1H), 7.552 (m, 1H), 7.473 (d, 1H, J ⫽ 8.7), 7.427 (d, 1H, J ⫽ 8.7), 7.142 (m, 1H), 4.65–3.00 (br,m, 13H), 1.436 (s, 6H), 1.335 (s, 3H); 13C NMR (DMSO-d6) ␦ 172.65, 171.49, 171.01, 170.01, 154.68, 146.54, 146.05, 130.81, 123.41, 79.99, 55.41, 54.98, (52.98, 52.43), (52.01, 51.83), 42.59, 38.043, (35.86, 35.43), (27.72, 27.42) conformational doubling; CI-MS (M⫹ ⫹ 1) 507, (M⫹ ⫹ NH3) 524. Anal. Calc. for C22H30N6O8: C, 52.16; H, 5.98; N, 16.59. Found: C, 52.25; H, 6.10; N, 16.19. 2.6. 2-(4-nitrobenzyl)-1, 4, 7, 10, 13pentaazacyclopentadecane-1, 4, 7, 10, 13,-hexaacetic acid (4) Dioxane (125 mL) in a 250 mL 3-necked round bottom flask was chilled in an ice bath and saturated with HCl(g) for 4 h. Macrocycle 2 (5.41 g, 10.7 mmol) was added and HCl(g) was bubbled through the reaction mixture for an additional hour. The reaction was stirred overnight (18 h) at room temperature. Diethyl ether (200 mL) was added and the reaction mixture chilled in the freezer for 6 h. The yellow-tan precipitate was collected on a Buchner, washed with diethyl ether, and vacuum dried overnight. The product was isolated as a pale, yellow solid and used without delay. 1 H NMR (D2O) ␦ 8.221 (d, 2H, J ⫽ 9.0), 7.479 (d, 2H, J ⫽ 8.7), 4.398 (t, 1H, J ⫽ 6.6), 3.975 (d, 1H, J ⫽ 17.7), 3.87–3.58 (m, 5H), 3.477 (d, 1H, J ⫽ 17.7), 3.40 –3.21 (m, 4H), 3.158 (dd, 1H, J ⫽ 14.1, 9.9); 13C NMR (D2O) ␦ 176.80, 174.43, 170.61, 169.64, 150.09, 146.75,133.39, 127.08,69.64, 52.70, 52.34, 45.17, 41.89, 40.07, 38.49. The above product was suspended in anhydrous THF (50 mL) in a 250 mL 2-necked round bottom flask and chilled in an ice bath. To this suspension was added 1 M BH3.THF (75 mL). The mixture was allowed to warm to room temperature and then heated at 50°C. After three days, additional 1 M BH3.THF was added (50 mL). The excess hydride was decomposed by addition of methanol and the mixture was evaporated to dryness with the residue dried under vacuum. This residue was transferred to a 250 mL

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3-necked flask and absolute EtOH (80 mL) was added. HCl(g) was bubbled through the reaction mixture for 2 h and then the reaction was heated at reflux for 17 h. The reaction mixture was then cooled to room temperature and the suspension was placed in the freezer for 48 hr. The pale yellow precipitate was collected on a Buchner funnel, washed with diethyl ether, and vacuum dried. The crude pentaamine (2.00 g) was taken up in DMF (50 mL) and anhydrous Na2CO3 (4.8 g, 45.3 mmol) and tertbutyl bromoacetate (4.42 g, 22.7 mmol) were added. The reaction was heated to ⬃90oC for 18 hr. The reaction mixture was cooled to room temperature, taken up in CH2Cl2 (200 mL), and washed with H2O (3 ⫻ 100 mL). The CH2Cl2 solution was dried over Na2SO4, filtered, and rotary evaporated to leave a thick, dark oil (FAB-MS (M⫹ ⫹ 1) 921). This residue was treated with trifluoroacetic acid (30 mL) for 18 hr. The acid was removed, and after drying under vacuum for 18 hr, the residue was taken up in H2O, and loaded onto an AG50wX8 cation-exchange resin (H⫹ form, 200 – 400 mesh) column (2.6 ⫻ 30 cm). The resin was washed with H2O until the eluant was neutral and then the crude product was eluted from the resin with 2M NH4OH (1 L). The ammonia solution was rotary evaporated, leaving the crude product as a dark, yellow-brown solid. The product was isolated in two portions by elution from AG1 (acetate form, 200 – 400 mesh) anion-exchange chromatography (1.6 ⫻ 35 cm) using a 0.0 – 4.0 M gradient and collecting the eluant in 88 18 x 150 test tubes. The product was found in tubes 40 – 66 by HPLC and the relevant fractions from each elution were combined, concentrated to ⬃30 mL, and finally lyophilized to leave the desired pentaacetic acid (695 mg). 1 H NMR (D2O) ␦ 8.231 (d, 2H, J ⫽ 8.7), 7.539 (d, 2H, J ⫽ 7.8), 3.52–2.70 (m, 21H); 13C NMR (D2O, pH 12.0) ␦ 183.29, 182.45(3C’s), 182.26, 152.39, 149.11, 133.39, 126.89, 65.03, 61.93, 61.44(2C’s), 58.65, 58.43, 55.61, 55.14, 54.95, 54.52, 54.09, 53.49(3C’s), 53.19; FAB-MS (M⫹ ⫹ 1) 641. Anal. Calc. for C27H40N6O12: C, 50.61; H, 6.31; N, 13.21. Found: C, 50.11; H, 6.32; N, 13.21. HPLC Rt ⫽ 11.63 min. 2.7. 2-(4-Isothiocynatobenzyl)-1, 4, 7, 10, 13pentaazacyclopentadecane-1, 4, 7, 10, 13,-hexaacetic acid (6) A Schlenk flask was charged with 10% Pd/C (100 mg) and H2O (5.0 mL) and fitted onto an atmospheric hydrogenator. The apparatus was flushed with H2(g) two times to fully saturate the catalyst. A solution of 4 (120 mg, 0.188 ␮mol) in H2O (3 mL), plus 1 drop of concentrated HCL, was injected via syringe into the flask. The hydrogenation was allowed to proceed until the uptake of H2(g) ceased. The reaction mixture was filtered through a bed of Celite 577 packed in a medium glass fritted funnel. The filtrate was reduced to ⬃10 mL by rotary evaporation. The HPLC of the aniline was a single peak, Rt ⫽ 11.63 min.

A solution of SCCl2 (22.0 ␮L) in CHCl3 (10 mL) was added to the aqueous solution of the crude aniline. The mixture was stirred rapidly for 2 h at room temperature. The volatile materials were removed by rotary evaporation and the aqueous layer was lyophilized to give the isothiocyanate as a pale yellow solid (115 mg). 1 H NMR (D2O) ␦ 7.33 (s, 4H), 3.85–2.60 (complex multiplet, 31H); Exact Mass measurement FAB-MS (M⫹/ noba) 653.2632; IR 2100 cm-1 (SCN). HPLC Rt ⫽ 15.87 min. The sample was incubated with excess n-butylamine and re-injected, Rt ⫽ 14.02 min. 2.8. Conjugation of B3 with BF_PEPA or CHX-A⬙ DTPA The isolation and characterization of mAb B3 has been described in detail in the literature and was provided by Dr I. Pastan [34]. The B3 was concentrated to 5 mg/mL and conjugated with either BF_PEPA or CHX-A⬙ DTPA [42] employing the linkage methods for aryl isothiocyanato groups that have been well described in the literature [31]. Unreacted or “free” ligand was separated from the conjugated antibody by dialysis in 0.15 M NH4OAc. The average number of chelates per antibody for the conjugation products was 1.2 and 1.0 for BF PEPA and CHX-A⬙ DTPA, respectively, as determined by the appropriate spectrometric method for the two classes of chelating agent [9,35]. 2.9. Radiochemistry, production of

205,206

Bi

The 205,206Bi was produced by bombarding an ultrapure Pb target at the National Institutes of Health cyclotron facility. A modification of the literature procedure was employed for the separation of Bi(III) from Pb(II) [25]. The target was dissolved in 7 M HNO3 (7 mL) and this solution was heated until the volume was reduced to ⬃1 mL. In order to precipitate the Pb completely, after approximately a 10 minute cooling period, 16 M HNO3 (10 mL) was added and the solution was heated reducing the volume to ⬃5 mL. This solution was permitted to cool to room temperature and the Pb(NO3)2 precipitate that formed was removed by filtration. Thereafter, the filtrate was heated to further reduce the volume to ⬃2 mL. If no precipitate was observed at this point, the solution was heated to dryness. Otherwise, the process was repeated until no precipitate was observed. The dry radioactivity was then dissolved in 0.1 M HNO3 (3 ⫻ 300 ␮L) and loaded onto AGMP-50 cation ion-exchange resin (100 –200 mesh, H⫹ form) (1 mL bed volume preequilibrated with 0.1 M HNO3) The resin was washed with 8 M HNO3 (2 mL) followed by 1 M HNO3 (2 mL) and finally with 0.1 M HNO3 (5 mL). The 205,206Bi (⬃1 mCi) was eluted with 0.1 M HI (600 ␮L) and the presence of the possible Pb contaminant was checked by radioassaying the eluant using the 279-KeV ␥-ray from 203Pb using a high resolution ␥-detector (EG&G Ortec, Oak Ridge, TN). This radioassay was done immediately after the elution of the

K. Garmestani et al. / Nuclear Medicine and Biology 28 (2001) 409 – 418

413

205,206

Bi to insure a complete separation of the desired isotope from Pb(II).

tion after the tumors had reached about 0.5 cm in maximal diameter (0.29 ⫾ 0.2 g).

2.10. Radiolabeling of antibody B3

2.14. Biodistribution study

The B3 conjugates were labeled with 205,206Bi by the previously reported procedure [29]. In brief, the above purified 205,206Bi was a mixture of 206Bi:205Bi at a ⬃3:2 molar ratio. The pH of the radionuclide solution was adjusted to 5.0 –5.5 using 5 M NH4OAc. The B3 conjugates were added to the above 205,206Bi solution, and after a 15 min incubation at room temperature for the B3-CHX-A⬙ (⬃400 ␮Ci of 205,206 Bi in 240 ␮L) or 25 min at 37oC for the B3-BF_PEPA (⬃600 ␮Ci of 205,206Bi in 360 ␮L), respectively. Each radioimmunoconjugate was purified from free 205,206Bi by HPLC size-exclusion chromatography using a TSK-3000 SW column (TosoHaas, Montgomeryville, PA), equilibrated in 100 mM sodium phosphate, pH 7.4 eluting at 1 mL/min. The specific activities of each of the radioimmunoconjugates was determined by measurements made with a Capintec dose calibrator (Model CRC 127R, Ramsey, NJ) for the two immunoconjugates, 205,206Bi-B3-BF_PEPA and 205,206 Bi-B3-CHX-A⬙ DTPA.

Tumor-bearing mice were injected via the tail vein with 5 ␮g of either the 205,206Bi labeled B3-PEPA or the 205,206Bi labeled B3-CHX-A⬙ conjugate while injecting ⬃5 ␮Ci of radioactivity. Groups of 5 mice were sacrificed at 0.5, 1, 2, 4 and 24 h after injection. Tumors and all major organs were removed, weighed and counted in a ␥-counter. All counting was performed with a 700-2000 KeV window and referenced to an external standard of the radiolabel. Counting of the carcasses was also performed to obtain the whole body clearance determined by accounting for the total radioactivity in the mice. The percentage of the injected dose per gram of tissue was calculated for each organ and normalized to a 20-g mouse. Statistical analysis was performed using Student’s t-test for unpaired data and one way ANOVA for multiple comparisons (Sigmastat, Jandel, San Rafael, CA).

2.11. Cell line A431, a human epidermoid carcinoma cell line that expresses the antigen recognized by B3, was used (courtesy of G. Todaro, NIH, Bethesda, MD). This cell line was used for immunoreactivity determination and for the development of tumor xenografts. Cells were grown in RPMI 1640 medium supplemented with 10% fatal calf serum, 2 mM l-glutamine, penicillin (100 IU/mL) and streptomycin (100 ␮g/mL) at 37oC in a moist atmosphere with 5% CO2. Cells were harvested with EDTA-trypsin and resuspended in PBS for immunoreactivity assay and mouse inoculation. 2.12. Immunoreactivity assay The immunoreactivity of the 205,206Bi labeled B3-PEPA and B3-CHX-A⬙ conjugates were determined using a cell binding assay based on Lindmo et al. [26]. In brief, a constant concentration of the conjugates (5 ng) labeled with 205,206 Bi was incubated with various concentration of A431 cells (20,000 –1,000,000 cells/tube) for 2 h at 4oC. The study was performed in triplicate. Cell-bound radioactivity was separated by centrifugation and the immunoreactive fraction was determined by dividing the counts in the cell pellet by the total counts added. 2.13. Tumor model Female athymic mice (nu/nu) were inoculated s.c. (0.1 mL) with 2 ⫻ 106 A431 cells in the right flank. The biodistribution study was performed 12 days post-inocula-

3. Results The synthesis of the bifunctional macrocyclic chelating agent BF_PEPA was initiated from BOC-p-nitrophenylalanine (Fig. 1). The chain length was extended by standard peptide synthesis methodology to form the ethyl ester of BOC-p-nitrophenylalaninylglycine [2]. Direct aminolysis with excess ethylenediamine as both reagent and solvent cleanly generated the mono-amide 1. Use of ethylenediamine eliminated production of bis(amide) and simplified purification. Acidic cleavage of the carbamate produced the diamine which was reacted with the bis(succinimidyl) ester of BOC-iminodiacetic acid under relatively high-dilution conditions with equimolar addition being effected by addition of the two components via syringe pump. After chromatography, the 15-membered ring was isolated without complication in 27.9% yield. The tert-butyl carbamate was cleaved with acid and the cyclic poly-amidoamine was reduced with BH3/THF complex. This reduction was performed with rigid control of temperature to eliminate concurrent reduction of the aryl nitro group. The cyclic polyamine was isolated from the BH3 reduction and was directly alkylated with excess tertbutyl bromoacetic acid. The tert-butyl groups were cleaved with acid, and the pent-acetic acid was then isolated by ion-exchange chromatography. The aryl nitro group was hydrogenated to produce the aniline that was in turn treated with thiophosgene to generate an isothiocyanate group for conjugation to protein [4]. The BF_PEPA and the CHX-A⬙ DTPA were conjugated to the mAB B3 using established methodology to introduce an average of ⬃1 chelating agent per protein molecule [31]. The number of chelates to protein was determined using the spectrophotometric methods developed in these labs to ac-

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Fig. 1. Synthesis of the bifunctional chelating agent, BF-PEPA.

curately measure the number of functional metal binding sites available on each conjugate [9,35]. The immunoreactivity of these preparations were 71.4% and 66.6% for the PEPA-B3 and the CHX-A⬙-conjugates, respectively. The 206Bi was obtained by irradiation of a high purity Pb target from which the formed Bi(III) was extracted and separated from the Pb by means of a modification of the literature method [25]. The desired Bi(III) radioisotopes were then finally purified by cation ion-exchange the generate a HI solution of the 205,6Bi suitable for immediate use for radiolabeling either of the immunoconjugates. Radiolabeling of B3-BF_PEPA or B3-CHX-A⬙ DTPA with 205,6Bi and purification thereafter was performed analogously to previous reports [30,37]. The immunoconjugates

Table 1 Biodistribution of mAb B3 radiolabeled with Chelate

Tissue

205,206

Bi using the bifunctional chelates BF_PEPA or CHX-A⬙:% ID/g

Time (h) 0.5

CHX-A⬙

BF_PEPA

Blood Liver Spleen Kidneys Lung Bone Tumor Blood Liver Spleen Kidneys Lung Bone Tumor

were labeled at specific activities of 0.61 ␮Ci/␮g and 0.90 ␮Ci/␮g, for 206Bi-B3-BF_PEPA and 206Bi-B3-CHX-A⬙ DTPA, respectively. Biodistribution studies in A431 xenograft tumor-bearing mice were performed. The results of the biodistribution study for 205,206Bi-B3-BF_PEPA and 205,206Bi-B3-CHX-A⬙ DTPA are shown in Table 1. Small differences in biodistribution were seen in the first four hours in some organs. The greatest differences were seen in the blood, kidney, and tumor. The 205,206Bi-BF_PEPA-B3 cleared from the blood more rapidly than the 205,206Bi-CHX-A⬙-B3. At 24 h, the retention of 205,206Bi-B3-PEPA was much less than that of the 205,206Bi-B3-CHX-A⬙, 7.30 ⫾ 1.10% ID/g versus 17.27 ⫾ 2.56% ID/g, respectively (p ⬍ 0.001). There was a

a

44.57 (3.54) 12.30 (1.75) 6.39 (0.82) 10.63 (1.36) 20.48 (2.68) 4.01 (0.73) 2.08 (0.50) 39.20 (1.9) 16.69 (2.0) 5.78 (0.78) 10.40 (0.73) 16.50 (0.81) 2.94 (0.32) 1.97 (0.12)

1

2

4

24

40.33 (1.48) 13.41 (2.08) 6.98 (1.15) 10.58 (0.83) 17.61 (2.13) 4.19 (0.51) 3.29 (1.33) 33.94 (2.2) 14.14 (2.81) 5.31 (0.86) 12.80 (0.68) 14.92 (1.36) 3.23 (0.45) 3.03 (0.34)

33.48 (2.85) 10.88 (1.85) 5.28 (1.46) 10.14 (0.98) 17.70 (4.34) 4.30 (0.72) 4.74 (1.05) 31.60 (1.52) 12.20 (2.03) 5.32 (0.99) 15.60 (0.68) 16.53 (2.39) 3.93 (0.37) 3.81 (1.33)

29.13 (0.87) 10.69 (0.87) 6.66 (1.14) 10.35 (0.57) 15.17 (3.51) 4.81 (0.46) 8.32 (3.24) 25.14 (2.5) 12.84 (1.20) 5.17 (1.33) 19.53 (2.4) 9.08 (2.49) 2.83 (0.29) 8.94 (2.52)

17.27 (2.56) 9.39 (1.40) 6.60 (1.82) 8.66 (0.62) 9.12 (1.97) 6.41 (0.68) 18.53 (1.41) 7.30 (1.1) 9.00 (2.56) 3.08 (0.60) 16.04 (1.2) 4.23 (0.59) 2.02 (0.19) 9.89 (2.0)

a A431 tumor bearing athymic mice (n ⫽ 5) were injected with ⬃5 ␮Ci/␮g of mAB B3 radiolabeled with 205.206Bi using either CHX-A⬙ or BF_PEPA. The mice were sacrificed at the indicated time points and the tumor and organs were harvested, weighed, and counted. The mean percent injected dose per gram (% ID/g) and the standard deviation were calculated.

K. Garmestani et al. / Nuclear Medicine and Biology 28 (2001) 409 – 418

Fig. 2. Comparative Whole Body Clearance of versus 205,206Bi-B3-CHX-A⬙ DTPA.

205,206

Bi-B3-BF

PEPA

continuous increase in accumulation in the kidneys with 16.04 ⫾ 1.2% ID/g for 205,206Bi-B3-PEPA versus 8.66 ⫾ 0.62% ID/g for the 205,206Bi-B3-CHX-A⬙ (p ⬍ 0.001). Moreover, the accretion of 205,206Bi in the kidney appeared to increase markedly at early time points with the 205,206BiBF_PEPA-B3 from 10.40 ⫾ 0.73%ID/g at 0.5 h, up to 12.80 ⫾ 0.68%ID/g at the 1 hr time point, finally peaking at 19.53 ⫾ 2.42%ID/g at 4 h (ANOVA p ⫽ 1.26 E-8). The activity in the kidney with 205,206Bi-CHX-A⬙-B3 essentially remained constant over the course of the experiment, declining slightly at the 24 h time point (ANOVA p ⫽ 0.016). Up to and through the 4 hr time point, the tumor uptake of either radioimmunoconjugates was comparable (8.94 ⫾ 2.52 vs. 8.32 ⫾ 3.24%ID/g) (t test p ⫽ 0.74). However, after 24 h there was significantly greater accumulation of the 205,206Bi-B3-CHX-A⬙ in the tumor than of the 205,206BiB3-PEPA, 9.9 ⫾ 2.0% ID/g vs 18.5 ⫾ 1.4% ID/g., respectively (p ⬍ 0.001). The whole body clearance showed small differences between the 2 reagents up to 4 h (⬍2%). Whereas, at 24 hr the whole body retention of 205,206Bi-B3 PEPA was 47.88% as compared to 70.69% of injected dose for 205,206Bi-B3-CHX-A⬙ (Fig. 2) (t test p ⬍ 0.001).

4. Discussion There are currently several ␣⫺emitting radionuclides that fit the profile for potential clinical application, i.e, 212Bi (T1/2 ⫽ 60 min), 213Bi (T1/2 ⫽ 47 min), and 211At (T1/2 ⫽ 7.2 h) [27]. The use of 212Bi is challenging due to high energy ␥-ray emissions (2.26 MeV, ⬃ 27% abundance) from its decay to 208Tl which requires substantial shielding. 213 Bi, with fewer intense ␥-ray emissions has been advocated as a viable alternative [27]. Furthermore, 213Bi is reasonably available, 225Ac/212Bi generator designs have been reported, and with a ␥-ray emission at 440 KeV,

415

scintigraphic imaging, might be obtained [28,43]. Finally, a Phase I clinical trial to treat myologenous leukemia has been performed to validate these characteristics [38]. The requirement for a clinically useful bifunctional chelating agent for Bi isotopes remains the rapid formation of an in vivo stable and kinetically inert complex [14]. The macrocycle DOTA had been shown to form stable and kinetically inert complexes with Bi(III), however, the formation kinetics were considered to be too slow [23,37]. Synthesis and evaluation of the acyclic CHX-A DTPA indicated this reagent met the requirements for use with Bi(III) isotopes [3,17,30,32]. A fourth ␣-emitting radionuclide proposed for radioimmunotherapy applications to obviate the short half-lives of 213 Bi and 211At and to take advantage of a 4 ␣-emission cascade has been 225Ac (T1/2 ⫽ 10 d) [15,16,27]. PEPA was noted to possess significant selectivity for 213Bi versus 225 Ac during screening candidates to develop suitable chelating agents for 225Ac. This in part provided the impetus for a synthesis of a bifunctional derivative of PEPA and its evaluation for in vivo sequestration of Bi(III) radionuclides. The possibility of obtaining a macrocyclic ligand for Bi(III) that would possess rapid formation kinetics and high stability in vivo with the Bi(III) isotopes was thought attractive. The cavity of PEPA was clearly adequate and perhaps even too large, considering the ionic radius of Bi(III) [39]. However, this larger ring size was thought to be less rigid than DOTA and with the increased flexibility to promote faster complex formation. Increasing the number of donors to the coordination sphere of the complexed Bi(III) was considered a potential counter to the cavity being overly large. Synthesis of the bifunctional PEPA was visualized as an expansion of the previously reported synthesis of bifunctional DOTA. Application of this methodology was expected to produce the desired substituted 15-membered ring, albeit in somewhat reduced yield due to the increase in ring size. The synthesis was initiated from the carbamate protected p-nitrophenylalanine (Fig. 1). The chain length was extended by forming the ethyl ester of BOC-p-nitrophenylalaninylglycine [2]. Direct aminolysis with excess ethylenediamine as both reagent and solvent served to further extend the molecule an additional two carbons and to provide four of the five amines of the pentaaza macrocycle without detectable production of any bis(amide) product. After cleavage of the carbamate and generation of the free base, the diamine was reacted with the bis(succinimidyl) ester of BOC-iminodiacetic acid under relatively high-dilution conditions. Precise equimolar addition of the two components was performed via syringe pump, a convenient and accurate method for such reactions [8,29]. This reagent was previously employed to close a 12-membered precursor to a bifunctional DOTA and closure of a three atom expanded analog was thought to be an equally suitable application. However, we have recently reported that use of the bis(succinimidyl) ester of BOC-iminodiacetic acid to close the homologous 18-membered ring met with complete lack of

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K. Garmestani et al. / Nuclear Medicine and Biology 28 (2001) 409 – 418

isolable product [8]. Thus, we were gratified to find the 15-membered ring macrocycle 2 was isolated without any significant complications in nearly 30% yield after chromatography. The carbamate of 2 was cleaved and the amides then reduced with BH3/THF. It was essential that this reduction be preformed with rigid control of temperature to eliminate concurrent reduction of the aryl nitro group. The cyclic polyamine was directly alkylated with excess tert-butyl bromoacetic acid. Rather than isolate the penta-ester, the tertbutyl groups were cleaved, and pent-acetic acid 4 was isolated by ion-exchange chromatography. The nitro group of 4 was hydrogenated producing aniline 5 which was immediately treated with thiophosgene to generate isothiocyanate 6, BF_PEPA. Monoclonal antibody B3 was chosen to evaluate BF_PEPA due to there being a significant body of literature concerning its biodistribution and conjugation behavior while fully recognizing that this antibody itself would not be suitable for delivery of ␣-emitting Bi(III) radionuclides [30 –32]. Conjugation of bifunctional ligands to B3 in excess amounts was known to decrease its binding and immunoreactivity. Therefore, this study strove to maintain conjugation product ratios at unity to produce immunoconjugates of comparable activity. Use of 205,206Bi as a tracer isotope for the ␣-emitters 212 Bi or 213Bi was chosen due to prior experience. Half-life (T ⁄ ⫽ 6.2 d/15d) and ␥-emission characteristics [25], permit an accurate and extended biodistribution study to detect late, as well as early differences between 205,206Bi-B3-BF_PEPA or 205,206 Bi-B3-CHX-A⬙ DTPA. Production and purification of 205,206 Bi was accomplished by modification of the literature methods and yielded adequate amounts of the isotope. Radiolabeling of the two immunoconjugates was accomplished via standard methods. The biodistribution experiment was performed with 205,206 Bi-B3-BF_PEPA and 205,206Bi-B3-CHX-A⬙ DTPA in A431 xenograft tumor-bearing athymic mice at 0.5, 1, 2, and 4 hr and at 24 hr. The faster blood clearance of the 205,206 Bi-B3-BF_PEPA versus 205,206Bi-B3-CHX-A⬙ DTPA indicated some early loss of 205,206Bi. After 24 hr, the disparity between the amounts of the two radioimmunoconjugates in the blood increased. Examination of activity in the kidneys is mandated to evaluate the in vivo stability of Bi(III) chelating agents as the kidney is the primary organ of accretion [13]. The data for the 24 hr time point for 205,206 Bi-B3-BF_PEPA was indicative of a significant loss of 205,206Bi and at the 1 hr time point there was already a notable increase in the amount of 205,206Bi in the kidney versus the control. The trend for tumor uptake of 205,206BiB3-BF_PEPA versus 205,206Bi-B3-CHX-A⬙ DTPA reflected the greater blood clearance and increased kidney uptake of the 205,206Bi-B3-BF_PEPA. In the early time points, there was comparable tumor uptake for the both radioimmunoconjugates, but at the late time point, the 205,206Bi-B3BF_PEPA failed to match the increase in the tumor that was

observed for the 205,206Bi-B3-CHX-A⬙ DTPA. A similar phenomenon was observed in the whole body clearance wherein after 24 hr, the level of 205,206Bi-B3-BF_PEPA had declined far more rapidly than the 205,206Bi-B3-CHX-A⬙ DTPA. Taken as a whole, the biodistribution results reflect that the 205,206Bi-B3-BF_PEPA failed to meet the standards set by the control, 205,206Bi-B3-CHX-A⬙ DTPA. In conclusion, despite apparent affinity and selectivity for Bi(III), the bifunctional macrocyclic ligand, BF_PEPA, was not suitable for sequestration of the Bi(III) isotopes as determined by the in vivo stability of the 205,206Bi complex formed with this ligand after conjugated to a monoclonal antibody. Expansion of the ring cavity, while increasing the denticity of the ligand, not only failed to provide adequate stability, but also failed to provide rapid, room temperature formation of the radio-metal complex. Efforts to examine both the fundamental Bi(III) coordination chemistry with PEPA as well as other possible applications for BF_PEPA continue to be explored.

Acknowledgments We would like thank Paul Plascjak of the NIH, Clinical Center, Nuclear Medicine Department cyclotron facility for his efforts towards the production of the 205,206Bi.

12

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