Synthesis of the First Diethylenetriaminepentahydroxamic Acid (DTPH

Feb 16, 2002 - Conjugation of this compound to the monoclonal antibody (MAb) ΔCh2HuCC49, used as a model protein, was carried out to evaluate the ...
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Bioconjugate Chem. 2002, 13, 327−332

327

Synthesis of the First Diethylenetriaminepentahydroxamic Acid (DTPH) Bifunctional Chelating Agent†,§ Ahmad Safavy,* Dale C. Smith Jr., Alireza Bazooband,‡ and Donald J. Buchsbaum Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, Alabama 35294. Received October 8, 2001; Revised Manuscript Received January 10, 2002

Synthesis of a new pentahydroxamic acid bifunctional chelating agent (BCA), constructed on the aminoazaalkyl core of diethylenetriaminepentaacetic acid (DTPA), is reported. Rational modifications in the structure of DTPA, which could result in an enhancement of its chelation properties, add to the collection of diagnostic and therapeutic metals bound by this chelator, and might implement significant improvements in the in vivo behavior of this compound, are described. Further improvements in the stability of the ligand-metal complexes of DTPA may improve both diagnostic and therapeutic outcomes such as tumor-to-normal tissue ratios and target-delivered radioactivity. A combination of hydroxamate functions with the azaalkyl backbone of DTPA might be a suitable approach to generate such higher stabilities. This rationale may be justified by the well-known affinity of hydroxamates against different transition metals and favorable properties of DTPA as a versatile chelator. Thus, the N4,NR,NR,N,N-pentakis[[((N-hydroxy-N-methyl]carbonyl)methyl]-2, 6-diamino4-azahexanoic hydrazide (5, DTPH) was designed and synthesized through a convergent synthesis and in 40.7% overall yield. Conjugation of this compound to the monoclonal antibody (MAb) ∆Ch2HuCC49, used as a model protein, was carried out to evaluate the efficiency of this molecule as a BCA. Radiolabeling of the DTPH-∆CH2HuCC49 conjugate with lutetium-177 (177Lu) and biodistribution of the labeled conjugate in athymic nude mice, bearing LS174T human colon carcinoma xenografts, are reported.

INTRODUCTION

Diethylenetriaminepentaacetic acid (DTPA1, 1, Figure 1) has been widely used as a metal chelating agent in both diagnostic and therapeutic procedures. Although the use of this agent as a single-molecule metal chelator has been reported, its application as a conjugate to TVs, for the transfer of metallic nuclides to the site of the disease, has been more prominent. The latter application includes radiolabeling of MAbs and peptides with different metallic radionuclides such as yttrium (88Y, 90Y) (1-3), indium (111In) (4-7), lead (203Pb) (8), samarium (152Sm, 153Sm) (9, 10), and lutetium (177Lu) (11, 12). Other applications of DTPA, such as in MRI (13-15) and luminescent lanthanide imaging (16), have also been demonstrated, † This article is dedicated to Professor W. Franklin Gilmore on the occasion of his 67th birthday. § Presented in part at the 14th International Symposium on Radiopharmaceutical Chemistry, Interlaken, Switzerland, June 10-15, 2001. * To whom correspondence should be addressed at 1824 6th Avenue South, WTI 674, Birmingham, AL 35294-6832. E-mail: [email protected]. ‡ Present address: University of Florida/Shands Jacksonville, Department of Pathology, 655 W 8th Street, Jacksonville, FL 32209. 1 Abbreviations: BCA, bifunctional chelating agent; BOC, tert-butyloxycarbonyl; DIEA, diisopropylethylamine; DPBS, Dulbecco’s phosphate-buffered saline; DTPA, diethylenetriaminepentaacetic acid; DTPH, diethylenetriaminepentahydroxamic acid; ESMS, electrospray mass spectrometry; HR, high-resolution; MAb, monoclonal antibody; MALDI, matrix-assisted laser desorption/ionization mass spectrometry; MRI, magnetic resonance imaging; OHA, 6-oxoheptanoic acid; Phth, pthalyl; SE, size-exclusion; TFA, trifluoroacetic acid; TLC, thin-layer chromatography; TV, targeting vehicle.

Figure 1. Chemical structure of DTPH and other DTPA derivatives.

which, collectively, point to the importance of this compound in medicine. It may be, therefore, of interest to explore possibilities for the development of more improved derivatives of this molecule. These improvements may be accomplished through either or both of the following approaches: (i) developing a bifunctional DTPA with higher chemical stability, and (ii) designing derivatives that could generate metal complexes of better kinetic/thermodynamic properties. A new bifunctional DTPA hydrazide, using Schiff base formation for MAb conjugation, has been reported by Safavy et al. (17). This molecule (2) showed a high shelf stability and efficient antibody conjugation under mild conditions, which is expected to be compatible with the structural and immunoreactivity features of most peptides and proteins. Improvement in the metal complex stability prevents the premature in vivo release of the complex metal, which could, in turn, result in high uptake in normal tissues as well as in low-efficiency treatment protocols. Furthermore, and in the case of immunoconjugates, this metal release is also a function of the BCA-TV bond stability. Readily cleavable covalent bonds, such as carboxylic

10.1021/bc010092x CCC: $22.00 © 2002 American Chemical Society Published on Web 02/16/2002

328 Bioconjugate Chem., Vol. 13, No. 2, 2002

esters, may be disrupted in vivo and in the presence of endogenous enzymes. The design of a potential BCA should, therefore, take into account both of these factors for an efficient site-directed delivery of the metal. Hydroxamic acids have been noted for their favorable metal-chelating features (18). This has been the basis for their utilization in nature as iron-solubilizing agents (siderophores) produced by microorganisms (19-23). In drug development, hydroxamic acids have been the subject of much research in the development of enzyme inhibitors (24, 25), as well as antithalassemic (26, 27), antifungal (28), and antibacterial (29) agents, while hydroxamate-derived enkephalinase inhibitors have been proposed as nonopioid analgesic drug candidates (3032). Furthermore, a trihydroxamate BCA, trisuccin, was reported by Safavy et al. as a radiometal-labeling bifunctional agent (33), and the use of this compound in radiolabeling of MAbs and peptides with rhenium-188 (188Re) through a post-conjugation protocol has been reported (34). The post-conjugation route of indirect radiolabeling, as opposed to the “preformed chelate approach” (35, 36), is a versatile techniques in preparation of rhenium and technetium radiopharmaceuticals that was not available as a routine procedure prior to the development of trisuccin. Thus, the potential of hydroxamic acids, along with the development of antibody conjugation and rhenium-labeling protocols using trisuccin, warranted studies on the design and synthesis of other hydroxamate BCAs. In this paper, we report the synthesis of a novel heterobifunctional DTPA pentahydroxamate, DTPH, containing a hydrazide sidearm as the TV-conjugating moiety. In contrast to the cyclic anhydride DTPA, this hydrazide function forms the prospective linkages through one of the core ethylenediamine segments, leaving all five hydroxamate groups available for metal chelation. The hydrazide group is also compatible with free (unprotected) hydroxamate functions and allows for efficient and group-specific antibody conjugation as described before by Safavy et al. (37). EXPERIMENTAL SECTION

General Procedures. Tetrahydrofuran (THF) was ketyl distilled under argon. All other reagents and solvents were obtained from commercial suppliers and were used as received. 1H NMR spectra were recorded on a Bruker ARX500 FT-NMR spectrometer. The chemical shifts were referenced to tetramethylsilane as internal calibrator. Mass spectra in electrospray mode (ESMS) were recorded either on an API VI triple quadruple mass spectrometer (PE-Sciex, Toronto, Ontario) or Micromass Platform LCZ mass spectrometer (Beverly, MA). TLC was performed with Whatman glass-backed silica gel plates (2.5 cm × 7.5 cm, coating thickness 250 µ, 60 A, 254 nm). Solvents for TLC were mixed on a v/v scale and are defined as follows: (A) chloroform/methanol, 90:10; (E) chloroform/methanol/acetic acid, 77:20:3. Metal-free purified water (18.2 MΩ) was obtained from a Milli-QF system (Millipore, Bedford, MA). Melting points were performed in open capillary tubes and are uncorrected. For analytical SE HPLC, a 7.5 mm × 25 cm G3000SW column (TosoHaas, Montgomeryville, PA) was used. A 10 mM PBS containing 10 mM Na2SO4 at pH 6.7 was used as solvent. Preparative SE chromatography was carried out using Sephadex G-25 columns (PD-10, Pharmacia Biotech AB, Uppsala, Sweden) eluted with DPBS. MALDI MS Procedure. Samples were analyzed in the positive mode on a Voyager Elite mass spectrometer

Safavy et al.

with delayed extraction technology (PerSeptive Biosystems, Framingham, MA). The acceleration voltage was set at 25 kV, and 50-100 laser shots were summed. The matrix was sinapinic acid, dissolved in a 50:50 (v/v) mixture of 0.1% TFA/acetonitrile. A 1 pmol/µL solution of bovine serum albumin was added as an internal standard. Equal volumes of sample and matrix were mixed on a smooth plate, and the average of three measurements were reported. For each reaction, the intact MAb was scanned as the control for evaluation of the following conjugation. The molecular weight increase of the OHA-MAb intermediate was calculated using the unconjugated MAb. The molecular weight of this intermediate then served as the control to measure the increase in the molecular weight of the final product conjugate. Each difference in molecular weight was divided by the molecular weight of the corresponding linker to measure the linker/MAb ratio. HR MS Procedure. The high-resolution liquid secondary ion mass spectra were acquired on a Micromass AutoSpec Magnetic Sector mass spectrometer (Beverly, MA) operated at a resolution of 10 000 (fwhm). All samples were run as approximately 1 µg/mL solutions in 3-nitrobenzyl alcohol using matrix clusters or added peptides as reference peak. 2-Bromo-N-benzyloxy-N-methylacetamide (19). A solution of O-benzylhydroxylamine‚HCl (6.57 g, 41.2 mmol) and K2CO3 (17.04 g, 123.5 mmol) in dioxane/water (1:1, 125 mL) was cooled to 0 °C, and a solution of ditert-butyl dicarbonate (9.02 g, 41.3 mmol) in dioxane was added dropwise over 20 min. The reaction was allowed to warm to ambient temperature and stirred vigorously for 20 h. The volatiles were removed in vacuo. The residue was taken up in ethyl acetate (100 mL) and extracted with water (3 × 50 mL), 10% aqueous citric acid (3 × 50 mL), and brine (1 × 20 mL), followed by drying over MgSO4. The filtered solution was concentrated in vacuo to yield O-benzyl-N-Boc-hydroxylamine (8.93 g, 97.3%) as a colorless oil. Rf 0.86 (A), 0.54 (CHCl3). 1H NMR (500 MHz, CDCl ) d 7.09-7.42 (m, 5H), 4.85 (s, 3 2H), 1.48 (s, 9H). A solution of O-benzyl-N-Boc-hydroxylamine (8.75 g, 39.2 mmol), iodomethane (14.5 mL. 235.25 mmol), and K2CO3 (5.82 g, 42.1 mmol) in acetone (75 mL) was heated at 50 °C with constant stirring. Reaction was complete after 8 days as monitored by TLC. The reaction mixture was filtered, and the volatiles were removed in vacuo. The oily residue was taken up in diethyl ether (100 mL) and extracted with NaHCO3 (3 × 50 mL) and brine (1 × 50 mL) and dried over MgSO4, and the solution was concentrated in vacuo to yield O-benzyl-N-Boc-N-methylhydroxylamine (9.13 g, 98.2%) as a colorless oil. Rf 0.90 (A), 0.68 (CHCl3). 1H NMR (500 MHz, CDCl3) d 7.347.41 (m, 5H), 4.83 (s, 2H), 3.02 (s, 3H), 1.50 (s, 9H). A flask was charged with the O-benzyl-N-Boc-Nmethylhydroxylamine (8.38 g, 35.32 mmol), purged with argon, and cooled to 0 °C. Trifluoroacetic acid (65 mL) was slowly added over 10 min, and the solution was stirred for 1 h (Caution: highly exothermic reaction). All volatiles were removed in vacuo to give O-benzyl-Nmethylhydroxylamine‚TFA (8.79 g, 93.3%) as an off-white crystalline solid. mp 62-64 °C. Rf 0.65 (A), 0.18 (CHCl3). 1H NMR (500 MHz, CDCl ) d 11.71 (s,1H); 7.32-7.37 (m, 3 5H), 5.06 (s, 2H), 2.96 (s, 3H). MS(ES+) 138. A solution of bromoacetyl bromide (0.66 mL, 7.5 mmol) in THF (25 mL) was cooled to -78 °C under an argon purge, and dry K2CO3 (0.753 g, 5.52 mmol) was added. The reaction was vigorously stirred, and a solution of DIEA (0.95 mL, 5.1 mmol) and O-benzyl-N-methylhydroxylamine (1.26 g, 5.02 mmol) in THF (10 mL) was

Synthesis of the First DTPH Bifunctional Chelating Agent

added over 15 min. The reaction mixture was allowed to warm to 0 °C over 4 h. The reaction mixture was filtered, and the volatiles were removed in vacuo at ambient temperature. The oily residue was taken up in diethyl ether (20 mL), and the solution was extracted with 10% aqueous NaHCO3 (3 × 5 mL) and brine (1 × 5 mL), dried over MgSO4, and filtered. The filtrate was concentrated in vacuo to yield 19 (1.21 g, 93.5%) as a colorless oil. Rf 0.85 (A) 0.24. 1H NMR (500 MHz, CDCl3) d 7.39-7.41 (m, 5H), 4.94 (s, 2H) 3.91 (s, 2H), 3.27 (s, 3H). MS(ES+) 258.0, 260.0. N′-BOC-N4,Na,Na,Ne,Ne-pentakis[[((N-benzyloxyN-methyl)amino)carbonyl]methyl]-2,6-diamino-4azahexanoic Hydrazide (20). A solution of N′-Boc-2amino-3-[[2-(amino)ethyl]amino] propionic hydrazide 8 (0.137 g, 0.311 mmol) (17) and DIEA (0.84 mL, 4.67 mmol) was dissolved in DMF (2 mL). A solution of O-benzyl-2-bromo-N-methylacetohydroxamic acid 19 (1.21 g, 4.67 mmol) dissolved in CH2Cl2 was added to the reaction mixture at -50 °C, and the mixture was allowed to warm to ambient temperature over 48 h. The solution was concentrated in vacuo to a yellow-orange oil that was dissolved in ethyl acetate (4 mL). This solution was extracted with 10% aqueous citric acid, 10% aqueous NaHCO3 (2 × 2 mL), and brine (1 × 2 mL) and was dried over MgSO4. Removal of the solvent afforded an oily residue that was triturated with petroleum ether redissolved in ethyl acetate (0.5 mL). The product was then precipitated into a diethyl ether: petroleum ether solution (1:1, 35 mL). The yellow-orange solid was separated by centrifugation and dried in vacuo to yield 20 (0.230 g, 64.5%). mp 93-98 °C. Rf 0.52 (A), 0.86 (E). 1H NMR (500 MHz, CDCl3) d 7.26-7.36 (25H, m); 4.80-4.82 (10H, m); 3.61-3.82 (10H, b); 3.14-3.09 (15H, b); 2.61-2.87 (7H, m); 1.44 (9H, s); HRMS (FAB+) 1147.5748 (C60H79N10O13 requires 1147.5828). MS ES+ 1147.2. N′-BOC-N4,Na,Na,Ne,Ne-pentakis[[((N-hydroxy-Nmethyl)amino)carbonyl]methyl]-2,6-diamino-4-azahexanoic Hydrazide (21). A solution of 20 (0.228 g, 0.199 mmol) in methanol (8 mL) was hydrogenated at ambient temperature over palladium on carbon (10%, 0.046 g) under a hydrogen balloon for 24 h. The catalyst was separated by filtration through a 0.2 µm Acrodisc syringe tip filter (Gelman Sciences, Ann Arbor, MI), and the filtrate was concentrated in vacuo to an off-white oil. The oil was dissolved in methanol (1 mL) and precipitated from diethyl ether (35 mL). The solid was isolated by centrifugation and dried in vacuo to yield 21 (0.112 g, 80.7%) as a hygroscopic yellow-white microcrystalline solid. mp 136-139 °C. 1H NMR (500 MHz, DMSO-d6) d 3.76-3.82 (b, 15 H), 2.78-3.16 (m, 17 H), 1.48 (s, 9 H). HRMS (FAB+) 697.3489 (C25H49N10O13 requires 697.3481). MS ES+ 697.5. N4,Nr,Nr,NE,NE-pentakis[[((N-hydroxy-N-methyl]carbonyl)methyl]-2,6-diamino-4-azahexanoic Hydrazide (5). TFA (5 mL) was added to the Boc-protected 21 (0.154 g, 0.221 mmol), and the homogeneous solution was stirred for 65 min. The solution was concentrated in vacuo and dissolved in methanol (1 mL), and the product was precipitated (×2) from diethyl ether (35 mL). The solid product was separated by centrifugation and dried in vacuo to yield 0.123 g (78.3%) of 5 as a hygroscopic orange-yellow microcrystalline solid. mp 127-133 °C 1H NMR (500 MHz, DMSO-d6) d 3.76-3.82 (15 H, b); 2.85-3.21 (17 H, m). HRMS (FAB+) 597.2964 (C20H41N10O11 requires 597.2956). MS ES+: 597.2. MAb Conjugation. The DTPA hydrazide 5 was conjugated to the MAb ∆CH2HuCC49 using ketone-hydrazide protocol described previously (37). Briefly, 6-oxoheptanoic

Bioconjugate Chem., Vol. 13, No. 2, 2002 329 Scheme 1

acid was conjugated to the MAb in PBS, pH 8.1. The intermediate conjugate was purified by SE filtration in Centricon-50 against 50 mM ammonium acetate (pH 5.5). Compound 5 was added in the same buffer and at a molar ratio of 300:1 relative to the MAb. The homogeneous solution was stirred at 4 °C for 16 h, and the product conjugate was purified by dialysis. The number of DTPH molecules per antibody (DTPH:MAB) was calculated by MALDI MS as described previously (37). This procedure resulted in a DTPH/MAb of 2.2. 177Lu-Labeling. The radioisotope was supplied (University of Missouri Research Reactor, St. Louis, MO) as lutetium chloride in 0.1 M HCl. The radiometal solution (3 µL) was incubated in 50 µL of acetate buffer (pH 5.5) at ambient temperature for 15 min and was added to the solution of the conjugate (120 µg) in the same buffer (63 µL). Radiolabeling yields (>96%) were assessed by SE HPLC, after 1 h incubation at ambient temperature. A control unconjugated MAb showed negligible nonspecific labeling under these conditions. The labeled conjugate was purified by gel filtration using a PD-10 column and phosphate-buffered saline as the eluant. One-milliliter fractions were collected with the conjugate eluting in fractions 3 and 4. Animal Model. Athymic nude female nu/nu mice with a BALB/c background, obtained from the National Cancer Institute Frederick Research Laboratory (Frederick, MD), kept under sterile conditions, were used. Procedures to minimize discomfort, pain, and distress were in accord with the Animal Resource Program at the University of Alabama at Birmingham, accredited by the American Association for Accreditation of Laboratory Animal Care. The LS174T human colon cancer cells (American Type Culture Collection, Manassas, VA) were harvested and suspended in sterile PBS at 7.5 × 108 viable cells/mL. Two groups of animals (6 mice per group) were injected intraperitoneally (i.p.) with 108 viable cells in PBS. At a tumor diameter of 5-10 mm, 2 µCi of 177LuDTPA-∆CH2HuCC49 was injected i.p. into the mice, and the animals were dissected 4 and 48 h postinjection. The radioactivity uptake in the tumor, and normal tissues were measured in a gamma-counter and the counts were used to calculate the %ID/g values. RESULTS AND DISCUSSION

A bifunctional DTPA molecule (2, Figure 1), containing a hydrazide function on the methylene backbone, has been recently prepared (17). The simplest approach to the synthesis of the target DTPH compound (5) seemed to be a direct reaction of the protected pentacarboxylate hydrazide 3, with an excess of hydroxylamine and in the presence of a coupling reagent. This turned out to be not a simple task as shown in Scheme 1. Reaction of 3 with O-benzylhydroxylamine in DCC/HOBT reactions formed

330 Bioconjugate Chem., Vol. 13, No. 2, 2002 Scheme 2

Safavy et al. Scheme 4

Scheme 5 Scheme 3

the cyclized compounds 6 and 7 as the major products and with no or negligible quantities of 4. Changing the reaction conditions, such as stoichiometry, solvent and temperature, did not improve the results, and longer reaction times converted 7 to 6. It was anticipated that a logical solution to this problem would be alkylation of the core diethylene triamine 8 by a reagent already equipped with a protected hydroxamic acid group as shown in Scheme 2. Thus, the functionalized triamine 8 (R ) CONHNHBoc) (17) was reacted with 2-bromo N-benzyloxy acetamide 9 under basic conditions. Again, formation of cyclic products 10 and 11, and not the desired product 4, was observed. These results demonstrated the high stabilities of the cyclic structures to the extent of eliminating hydroxylamine and O-benzylhydroxylamine as two otherwise poor leaving groups. In the course of the synthesis of the triamine 8, formation of a 1,4-disubstituted dihydropyrazine 14, and not the desired aldehyde, was noted as the major product of the amino aldehyde 13 deprotection as shown in Scheme 3 (17). This occurred only when the protected amine in 13 carried an amide proton. Removal of this proton, using phthalyl as the amine-protecting group (15), produced exclusively the target aldehyde 16 and through the same deblocking process. N-Alkyl hydroxamates have been known to maintain their metal-chelating abilities (19, 21, 27). The bacterial siderophore, desferrioxamine, as well as some other natural-product siderophores, are potent N-alkylated hydroxamic acid chelators (20), both in the form of openchain and cyclic compounds where the hydroxamic nitrogen is incorporated into the ring structure. We anticipated, therefore, that replacing the amide proton of the alkylating agent 2-bromo N-bezyloxyacetamide by a small alkyl group should prevent the unwanted cyclizations and still preserve the metal-chelating abilities of the molecule. Thus, 2-bromo-O-benzyloxy-N-methylacetamide (BNMA, 19) was prepared by the procedure of Esteves et al. (38) with slight modification (Scheme 4). This modification afforded the O-benzyl-2-bromo-N-methylhydroxylamine

in a significantly higher overall yield than reported previously. The improved method involves the N-protection of O-benzylhydroxylamine with Boc, instead of 2,2,2trichloroacetylation, followed by methylation of N-BocO-benzylhydroxylamine with iodomethane at elevated temperature and for 8 days. Additionally, the reaction of O-benzyl-N-methylhydroxylamine with bromoacetyl bromide was improved by lowering the temperature to -78 °C and carrying out the reaction for 4 h to give the analytically pure product 19 at a higher yield than the one reported previously (93.5 versus 36% reported) (37). Alkylation of the triamine 8 (17) with BNMA afforded the product 20 with only minor amounts of the cyclized compounds (Scheme 5). The orthogonally protected 20 was stable and withstood chromatographic purification procedures with no later cyclizations. Removal of the protecting groups by successive hydrogenation/acidolysis proceeded smoothly and with no effect on the structure of the molecule. This synthesis afforded the target product 5 in the form of its TFA salt and at 40.7% overall yield. MAb Conjugation. A conjugation procedure has been reported for the covalent attachment of unprotected hydroxamic acids to antibodies, using a hydrazide conjugating function (37). In this procedure, a ketone-derived linker (6-oxoheptanoic acid) is first implanted into the MAb molecule by an activated ester coupling. With the ketone groups serving as hydrazide anchors, the BCA is then conjugated to the MAb, at a low pH and in a groupselective (ketone-directed) manner, and with no conflict with the native amine groups of the protein. This protocol was used here to conjugate the DTPH hydrazide 5 to the CH2 domain-deleted anti-TAG-72 antibody ∆CH2HuCC49. MALDI MS analyses of the conjugation products and, using MWs of the unconjugated starting MAb, the linker, and 5, indicated a DTPH-to-MAb ratio of 2.2 for this conjugation. Radiolabeling. Radiolabeling of the DTPH-∆CH2HuCC49 conjugate with 177Lu proceeded at high yields of

Synthesis of the First DTPH Bifunctional Chelating Agent

Bioconjugate Chem., Vol. 13, No. 2, 2002 331

phase of this investigation, the evaluation of the molecule with other radioactive metals and its comparison with DTPA as a multimetal chelator. ACKNOWLEDGMENT

Figure 2. Biodistribution of 177Lu-DTPH-∆Ch2HuCC49 in athymic nude mice bearing i.p. LS174T human colon cancer xenografts at 4 h (9) and 24 h (0) postinjection. Table 1. Tumor-to-Normal Tissue Ratios for 177Lu-DTPH-∆CH2HuCC49 in LS174T-Bearing Athymic Nude Mice at 4 and 24 h Post-Injection tissue

4h

24 h

blood heart liver lung stomach spleen kidney bone pancreas

6.4 ( 1.1 14.2 ( 2.7 8.5 ( 0.5 2.3 ( 1.3 1.9 ( 0.3 4.5 ( 0.6 2.0 ( 0.3 2.6 ( 0.3 5.1 ( 1.1

80.9 ( 18.9 27.0 ( 4.7 10.4 ( 0.3 1.7 ( 2.4 5.9 ( 2.2 3.8 ( 0.4 1.6 ( 0.2 2.5 ( 0.2 3.6 ( 0.7

radiolabeling (>96%) confirming the ability of the ligand to maintain its metal-chelating properties after conjugation to the MAb. The SEC-HPLC and Sephadex-25 (PD10) showed the expected IgG elution patterns. Biodistribution. Tumor and normal tissue uptake of the 177Lu-DTPH-∆CH2HuCC49 were studied in athymic nude mice, implanted i.p. with LS174T human colon carcinoma cells, at two time points as shown in Figure 2. The %ID/g values for this conjugate showed tumor uptake of 15.3 ( 6.5 and 13.0 ( 9.1 at 4 and 24 h timepoints, respectively. The respective blood %ID/g values were 2.4 ( 0.7 and 0.16 ( 0.05 at these time-points. The tumor-to-normal tissue ratios (Table 1) indicated positive values for all organs with the tumor-to-blood ratio increasing significantly from the 4-h to the 24-h time points. These results demonstrate that the immunoreactivity of the labeled conjugate was not affected by the conjugation and radiolabeling procedures and the conjugate had a rapid blood clearance pattern characteristic of this antibody. It is important to note, however, that the in vivo stability of the 177Lu complex of DTPH may not be as high as that of its pentacarboxylate counterpart. This is based on the observation that the 24-h bone concentration of 177Lu-DTPH-∆CH2HuCC49 (Figure 2) is over three times that of a 177Lu-labeled DTPA conjugate (17). Conclusion. In an attempt to evaluate the possibilities of designing derivatives with improved properties, a heterobifunctional pentahydroxamate version of the routinely used chelating agent, DTPA, was synthesized. The rationale was the study of a combination of the azaalkyl backbone of DTPA with the metal-chelating hydroxamic acid functionality. The new DTPH molecule, carrying a hydrazide arm on the core chain, formed a covalent conjugate with the ∆Ch2HuCC49 as a model antibody and at a DTPH/MAb ratio of 2.2. The 177Lu-labeled conjugate showed tumor uptake in LS174T-bearing nude mice both at 4 and 24 h postinjection timepoints. This study demonstrates the feasibility of synthesis, antibody conjugation, and radiolabeling of this novel BCA and provides the information needed for moving into the next

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