and 177Lu-Labeling of a DOTA-Conjugated Nonpeptide Vitronectin

Recently, we reported the 90Y- and 177Lu-labeling of a DOTA-conjugated cyclic peptide vitronectin receptor antagonist (Figure 1: SU015). Through a ser...
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Bioconjugate Chem. 2003, 14, 1030−1037

TECHNICAL NOTES Anaerobic 90Y- and 177Lu-Labeling of a DOTA-Conjugated Nonpeptide Vitronectin Receptor Antagonist Shuang Liu,* Thomas D. Harris, Charles E. Ellars, and D. Scott Edwards Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received September 23, 2002; Revised Manuscript Received April 7, 2003

This study describes the discovery and development of an anaerobic formulation for the routine preparation of 90Y and 177Lu complexes (90Y-TA138 and 177Lu-TA138) of a DOTA-conjugated nonpeptide vitronectin receptor antagonist (TA138: 3-sulfon-N-[[4,7,10-tris(carboxymethyl)1,4,7,10-tetraazacyclododec-1-yl]acetyl]-L-alanyl-N-[2-[4-[[[(1S)-1-carboxy-2[[[1,4-dihydro-7-[(1H-imidazol-2-ylamino]methyl]-1-methyl-4-oxo-3-quinolinyl]carbonyl]amino]ethyl]amino]-sulfonyl]-3,5-dimethylphenoxy]-1-oxobutyl]amino]ethyl]-3-sulfo-L-alaninamide). Since 90Y-TA138 and 177Lu-TA138 are very sensitive to radiolytic degradation, exclusion of oxygen is necessary during the radiolabeling. Using the anaerobic formulation, 90 Y-TA138 and 177Lu-TA138 can be prepared in high yield and high specific activity. The anaerobic formulation described in this study is particularly useful for 90Y- and 177Lu-labeling of DOTA-conjugated small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling.

INTRODUCTION

We have been interested in the development of diagnostic and therapeutic radiopharmaceuticals based on small biomolecules (1-17). Recently, we reported the 90Yand 177Lu-labeling of a DOTA-conjugated cyclic peptide vitronectin receptor antagonist (Figure 1: SU015). Through a series of radiolabeling experiments, we found that there are many factors influencing the 90Y-chelation rate of the DOTA conjugate (16). These include the purity of the DOTA conjugate (SU015), the pH, reaction temperature, and heating time, as well as the presence of trace metal contaminants. The 90Y-chelation rate of SU015 is slow room temperature and can be accelerated by raising the pH of the reaction mixture or/and elevating the heating temperature. Under optimized conditions (pH 7.2-7.8 and heating at 50-100 °C for 5-10 min), the minimal amount of SU015 required to achieve RCP of 95% for the complex 90Y-SU015 is ∼25 µg for 20 mCi of 90 YCl3 corresponding to a SU015:90Y ratio of ∼30:1 (16). As a continuation of our interest in the development of new diagnostic and therapeutic radiopharmaceuticals, we now present the synthesis of 90Y and 177Lu complexes of a DOTA-conjugated nonpeptide vitronectin receptor antagonist (Figure 1: TA138). It was found that complexes 90Y-TA138 and 177Lu-TA138 are extremely prone to radiolytic degradation. Exclusion of oxygen is required for the successful 90Y- and 177Lu-labeling of TA138. Through a series of experiments, we have developed an anaerobic formulation for routine preparation of 90YTA138 and 177Lu-TA138. This formulation is particularly * To whom correspondence should be addressed. Current address: Department of Industrial and Physical Pharmacy, Division of Nuclear Pharmacy, School of Pharmacy, Purdue University, 575 Stadium Dr., West Lafayette, IN 47907-2051. Tel: 765-494-0236 (S.L.); Fax: 765-496-3367; E-mail: shuang.liu@ pharmacy.purdue.edu.

useful for radiolabeling of small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling. EXPERIMENTAL SECTION

Materials. Acetic acid (ultrapure), ammonium hydroxide (ultrapure), diethylenetriaminepentaacetic acid (DTPA), sodium ascorbate (AA), and sodium gentisate (GA) were purchased from either Aldrich or Sigma Chemical Co. and were used as received. 90YCl3 (in 0.05 N HCl) was purchased from NEN Life Sciences, N. Billerica, MA. High specific activity 177LuCl3 was obtained from University of Missouri Research Reactor, Columbia, MO. DOTA tris(tert-butyl) ester was obtained from Macrocyclics, Richardson, TX. The synthesis of 2-({[4-(3{N-[2-((2R)-2-{(2R)-2-[(tert-butoxy)carbonylamino]-3-sulfopropyl}-3-sulfopropyl)ethyl]carbamoyl}propoxy)-2,6dimethylphenyl]sulfonyl}amino)(2S)-3-{[1-methyl-4-oxo7-({[1-(triphenylmethyl)imidazol-2-yl]amino}methyl)(3hydroquinolyl)]carbonylamino}propanoic acid (DPC-AG1522) will be reported elsewhere (18). Analytical Methods. The LC-MS data were collected using a HP1100 LC/MSD system with an API-electrospray interface. The LC-MS method used a Zorbax SBC18 column (4.6 mm × 150 mm, 3.5 µm particle size). The flow rate was 1 mL/min with mobile phase gradient starting from 95% solvent A (25 mM ammonium acetate buffer, pH ) 6.8) and 5% solvent B (acetonitrile) to 85% A and 15% B at 40 min. The HPLC method 1 used a HP-1100 HPLC system with a UV/visible detector (λ ) 230 nm), an IN-US radiodetector, and a Zorbax C18 column (4.6 mm × 250 mm, 80 Å pore size). The flow rate was 1 mL/min with a gradient mobile phase starting from 92% solvent A (0.025 M ammonium acetate buffer, pH 6.8) and 8% solvent B (acetonitrile) to 90% solvent A and 13% solvent B at 18

10.1021/bc020061h CCC: $25.00 © 2003 American Chemical Society Published on Web 07/11/2003

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Figure 1. Structures of DOTA-conjugated vitronectin receptor antagonists (SU015 and TA138).

min. The mobile phase was isocratic using 40% of solvent A and 60% solvent B from 19 to 25 min. The HPLC method 2 used a HP-1050 HPLC system with a Rainin Dynamax UV/visible detector (model UVC, λ ) 230 nm) and a Zorbax C18 column (4.6 mm × 250 mm, 80 Å pore size). The flow rate was 1 mL/min with the mobile phase starting 10% of solvent A (0.1% TFA in water) and 90% solvent B (0.1% TFA in acetonitrile) to 20% solvent A and 80% of solvent B at 20 min, followed by an isocratic wash using 40% of solvent A and 60% solvent B from 21 to 26 min. The TLC method used the C18 reverse phase glass plates and a mobile phase containing methanol, acetone, and saline (2:1:1 ) v:v:v). By this method, 90Y-TA138 and 177Lu-TA138 migrate to the solvent front while [90Y/ 177 Lu]colloid and [90Y/177Lu]acetate remain at the origin. The corrected RCP was calculated by subtracting the percentage of [90Y/177Lu]colloid and [90Y/177Lu]acetate obtained by TLC from that obtained by radio-HPLC. Synthesis of 2-{[(4-{3-[N-(2-{(2R)-2-[(2R)-3-Sulfo2-(2-{1,4,7,10-tetraaza-4,7,10-tris[(tert-butoxycarbonyl)methyl]cyclododecyl}acetylamino)propyl]-3sulfopropyl}ethyl)carbamoyl]propoxy }-2,6dimethylphenyl)sulfonyl]amino}(2S)-3-{[1-methyl4-oxo-7-({[1-(triphenylmethyl)imidazol-2-yl]amino}methyl)(3-hydroquinolyl)]carbonylamino}propanoic Acid (DPC-AG1613). DPC-AG1522 (2.95 g, 2.20 mmol) was dissolved in 25/75 TFA/DCM (40 mL). The solution was stirred at room temperature for 60 min and was then concentrated under vacuum to give the free amine as an amber oil. Trituration with anhydrous diethyl ether (6 × 50 mL) gave a colorless solid. The solid was dried under vacuum for 60 min to give 3.07 g of the title compound. LC-MS: m/z ) 1241.3 for [M + H]+ (25%) and 999.3 for [M + H - Trt]+ (42%). The free amine was dissolved in anhydrous DMF (30 mL) and DIEA (0.128 mL). In a separate flask, DOTA tris(tert-butyl) ester (2.10 g, 3.67 mmol) was dissolved in anhydrous DMF (21 mL)

and DIEA (0.85 mL). The solution was treated with HBTU (1.16 g, 3.06 mmol) and stirred under nitrogen at room temperature for 15 min. The activated DOTA tris(tert-butyl) ester was added to the solution of the free amine prepared above. The reaction mixture was stirred at room temperature for 1.5 h. DMF was removed under vacuum. The oily residue was triturated with EtOAc (5 × 50 mL) to afford a pale yellow solid (4.54 g). This solid was purified in four separate runs by HPLC on a Vydac C18 Pharmaceutical column (50 × 250 mm) using a gradient mobile phase from 65% A (0.1 M NaOAc, pH 5.0) and 35% B (ACN) to 53% A and 47% B at 60 min and a flow rate of 80 mL/min. The collected fractions were diluted with two volumes of water and desalted by HPLC using the same Vydac C18 column. The column was equilibrated with 13.5% ACN containing 0.1% TFA, and the diluted product fractions were pumped onto the column. The column was desalted by eluting isocratically using 22.5% ACN containing 0.1% TFA for 10 min at 88 mL/min. Product was eluted using a 1.8%/min gradient of 22.5 to 31.5% ACN containing 0.1% TFA followed by a 0.45%/min gradient mobile phase of 31.5 to 49.5% ACN containing 0.1% TFA. The collected fractions were combined and lyophilized to give the title compound as a colorless solid (2.58 g, 65.3%). LC-MS: m/z ) 1795.6 for [M + H]+ (60%) and 1553.5 [M + H - Trt]+ (50%). Highresolution MS: m/z ) 1795.744 for C85H115N14O23S3, [M + H]+ (calcd 1795.7422). Synthesis of 3-Sulfon-N-[[4,7,10-tris(carboxymethyl)1,4,7,10-tetraaza-cyclododec-1-yl]acetyl]-L-alanylN-[2-[4-[[[(1S)-1-carboxy-2[[[1,4-dihydro-7-[(1H-imidazol-2-ylamino]methyl]-1-methyl-4-oxo-3quinolinyl]carbonyl]amino]ethyl]amino]sulfonyl]3,5-dimethylphenoxy]-1-oxobutyl]amino]ethyl]-3sulfo-L-alaninamide (TA138). A solution of DPCAG1613 (2.50 g, 1.39 mmol) in 94/6 TFA/Et3SiH (75 mL) was heated at 70 °C under nitrogen for 60 min and concentrated under vacuum. The resulting oily solid was

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partitioned between diethyl ether (40 mL) and 5% aqueous ACN. The ether layer was extracted with 5% aqueous ACN (2 × 30 mL). The combined aqueous extracts were lyophilized to give crude TA138 (1.99 g) as a pale yellow solid. TA138 was purified in seven separate runs by HPLC on a Vydac C18 Pharmaceutical column (50 × 250 mm) using a 0.45%/min gradient of 4.5 to 22.5% ACN containing 0.1% TFA at a flow rate of 80 mL/min. The collected product fractions from the seven runs were combined and lyophilized to give the title compound as its TFA salt. A second lyophilization from neutral ACN/ water gave TA138 (1.55 g, 80.5%) as a fluffy solid. MS: m/z ) 1385.4 for [M + H]+ (29%) and 693.3 for [M + 2H]2+ (100%). High-resolution MS: m/z ) 1385.442 for C54H77N14O23S3, [M + H]+ (calcd 1385.4448). A General Procedure for Synthesis of 90Y-TA138 and 177Lu-TA138. To a shielded, clean 5 mL vial were added 10-100 µg of TA138, 2-10 mg of sodium gentisate (GA), and 0-20 mg of sodium ascorbate (AA) dissolved in 1.0 mL of 0.5 M ammonium acetate buffer (pH ) 6.08.0). The mixture was degassed under vacuum ( 98% by HPLC (Method 1). LC-MS: m/z ) 1472.1 for C54H73N14O23S3Y, (M + H)+, 736.2 for C54H74N14O23S3Y, (M + 2H)2+. RESULTS

Discovery of the Anaerobic Formulation. We first tried to prepare 90Y-TA138 using the procedure described in our previous communication (16). TA138 was allowed to react with 90YCl3 in the ammonium acetate buffer (0.5 M, pH ) 7.5) at 100 °C for 5 min. GA (10 mg for 20 mCi of 90YCl3) was used as the stabilizer to prevent radiolytic degradation of 90Y-TA138 during radiolabeling. The total volume was 0.5 mL, and the 90YCl3 concentration was 40 mCi/mL. We used 100 µg of TA138 for 20 mCi of 90 YCl3. The yield for 90Y-TA138 prepared in the presence of oxygen was only 85-90% with a large wash peak (58%) at ∼23 min. We also found that 90Y-TA138 was not stable in the HPLC autosampler vial in the presence of air unless GA or AA (10 mg/mL) is added. If the autosampler vial is sealed, 90Y-TA138 remains relatively stable for 2 h. These observations suggest that the instability and low yield of 90Y-TA138 might be caused by the presence of oxygen dissolved in solution. That led to the discovery of an anaerobic formulation for routine preparations of 90Y-TA138 and 177Lu-TA138.

Figure 2. Radio-HPLC chromatograms (method 1) of TA138 (top) and 177Lu-TA138 (bottom).

90Y-

Synthesis of 90Y-TA138 and 177Lu-TA138. 90Y-TA138 can be readily prepared in high yield (RCP > 95%) using the anaerobic formulation, in which TA138 is allowed to react with 90YCl3 in the degassed buffer solution (pH ) 6.0-7.5) at 100 °C for 5-30 min. Exclusion of oxygen is required for successful radiolabeling, and can be achieved either by degassing under vacuum or by bubbling nitrogen through the reaction mixture before the addition of 90 YCl3. GA (2 mg/20 mCi) and AA (20 mg/20 mCi) are used as stabilizers to prevent radiolytic degradation of 90 Y-TA138 during radiolabeling. Both 0.5 M ammonium acetate and 0.1 M TRIS (tris(hydroxymethyl)aminomethane) can be used as buffers for the radiolabeling. The heating time depends on the pH of the buffer solution. Heating at 100 °C for 30 min is needed for successful radiolabeling if the pH is at 6.0. Heating at 100 °C for 5 min is usually sufficient to achieve RCP > 95% if the pH is > 7.5. We typically use 100 µg of TA138 for 20 mCi of 90YCl3 (TA138:90Y ∼170:1) for most of the radiolabeling experiments. However, under optimized conditions 90Y-TA138 can be prepared in high yield (RCP > 95%) using 20 µg of TA138 for 20 mCi of 90YCl3 corresponding to a TA138:90Y ratio of ∼32:1. 177Lu-TA138 can also be prepared in high yield using the same anaerobic formulation. HPLC Analysis of 90Y-TA138 and 177Lu-TA138. 90YTA138 and 177Lu-TA138 were analyzed by a reversed phase HPLC method using a gradient mobile phase. Figures 2 show typical radio-HPLC chromatograms of 90 Y-TA138 and 177Lu-TA138. There are some small peaks (the void-volume peak at ∼2.5 min and the wash-peak at ∼23 min) due to radioimpurities in 90Y-TA138 and 177 Lu-TA138 preparations. Since these radioimpurities are less than 1.0%, no further characterization was performed. The HPLC retention time of 177Lu-TA138 is almost identical to that of 90Y-TA138. Synthesis of 89Y-TA138. 89Y-TA138 was prepared by reaction TA138 with excess of yttrium(III) nitrate tetrahydrate and was separated from the reaction mixture by HPLC purification. 89Y-TA138 has been characterized by HPLC and LC-MS methods. The HPLC concordance

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Figure 5. Effect of the buffering agent on RCP of 90Y-TA138.

Figure 3. HPLC concordance of 90Y-TA138 (bottom, β-detector) and 89Y-TA138 (top, UV detector).

Figure 4. Effect of sodium gentisate (GA) concentration on RCP of 90Y-TA138.

experiment (Figure 3) showed that 90Y-TA138 and 89YTA138 have the same HPLC elution profiles under identical chromatographic conditions. The LC-MS shows the monoprotonated molecular ion, (M + H)+, peak at m/z ) 1472.1 for [C54H73N14O23S3Y]+ and diprotonated molecular ion, (M + 2H)2+, peak at m/z ) 736.2 for [C54H74N14O23S3Y]2+. Stabilizer Concentration. In this experiment, we prepared 90Y-TA138 using 100 µg of TA138, 2-20 mg of GA, 20 mCi of 90YCl3, and 0.5 M ammonium acetate buffer (pH ) 7.5). The reaction mixture was heated at 100 °C for 5 min. Figure 4 shows the effect of GA concentration on RCP of 90Y-TA138. Lower GA concentration gives better RCP for 90Y-TA138. However, 2 mg of GA is not sufficient to maintain the solution stability of 90Y-TA138. We have to use AA (20 mg for 20 mCi of 90 YCl3) as the second stabilizer. AA can be added to the reaction mixture before addition of 90YCl3 or after radiolabeling. Buffer Agent. The purpose of this experiment is to see if other buffer agents can be used to replace ammonium acetate for 90Y-labeling of DOTA-biomolecule conjugates at pH 6.0-8.5. The buffer agents tested include tricine [N-[tris(hydroxymethyl)methyl]glycine] (pKa ) 8.15 at 20 °C) and TRIS (pKa ) 8.3 at 20 °C). We

Figure 6. Effect of the pH and heating time on RCP of TA138.

90Y-

prepared 90Y-TA138 using 100 µg of TA138, 2 mg of GA, 20 mg of AA, and 20 mCi of 90YCl3 for the radiolabeling. The buffer concentration was 0.5 M for ammonium acetate and 0.1 M for tricine and TRIS. The pH in the mixture was 7.5 before addition of 90YCl3. The reaction mixture was heated at 100 °C for 5 min. Figure 5 shows that TRIS and ammonium acetate are comparable as buffer agents for the preparation of 90Y-TA138. Tricine is not a good buffer agent because it is a stronger chelator and may interfere with the 90Y-chelation of the DOTA conjugate. Since it has been used for the radiolabeling of various biomolecules, we chose 0.5 M ammonium acetate as the buffer agent for most radiolabeling experiments in this study. Buffer pH and Heating Time. We prepared 90YTA138 using 100 µg of TA138, 2 mg of GA, 20 mg of AA, and 20 mCi of 90YCl3 in 1.0 mL of 0.5 M ammonium acetate buffer. The pH in the reaction mixture was 6.0 or 7.5 before addition of 90YCl3. The reaction mixture was heated at 100 °C for 5 or 30 min. Figure 6 shows the effect of pH and heating time on RCP of 90Y-TA138. Obviously, 30 min heating at 100 °C is needed for successful radiolabeling (RCP > 95%) if the pH is 6.0. Heating at 100 °C for 5 min is sufficient to achieve 95% RCP if the pH is 7.5. At pH > 7.5, longer heating time often results in more degradation of 90Y-TA138. We also found that 90 Y-TA138 has less degradation at pH 6.0 during storage postlabeling. 90 Y- and 177Lu-Labeling Efficiency of TA138. We studied the 90Y- and 177Lu-labeling efficiency of TA138 by determining the minimal amount of TA138 required to achieve 95% RCP for 90Y-TA138 and 177Lu-TA138. We prepared 90Y-TA138/177Lu-TA138 using 10, 20, 50, or 100 µg of TA138, 2 mg of GA, 20 mg of AA for 20 mCi of 90 YCl3/177LuCl3 in 1.0 mL of 0.5 M ammonium acetate buffer (pH ) 6.0). The heating temperature was 100 °C, and the heating time was 30 min. Figure 7 shows the effect of TA138 concentration on RCP for 90Y-TA138. At pH 6.0, the minimal amount of TA138 required to achieve

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Figure 7. Effect of the TA138 level (µg/20 mCi 90Y) on RCP of 90Y-TA138 (left) and 177Lu-TA138 (right).

Figure 8. Solution stability of 90Y-TA138 at different activity levels.

95% RCP for 90Y-TA138 is about 20 µg for 20 mCi of 90 YCl3 corresponding to a TA138:90Y ratio of ∼32:1. The same labeling efficiency can also be achieved at pH 7.5 except that the reaction mixture is heated at 100 °C for only 5 min. 177Lu-TA138 can also be prepared in high yield (RCP > 95%) under the same anaerobic conditions. Solution Stability of 90Y-TA138 at -78 °C. We prepared 90Y-TA138 using 100 µg of TA138, 2 mg of GA, 20 mg of AA, and 20 mCi of 90YCl3 in the 1.0 mL of 0.5 M ammonium acetate buffer (pH ) 6.0). For the vials containing 100 mCi and 200 mCi of activity, all the component levels were increased proportionally so that the concentration of each component in the reaction mixture is constant. The reaction mixture was heated at 100 °C for 30 min. After radiolabeling, all the vials containing 90Y-TA138 were kept in a dry ice box (-78 °C). Samples of the reaction mixture were analyzed by radio-HPLC at 0, 24, 56, and 144 h. Figure 8 shows the RCP change over 6 days for 90Y-TA138. Apparently, 90YTA138 remains stable in the kit matrix when stored at -78 °C. DISCUSSION

DOTA Conjugate Design. Angiogenesis is a requirement for tumor growth and metastasis (19-21). The angiogenic process depends on vascular endothelial cell migration and invasion and is regulated by cell adhesion receptors (22-31). The integrin Rvβ3 (vitronectin receptor) is such a cell adhesion receptor and interacts with proteins and peptides containing the RGD tripeptide sequence. A number of peptide and nonpeptide vitronectin receptor antagonists have been synthesized and studied for their antitumor activity (25-35). Radiolabeled vitronectin receptor antagonists (peptides and peptidomimetics) have been studied as new radiotracers for noninvasive tumor imaging and monitoring Rvβ3 expression (36-42).

We are interested in the quinolone-based nonpeptide vitronectin receptor antagonist (Figure 1: TA138) due to its high binding affinity and specificity for the integrin Rvβ3 receptor (18). DOTA is chosen for 177Lu- and 90Ychelation due its ability to form lutetium and yttrium complexes with high thermodynamic stability and kinetic inertness (42-46). The dicysteic acid linker (Figure 1) is used as a pharmacokinetic modifier to increase the hydrophilicity and to improve the renal clearance of the 177 Lu- and 90Y-labeled DOTA conjugates (46). Anaerobic Synthesis of 90Y-TA138. Radiopharmaceuticals comprising β-emitting radionuclides may undergo autoradiolysis during preparation and storage. Radiolysis is caused by the formation of free radicals such as hydroxyl and superoxide radicals (47) and is dependent on the concentration of the radionuclide and oxygen dissolved in the reaction mixture (48). To prevent radiolysis, a radiolytic stabilizer is often used during or/and after the radiolabeling (17, 49, 50). However, the use of the stabilizer cannot totally eliminate the oxygen dissolved in the reaction mixture. The combination of oxygen and the high-energy β-particles will result in formation of a large number of superoxide radicals, which are very reactive toward organic molecules. It should be noted that different biomolecules have different sensitivity toward radiolysis. For the 90Y-labeled cyclic peptide (90Y-SU015), it can be prepared in high yield in the presence of oxygen (16). The HPLC-purified 90 Y-SU015 remains stable for several hours in saline. For 90 Y-TA138, however, exclusion of oxygen is necessary during the radiolabeling. Addition of a stabilizer (GA or AA) in the HPLC autosampler vial is also needed to maintain the solution stability of the 90Y-TA138 sample. 90 Y- and 177Lu-Labeling Kinetics. The 90Y-chelation kinetics of DOTA conjugates is largely dependent on the radiolabeling conditions, such as the pH in the reaction mixture, reaction time, and heating temperature (46, 5156). Meares and co-workers (53) recently reported optimized conditions for 90Y-chelation of DOTA immunoconjugates. It was found that the time required to chelate 94% of 90Y was 17-148 min at pH 6.5, but it was only 1-10 min at pH 7.5 when the concentration of DOTA conjugate was 97-870 µM and 90Y concentration was in the range of 0.83-6.1 µM (53). In this study, we found that heating the reaction mixture at 100 °C for 30 min is needed to achieve 95% RCP for 90Y-TA138 if the pH is 6.0 while 5 min heating at 100 °C is sufficient if the pH is 7.5. In addition, the choice of radiolabeling conditions also depends on the chemical and radiolytic stability of the specific DOTA conjugate. A major advantage in using DOTA for 90Y- and 177Luchelation is its capability to form yttrium and lutetium chelates with extremely high solution stability (43-46). However, studies have shown that the 90Y-chelation efficiency of DOTA derivatives is much lower than that of acyclic chelators such as DTPA (51, 54). The trace metal (Ca2+, Fe2+, and Zn2+) contamination also has the more significant effect on the radiolabeling efficiency of the DOTA conjugate (51, 52, 55). Therefore, excess DOTA conjugate is often used to compensate for the presence of the trace metals and to achieve a high yield radiolabeling (RCP > 95%). As demonstrated in this study, 90YTA138 can be prepared in high yield under optimized conditions using 20 µg of TA138 for 20 mCi of 90YCl3. Although the specific activity of 177Lu (∼20 Ci/mg) is much lower than that of 90Y (∼500 Ci/mg, carrier free), 177 Lu-TA138 can be prepared in high yield (RCP > 95%) using the same anaerobic formulation.

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In addition to the anaerobic condition, a stabilizer is needed during the radiolabeling. In our previous communication (16), we found that the GA level has minimal effect on the RCP of 90Y-SU015. In this study, however, we found that a high level of GA (20 mg/20 mCi) gives lower RCP for 90Y-TA138. Therefore, we have to use a combination of 2 mg of GA and 20 mg of AA for 20 mCi of 90Y. We also noticed that GA and ammonium acetate buffer can be totally replaced with AA (20-100 mg/mL or 100-500 mM) due to the fact that AA is not only a radiolytic stabilizer but also a buffer agent at pH 4-6. That led to the discovery of the anaerobic AA formulation, details of which will be reported as a separate communication (57). Controlling pH is very important in the manufacturing process in order to have consistency and reproducibility. Although ammonium acetate has been used as a buffer agent for radiolabeling of biomolecules, including antibodies (44, 50, 51) and small peptides (14-17, 56), its buffer capacity is low if the pH is > 6.0. Therefore, a better buffer agent is needed for the 90Y-labeling of a DOTA conjugate at pH 6.0-8.0. In this study, we tested tricine (pKa ) 8.15 at 20 °C) and TRIS (pKa ) 8.3 at 20 °C), both of which have been used in various pharmaceutical compositions (58). Results from radiolabeling experiments show that TRIS is comparable to ammonium acetate as a buffer agent for the 90Y-labeling of the DOTA conjugate while tricine is not a good buffer agent due to its stronger chelating capability. Ammonium citrate has also been reported to interfere with the 90Y-labeling of DOTA immunoconjugates (53). Solution Stability of 90Y-TA138. Unlike diagnostic radiopharmaceuticals, therapeutic radiopharmaceuticals have to be manufactured and released under GMP (good manufacturing practice) conditions and delivered for clinic applications. Therefore, the new therapeutic radiopharmaceutical must retain its chemical and biological integrity during release and transportation. In this study, we used GA and AA as radiolytic stabilizers in the anaerobic formulation and stored 90Y-TA138 at low temperature (-78 °C/dry ice). Under these conditions, 90 Y-TA138 remains stable for at least two half-lives of 90 Y. CONCLUSIONS

In this study, we describe the discovery and development of an anaerobic formulation for the 90Y- and 177Lulabeling of a DOTA-conjugated nonpeptide vitronectin receptor antagonist (TA138). It was found that difference biomolecules have different sensitivity toward radiolytic degradation and require different radiolabeling conditions. Using the anaerobic formulation, 90Y-TA138 can be prepared in high yield and high specific activity (20 µg of TA138 for 20 mCi of 90Y corresponding to a TA138: 90 Y ratio of ∼30:1). 177Lu-TA138 can be prepared in high yield using the same anaerobic formulation. 90Y-TA138 remains stable in the formulation matrix when stored at -78 °C. The amount of radiolytic stabilizer used in the formulation and storage temperature should be adjusted according to the sensitivity of a specific radiolabeled DOTA conjugate toward radiolytic decomposition. The anaerobic formulation described in this study is extremely useful for 90Y- and 177Lu-labeling of DOTAconjugated small biomolecules, which are sensitive to the radiolytic degradation during radiolabeling. LITERATURE CITED (1) Liu, S., Edwards, D. S., Looby, R. J., Harris, A. R., Poirier, M. J., Barrett, J. A., Heminway, S. J., and Carroll, T. R. (1996)

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