Identification of the Soluble in Vivo Metabolites of Indium-111-Diethylenetriaminepentaacetic Acid-D-Phe 1 -Octreotide Laura A. Bass, † Margaret V. Lanahan, † James R. Duncan, † Jack L. Erion, ‡ Ananth Srinivasan, ‡ Michelle A. Schmidt, ‡ and Carolyn J. Anderson* ,† Division of Radiological Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, and Mallinckrodt Inc., St. Louis, Missouri 63134. Received August 13, 1997; Revised Manuscript Received December 23, 1997 Indium-111-diethylenetriaminepentaacetic Acid-D-phenylalanine 1 -octreotide ( 111 In-DTPA-octreotide) is a cyclic eight amino acid somatostatin analogue which is approved for gamma scintigraphy of neuroendocrine tumors. To address the factors that contribute to liver and kidney retention of this radiopharmaceutical, its metabolism was evaluated in normal and tumor-bearing rats. The soluble fractions from nontarget (liver and kidney) and target (tumor, pancreas, adrenals) organ homogenates were analyzed out to 21 h postinjection, and urine was analyzed out to 12 h postinjection. The blood was analyzed at shorter time intervals due to the rapid clearance of 111 In-DTPA-octreotide. Radio- TLC and HPLC were used to analyze organ homogenates, blood, and urine. By TLC, intact 111 In- DTPA-octreotide was resolved from the soluble metabolites, and a similar apparent rate of metabolism was observed in the liver, kidney, tumor, and pancreas with ∼30% intact 111 In-DTPA-octreotide at 4 h postinjection. In the adrenals, metabolism occurred more slowly with ∼60% intact 111 In-DTPA- octreotide at 4 h postinjection. At 4 h postinjection, the activity excreted in the urine consisted of 85% intact 111 In-DTPA-octreotide. HPLC provided resolution of the individual extractable metabolites. In an attempt to identify these metabolites, two DTPA-amino acid sequences were synthesized: DTPA- D-Phe-Cys and DTPA-D-Phe. Under the conditions used for metabolite analysis, 111 In-DTPA-D-Phe- Cys-OH eluted at 14.6 min and 111 In-DTPA-D-Phe-OH eluted at 7.0 min. Each of these standard sequences was combined with the soluble portion of the organ homogenate and was shown by HPLC to coelute with the metabolites. These data suggest that 111 In-DTPA-octreotide was initially degraded to 111 In-DTPA-D-Phe-Cys-OH and 111 In-DTPA-D-Phe-OH. The 111 In-DTPA-D-Phe-Cys-OH was further degraded to 111 In-DTPA-D-Phe-OH, which appeared to be the final metabolite that was extracted from the organs. From these results, it can be concluded that at longer time points (>2 h postinjection) a significant amount of 111 In was retained in nontarget organs as 111 In-DTPA-D-Phe-OH and 111 In-DTPA- D-Phe-Cys-OH and not as intact 111 In-DTPA-octreotide. INTRODUCTION Somatostatin is a naturally occurring peptide hormone consisting of 14 or 28 amino acids. It is present in the hypothalamus, the cerebral cortex, the brain stem, the gastrointestinal tract, and the pancreas and exerts an inhibitory effect on several cell functions such as secretion of peptide hormones and growth factors (1-4). Neuroen- docrine tumors have been found to have an increased number of somatostatin receptors. Other common types of tumors also contain somatostatin receptors, such as those of the CNS, breast, and lung (5-10). The clinical value of somatostatin is limited due to its very short half- life in vivo (t 1/2 < 3 min) (11). Octreotide, an eight amino acid peptide, is an analogue of somatostatin that is highly resistant to degradation by enzyme attack and more effective than the native hormone in the suppression of growth hormone secretion (12). Somatostatin analogues, including octreotide have been labeled with a variety of radiometals such as 111 In (13, 14), 64 Cu (15), 67/68 Ga (16, 17), 90 Y(18), and 188 Re (19) for targeted radiotherapy or for imaging of somatostatin receptor positive tumors by SPECT or PET. Radiometals are linked to octreotide via a bifunctional chelate. The bifunctional chelate contains a functional group through which it is conjugated to octreotide. The chelate is chosen such that it binds the metal strongly to prevent release of the metal in vivo. The biodistribution of a variety of octreotide conjugates has been investigated in tumor- bearing rodents, and although receptor-mediated tumor uptake was observed, many of these agents exhibited high nontarget uptake in the kidney and liver. To alleviate nontarget accumulation, research efforts have focused on altering the stability of the metal-chelate complex or the chemistry of the chelate-octreotide bond. Understanding the mechanism of uptake, degradation and retention of octreotide conjugates is key to designing optimal somatostatin analogues. Since 111 In-DTPA-oct- reotide 1 (Figure 1) is clinically used for the scintigraphic imaging of neuroendocrine tumors (20-23) and, more recently, has been studied for its therapeutic efficacy (11, * Corresponding author. Division of Radiological Sciences, 510 S. Kingshighway, Box 8225, St. Louis, MO 63110. Phone: 314- 362-8427. Fax: 314-362-9940. E-mail: andersoncj@ mirlink.wustl.edu. † Washington University School of Medicine. ‡ Mallinckrodt, Inc. 192 Bioconjugate Chem. 1998, 9, 192-200 S1043-1802(97)00158-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/14/1998