1. Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs
W B Edwards, C G Fields, C J Anderson, T S Pajeau, M J Welch, G B Fields J Med Chem. 1994 Oct 28;37(22):3749-57. doi: 10.1021/jm00048a011.
Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. We have developed a generally applicable, convenient stepwise solid-phase synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-threoninol). [Cys(Acm)2,D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6,Cys(Acm)7, des(threoninol)]-octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [Tl(Tfa)3]. Side-chain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)3 treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP-HPLC. Isolated [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide had the correct molecular mass ([M+H]+ = 1275 Da) and sequence and was obtained in 14% yield at > 98% purity. [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide was utilized for the solution-phase synthesis of CPTA-D-Phe1-octreotide, where CPTA is 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid. Cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA) was coupled to a portion of the protected peptide-resin following disulfide bond formation. The DTPA-conjugated, side-chain-protected peptide was cleaved from the resin by aminolysis with threoninol, side-chain deprotected with trifluoroacetic acid, and purified by semipreparative RP-HPLC. The isolated DTPA-D-Phe1-octreotide had the correct molecular mass ([M+H]+ = 1395 Da) and was obtained in 5% yield at > 90% purity. The efficiency of aminolysis was partially dependent upon the linkage between 4-(hydroxymethyl)phenoxy (HMP) handles and the resin and/or resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-Phe1-octreotide and CPTA-D-Phe1-octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with 111In and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant (Kd) for our synthetic [111In]DTPA-D-Phe1-octreotide was 4.31 nM, which is comparable to a Kd = 5.57 nM obtained with commercially available DTPA-D-Phe1-octreotide. The Kd for [64Cu]CPTA-D-Phe1-octreotide was 78.5 pM. On the basis of the criteria of molecular mass, RP-HPLC elution time, sequence analysis, and somatostatin receptor binding affinity, our synthetic octreotide is identical to commercially available octreotide. The aminolysis protocol used here has distinct advantages over either reductive cleavage or preformed linker methods described previously for the preparation of octreotide.
2. Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium-111 labeling of polypeptides using a newly synthesized monoreactive DTPA derivative
Y Arano, T Uezono, H Akizawa, M Ono, K Wakisaka, M Nakayama, H Sakahara, J Konishi, A Yokoyama J Med Chem. 1996 Aug 30;39(18):3451-60. doi: 10.1021/jm950949+.
Previous studies on indium-111 (111In) labeling of polypeptides and peptides using cyclic diethylenetriaminepentaacetic dianhydride (cDTPA) as a bifunctional chelating agent (BCA) have indicated that DTPA might be a useful BCA for 111In labeling of polypeptides at high specific activities when DTPA can be incorporated without inducing intra- or intermolecular cross-linking. To investigate this hypothesis, a monoreactive DTPA derivative with a maleimide group as the peptide binding site (MDTPA) was designed and synthesized. A monoclonal antibody (OST7, IgG1) was used as a model polypeptide, and conjugation of MDTPA with OST7, 111In radiolabeling of MDTPA-OST7, and the stability of 111In-MDTPA-OST7 were investigated using cDTPA and benzyl-EDTA derivatives as references. SDS-PAGE analysis demonstrated that while cDTPA induced intramolecular cross-linking, no such undesirable side reactions were observed with MDTPA. MDTPA generated 111In-labeled OST7 with high radiochemical yields at higher specific activities than those produced using cDTPA and benzyl-EDTA derivatives as the BCAs. Incubation of each 111In-labeled OST7 in human serum indicated that MDTPA generated 111In-labeled OST7 of much higher and a little lower stability than those derived from cDTPA and benzyl-EDTA derivatives, respectively. These findings indicated that the low in vivo stability of cDTPA-conjugated antibody reported previously is not attributable to low stability of 111In-DTPA but to formation of intramolecular cross-linking during cDTPA conjugation reactions. The present study also indicated that MDTPA and its precursor, the tetra-tert-butyl derivative of DTPA, would be useful BCAs for 111In radiolabeling of polypeptides that have rapid blood clearance with high specific activities.
3. Renal metabolism of 111In-DTPA-D-Phe1-octreotide in vivo
H Akizawa, Y Arano, T Uezono, M Ono, Y Fujioka, T Uehara, A Yokoyama, K Akaji, Y Kiso, M Koizumi, H Saji Bioconjug Chem. 1998 Nov-Dec;9(6):662-70. doi: 10.1021/bc9702258.
The persistent localization of radioactivity in the kidney after administration of 111In-DTPA-D-Phe1-octreotide impairs the diagnostic accuracy of this radiopharmaceutical. To better understand the mechanisms responsible for the renal radioactivity levels of 111In-DTPA-D-Phe1-octreotide, the renal metabolism of this compound was compared with 111In-DTPA-L-Phe1-octreotide, where the N-terminal D-phenylalanine was replaced with L-phenylalanine to facilitate metabolism. DTPA-D-Phe1-octreotide and DTPA-L-Phe1-octreotide were synthesized by solid-phase methods. Both 111In-DTPA-conjugated octreotide analogues were prepared with radiochemical yields of over 96%, and both remained stable after a 3 h incubation in murine serum at 37 degreesC. When injected into mice, the two 111In-DTPA-conjugated octreotide analogues showed similar radioactivity elimination rates from the blood and accumulation in the kidney with about 60% injected radioactivity being excreted in the urine by 24 h postinjection. Over 85% of the radioactivity in the urine existed as intact peptides for both analogues. Despite the similar renal radioactivity levels, significant differences were observed in the radiolabeled species remaining in the kidney between the two; while 111In-DTPA-L-Phe1-octreotide was rapidly metabolized to the final radiometabolite, 111In-DTPA-L-Phe, the metabolic rate of 111In-DTPA-D-Phe1-octreotide was so slow that various intermediate radiolabeled species were observed. However, both 111In-DTPA-D-Phe and 111In-DTPA-L-Phe remained in the lysosomal compartment of the renal cells as the final radiometabolites for long periods. These findings indicated that although the metabolic stability of 111In-DTPA-D-Phe1-octreotide in the renal cells may be partially involved, the slow elimination rate of the radiometabolite derived from 111In-DTPA-D-Phe1-octreotide from the lysosomal compartment of renal cells would be predominantly attributable to the persistent renal radioactivity levels of 111In-DTPA-D-Phe1-octreotide.
