Tomoya Uehara

Effect of molecular charges on renal uptake of 111In-DTPA-conjugated peptides

The effect of molecular charges on renal accumulation of 111In-DTPA-labeled low molecular weight (LMW) peptides was investigated using 111In-DTPA-octreotide derivatives as models to design radiolabeled peptides that are taken up less by renal cells. The N-terminal D-phenylalanine (Phe) of 111In-DTPA-D-Phe(1)-octreotide was replaced with L-aspartic acid (Asp), L-lysine (Lys), L-methionine (Met) or L-Phe. Cellulose acetate electrophoresis indicated that both 111In-DTPA-L-Phe(1)-octreotide and 111In-DTPA-L-Met(1)-octreotide showed similar net charges, whereas 111In-DTPA-L-alphaLys(1)-octreotide and 111In-DTPA-L-Asp(1)-octreotide had more positive and negative charges, respectively, at pH values similar to those in blood and glomerular filtrate. When injected into mice, significant differences were observed in the renal radioactivity levels. 111In-DTPA-L-alphaLys(1)-octreotide showed the highest radioactivity levels from 10 min to 6 h postinjection, whereas the lowest radioactivity levels were observed with 111In-DTPA-L-Asp(1)-octreotide at all the postinjection intervals. These findings indicated that the replacement of only one amino acid in 111In-DTPA-D-Phe(1)-octreotide significantly altered net molecular charges of the resulting peptides and that the net charges of the 111In-DTPA-octreotide derivatives significantly affected their renal uptake. Thus, an increase of negative charges in peptide molecules may constitute a strategy for designing 111In-DTPA-conjugated LMW peptides with low renal radioactivity levels.

Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins

Radiolabeled anti-CD20 antibodies have demonstrated impressive efficacy in the treatment of relapsed non-Hodgkin lymphoma. This encourages the treatment of solid tumor with radiolabeled antibody fragments and peptides. However, both preclinical and clinical studies revealed that persistent localization of radioactivity in the kidney constitutes a major obstacle that compromises therapeutic efficacy. Recent extensive studies show that long residence times of radiolabeled end products from lysosomes are responsible for the renal radioactivity levels. Recent studies have also elucidated the involvement of megalin-cubilin in renal tubular reabsorption of radiolabeled antibody fragments and peptides. In light of these findings, efforts are being made to block tubular reabsorption of radiolabeled antibody fragments and peptides by competitive inhibitors, charge modification, and PEGylation. An interposition of an enzyme-cleavable linkage between antibody fragments and radiolabels would constitute an alternative approach to reduce renal radioactivity levels. Recent findings of these studies will be described.

Detection of Experimental Autoimmune Myocarditis in Rats by 111In Monoclonal Antibody Specific for Tenascin-C

Background—Although the identification of inflammatory infiltrates in endomyocardial biopsy specimens is necessary for the definite diagnosis of myocarditis, the biopsy test is invasive and is not sensitive. Therefore, a new diagnostic technique for the early and noninvasive evaluation of myocarditis has been awaited. Expression of tenascin-C (TNC), one of the oligometric extracellular glycoproteins, is induced in various pathological states, including inflammation, suggesting that TNC can be a molecular marker of myocarditis. Methods and Results—An 111In anti-TNC monoclonal antibody Fab′ fragment was injected intravenously into rats with experimental autoimmune myocarditis (EAM), and the biodistribution of this radiotracer was measured. Rapid clearance of radioactivity from the blood was observed in both EAM and control rats (<1% at 6 hours after injection). Myocardial uptake of the tracer was much higher in EAM rats than in control rats (7.54-, 4.39-, and 3.51-fold at 6, 24, and 48 hours after injection, respectively). By autoradiography, high radioactivities were clearly observed in the regions indicative of inflammation in EAM rats. Single-photon emission CT imaging demonstrated the focal myocardial uptake of 111In anti-TNC Fab′ in vivo. Conclusions—Radiolabeled anti-TNC Fab′ may be useful for the noninvasive diagnosis of myocarditis.

Plasma protein binding of 99mTc-labeled hydrazino nicotinamide derivatized polypeptides and peptides

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine coligands with plasma proteins.

99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ((99m)Tc)-labeled polypeptides with tricine as a coligand. However, (99m)Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [(99m)Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare (99m)Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [(99m)Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [(99m)Tc](HYNIC-Fab)(tricine)(2.) When injected into mice, [(99m)Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [(99m)Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [(99m)Tc](HYNIC-NGA)(tricine)(2.) In addition, [(99m)Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [(99m)Tc](HYNIC-NGA)(tricine)(2.) Similar results were observed with (99m)Tc-labeled Fab fragments where [(99m)Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [(99m)Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [(99m)Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [(99m)Tc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging.

γ-Glutamyl PAMAM Dendrimer as Versatile Precursor for Dendrimer-Based Targeting Devices

Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a gamma-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were gamma-glutamylated to prepare Glu-P (pI 7.1). The alpha-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or Suc-Glu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[(125)I]I. When injected intravenously to mice, both [(125)I]Ac-P and [(125)I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [(125)I]Phe-P. Both [(125)I]Glu-P and [(125)I]Ac-Glu-P showed residence times in the blood similar to those of [(125)I]Ac-P and [(125)I]Suc-P. However, [(125)I]Glu-P also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [(125)I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that gamma-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of alpha-amine residues available for conjugation of targeting molecules and drugs/imaging agents.

C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model.

INTRODUCTION Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. METHODS A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. (125)I- and (111)In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. RESULTS Both (125)I- and (111)In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10(9) M(-1)). Internalization assay showed that (125)I-labeled antibodies were rapidly internalized and dehalogenated, with the release of (125)I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, (111)In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of (125)I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of (111)In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of (111)In-labeled antibody. CONCLUSION The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

Antiproliferative activity induced by the somatostatin analogue, TT-232, in human pancreatic cancer cells

Somatostatin analogues have been developed as antiproliferative agents, but their administration as general antitumour agents is limited, mainly because of the wide distribution of somatostatin receptors throughout the human body. TT-232, a new somatostatin structural analogue, was reported to have tumour-selective antiproliferative activity without an antisecretory action. We examined whether TT-232 had antiproliferative activity in human pancreatic cancer cell lines, and compared its antiproliferative activity with that of RC-160 and other TT-232 derivatives. TT-232 inhibited the growth of all of the cell lines used in this study and induced apoptotic cell death. RC-160 showed no such growth inhibition. TT-232 also inhibited tumour formation in a xenograft model. A competitive binding assay was performed using the cell membrane fraction and 111In-DTPA-TT-232 in order to show the existence of a specific binding site on the cells. A specific binding site was detected in MIAPaCa-2 cells. It has been shown that the activation of protein tyrosine phosphatase (PTPase) is one of the main intracellular pathways responsible for somatostatinergic inhibition of cell growth. We found a significant PTPase stimulation after TT-232 administration using an immunoblot analysis assessing the level of protein tyrosine phosphorylation, and also a direct measurement of the PTPase activity. We also demonstrated that PTPase stimulation by TT-232 was involved in its antiproliferative activity as this activity was reversed by the addition of sodium orthovanadate, a PTPase inhibitor. Our results indicate that TT-232 could be a potentially useful therapeutic agent if these data are translated into clinical practice.

Significance of 111In-DTPA chelate in renal radioactivity levels of 111In-DTPA-conjugated peptides

Metabolic studies of (111)In-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated (111)In-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated. However, a recent study suggested that metal-chelate-methionine (Met) might possess in vivo behaviors different from metal-chelate adducts of other amino acids. In this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of (111)In-DTPA-adduct of Met into urine, (111)In-DTPA-Met(1)-octreotide was synthesized and the renal handling of (111)In-DTPA-Met was investigated using (111)In-DTPA-L-Phe(1)-octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both (111)In-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37 degrees C. Both (111)In-DTPA-octreotide analogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. No significant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studies indicated that (111)In-DTPA-Met(1)-octreotide and (111)In-DTPA-L-Phe(1)-octreotide generated (111)In-DTPA-adducts of Met and Phe, respectively, as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues after administration of (111)In-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of (111)In-DTPA chelate.

Elacridar enhances the cytotoxic effects of sunitinib and prevents multidrug resistance in renal carcinoma cells

Intrinsic drug resistance occurs in many renal carcinomas and is associated with increased expression of multidrug resistant proteins, which inhibits intracellular drug accumulation. Multidrug resistant protein 1, also known as P-glycoprotein, is a membrane drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC Sub-family B Member 2 (ABCG2) is widely distributed and is involved in the multidrug resistant phenotype. Sunitinib is a tyrosine kinase inhibitor used to treat kidney cancer that disrupts signaling pathways responsible for abnormal cancer cell proliferation and tumor angiogenesis. Multiple drug resistance is important in tyrosine kinase inhibitor-induced resistance. We hypothesized that inhibition of multidrug resistant transporters by elacridar (dual inhibitor of P-glycoprotein and ABCG 2) might overcome sunitinib resistance in experimental renal cell carcinoma. Human renal carcinoma cell lines 786-O, ACHN, and Caki-1 were treated with sunitinib or elacridar alone, or in combination. We showed that elacridar significantly enhanced sunitinib cytotoxicity in 786-O cells. P-glycoprotein activity, confirmed by P-glycoprotein function assay, was found to be inhibited by elacridar. ABCG2 expression was low in all renal carcinoma cell lines, and was suppressed only by combination treatment in 786-O cells. ABCG2 function was inhibited by sunitinib alone or combination with elacridar but not elacridar alone. These findings suggest that sunitinib resistance involves multidrug resistance transporters, and in combination with elacridar, can be reversed in renal carcinoma cells by P-glycoprotein inhibition.