An estradiol-conjugate for radiolabelling with 177Lu: an attempt to prepare a radiotherapeutic agent

177Lu is presently being considered as one of the most promising radionuclide for targeted therapy owing to its suitable decay characteristics. 177Lu in high radionuclidic purity (99.99%) and moderate specific activity (100-110 TBq/g) was produced using enriched (60.6% 176Lu) Lu2O3 target. The macrocycle 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is known to form stable complexes with lanthanides. Herein, we describe a novel attempt to introduce 177Lu in the estradiol moiety through a steroidal-BFCA (Bifunctional Chelating Agent) conjugate. The preparation of a steroid conjugate via coupling of 6alpha-amino-17beta-estradiol with a C-functionalized DOTA derivative viz. p-NCS-benzyl-DOTA as a BFCA and thereafter the radiolabelling of the conjugate with 177Lu is reported. Biological activity of the resultant estradiol-DOTA conjugate after radiolabelling was studied by carrying out preliminary in vitro cell uptake studies with MCF-7, human breast carcinoma cell line expressing estrogen receptors as well as binding studies with anti-estradiol antibodies.     

Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.     

Radiosynthesis and evaluation of a 99mTc-folic acid radiotracer prepared using [99mTcN(PNP)]2+ metal fragment

Folate receptors (FR) are over-expressed on a wide variety of tumor cells and are a potential molecular target for radiolabeled folates. In this respect, several SPECT and PET based radiofolates have been evaluated in the past albeit with their high renal uptake posing limitation towards their clinical use. To overcome this, a new ^99mTc labeled folic acid was synthesized via the use of [^99mTcN(PNP)]²⁺ metal fragment, where the presence of the latter pharmacophore redirects in vivo clearance via the hepatobiliary pathway. In this respect, folic acid was derivatized at the γ-acid group with a cysteine BFCA (bifunctional chelating agent) and subsequently reacted with the preformed [^99mTcN]²⁺ intermediate in presence of PNP2 (bisphosphine) ligand, to yield the final complex. While preliminary, in vivo distribution of the complex exhibited high association of activity with liver and intestines and provided support to the rationality of the present design as clearance of labeled folic acid could be effected via the hepatic route, the in vitro studies of the folic acid-cysteine conjugate carried out in KB-31 cells, did not show much promise with reduction in receptor affinity in comparison with the native folic acid. The route followed herein to prepare a folic-acid based radiotracer constitutes the first report of radiolabeling folic acid using the [^99mTcN(PNP)]²⁺ as a radiosynthon. Modification in the structure of conjugate by linking the BFCA through a long-chain linker can be envisaged to improve the affinity of [^99mTcN(PNP)]-folic acid complex towards FRs.     

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V)beta(3) integrin for tumor imaging

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.     

Expression of airway secretory epithelial functions by lung carcinoma cells

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.     

Poly(ethylene glycol)-conjugated anti-EGF receptor antibody C225 with radiometal chelator attached to the termini of polymer chains.

Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, S-acetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimide-activated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225's binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH(2) substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using (111)In-labeled compounds revealed narrower steady-state distribution of (111)In-DTPA-PEG-C225 than (111)In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t(1/2,)(gamma)) of (111)In-DTPA-PEG-C225, 21.1 h, was shorter than that of (111)In-DTPA-C225, 52.9 h. These data suggest that (111)In-DTPA-PEG-C225 may provide better imaging characteristics than (111)In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents.     

Polymer-supported organotin reagent for prosthetic group labeling of biological macromolecules with radioiodine

In this study, we investigated the use of poly-mer-bound precursor for generating a radiolabeled prosthetic group to be used for conjugate labeling of biological macromolecules. For the approach, a trialkyltin chloride in which the tin was bound to a hydrophilic PEG-based resin support via one of the alkyl groups was synthesized. This resin was then used to prepare a resin-bound trialkyltin benzoic acid, which in some cases was further derivatized on-resin by converting it to a succinimidyl ester. Exposure of the resin-bound compounds to electrophilic radioiodine (12?I) in either an aqueous or methanol solvent liberated either free radiolabeled [12?I]iodobenzoic acid or its succinimidyl ester without co-release of the resin-bound precursors. Radiochemical yield was between 35% and 75%, depending on the solvent system and precursor. As example applications for the released compounds, the amine-reactive N-succinimidyl-[12?I]iodobenzoate prosthetic group was used for conjugate radiolabeling of a peptide, tomato plant systemin, and two proteins, albumin and IgG antibody. These results demonstrate that resin-bound organotin precursors in which the compound to be labeled is tethered to the support via the tin group to be substituted can be used to produce radioiodine-labeled aromatic prosthetic groups in good specific activity without the need for HPLC purification. This solid-phase approach is potentially adaptable to kit-formulation for performing conjugate radiolabeling of biological macromolecules.     

Thrombospondin binding by human squamous carcinoma and melanoma cells: relationship to biological activity

Human squamous carcinoma cells attach and spread on thrombospondin (TSP)-coated culture dishes but exhibit significant variability among individual cell lines in their degree of responsiveness. Using a highly responsive squamous carcinoma line and a cell line which is much less responsive (as well as a human melanoma cell line which does not respond at all in the adhesion assay), we have examined binding of exogenous radiolabeled TSP. The cells which were the most responsive to TSP in the adhesion assay bound the greatest amount of radiolabeled ligand. Binding was time- and dose-dependent, saturable, inhibitable with excess unlabeled TSP, reversible, and specific. The less-responsive squamous carcinoma cells bound only 25-30% of the amount of TSP bound by the highly responsive cells while the nonresponsive melanoma cells bound less than 10% of the amount bound by the highly responsive squamous carcinoma cells. Our previous studies (J. Varani et al. (1986) Exp. Cell Res. 167, 376) have shown that the highly responsive squamous carcinoma cells also synthesized the greatest amount of TSP as indicated by biosynthetic labeling studies. The less-responsive squamous carcinoma cells were intermediate in synthetic activity and no synthetic activity was seen with the melanoma cells. These findings suggest that the amount of ligand bound may determine the degree of biological responsiveness and that endogenously synthesized TSP may be the source of that ligand.     

RGD targeted poly(L-glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting angiogenesis biomarker alpha(v) beta3 integrin with MRT, mapping

Cyclic Arg-Gly-Asp-D-Phe-Lys [c(RGDfK)] targeted poly(L-glutamic acid) (PGA)-(Gd-DO3A) conjugate with a biodegradable cystamine spacer was prepared and evaluated for in vivo detection of an angiogenesis biomarker, alpha(v)beta3 integrin, in neoplastic tissues with T1 mapping, a quantitative magnetic resonance imaging (MRI) technique. The binding activity of the c(RGDfK) containing conjugate was investigated using in vitro vitronectin assay with human prostate carcinoma DU145 cell line and Kaposi's sarcoma SLK cell line. The peptide c(RGDfK) and PGA-cystamine-(Gd-DO3A) conjugate were used as controls. The binding affinity of polymer bound c(RGDfK) was slightly lower than free c(RGDfK) peptide. The RGD targeted conjugate had higher binding affinity to the DU145 cells than the SLK cells, which was consistent to free c(RGDfK). The imaging of alpha(v)beta3 integrin with targeted PGA-cystamine-(Gd-DO3A) was evaluated in nude mice bearing DU145 and SLK xenografts at a dose of 5 micromol-Gd/kg. The targeted conjugate demonstrated higher in vivo binding affinity to the DU145 xenografts than the SLK xenografts, resulting in a significant decrease of T1 values of water protons in the periphery of the DU145 tumors as shown in the MR T1 maps. No significant decrease of T1 values was observed in the SLK tumor with the targeted conjugate and in both tumors with the non-targeted conjugate. The targeted polymeric Gd(III) chelate conjugate with a degradable spacer has the potential to be a new paradigm for safe and effective probes in molecular imaging with quantitative MR T1 mapping.     

High affinity binding of VIP to human lung cancer cell lines

The binding of ¹²⁵I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. ¹²⁵I-VIP bound to membranes in a specific and time dependent manner. ¹²⁵I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP^10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer.     

Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.     

Recombinant complement receptor 2 radiolabeled with [99mTc(CO)3]+: a potential new radiopharmaceutical for imaging activated complement

We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [(99m)Tc(CO)(3)(OH(2))(3)](+)). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d(+) red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d(+), in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a K(D)≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [(99m)Tc(CO)(3)(OH(2))(3)](+) at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d(+) RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d(+) RBCs. We conclude that rCR2-Tc(99m) has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

Stabilization of (90)y-labeled DOTA-biomolecule conjugates using gentisic acid and ascorbic acid.

Radiolytic degradation of radiolabeled compounds is a major challenge for the development of new therapeutic radiopharmaceuticals. The goal of this study is to explore the factors influencing the solution stability of a (90)Y-labeled DOTA-peptide conjugate (RP697), including the amount of total activity, the activity concentration, the stabilizer concentration, and the storage temperature. In general, the rate of radiolytic decomposition of RP697 is much slower at the lower activity concentration (<4 mCi/mL) than that at the higher concentration (>10 mCi/mL). RP697 remains relatively stable at the 20 mCi level and room temperature while it decomposes rapidly at the 100 mCi level under the same storage conditions. Radical scavengers, such as gentisic acid (GA) and ascorbic acid (AA), were used in combination with the low temperature (-78 degrees C) to prevent the radiolytic decomposition of RP697. It was found that RP697 remains stable for at least 2 half-lives of (90)Y when GA or AA (10 mg for 20 mCi of (90)Y) is used as a stabilizer when the radiopharmaceutical composition is stored at -78 degrees C. The stabilizer (GA and AA) can be added into the formulation either before or after radiolabeling. The post-labeling approach is particularly useful when the use of a large amount of the stabilizer interferes with the radiolabeling. The radiopharmaceutical composition developed in this study can also apply to other (90)Y-labeled DOTA-biomolecule conjugates. The amount of the stabilizer used in the radiopharmaceutical composition and storage temperature should be adjusted according to the sensitivity of the radiolabeled DOTA-biomolecule conjugate toward radiolytic decomposition.     

A specific, high-affinity binding site for the hepta-beta-glucoside elicitor exists in soybean membranes.

The presence of a specific binding site for a hepta-beta-glucoside elicitor of phytoalexin accumulation has been demonstrated in soybean microsomal membranes. A tyramine conjugate of the elicitor-active hepta-beta-glucoside was prepared and radiolabeled with 125I. The labeled hepta-beta-glucoside-tyramine conjugate was used as a ligand in binding assays with a total membrane fraction prepared from soybean roots. Binding of the radiolabeled hepta-beta-glucoside elicitor was saturable, reversible, and with an affinity (apparent Kd = 7.5 x 10(-10) M) comparable with the concentration of hepta-beta-glucoside required for biological activity. A single class of hepta-beta-glucoside binding sites was found. The binding site was inactivated by proteolysis and by heat treatment, suggesting that the binding site is a protein or glycoprotein. Competitive inhibition of binding of the radiolabeled hepta-beta-glucoside elicitor by a number of structurally related oligoglucosides demonstrated a direct correlation between the binding affinities and the elicitor activities of these oligoglucosides. Thus, the hepta-beta-glucoside-binding protein fulfills criteria expected of a bona fide receptor for the elicitor-active oligosaccharin.     

The role of glutathione conjugate metabolism and cysteine conjugate beta-lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PK1 cells

A cell line derived from pig kidney, LLC-PK1, was grown in a culture system in which the cells express morphological and biochemical characteristics of the proximal tubule. This model was used to investigate the mechanism of S-cysteine conjugate toxicity and the role of glutathione conjugate metabolism. LLC-PK1 cells have the degradative enzymes of the mercapturate pathway, and S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-glutathione are toxic. S-(1,2-Dichlorovinyl)-L-glutathione is not toxic when the cells are pretreated with AT-125, an inhibitor of gamma-glutamyl transpeptidase. The cells respond to a variety of toxic cysteine conjugates. Cysteine conjugate beta-lyase activity is not detectable by standard assays, but can be measured using radiolabeled S-(1,2-dichlorovinyl)-L-cysteine. Pyruvate stimulates the beta-elimination reaction with S-(1,2-dichlorovinyl)-L-cysteine as substrate 2-3-fold. The data suggest that a side transamination reaction regulates the flux of substrate through the beta-elimination pathway; therefore, cysteine conjugate beta-lyase in LLC-PK1 cells may be regulated by transamination, and measurement of lyase activity in some systems may require the presence of alpha-ketoacids. Aminoxyacetic acid blocks both the metabolism of S-(1,2-dichlorovinyl)-L-cysteine to a reactive species which covalently binds to cellular macromolecules and toxicity. Glutathione inhibits the binding of the sulfur containing cleavage fragment to acid insoluble material in vitro. The data provide direct evidence that S-(1,2-dichlorovinyl)-L-cysteine is metabolized to a reactive species which covalently binds to cellular macromolecules, and the binding is proportional to toxicity.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.