Biomedical subjects

M Rusckowski

Publications and source records attributed to M Rusckowski.

At least 19 recordsLinked to original sources

PubMed2002

The influence of chain length and base sequence on the pharmacokinetic behavior of 99mTc-morpholinos in mice.

BACKGROUND: Despite in vivo use now over several years, in particular for nuclear medicine imaging, the influences on pharmacokinetics of chain length and base sequence of radiolabeled oligomers has not been investigated. METHODS: As test oligomer, morpholinos (MORFs), a DNA analogue, were radiolabeled with 99mTc via MAG3 and the pharmacokinetics in normal mice determined for 3 chain lengths (15, 18 and 25 mer) and 2 base sequences (MORF and its complement cMORF). In addition, LS174T-tumor bearing nude mice received the anti-CEA antibody MN14 (Immunomedics) conjugated either with MORF15 or MORF18 and subsequently received 99mTc-labeled cMORF15 or cMORF18 respectively in a pretargeting strategy. RESULTS: In normal mice, after 1 hr, regardless of chain length or sequence, all labeled MORFs and cMORFs accumulated only slightly in all tissues (e.g. at 3 hr <0.15 ID%/g) except in kidneys. Besides being excessive, the kidneys were the only tissue with levels dependent upon chain length (e.g. at 1 hr, 5, 7 and 22 ID%/g for MORF15, 18 and 25, respectively) and sequence (e.g. at 3 hrs 9 ID%/g for MORF25 and 21 ID%/g for cMORF25). Identical biodistribution trends were observed in tumored mice with all tissues including tumor showing levels independent of chain length or base sequence except for kidneys. Furthermore, while all other tissues cleared in the interval from 1-3 hrs, kidney levels remained constant in both normal and tumored animals. Largely because of these differences in kidneys, images obtained by pretargeting with 99mTc-cMORF15 were superior compared to 99mTc-cMORF18 (images of control animals not receiving the antibody showed no tumor at all). CONCLUSIONS: Judged by radiolabel accumulations in tissue, the pharmacokinetics of 99mTc labeled morpholinos were independent of chain length and base sequence. The only obvious exception was kidneys in which accumulations were significantly higher for the longer chain lengths and significantly different for cMORF vs MORF. These results show that chain length and base sequences may be varied to alter the pharmacokinetics of radiolabeled oligomers in nuclear medicine imaging studies.

Animals↗

PubMed2001

Initial investigations of 99mTc-labeled morpholinos for radiopharmaceutical applications.

This laboratory is evaluating phosphorothioate deoxyribonucleic acids (DNAs) and peptide nucleic acids (PNAs) for a variety of nuclear medicine applications. Morpholinos (MORFs) are a new class of oligomers with a nuclease-resistant, nonionic and water-soluble phosphorodiamidate backbone. We now report on the in vitro and in vivo properties of MORFs labeled with technetium-99m. Both 15-mer and 18-mer MORFs were obtained, each with a primary amine attached to the 3' equivalent end via a three-carbon beta-alanine linker. The amine was used to conjugate with NHS-MAG3 for 99mTc radiolabeling. By surface plasmon resonance at room temperature, the association rate constant for hybridization of the 18-mer MORF to its complementary oligomer (cMORF) was equivalent to that of DNAs and PNAs of comparable length. Hybridization of 99mTc-MORF in vitro to free cMORF, to a cMORF polymer and to cMORF beads was nearly quantitative under a variety of conditions. Kinetic studies in vitro at room temperature showed rapid (2-5 min) and nearly quantitative (90%) binding to cMORF beads. Using size-exclusion high-performance liquid chromatography, the stability of the 99mTc-MORF was found to be greater than 85% over 24 h in 37 degrees C serum with minimal protein binding. In normal mice, the 99mTc-MORF showed rapid pharmacokinetics, with only 21% and 8% remaining in the whole body at 3 and 24 h post administration, respectively. In vivo targeting with 99mTc-MORF of cMORF beads in one thigh of normal mice compared to control beads in the other thigh showed target/control thigh ratios of 2-10 between 3 and 24 h. These results demonstrate that MORF oligomers are capable of in vivo hybridization. Their properties of hybridization affinity and kinetics and their in vivo stability and pharmacokinetics make them suitable subjects for in vivo studies.

Animals↗

PubMed2001

A novel and simplified route to the synthesis of N3S chelators for 99mTc labeling.

As one example of a N(3)S chelator, MAG(3) has been used successfully for labeling peptides, proteins, DNAs and other carriers with 99mTc. We now report on a simplified route to the synthesis of N(3)S chelators. As a test of the approach, we have synthesized the succinimidyl ester of S-acetylmercaptoacetyl-(L)-glutamyl(gamma-O-t-Bu)glycylglycolic acid (MAGluG(2)) (thus MAG(3) with a t-butyl protected carboxyl group on the backbone via an ethylene linker) and the succinimidyl ester of S-acetylmercaptoacetyl-phenylalanyl-glycylglycolic acid (MAPheG(2)) (thus MAG(3) with a benzyl group on the backbone). The first chelator was selected to provide a free carboxyl group in the backbone after conjugation to peptides and after t-butyl deprotection whereas the second chelator was selected for its expected lipophilicity. The Fmoc protected NHS ester of the corresponding glutamic acid and phenylalanine were purchased and each was reacted with diglycine followed by Fmoc deprotection to provide the tripeptide. This was reacted with SATA and the NHS ester added via DCC to provide the final NHS ester of MAGluG(2) or MAPheG(2). After purification, both NHS-derivatives were conjugated to HNE2 (a 7 kDa neutrophil elastase inhibitor) as a test polypeptide. In the MAGluG(2) case, t-butyl deprotection was performed after peptide conjugation. Both of the conjugated HNE2 peptides were radiolabeled with 99mTc by transchelation from tartrate as is routine for the labeling of MAG(3)-conjugated carriers. Labeling efficiencies and stability of the chelated 99mTc towards cysteine transchelation were identical for HNE2 labeled via MAGluG(2), MAPheG(2) and MAG(3). A 3 hr biodistribution of 99mTc radiolabels in normal mice showed significant differences between the three labeled HNE2, especially in major organs (liver and kidneys). We conclude that this synthesis route provides a simplified path to the synthesis of N(3)S chelators which in principle may be used to incorporate any natural or unnatural amino acid.

Animals↗

PubMed2001

Cationic liposomes enhance cellular/nuclear localization of 99mTc-antisense oligonucleotides in target tumor cells.

UNLABELLED: Efforts are underway to apply strategies developed in connection with antisense chemotherapy to antisense imaging in nuclear medicine. One such strategy is the use of cationic liposome to enhance the cellular uptake of antisense oligonucleotides. METHODS: Using a 99mTc-labeled 18-mer uniformly phosphorothioate DNA antisense to the mRNA of the RI alpha subunit of PKA, the effects of a cationic liposome as carrier on cell uptake and efflux kinetics in tissue culture was evaluated in a RI alpha mRNA positive ACHN cell line. The sense DNA was used as control. RESULTS: Cell uptake was increased 4-5 fold using the liposome carrier compared to the same dosage of naked DNA. Whether naked or liposome-bound, the antisense DNA showed slower efflux from cells compared to the control, resulting in statistically higher accumulation of the antisense compared to the control DNA and suggesting an antisense effect. The internalization and increased cellular accumulation for both antisense and control DNAs with liposomes were demonstrated by microautoradiography and by subcellular fractionation. Finally, using 99mTc-labeled 15-mer antisense DNA against the c-myc oncogene mRNA in MDA-MB-231 cells, significantly more radiolabel was found in total mRNA for the antisense compared to the sense control DNA, both with and without liposome carrier. In conclusion, in tissue culture, the use of a cationic liposome carrier greatly increased cellular uptake and target mRNA binding of 99mTc-labeled antisense DNA.

Autoradiography↗

PubMed2001

Different chelators and different peptides together influence the in vitro and mouse in vivo properties of 99Tcm.

Relatively few studies comparing different methods of labelling peptides with 99Tcm have been reported. In this investigation, we evaluated the influence of three chelators on the in vitro and in vivo properties of two small, similar peptides (HNE2 and HNE4) labelled with 99Tcm. Both peptides were labelled with hydrazinonicotinamide (HYNIC) (tricine) at pH 5-6 and with diethylenetriaminepentaacetic acid (DTPA) and mercaptoacetyltriglycine (MAG3) at both pH 5-6 and 7-8. All ten preparations were brought to pH 7.2 immediately after labelling. Each preparation labelled well and control labelling showed each label to be attached specifically at chelation sites. Analysis of 37 degrees C human serum incubates showed little evidence of label instability but high protein binding in several cases. The stability of 99Tcm to cysteine challenge for labelled DTPA- and MAG3-peptides was similar but lower than that for the HYNIC-peptides. Reverse phase HPLC of the DTPA-peptides, but not the MAG3-peptides, showed different 99Tcm species depending on labelling pH. The 3 h biodistributions in normal mice were generally independent of labelling pH for both MAG3-peptides but were heavily influenced by labelling pH for both DTPA-peptides. While significant differences in biodistribution for the same labelling method were evident between peptides, as expected, far larger differences in the case of both peptides resulted from changing chelators and, in the case of DTPA, changing the labelling method. In summary, the chelators and labelling methods influenced the biodistribution of 99Tcm in a characteristic fashion common to both peptides. Differences in biodistribution due to the different peptides were relatively small and generally lost in the much larger differences due to chelator and labelling method. In conclusion, it may be important to compare chelators and labelling methods before selecting a 99Tcm labelling method for any particular peptide.

Animals↗

PubMed2001

In vitro investigations of tumor targeting with (99m)Tc-labeled antisense DNA.

UNLABELLED: One objective of this investigation was to determine whether chemical modifications of oligonucleotides to permit radiolabeling with gamma- or positron emitters interferes with hybridization and target cell accumulation. A second objective was to establish to a reasonable extent whether cellular accumulation of radiolabeled oligonucleotides can be explained by an antisense mechanism. METHODS: An 18mer uniform phosphorothioate DNA antisense to the messenger RNA (mRNA) of the type I regulatory subunit alpha of cyclic adenosine monophosphate-dependent protein kinase A (RI alpha) was conjugated with the N-hydroxysuccinimidyl derivative of S-acetylmercaptoacetyltriglycine (MAG3) through a primary amine/linker and investigated in vitro in cell culture. RESULTS: By surface plasmon resonance, the association kinetics between native (i.e., without amine/linker) DNA and MAG3-amide/linker-DNA were identical. Melting temperatures were also identical for native DNA, amine/linker-DNA, and MAG3-amide/linker-DNA, indicating that these chemical modifications had no detectable influence on hybridization. However, cellular accumulation of (99m)Tc-MAG3-DNA was lower than that of (35)S-MAG3-DNA, suggesting that chemical modifications can have an important influence on cellular accumulation. In tissue culture studies of ACHN tumor cells (a human renal adenocarcinoma cell type), an antisense effect was suggested by 3 findings: an increased accumulation of (35)S- or (99m)Tc-labeled antisense versus sense DNA, an increased accumulation of (99m)Tc-antisense DNA in another RI alpha-positive tumor cell line (LS174T) but not in a murine transfected control cell line (HC-2), and the disappearance of the increased cellular accumulation of (99m)Tc-antisense DNA with increasing dosage of antisense DNA. Higher than expected cellular accumulations of about 10(5) antisense DNAs per cell over 24 h suggest stabilization of the target mRNA or increased mRNA production by the presence of the antisense DNA. In support of this suggestion, we observed, first, an increased incorporation of uridine-5'-triphosphate into RNA in cells exposed to the antisense DNA but not to the control DNA and, second, an increase in target mRNA expression in cells exposed to the antisense DNA but not to the control DNA. CONCLUSION: This evidence suggests tumor cell accumulation by an antisense mechanism. Moreover, the high level of DNA accumulation suggests that a rapid target mRNA turnover or transcription rate may be an important determinant of tumor counting rates.

Autoradiography↗

PubMed2001

A comparison in monkeys of (99m)Tc labeled to a peptide by 4 methods.

UNLABELLED: Although a number of different strategies for labeling peptides with (99m)Tc have been developed, only a few studies have compared the in vivo properties of (99m)Tc when attached to different chelators. Furthermore, these comparisons are usually in mice, whereas results obtained in nonhuman primates may be expected to be more relevant to the clinical situation. METHODS: We evaluated the influence of 4 common chelators on the biodistribution in monkeys of (99m)Tc-labeled HNE-2, a 6.7-kDa peptide being investigated as an inflammation/infection imaging agent. The peptide was conjugated with the N-hydroxysuccinimide ester of mercaptoacetyltriglycine (MAG3), mercaptoacetyltriserine (MAS3), hydrazinonicotinamide (HYNIC), and the cyclic anhydride of diethylenetriaminepentaacetic acid (DTPA). After radiolabeling, each peptide was administered intravenously to rhesus monkeys with a Staphylococcus aureus-induced focal inflammation/infection. RESULTS: Quantification of radioactivity accumulation by regions of interest over 3 h after administration in monkeys showed important differences among labeling methods: For example, at 3 h, kidney accumulation varied in percentage injected dose per organ (%ID per organ) from 31 %ID per organ (HYNIC) to 18 %ID per organ (MAG3), whereas liver varied from 7.8 %ID per organ (MAG3) to 2.8 %ID per organ (MAS3). Radioactivity accumulation in the lesion was independent of labeling method. These organ accumulations were compared with that obtained earlier in mice by sacrifice and dissection also at 3 h and at the same administered dosage. In the rodent, kidney levels varied from 45 %ID per organ (HYNIC) to 12 %ID per organ (MAS3) and liver levels varied from 6.5 %ID per organ (DTPA) to 2.0 %ID per organ (MAS3). CONCLUSION: In agreement with previous work from this laboratory and elsewhere, the method of radiolabeling had an important effect on the biodistribution of (99m)Tc. Furthermore, although biodistribution results in mice should be used with caution to predict biodistributions in primates, in major organs, these results in mice and monkeys were similar.

Animals↗

PubMed2000

Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA.

We have shown recently that cell accumulation in culture of antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one antisense phosphorothioate DNA by three different methods. The 18-mer antisense DNA against the RIalpha subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37 degrees C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIalpha mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine) >MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the results suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling.

Animals↗

PubMed2000

Biodistribution and metabolism of a mixed backbone oligonucleotide (GEM 231) following single and multiple dose administration in mice.

Biodistribution and metabolism of a mixed backbone oligonucleotide (MBO), GEM 231, targeted to the RIalpha subunit of protein kinase A has been studied in normal and tumor xenografted mice. The study has been carried out using [35S]-labeled MBO following single and multiple administrations of doses varying from 2 to 50 mg/kg. MBO showed wide tissue distribution following intravenous and subcutaneous administration. The highest concentration of MBO was in the kidney and liver. The general disposition of MBO was followed by digitized autoradiographic pictures of tumored mice and further confirmed wide tissue disposition and also showed defined intratumor uptake of MBO. Multiple dose administration showed increased disposition in the majority of the tissues/organs, with the exception of the kidneys. Analysis of the extracted MBO by polyacrylamide gel electrophoresis (PAGE) showed the presence of primarily intact MBO along with its degraded forms. Based on our radioactivity levels, the primary route of excretion was in urine, analysis of which showed mainly degraded forms of MBO.

Animals↗

PubMed2000

Inflammation and infection imaging with a 99mTc-neutrophil elastase inhibitor in monkeys.

UNLABELLED: A radiolabeled human neutrophil elastase inhibitor (EPI-HNE-2) may represent an improved nuclear medicine imaging agent for inflammation and infection. This peptide displays rapid pharmacokinetics due to its low molecular weight and localizes specifically on neutrophil elastase released in inflammatory sites by activated neutrophils. METHODS: In this investigation, the peptide was radiolabeled with 99mTc using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) as a bifunctional chelator and was administered on 18 occasions to 5 rhesus monkeys with inflammation/infection. RESULTS: Plasma clearance was rapid, with liver and kidneys representing the major organs of accumulation. No evidence of toxicity, dosage effects, or circulating antiMAG3-EPI-HNE-2 antibodies was observed. Specificity of localization was established using radiolabeled bovine pancreatic trypsin inhibitor (a non-hNE-binding peptide of similar size) as a nonspecific negative control peptide and by predosing with unlabeled EPI-HNE-2 to block receptor sites before the administration of radiolabeled EPI-HNE-2. The ability of radiolabeled EPI-HNE-2 to image inflammation/infection was evaluated in 12 studies in monkeys receiving only radiolabeled EPI-HNE-2 and with lesions in the arm, shoulder, or lower back. Positive images were obtained in all studies, uptake was apparent almost immediately, and images were still positive 24 h later. As a positive control, animals also received nonspecific IgG antibody radiolabeled with 99mTc either directly or by NHS-MAG3. Compared with labeled antibody, plasma clearance of 99mTc was faster with labeled EPI-HNE-2 and accumulation in liver and heart was lower. Uptake of radioactivity in the inflammation was higher during the first hour with EPI-HNE-2 versus antibody but lower thereafter. CONCLUSION: When radiolabeled with 99mTc, EPI-HNE-2 localized specifically in inflammations in a monkey model and provided early images of diagnostic quality.

Animals↗

PubMed1999

NHS-MAS3: a bifunctional chelator alternative to NHS-MAG3.

This laboratory uses an N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS-MAG3) to conjugate amines for subsequent labeling with 99mTc. However, the synthesis from triglycerine is general and not restricted to this tripeptide. We had earlier selected a small number of alternative tripeptides and synthesized the corresponding NHS derivatives. Each was then evaluated in a search for bifunctional chelators with properties superior to NHS-MAG3, such as lower serum protein binding or improved stability to cysteine challenge. Based on these preliminary results, NHS-S-acetylmercaptoacetyltriserine (NHS-MAS3) was selected for further investigation. We have now conjugated this bifunctional chelator to an biocytin and to an amine-derivatized peptide nucleic acid (PNA). Both carriers were also conjugated with NHS-MAG3 under identical conditions and all were labeled with 99mTc at neutral pH and at boiling temperature while the conjugated PNAs were radiolabelled at neutral pH and at room temperature. Regardless of the chelator, reverse phase HPLC radiochromatograms of the labeled biotins and PNAs after purification showed a single peak. However, by size exclusion HPLC, the radiochromatograms always showed several peaks even after purification, but the MAS3 radiochromatograms were less complicated. For biotin and PNA both, radiolabeling via MAS3 showed improved 99mTc stability in 37 degrees C serum and in cysteine solution. The four preparations were administered to mice implanted in one thigh with avidin beads (biotins) or complementary PNA beads (PNAs). At 5 h post-administration, no significant differences were observed in the targeting of PNA beads between the two chelators, however the target thigh/normal thigh ratio was significantly higher for MAS3-biotin compared to MAG3-biotin. We conclude that labeling biocytin and amine-derivatized PNA with NHS-MAS3 compared to NHS-MAG3 provides simpler radiochromatographic profiles, improved stability of the label in serum and cysteine solution and can improve in vivo targeting.

Animals↗

PubMed1999

Technetium-99m labeled peptides--an investigation of multiple HPLC peaks.

This laboratory, and others, have reported multiple radioactive peaks in the size exclusion high performance liquid chromatographic (HPLC) analysis of 99mTc-labeled peptides. In the case of one 99mTc-MAG3-labeled peptide studied in this laboratory, human neutrophil elastase inhibitor, all five radioactive peaks were shown to be due to active peptide rather than radiocontaminants. By a variety of experiments, the nature of these peaks have now been examined. A high molecular weight UV peak could be generated by heating the MAG3 coupled, but not the native, peptide. Furthermore, this UV peak did not appear upon heating the peptide if the sulfur within the MAG3 chelator was replaced with oxygen. This peak may therefore be due to polymers resulting from intermolecular disulfide bond formation between sulfurs in the MAG3 chelate and the peptide. Several peaks with apparent lower molecular weights were absent on analysis with a different size exclusion column with superior resolution in their molecular weight range. More importantly, they were also absent on analysis by SDS polyacrylamide gel electrophoresis. These "low" molecular weight radioactive peaks may therefore be due to interactions between the 99mTc-MAG3 chelate and the peptide which produce multiple molecular configurations of identical molecular weight but differing in shape, charge, isomerism or lipophilicity such that they are resolved under the conditions of certain analyses. In support of this possibility, lengthening the linker between MAG3 and the peptide reduced the number of radioactive peaks, while encouraging the interaction by replacing MAG3 with the shorter MAG2 seemed to increase the number of radioactive peaks. Finally, that the three "low" molecular weight radioactive peaks reappeared when a single peak fraction was reanalyzed suggests that the species responsible are in rapid equilibrium. One conclusion from this investigation is that the appearance of a single peak by any HPLC analysis offers no assurance that multiple peaks would not appear on alternative HPLC analyses. Evidence that each species is due to radiolabeled active peptide and not to radiocontaminants is therefore potentially more important than evidence of a single peak.

Chelating Agents↗

PubMed1999

Pretargeting of bacterial endocarditis in rats with streptavidin and 111In-labeled biotin.

UNLABELLED: A radioimaging approach for the detection of endocarditis has been investigated using two-step pretargeting with streptavidin and radiolabeled biotin. METHODS: Hemodynamic alterations within the rat heart were induced by placing an in-dwelling catheter into the left ventricle through the aortic valves. The animals were subsequently infected with Staphylococcus aureus through a tail vein. After an incubation period, rats were first injected with streptavidin and, 2 h later, with 111In-labeled ethylene-diaminetetraacetic acid-biotin. Whole-body gamma camera images were taken 4-5 h postinjection of the radiolabeled biotin. Control animals consisted of catheterized but uninfected, infected but uncatheterized and normal untreated rats. As a further control, the labeled biotin was administered to a study animal without the preadministration of streptavidin. RESULTS: Histology showed typical endocarditic changes in the hearts of study animals with massive deposition of gram-positive cocci. Catheterized but uninfected animals showed alterations corresponding to nonbacterial thrombotic endocarditis. Macroautoradiography showed accumulation of radiolabel in the endocarditic vegetations of study animals. Whole-body gamma camera images showed important cardiac uptake in 7 of 8 catheterized and infected animals and in 3 of 6 catheterized but uninfected animals. Normal rats and those infected but not catheterized showed negative results by histology, autoradiography and imaging. The percent uptake of the injected dose in the heart was 0.20 (SD = 0.13) in catheterized and infected animals, 0.12 (SD = 0.10) in catheterized but uninfected animals, 0.10 (SD = 0.04) in infected but uncatheterized animals and 0.04 (SD = 0.01) in normal control animals. CONCLUSION: The two-step pretargeting approach using streptavidin and 111In-labeled biotin was used successfully to detect S. aureus-induced bacterial endocarditis in rats.

Animals↗

PubMed1998

Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine.

UNLABELLED: A modified mercaptoacetyltriglycine (MAG3) chelator, which has acetyl S-protection and which is derivitized with N-hydroxysuccinimide (NHS) ester for conjugation, has been used to radiolabel four small (approximately 6- to 7-kDa) peptides, bovine pancreatic trypsin inhibitor, epidermal growth factor, human neutrophil elastase inhibitor and plasmin inhibitor, with 99mTc. METHODS: Each peptide was specifically labeled at the MAG3 chelation sites at ambient temperature and neutral pH. Specific activities of 100-150 mCi/mg were achieved at labeling efficiencies of about 50%, but specific activities of 3500 mCi/micromol could be attained. RESULTS: By a variety of assays, protein activity was unimpaired by the conjugation and labeling for two of the four peptides. The activities for plasmin of the plasmin inhibitor and bovine pancreatic trypsin inhibitor were reduced by conjugation, presumably because of a sensitive lysine residue in the structure of each of these two peptides. Multiple peaks were present in the high-performance liquid chromatography radiochromatograms, especially of human neutrophil elastase inhibitor; however, most peaks could be shown to be labeled active peptide. Stability during cysteine challenge at modest cysteine-to-peptide molar ratios and during incubation in serum was observed in each case. Large differences among the labeled peptides were apparent in the 3-hr biodistributions of 99mTc in normal mice. CONCLUSION: The use of NHS-S-acetyl-MAG3 may be a convenient method of radiolabeling peptides with 99mTc.

Animals↗

PubMed1997

Early results in the irrational design of new bifunctional chelators.

BACKGROUND: The development of a simple route for the synthesis of the N-hydroxysuccinimide (NHS) ester of S-acetyl-protected mercaptoacetyltriglycine (MAG3) has opened the possibility of preparing novel bifunctional N3S chelators for technetium-99m (99mTc) and other radionuclides. In particular, the synthesis may be applied to a vast number of tripeptides in place of triglycine, to provide a "library" of bifunctional N3S chelators, each with unique properties related to the particular amino acid residues within each tripeptide. METHODS: The authors have synthesized by this simple route the NHS esters of four N3S chelators by reacting NHS-S-acetylthioglycolic acid with ala-gly-gly, phe-gly-gly, pro-gly-gly, and ser-ser-ser, in addition to gly-gly-gly. Each bifunctional chelator was conjugated to biocytin as a model primary amine and radiolabeled with 99mTc. The properties of the four chelators were compared with MAG3 with respect to the stability of the label in saline and serum, the extent of serum protein binding, and the instability to cysteine challenge. RESULTS: A range of values was observed. Labeled mercaptoacetyltriserine showed stability towards transchelation to cysteine similar to that of MAG3 as well as lower serum protein binding; labeled mercaptoacetylalanyldiglycine showed slightly higher serum protein binding than labeled MAG3 but greater stability to cysteine challenge. CONCLUSIONS: The authors concluded that this simple synthesis and evaluation scheme may be used to prepare and screen a large library of bifunctional chelators for those with useful properties.

Blood Proteins↗

PubMed1997

Pretargeting using peptide nucleic acid.

BACKGROUND: Pretargeting studies in animals and humans have usually involved (strept)avidin and biotin. Depending on the particular strategy, endogenous biotin can adversely influence localization when these molecules are used. METHODS: As an alternative to (strept)avidin and biotin, we have explored the use of a single-stranded peptide nucleic acid (PNA) bound to a protein administered first and followed by the complementary single-stranded PNA radiolabeled with 99mTc. Target localization of the PNA-bound protein in a mouse infection and a mouse tumor model occurred by passive diffusion while the radiolabeled complementary PNA localized by in vivo hybridization. The PNA-streptavidin was prepared by adding biotin-conjugated PNA to streptavidin; the complementary PNA, derivatized with a primary amine, was conjugated with acetyl S-protected NHS-MAG3 bifunctional chelator and radiolabeled with 99mTc. RESULTS: In both the infection and tumor mouse models, increased localization of radiolabel was achieved in animals receiving both injectates compared with control animals receiving only the radiolabeled PNA. In the infection model, the infected to normal thigh radioactivity ratio was 3.5 for the study animals compared with 1.7 for control animals (P = 0.0001). In the tumor model, these values were 1.7 versus 1.2 (P = 0.003). CONCLUSIONS: We conclude that PNA may be considered an alternative to (strept)avidin and biotin for pretargeting studies.

Animals↗

PubMed1997

The influence of temperature and alkaline pH on the labeling of free and conjugated MAG3 with technetium-99m.

Benzoyl-protected mercaptoacetyltriglycine (S-benzoyl MAG3) is radiolabeled by tartrate transchelation at elevated temperatures or basic pH. The object of this investigation was to establish whether the same 99mTc labeled species are formed when S-acetyl (S-acetyl MAG3)-conjugated compounds are radiolabeled by tartrate transchelation at ambient temperature and neutral pH in contrast to labeling at 95 degrees C and pH 11. S-acetyl MAG3 was conjugated to biocytin and to the amine-derivitized oligomers DNA and PNA. Along with free S-acetyl MAG3, these were radiolabeled under the different conditions. Although labeling efficiencies were always lower when labeled at ambient temperature and neutral pH relative to labeling at 95 degrees C or pH 11 (free S-acetyl MAG3 could not be labeled at all), size exclusion and reverse phase HPLC showed no difference with labeling conditions in the radiochemical profiles for labeled DNA and biocytin. In the case of DNA, a cysteine challenge also failed to demonstrate a difference. However, in the case of PNA, some important differences were observed in the size exclusion HPLC radiochromatograms. In addition, PNA labeled at ambient temperature and neutral pH was less stable to transchelation to cysteine. In conclusion, S-acetyl MAG3 conjugated compounds may be radiolabeled at ambient temperature and neutral pH. In most cases, the radiochemical species produced appear to be identical to those formed when labeling is accomplished at 95 degrees C or pH 11.

Chelating Agents↗

PubMed1997

Preparation and use of NHS-MAG3 for technetium-99m labeling of DNA.

The chelator mercaptoacetylglycylglycylglycine (MAG3) is on of several amidothiols that have been used successfully to radiolabeled proteins and other molecules with 99mTc. Prior to radiolabeling, the sulfur in these amidothiols is usually protected by a benzoyl group (i.e. S-benzoyl MAG3) which requires extreme alkaline pH or boiling water temperatures for rapid deprotection. As a result, the benzoyl-protected chelator is radiolabeled prior to conjugation (i.e. preconjugation labeling) in the case of carriers such as proteins or polypeptides which cannot withstand harsh conditions. We have employed a simple, two-step, synthesis of the N-hydroxysuccinimide ester of MAG3 in which the sulfur is protected with an acetyl group (i.e. S-acetyl NHS-MAG3). A single-stranded amine-derivitized DNA was coupled with NHS-S-acetyl MAG3. Radiolabeling was accomplished at room temperature and neutral pH by transchelation from 99mTc-tartrate. In comparison to labeled SHNH-DNA, the labeled MAG3-DNA was unstable to cysteine transchelation, however, in contrast to SHNH-DNA, no evidence for serum protein binding of the labeled MAG3-DNA was observed. We conclude that the S-acetyl NHS MAG3 bifunctional chelator may prove to be an attractive alternative method of radiolabeling DNA and other biologically important molecules with 99mTc.

Chelating Agents↗