Paramagnetic contrast agents in magnetic resonance imaging: research at Vanderbilt University

Experimental studies in animals have demonstrated the application of particulate and chelated paramagnetic oral contrast agents in magnetic resonance imaging (at 0.5 tesla). The ability of a soluble paramagnetic species, ferrous gluconate, to improve imaging studies of the pancreas presently is being evaluated in clinical trials. Two paramagnetic metal ion chelates, Cr EDTA and Gd DTPA, have been evaluated extensively as potential intravascular contrast agents. Renal function, tissue vascularity, abnormalities of the blood-brain barrier, and infarction of myocardial tissue may all be assessed with IV contrast enhanced magnetic resonance imaging. The contrast materials tested all represent first generation compounds. Improved relaxation characteristics, toxicity, distribution, and flexibility will result from development of second generation agents, primarily within the particulate and chelate classes.     

Terminal-modified polylysine-based chelating polymers: highly efficient coupling to antibody with minimal loss in immunoreactivity.

A method is suggested for the preparation of chelating polymers containing a single terminal reactive group capable of interaction with proteins. These polymers were synthesized from N-CBZ-polylysine and DTPA and contain a terminal SH or pyridyldisulfide group. A polymer molecule with MW 13,500 is able to carry up to 40 DTPA residues. Polymers easily and quantitatively bind with antibodies (Fab fragments of antimyosin antibodies R11D10) with minimal effect on antibody immunoreactivity as revealed in ELISA assay and in direct immunoanalysis. Conjugates prepared can chelate radioactive metal ions reaching very high specific radioactivity (greater than 1 mCi 111In/10 micrograms of protein). Perspectives for their application are discussed.     

Effects of Chelates in Chemotherapy of Experimental Gas-Gangrene Toxemia

The relative ability of the calcium chelates of calcium disodium ethylenediaminetetraacetate (EDTA) and calcium trisodium ethylenetriaminepentaacetate (DTPA) to protect mice against lethal doses of Clostridium perfringens alpha-toxin was investigated. Their protective ability was assayed by the increase in survival time of mice which had been given large doses of toxin, and by determining the median protective dose of chelate that would protect mice against toxin at a minimum lethal dose of two. In both assay procedures, intraperitoneal, intravenous, and intracutaneous injections of toxin were utilized, and with each toxin injection route the protective ability of the chelate was determined with the three routes of injection. DTPA was 10 to 20 times more effective than EDTA with both types of assay procedure and with all injection routes. DTPA may be superior to EDTA as a protective agent not only because it binds zinc to a greater extent, but also because of its greater retention in the body and its ability to gain entrance into cells. It appears that DTPA may be of value as a therapeutic agent in gas-gangrene.     

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.     

A gadolinium(III) complex of a carboxylic-phosphorus acid derivative of diethylenetriamine covalently bound to inulin, a potential macromolecular MRI contrast agent

A novel conjugate of a polysaccharide and a Gd(III) chelate with potential as contrast agent for magnetic resonance imaging (MRI) was synthesized. The structure of the chelate was derived from H5DTPA by replacing the central pendant arm by a phosphinic acid functional group, which was covalently bound to the polysaccharide inulin. On the average, each monosaccharide unit of the inulin was attached to approximately one (0.9) chelate moiety. The average molecular weight is 23110 and the average number of Gd3+ ions per molecule is 24. The ligand binds the Gd3+ ion in an octadentate fashion via three nitrogen atoms, four carboxylate oxygen atoms, and one P-O oxygen atom, and its first coordination sphere is completed by a water molecule. This compound shows promising properties for application as a contrast agent for MRI thanks to a favorable residence lifetime of this water molecule (170 ns at 298 K), a relatively long rotational correlation time (866 ps at 298 K), and the presence of two water molecules in the second coordination sphere of the Gd3+ ion. Furthermore, its stability toward transmetalation with Zn(II) is as high as that of the clinically used [Gd(DTPA)(H2O)]2-.     

Water-soluble polysaccharides as carriers of paramagnetic contrast agents for magnetic resonance imaging: synthesis and relaxation properties.

Water-soluble, carbohydrate-based, paramagnetic metal chelate derivatives have been investigated as potential organ-selective contrast media for magnetic resonance imaging (m.r.i.). The in vitro proton spin-lattice relaxation properties of compounds with different paramagnetic metals, chelating agents, and carbohydrate matrixes have been studied. Typically, these complexes were 60-260% more efficient proton-relaxation agents than the corresponding low-molecular-weight metal chelates at 10 MHz, but less efficient than the corresponding protein derivatives. As expected, carbohydrates that contained manganese or gadolinium were more effective relaxation agents than iron, copper, erbium, or nickel derivatives.     

Backbone-substituted DTPA ligands for 90Y radioimmunotherapy

Four new bifunctional diethylenetriaminepentaacetic acid (DTPA) ligands were synthesized to provide an improved chelating agent for radioimmunotherapy with 90Y. The new DTPA ligands contained a 4-isothiocyanatobenzyl group (pSCNBz) substituted onto the carbon backbone of DTPA for use in linkage to immunoprotein. Methyl groups were strategically incorporated onto the backbone of the ligands via a peptide route to provide 2-pSCNBz-5-Me-DTPA (2) and 3-Me-6-pSCNBz-DTPA (3). Addition of these functionalities was expected to sterically hinder the release of radiometal from the chelate. A new monosubstituted ligand, 3-pSCNBz-DTPA (4), was also prepared in order to determine whether a shift in position of the linking group had an effect on the in vivo stability of the yttrium complex. Additionally, by modification of known methods, a disubstituted DTPA ligand, 2-pSCNBz-6-Me-DTPA (1), was prepared.     

Influence of Complexation on the Uptake by Plants of Iron, Manganese, Copper and Zinc: II. EFFECT OF DTPA IN A MULTI-METAL AND COMPUTER SIMULATION STUDY

Barley (Hordeum vulgare L.) plants were grown in nutrient solutionscontaining the chelating agent, DTPA. The experiments replicatedthose reported in the preceding paper in which EDTA was thechelating agent used. The concentrations of all the chemicalspecies of metals were stimulated using the program NUTRIENT.The concentrations of DTPA used were chosen to give a similarrange of complexation as used in the EDTA experiments. The effectof complexation by DTPA on the uptakes of the metal ions Fe³⁺,Mn²⁺, Cu²⁺, and Zn²⁺ and on plant growth were sufficiently differentfrom those with EDTA to indicate some dependence on the natureof the chelating agent used. The biggest difference betweenthe EDTA and DTPA experiments occurred in the solutions containingthe largest concentrations of these reagents. With DTPA, chlorosiswas seen only in the early stages; otherwise the plants showednormal growth. A simple chemical model for metal uptake is discussed. Key words: DTPA, EDTA, micronutrients, trace metals, computer simulation, plants, absorption, iron, manganese, copper, zinc     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Application of a new surface labeling reagent, EDTA derivative, on erythrocytes and platelets.

The modes of binding of a new class of impermeant metal-chelating probe, the complex of 111In3+ to 1-(p-benzenediazonium) ethylenediamine tetraacetic acid (azo-phenyl-EDTA), to human and rabbit erythrocyte membranes and the effect of binding on the function of rabbit platelets have been studied. The metal chelate, azo-phenyl-EDTA.[111In3+] bound covalently to membrane proteins following reaction with intact erythrocytes. The amount and the pattern of labeling was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide disc and slab gels for radioactivity. The pattern of labeling of intact human erythrocytes by azo-phenyl-EDTA.[111In3+], by pyridoxal phosphate-NaB3H7 and by galactose oxidase-NaB3H4 was also compared. The following results were obtained: (a) The pattern of labeling of intact human erythrocyte by azo-phenyl-EDTA.[111In3+] differed from other commonly used probes for labeling external membrane surfaces. Five polypeptides were labeled by the metal chelates. In addition to the known major proteins (protein band III, PAS-1, PAS-2 and PAS-3 of Fairbanks et al. (1972) Biochemistry 10, 2606--2617) a protein (radioactive band 4) which migrated slightly slower than PAS-3 in SDS gel was labeled heavily by the metal chelate. This protein has an apparent molecular weight of 37,500 in 8.4% acrylamide-SDS gel. About 40% of bound radioactivity was found in this protein. The diazo linkage of the metal chelate to this protein was found to be especially unstable to heat. (b) In rabbit erythrocyte membranes, the metal chelate bound to three polypeptides with apparent molecular weights of 96,000, 43,000 and 33,000 in 8.4% acrylamide gel. They are probably glycoproteins in nature. (c) The binding of the probe to platelets did not affect the platelet aggregability induced by adenosine diphoshpate. In vivo studies indicated that the labeled platelets accumulated at the plague of atherosclerotic rabbits. (d) The bifunctional analog of EDTA may permit new applications of metals with useful physical properties for studies of cell membranes.     

Complexation of epigallocatechin gallate (a green tea extract, egcg) with Mn2+: nuclear spin relaxation by the paramagnetic ion

Metal-binding sites in epigallocatechin gallate (egcg) were identified by measuring (1)H nuclear spin relaxation time T(1) under the coexistence of paramagnetic Mn(2+). The T(1) of the proton of gallate ring (D-ring) was shortened by the paramagnetic ion to a greater extent than the proton in gallocatechin ring (B-ring); the D-ring OH groups occupy the first coordination sphere around a metal ion to form a diolate chelate ring, while B-ring OH groups have a weak interaction with the metal ion. Comparison of changes in T(1) of egcg and epigallocatechin (egc) indicates that the gallate ring has a higher coordination capability than that of the gallocatechin ring so that the former plays a predominant role in the complex formation of egcg.     

A derivative of diethylenetriaminepentaacetic acid for europium labeling of proteins.

Preparation of a reagent that will incorporate diethylenetriaminepentaacetic acid (DTPA) into proteins under mild conditions and make a strong europium chelate is described. Aminoacetaldehyde diethyl acetal was reacted with DTPA dianhydride, and mono- and disubstituted products as well as unsubstituted DTPA were separated by gel filtration. The monosubstituted product, after conversion into the corresponding aldehyde by mild acid hydrolysis, is conjugated to protein or other amino-containing compounds via reductive amination at neutral pH. Although the DTPA-Eu-labeled proteins are themselves not fluorescent, a strong fluorescence of europium can be generated easily by the dissociation-enhancement mechanism. A direct measurement of lectin-ligand interaction using Eu-labeled ligand and lectin immobilized on 96-well plate illustrates that the assay utilizing Eu fluorescence is as sensitive as the radioactive assays.     

Effective chelation therapy after incorporation of neptunium-239 in rats

It was demonstrated in rats that it is possible to reduce the retention of 239Np in all body tissues by an early combined treatment with small doses of DTPA and DFOA. The content of 239Np can be decreased in soft tissues even if treatment is delayed. Promptly administered LICAM(C) proved more effective than the above chelate combination in reducing 239Np retention in the bones but increased that in the muscles and especially in the kidneys. This side effect of LICAM(C) could be partly prevented by simultaneous treatment with DTPA.     

The effect of X rays, DTPA, and aspirin on the absorption of plutonium from the gastrointestinal tract of rats

To measure the effect of radiation on plutonium transport, rats that were exposed to 250-kVp X rays were given 238Pu 3 days afterwards by either gavage or injection into a ligated segment of the duodenum. In a second group of experiments, rats were either injected intraduodenally with 238Pu-DTPA or administered the chelate intravenously and the 238Pu by gavage. In a third experiment, rats that had been gavaged with 200 or 400 mg/kg/day of aspirin for 2 days were injected intragastrically with 238Pu nitrate. Results of the first experiment showed a dose-dependent increase in 238Pu absorption between 800 and 1500 rad of lower-body X irradiation. Intravenous or intraduodenal injections of DTPA caused a marked increase in 238Pu absorption but resulted in decreased plutonium deposition in the skeleton and liver. Retention of 238Pu in the skeleton of rats given aspirin was double that of controls, but the effect on plutonium absorption was less marked than that of DTPA.     

Nuclear magnetic spin-lattice relaxation of water protons caused by metal cage compounds.

The nuclear magnetic spin-lattice relaxation rates of water protons are reported for solutions of manganese(II), copper(II), and chromium(III) cage complexes of the sarcophagine type. As simple aqueous solutions, the complexes are only modest magnetic relaxation agents, presumably because they lack protons on atoms in the first-coordination-sphere protons that are sufficiently labile to mix the large relaxation rate at the metal complex with that of the bulk solvent. The relaxation is approximately modeled using spectral density functions derived for translational diffusion of the interacting dipole moments with the modification that the electron spin relaxation rate is directly included as a contribution to the correlation time. In all cases studied, the electron spin relaxation rate is sufficiently large that it contributes directly to the water-proton spin relaxation process. The poor relaxation efficiency of the cage compound may, however, be improved dramatically by binding the complex to a protein. The efficiency is improved even further if the rotational motion of the protein is reduced drastically by an intermolecular cross-linking reaction. The relaxation efficiency of the cross-linked protein-cage complexes rivals that of the best first-coordination-sphere relaxation agents like [Gd(DTPA)(H2O)]2- and [Gd(DOTA)(H2O)]-.     

Synthesis, characterization, and relaxivity of two linear Gd(DTPA)-polymer conjugates

Two linear polyamide conjugates of Gd(DTPA)2- were synthesized and characterized by high-resolution nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). DTPA was copolymerized with two different diamines, 1,6-hexanediamine and trans-1,4-cyclohexanediamine, yielding the polymers DTPA-HMD and DTPA-CHD, with low polydispersity. Their molecular flexibility in solution was studied using 13C spin-lattice relaxation time measurements, indicating that the cyclohexanediamine linking moiety of the DTPA-HMD polymer is more rigid than that of DTPA-CHD. The influence of the flexibility of the linking functionalities on the relaxivity of the Gd3+-DTPA-polymer conjugates was studied by water nuclear magnetic relaxation dispersion (NMRD). The relaxivity of the Gd(DTPA-CHD) polymer was only slightly higher than that of the Gd(DTPA-HMD) polymer, and only two times higher than the usual values for small Gd-DTPA-like chelates. The low relaxivities obtained for both polymers, much lower than expected from the polymer apparent molecular weights, result from their substantial residual flexibility, and also from a too long, nonoptimal, value of the inner-sphere water exchange rate. These polymeric compounds are also cleared very quickly from the blood of rats, indicating that they are of limited value as blood pool contrast agents for MRA.     

Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.     

High-level conjugation of chelating agents onto immunoglobulins: use of an intermediary poly(L-lysine)-diethylenetriaminepentaacetic acid carrier

Diethylenetriaminepentaacetic acid (DTPA), a strong chelating agent, was covalently linked to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of poly(L-lysine) (Mr 14,000) as a multivalent, intermediary carrier, via thiol-disulfide exchange reaction. The conjugates contained up to 42.5 mol DTPA per mol antibody, and retained over 90% of their antibody activity in vitro. The conjugates incorporated gadolinium (Gd) through an exchange reaction with Gd-EDTA, used to prevent colloid formation and nonspecific binding of the free metal. The IgG-poly(L-lysine)-DTPA-Gd had a greater effect per mol on proton relaxation rates than DTPA-Gd itself. Use of poly(L-lysine) as an intermediary carrier for attachment of chelating agents to IgG thus offers great potential for achieving high-specific-activity conjugates, particularly for use as biologically specific contrast agents in nuclear magnetic resonance imaging.     

Polymeric gadolinium chelate magnetic resonance imaging contrast agents: design, synthesis, and properties.

We have synthesized and evaluated five series of polymeric gadolinium chelates which are of interest as potential MRI blood pool contrast agents. The polymers were designed so that important physical properties including molecular weight, relaxivity, metal content, viscosity, and chelate stability could be varied. We have shown that, by selecting polymers of the appropriate MW, extended blood pool retention can be achieved. In addition, relaxivity can be manipulated by changing the polymer rigidity, metal content affected by monomer selection, viscosity by polymer shape, and chelate stability by chelator selection.     

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.