Preparation and biodistribution of copper-67-labeled porphyrins and porphyrin-A6H immunoconjugates.

The synthetic porphyrins, N-benzyl-5,10,15,20-tetrakis (4-carboxyphenyl) porphine (N-bzHTCPP) and N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P), represent excellent radiocopper chelating agents that may find utility in antibody-mediated diagnosis and/or therapy. N-bzHCS3P was conjugated to an anti-renal cell carcinoma (RCC) antibody, A6H, and labeled with copper-67. 67CuCS3P-A6H was studied for its biodistribution in human RCC xenograft-bearing nude mice, along with the radiolabeled free porphyrins. The porphyrins resulted in tumor:blood ratios in the range of 3 to 4 after 48 h. The radiolabeled antibody achieved a tumor:blood ratio of over 16 after 45 h, indicating accumulation at the desired site. However, unwanted localization also occurred in the liver and spleen, which will have to be rectified before realizing the full potential of this approach.

Using starburst dendrimers as linker molecules to radiolabel antibodies.

Starburst dendrimers, spherical polymers constructed from methyl acrylate and ethylenediamine, were successfully used to covalently couple synthetic porphyrins to antibody molecules. The dendrimers, as linker molecules, have great potential for increasing the specific activity of radiolabeled antibodies for tumor therapy and diagnosis.

Mn (III) hematoporphyrin. A potential MR contrast agent.

Manganese (III) hematoporphyrin (MnHP), a new and stable complex, was prepared, and its toxicity and magnetic resonance (MR) imaging properties were evaluated. In tests of acute and subacute toxicity, no deaths resulted from bolus intravenous injections of 13 or 19 mumols/kg of MnHP, but there was a 33% mortality when the dose was 38 mumols/kg. Laboratory results were normal in the surviving rats. Ultraviolet- visible spectroscopy of the urine and serum of two rats injected 24 hours previously with 38 mumols/kg MnHP revealed no free HP, suggesting in vivo stability of MnHP. Finally, using a standardized imaging protocol, there was a mean increase of 37% in the liver-to-muscle intensity ratios in four rats injected 24 hours previously with 25 mumols/kg MnHP when compared to paired controls (P less than .005). In addition, obvious visual increase in the signal intensity of the liver on T1-weighted images was seen in animals tested with 13 and 19 mumols/kg of MnHP. The results suggest that further evaluation of MnHP as an MR contrast agent for the liver is warranted.

The biological characteristics of a water soluble porphyrin in rat lymph nodes.

The biological characteristics of a radiolabeled metalloporphyrin, 5,10,15,20-tetrakis(4-carboxyphenyl)-porphinato [67Cu]copper (II) [( 67Cu]TCPP), in rat lymph nodes, surrounding muscle, fat, and blood were determined. Lymphatic tissue localized greater amounts of [67Cu]TCPP than did surrounding muscle and fat. Inflamed lymph nodes localized greater amounts of [67Cu]TCPP than did noninflamed lymph nodes. Time course studies suggest that the uptake of [67Cu]TCPP in noninflamed and in inflamed lymph nodes may involve different biological processes. The affinity of [67Cu]TCPP for inflamed lymph nodes may be influenced by the degree of inflammation. If further studies confirm these results, [67Cu]TCPP may be useful as a potential radiopharmaceutical for imaging inflamed lymph nodes.

Labeling antibodies with copper radionuclides using N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine.

Antibody conjugates labeled with copper-64 and -67 (64Cu and 67Cu) were prepared using the porphyrin chelator N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P). N-bzHCS3P was chosen because it has only one carboxylate group available for activation and coupling to antibody. The conjugates were characterized with respect to (1) the location of the porphyrin on the antibody, (2) the retention of immunoreactivity, and (3) the serum stability of the amide bond linking porphyrin to antibody. These studies showed that porphyrin attachment on the antibody surface is random. The conjugates exhibited high retention of immunoreactivity and reasonable serum stability for potential application in nuclear medicine.

The biodistribution of radiocopper-labeled compounds.

Porphyrins form extremely stable chelates with Cu2+. Two copper radionuclides, 67Cu and 64Cu, have attractive nuclear decay properties for use in nuclear medicine applications. We have investigated the use of radiocopper-labeled porphyrins for localization in inflamed tissue and for attachment to antibodies for tumor imaging and therapy. We have examined the biodistribution of a 67Cu labeled porphyrin, [5, 10, 15, 20-tetrakis(4-carboxyphenyl) porphinato [67Cu] copper (II)], 67 CuTCPP. The 67CuTCPP was intravenously injected into the tail vein of Fischer F344 male rats. The kidneys, liver, and spleen localize the greatest amounts of 67CuTCPP. The elimination of 67CuTCPP from the body is described by a normal exponential decay curve with a biological half-life of 108 hours and an effective half-life of 32 hours. We have also examined the biodistribution of 5-(4-carboxyphenyl)-10, 15,20-tris(4-sulfophenyl) porphinato [67Cu] copper (II) anti-Thy 1.2 antibody conjugates in normal and tumor-bearing male AKR/J mice. The liver, kidney, and tumor have the highest uptake of the 67Cu labeled antibody conjugate. In all 67Cu labeled compounds studied, the blood clearance was rapid and the bone concentration of the radiolabeled species was low.

Copper-67 labeled porphyrin localization in inflamed tissue.

A series of experiments compared the uptake of 5,10,15,20 tetrakis(4-carboxyphenyl) porphinato [67Cu] copper (II), 67CuTCPP, by the lymph nodes of inflamed and two sets or control rats. The results demonstrate that 67CuTCPP localizes in greater concentration in inflamed lymph nodes than in noninflamed control lymph nodes. This enhanced uptake of 67CuTCPP by inflamed lymph nodes was 3.6 times greater than was the uptake by control lymph nodes. A time course study demonstrated that the uptake of 67CuTCPP by inflamed lymph nodes reached the maximum level by 24 hours post-injection of 67CuTCPP and remained constant throughout the 96 hours examined. It was also found that the uptake of 67CuTCPP by inflamed lymph nodes was not exclusively dependent upon an increase in the weight of inflamed lymph nodes. These studies show that 67CuTCPP has potential as a lymphoscintigraphy agent.

Preparation and characterization of copper-67 porphyrin-antibody conjugates.

Methods were developed to label antibodies with copper-67, a potentially useful medical radioisotope, using the porphyrin chelating agent N-benzyl-5,10,15,20-tetrakis(4-carboxyphenyl) porphine. The porphyrin was activated for coupling using either (1) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl and N-hydroxysuccinimide or (2) 1,1'-carbonyldiimidazole. The coupling reactions were optimized as a function of activation time, coupling time, coupling pH, and reagent concentrations to achieve maximum coupling to IgG monomer. Sodium dodecylsulfate polyacrylamide gel electrophoresis was used to determine coupling yields. After purification by gel filtration, the antibody-porphyrin conjugates were labeled with copper-67 in aqueous solution. The coupling protocols were used to label antibodies from several species, demonstrating the general utility of these methods. Characterization of the conjugates indicated that the porphyrin label was attached randomly to the IgG molecule. Antigen binding capacities after conjugation were unaltered or slightly lowered as determined by a competitive ELISA.

Compensation by zinc chelation for fluorescent background in determining coproporphyrin and uroporphyrin in urine.

An improved method for separation and fluorescent analysis of coproporphyrin and uroporphyrin in urine was presented by Sobel, Cano, and Thiers [Clin. Chem. 20, 1397 (1974)]. Accurate determination of porphyrin concentrations by this method is impeded by the fluorescent background in urine samples, which varies from one specimen to another. By use of a straightforward procedure, zinc may be incorporated into coproporphyrin and uroporphyrin. Measurement of the difference in the fluorescent emission intensity at 650 nm for the porphyrin solutions before and after zinc incorporation compensates for fluorescent background and gives a more accurate determination.