Thomas R. Sykes

INDIRECT IODOMETRIC PROCEDURE FOR QUANTITATION OF SN(II) IN RADIOPHARMACEUTICAL KITS

Abstract A method of quantitating stannous ion [Sn(II)] suitable for radiopharmaceutical kits, based on indirect iodometric titration, is described. The method is based on the oxidation of Sn(II) using a known excess of iodine and the excess unreacted iodine determined using thiosulphate by potentiometric titration. The titration cell is a beaker and the titrations are done conveniently under air using an autotitrator in approx. 4 min. The method is accurate and is linear in the range of approx. 10 μg to approx. 6 mg of Sn(II). Several radiopharmaceutical kits were analysed for their Sn(II) content using the method including those containing antibodies or other proteins. The studies indicate that the procedure is rapid, simple and accurate for routine quantitative estimation of Sn(II) in radiopharmaceutical preparations during development, manufacture and storage.

Reactor production and detection of radiolabeled cis-platinum

Small quantities of radiolabeled cis-platinum were prepared by two methods. The thermal neutron irradiation of potassium tetrachloroplatinite followed by radiochemical synthesis was compared to the direct irradiation of the cis-platinum itself. Both methods produced usable quantities of tracer, but the direct method proved most practical. Radiochemical purification and analysis data are also presented. Liquid scintillation and NaI(Tl) counting techniques were evaluated. Counting the numerous x-rays produced from several platinum radioisotopes in a well-type NaI(Tl) detector proved most convenient for routine use. It is concluded that these methods provide a useful route for the preparation, analysis and determination of measurable quantities of radiolabeled cis-platinum for small scale applications.

Radiolabeling of monoclonal antibody B43.13 with rhenium-188 for immunoradiotherapy.

In this study we report a novel method for direct radiolabeling of monoclonal antibody B43.13 (MAb-B43.13) with 188Re and have evaluated the products radiochemical, biochemical, immunochemical and selected biological properties. 188Re-MAb-B43.13 was readily prepared by the addition of generator produced perrhenate to a preformulated antibody vial after an optimal amount of supplemental stannous ion, in the form of stannous tartrate, was added. The final radiolabeled product retained its biochemical purity (as determined by size-exclusion HPLC and R/NR-SDS-PAGE), its immunoreactivity (as determined by immunoassay) and presented with a typical stability (in the presence of serum and cysteine) and biodistribution (in tumored mice) profile. The evaluation of the product for immunoradiotherapy of ovarian cancer in a clinical setting requires further studies.

Comparative radiolabeling and distribution of a tumour-directed monoclonal antibody

The monoclonal antibody (MAb) 155H.7, raised against a synthetic beta-anomer of the Thomsen-Friedenreich antigen (S-TAG), was radioiodinated using iodine monochloride, chloramine-T and Iodogen and radiolabeled with 111In using the bromoacetamido-derivative of benzyl-EDTA. The in vitro immunoreactivity of the MAb was assessed using an ELISA with the S-TAG and the in vivo distribution of the radioiodinated and radiochelated MAb was determined in the murine mammary carcinoma TA3/Ha tumour model. Both chloramine-T and iodine monochloride radioiodination greatly reduced the immunoreactivity of the MAb compared to radioiodination using Iodogen. Bifunctional chelate labeling was comparable to Iodogen in reducing the immunoreactivity of the MAb and subsequent chelation of 111In did not further compromise the immunoreactivity of the MAb. The in vivo distribution data showed significantly different distributions of the radiolabels after injection of the radioiodinated and radiochelated MAb. The 131I-MAb showed some tumour association as compared to the distribution of an 125I-non-specific protein and the data also indicates that there is preferential dehalogenation of the radioiodinated MAb. 111In from the radiochelated MAb showed significantly higher uptake in the tumour than 131I from the 131I-MAb. It is suggested that the differing fates of the two radiolabels within the tumour cell is responsible for the difference in retention observed and not necessarily due to the lack of MAb uptake by the tumour. Overall, the radiochelate label for MAb 155H.7 appears to be superior to radioiodine for in vivo use.

Assay of platinum and gold in biological material by neutron activation analysis

Compounds of both gold and platinum are used in medicine, the former as salts to treat arthritides and the latter as the metal complex cisplatin to treat cancer. We have investigated neutron activation analysis with the Slowpoke II reactor as an assay method for both elements using human blood plasma as a matrix. Neutron activation of platinum gives rise to 3.15 day199Au while that of gold produces 2.7 day198Au. Activated samples are dissolved during heating in test tubes and the gold extracted by adding dibutyl sulphide to the same tube. The latter is formed to able to quantitate Pt down to 60 ng and Au down to 60 pg. The dissolution technique and possible interferences in the assay are discussed.

An improved method for analysis of technetium radiolabeled antibody by size exclusion HPLC

A quantitative radio HPLC method for the analysis of radiolabeled 99mTc compounds, in particular labeled antibodies or proteins, is described. The method is based on the quantitative re-oxidation of the reduced column absorbed 99mTc species to pertechnetate using hydrogen peroxide. The pertechnetate is quantitatively eluted from the column. The method can be used as a stand alone technique for determining the true radiolabeling yield of labeled proteins/antibodies. The method has been automated for routine use and the typical analysis time is 30 min.

The Potential of Synthetic Tumor-Associated Glycoconjugates (S-TAGs) for Generating Monoclonal Antibodies for Breast Cancer Imaging and for Specific Immunotherapy

The Thomsen Friedenreich (TF) antigen may be important for the detection and immunotherapy of a number of common cancers including breast cancer. Revealed on normal human erythrocytes by neuraminidase treatment, TF has been characterized as: β-D-Gal-(1–3)- α-GalNAc, attached to glycophorin or other glycoproteins through O-serine or O-threonine linkages (1). Tn, the TF precursor, is reported to be α-GalNAc-O-serine/ threonine. While TF is normally cryptic due to the presence of a terminal sialic acid residue, Tn is exposed in individuals with a recessive genetic disorder (2). Springer (1) has claimed expression of TF and Tn antigens on over 90% of cancers of the breast, lung and pancreas, although the nature of the molecules which bear these antigens and their exact structures has not been defined.

Optimization of protein conjugation using an aldehyde derivatized bifunctional chelating agent

Abstract The covalent attachment of the bifunctional chelating agent 2-( p -aminobenzyl)-1,3-propanediamine-N,N,N′,N′-tetraacetic acid to proteins through a new linking arm containing an aldehyde function for the purpose of labeling proteins and monoclonal antibodies with radiometals was studied. The number of protein-bound chelating groups was measured by various techniques after labeling the conjugate with 111 In. The effect of various parameters such as aldehyde-chelate concentration, pH, temperature, protein concentration and concentration of sodium cyanoborohydride on the rate and extent of protein conjugation have been examined.

Effect of cis-platinum on the protein binding and pharmacokinetics of 67Ga-citrate.

Cis-platinum, a widely used chemotherapeutic drug, and K2PtCl4, a related complex, were found to inhibit the in vitro protein binding of 67Ga to human transferrin in isolated solution but not to whole serum. Cis-platinum treatment of rabbits resulted in enhanced protein binding and delayed blood clearance of subsequently injected 67Ga-citrate. These effects are related to the toxicity of the drug in vivo.

Preparation and biological evaluation of radiolabelled antibodies with selected carbohydrate modifications

Two carbohydrates, N-acetylgalactosamine (GalNAc) and galactose-beta-1,3-GalNAc have been attached to human IgG (hIgG) by a novel linking reagent, hexafluoroglutaric acid dimethyl ester. Fluorine-19 NMR signals were used for the determination of the conjugation ratio. A third carbohydrate, sialic acid, was conjugated via reductive amination and the conjugation ratio determined by a resorcinol assay. The biological behaviour of these radioiodinated antibodies with carbohydrate modifications in normal mice indicates an enhanced liver uptake at 15 min post-injection with an associated change in circulating blood levels occurs for the galactose-based hIgG preparations. However, no significant differences in the biodistribution were observed for the sialic acid conjugate. These studies confirm the potential of carbohydrate-antibody conjugation for modifying the behaviour of antibodies in immunoscintigraphy and radioimmunotherapy.