Formulation development of an antifibrin monoclonal antibody radiopharmaceutical.

These studies have shown that formulation development of a monoclonal antibody radio-immunoscintigraphy agent is a challenging task involving a number of issues related to the radiochemistry of labeling as well as the stability of the antibody. Through a systematic approach, as described in this study, a stable and efficacious product of high quality can be developed in a rational and efficient manner. In developing an optimized formulation of Tc-99m-antifibrin Fab' for use in immunoscintigraphy of DVT, a number of critical components were examined in regard to technetium-labeling chemistry and the ability of the formulation to support the long-term stability of the product. It was found that the addition of glucarate as a transfer ligand, SnCl2 as a reducing agent, and neutral-to-acidic pH of the solution were essential for optimum radiolabeling of 0.5 mg of antifibrin Fab' to a desired activity of 25 mCi of Tc-99m. The lyophilization of the final product was also required to further stabilize both the antifibrin Fab' fragment and the reducing agent. The addition of carbohydrate as bulking agent and lyoprotectant and inclusion of EDTA as a chelating agent further improved the performance of the formulations, resulting in products with long shelf-life. Many of the principles described in this study are not only useful in developing a technetium-based immunoscintigraphic agent but are also applicable to other immunopharmaceuticals, including products involving delivery of radionuclides, drugs, and toxins for immunotherapy of cancer and other diseases.

Gram scale purification and preparation of rabbit liver zinc metallothionein.

A simplified procedure for purifying gram quantities of rabbit liver metallothionein (MT) using gel filtration and anion exchange chromatography is presented. The MT purification made use of anion exchange batch elution chromatography which greatly shortened the procedure. Quantitation techniques for use with crude and purified MT are discussed. This paper also describes the preparation of large amounts of ZnMT from Cd,ZnMT.

Pharmacokinetic evaluation of technetium-99-metallothionein-conjugated mouse monoclonal antibody B72.3 in rhesus monkeys.

These studies were conducted to determine the biodistribution and pharmacokinetics of [99mTc]metallothionein-conjugated B72.3 ([ Tc]MT-B72.3) in Rhesus monkeys (Macaca mulatta) that were performed as part of the preclinical evaluation of [Tc]MT-B72.3. The B72.3-MT conjugate was studied at three doses of B72.3 ranging from 0.03 mg/kg to 1 mg/kg to determine whether a relationship existed between the dose of total antibody administered intravenously and the biodistribution and clearance of the radiolabeled protein. Results indicated that [Tc]MT-B72.3 distributes rapidly to central body cavity organs and that there was no difference in the rate of blood elimination for the three doses of B72.3 studied. The terminal phase of blood elimination was found to be 26.2 +/- 6.1 hr for the combined groups of monkeys. Approximately one-half of injected 99mTc activity was recovered in the urine within 24 hr. A second purpose of these studies was to evaluate the overall immunogenicity of the mouse monoclonal B72.3 IgG1 antibody in Rhesus monkeys. These results demonstrated that a single i.v. exposure to mouse monoclonal B72.3 at doses of 0.3 mg/kg or greater elicited antibody production to B72.3 in Rhesus monkeys within 3 wk. Analysis of [Tc]MT-B72.3 biodistribution and clearance in monkeys with circulating levels of antibodies to B72.3 (immunized monkeys) revealed that the liver was the primary site of clearance of the presumed immune complex and that blood elimination was greatly accelerated.

Conjugation of metallothionein to a murine monoclonal antibody.

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.

Metallothionein: a bifunctional chelator for the radiolabeling of biologically active molecules.

Cysteine-rich metallothionein has been shown to bind as many as seven TcO3+ cores in TcO (Cys) units which adopt square-base pyramidal geometry. The Tc atoms in 99mTc,Zn-MT and 99mTc,Zn-MT-MAb species formed in the exchange reaction with the labile 99mTcO (glucoheptonate) complex are stable to oxidation and transchelation in vivo. The kinetics of labeling with the carrier-free radionuclide 99mTc are diffusion-limited at MT concentrations below 10(-5) M. Zinc-MT-MAb conjugates can be efficiently radiolabeled at concentrations greater than or equal to 10(-5) M. The conjugation of MT to tumor specific MAb's has been accomplished with the preservation of the in vivo tumor targeting capability of the MAb and the excellent metal-binding properties of MT. The success achieved in this research demonstrates the potential of metallothionein as a carrier of medically useful metallic radionuclides in labeling of important biologically active molecules.

Flavin 1, N 6 -ethenoadenine dinucleotide: dynamic and static quenching of fluorescence.

Flavin 1,N(6)-ethenoadenine dinucleotide (epsilonFAD) was prepared by the action of chloroacetaldehyde on flavin adenine dinucleotide. This compound, which has two potential fluorophores, epsilon-adenine and isoalloxazine, shows extremely efficient energy transfer from the former to the latter. The fluorescences of both moieties are greatly diminished in the intact molecule. Determination of the fluorescence yields and lifetimes leads to the conclusion that at 20 degrees in neutral aqueous solution epsilonFAD exists mainly (90%) as an internally complexed or stacked form.