Pretreatment with drug-specific antibody reduces desipramine cardiotoxicity in rats

The effect of a drug-specific antibody on disipramine (DMI) cardiotoxicity was studied in rats. Animals were pretreated i.v. with 4.2 g/kg of a monoclonal antibody (anti-TCA) followed by DMI HCl 30 mg/kg i.p. (molar ratio of anti-TCA binding sites to DMI = 0.56). Peak QRS complex prolongation was substantially lower after pretreatment with anti-TCA than after control antibody (70 ± 14 v. 21 ± 4%, p < 0.001). Time to peak toxicity was the same in both groups. Binding of DMI by anti-TCA was demonstrated by a higher serum total DMI concentration and increased DMI binding in serum after anti-TCA compared to controls. The DMI concentration in anti-TCA treated animals was lower in some organs (brain, lung, liver, spleen), but not in others (heart, muscle, kidney, fat). The calculated fraction of the DMI dose bound by anti-TCA was 19.9%. The steepness of the DMI dose-response curve was examined by administering DMI alone (without antibody) at various doses to rats. Compared to 30 mg/kg DMI, a dose reduction of 30-50% was needed to reduce QRS duration to the same extent as anti-TCA pretreatment. We conclude that DMI cardiotoxicity was markedly reduced by the binding of a relative small fraction of the DMI body burden to anti-TCA. This disproportionate effect of DMI binding was not due to the steepness of the DMI dose-response curve, nor to slowing of the rate of DMI distribution to tissues

Cloning, expression, and purification of an anti‐desipramine single chain antibody in NS/0 myeloma cells

Drug‐specific monoclonal antibodies and their antigen‐binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/0 myeloma expression system was developed. The variable light (V) and variable heavy (V) domains of a murine anti‐desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid V‐(GlySer)‐V sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of V with heavy chain leader peptide, V, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SRα promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T‐cell leukemia virus type I long terminal repeat and also containing the Escherichia coli xanthine–guanine phosphoribo‐syltransferase gene for selection. NS/0 myeloma cells were transfected by electroporation. Stable recombinant NS/0 clones were screened for expression of sFv using reverse transcriptase‐PCR to detect mRNA and an enzyme‐linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2–4 × 10 viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12–39 mg/L of purified sFv. Affinity‐purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA. Copyrigh