New modular delivery system for diagnostic and therapeutic pre-targeting using tautomer-specific monoclonal antibody EM-6-47 and 3-substituted adenines.

We have developed a new modular affinity system for the 2-step delivery of functional molecules to target cells. The system is based on the tautomer-specific monoclonal antibody (MAb) EM-6-47, which binds to 3- and 3,8-substituted adenines with high affinity (Ka > 10(9) l/mol) without cross-reacting with naturally occurring purine derivatives. This MAb serves as the hapten-specific fusion partner to produce bispecific MAbs (bs-MAbs) recognizing a target cell antigen and a low-m.w. hapten as carrier molecule for, e.g., radionuclides. Either the C-8 or the N-3 position of adenines can be used for conjugation with effector molecules; the remaining position may be substituted with different moieties to modulate the pharmacokinetics of the haptens. Different 3- and 3,8-substituted adenines conjugated to the chelates DOTA and DTPA or to the drug daunomycin were synthesized. Adenine-chelate derivatives were efficiently labeled with (111)In and 90Y, while high-affinity binding of 3-substituted adenines to MAb EM-6-47 remained almost unaffected by the conjugation to radiochelates. To confirm the validity of the delivery system, a prototype bs-MAb, EM-168-47, was generated by somatic cell fusion of MAb EM-6-47 and MAb EM-168-2, the latter recognizing a surface antigen on canine hematopoietic cells. Two-step targeting assays in vitro verified the bs-MAb-mediated, dose-dependent delivery of (111)In-labeled adenine-chelate derivatives to myeloid cells. This system represents a powerful tool for new pre-targeting approaches relying on bs-MAbs and low-m.w. haptens. Suitable cellular antigens can be targeted by fusing the appropriate MAbs with hapten-specific MAb EM-6-47, and tailor-made 3-substituted adenines may be labeled with diagnostic or therapeutic radionuclides, cytotoxic drugs or other functional molecules.

Determination of N7- and O6-methylguanine in rat liver DNA after oral exposure to hydrazine by use of immunochemical and electrochemical detection methods.

Hydrazine belongs to a group of compounds for which there is evidence that the in vivo genotoxic effects become manifest only upon exposure to toxic dose levels. The present study was performed to investigate whether this phenomenon is also reflected in the pattern of DNA methylation. The induction of N7- and O6-methylguanine (MeGua) was studied in liver DNA of rats, 16 hr after treatment with various doses of hydrazine. After DNA isolation, the presence of N7-MeGua in DNA was assessed with an immunochemical method and with a physicochemical technique (HPLC with electrochemical detection). Application of these two methods resulted in almost identical patterns of dose-dependent induction of guanine N7-methylation in rats dosed orally with 0.1 to 10 mg hydrazine per kilogram of body weight, increasing from 1.1-1.3 to 39-45 N7-MeGua per 10(6) nucleotides. At lower dosages a constant adduct level was observed, equivalent to that in untreated rats (background level). The O6-MeGua level was analyzed by a combination of HPLC separation and competitive radioimmunoassay. A background level was observed for untreated rats and no increase was visible up to the 0.2 mg/kg dose group. After hydrazine doses from 0.2 to 10 mg/kg, O6-MeGua increased from 0.29 to 134 per 10(9) nucleotides. These data show that even at dosages below the maximum tolerated dose (0.6 mg/kg/day), for which carcinogenic effects have not been described, DNA adducts are formed. A comparison is made of the data obtained in this study with models that describe the mechanism of hydrazine-induced DNA methylation.

Urinary excretion of 3-methyladenine and 3-ethyladenine after controlled exposure to tobacco smoke.

The urinary excretion of the DNA alkylation products 3-methyladenine (3-MeAde) and 3-ethyladenine (3-EtAde) after controlled exposure to cigarette smoke over a period of 4 days was determined by competitive radioimmunoassay after separation by HPLC. Twenty-four hour urine samples were collected from five smokers and five non-smokers. Days 1 and 3 (control days) were without smoking, on days 2 and 4 smokers consumed 24 cigarettes each within 8 h in an unventilated room (45 m3) in the presence of non-smokers. Average levels of carbon monoxide during exposure were 15-20 p.p.m., 2.8-3.5 mg/m3 of respirable suspended particles and 75-86 micrograms/m3 of nicotine. Carboxyhemoglobin levels increased by 9.0 and 1.8% in smokers and passive smokers respectively. On control days, urinary excretion of 3-MeAde was similar in smokers and non-smokers (4.7-6.2 micrograms/24 h). Smoking resulted in a significant increase (P < 0.01) in 3-MeAde excretion (13.6-14.8 micrograms/24 h); no change was observed in the average excretion of 3-MeAde by passive smokers (4.8-4.9 micrograms of 3-MeAde/24h). Baseline 3-EtAde excretion on control days was similar in smokers and passive smokers (13.7-32.8 ng/24 h). In smokers, the amount of urinary 3-EtAde was increased > 5-fold (119.3-138.5 ng/24 h) on smoking days; no effect on 3-EtAde excretion was observed on average in passive smokers (18.0-25.2 ng/24 h). The nature of the DNA-reactive agent(s) responsible for the increased urinary excretion of 3-alkyladenines, in particular of the sensitive indicator 3-EtAde, remains to be determined.