For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([ 3 H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% 6 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% 6 3.12%) and through urine only to a minor extent (4.15% 6 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be identified in serum and excreta.
The synthesis of a tritiated benzopyran‐type opener of the ATP‐dependent K+ channel (KATP channel), [3H]‐PKF217 – 744 {(3S,4R)‐N‐[3,4‐dihydro‐2,2‐dimethyl‐3‐hydroxy‐6‐(2‐methyl‐4‐pyridinyl)‐2H‐1‐benzopyran‐4‐yl]‐3‐[2,6‐3H]pyridinecarboxamide} with a specific activity of 50 Ci mmol−1 is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively.
PKF217 – 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]‐P1075, to SUR2B (Ki=26 nM).
The kinetics of [3H]‐PKF217 – 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9×106 M−1 min−1 and 0.09 min−1, respectively.
Binding of [3H]‐PKF217 – 744 to SUR2B/2A was activated by MgATP (EC50∼3 μM) and inhibited (SUR2B) or enhanced (SUR2A) by MgADP.
Binding of [3H]‐PKF217 – 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]‐P1075 as the radioligand.
Glibenclamide accelerated dissociation of the SUR2B‐[3H]‐PKF217 – 744 complex.
The data show that the affinity of [3H]‐PKF217 – 744 binding to SUR2B is ∼6 times lower than that of [3H]‐P1075. This is due to a surprisingly slow association rate of the benzopyran‐type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.
British Journal of Pharmacology (2001) 133, 275–285; doi:10.1038/sj.bjp.0704071
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