Leon van Haandel , Division of Clinical Pharmacology, Toxicology & Therapeutic Innovation, Children's Mercy Hospital, Kansas City, MO
Ryan Funk , University of Kansas Medical Center, Lawrence, KS
J. Steven Leeder , Division of Clinical Pharmacology, Toxicology & Therapeutic Innovation, Children's Mercy Hospital, Kansas City, MO
Jennifer L Goldman , Division of Clinical Pharmacology, Toxicology & Therapeutic Innovation, Children's Mercy Hospital, Kansas City, MO
Background: The combination antibiotic trimethoprim-sulfamethoxazole (TMP-SMX) is effective, inexpensive, and widely prescribed, yet it also causes a high rate of idiosyncratic adverse drug reactions (IADRs). To date, SMX is considered to be the causative agent behind these reactions due to its ability to form reactive metabolites. Recently it has been recognized that similar to SMX, TMP can also be bioactivated. The reactive intermediate is an imminoquinone methide species capable of binding to thiols. By performing in vitro studies using human liver microsomes with an in house synthesized 14C radiolabeled form of TMP we were able to show that significant protein covalent binding by TMP was occurring. We have also shown that mercapturic acids of TMP are present in the urine of children taking TMP-SMX, suggesting that processes of bioactivation and detoxification are occurring in vivo. However, there have been no reports of the detection of TMP-protein adducts in vivo. Here we hypothesize that TMP-protein adducts can be detected in the plasma of patients receiving TMP-SMX. Methods: To identify TMP-protein adducts in plasma, a polyclonal antibody was generated by coupling Cα-NAC-TMP to keyhole limpet hemocyanin via carbodiimide coupling. Two rabbits were immunized with TMP antigen per a standard 70-day immunization protocol (Thermo Scientific). Sensitivity and specificity of the TMP directed antiserum was performed by ELISA confirming antibody activity. Plasma samples obtained from 5 subjects taking and tolerating TMP-SMX and 3 subjects who were not TMP-SMX exposed (negative controls) were probed with the TMP antiserum via immunoblotting to determine whether TMP-protein adducts could be found in the plasma. Human serum albumin (HSA) covalently modified at cys34 with TMP was utilized as the positive control during immunoblotting. Results: Patient plasma samples and controls were loaded onto a nitrocellulose membrane and probed with the anti-TMP-protein serum. Positive signal consistent with the formation of covalent protein adducts of TMP was found in all patients taking TMP-SMX. Western blot analysis of the patient samples and controls using the anti-TMP-protein serum revealed two bands that were consistently labeled within the patient samples. The heaviest band had a molecular weight slightly over 50kDa, which corresponds to the labeled HSA standard. The band intensity greatly diminished upon albumin stripping. The second band has a molecular weight of around 37kDa and appears as a doublet and is resistant to albumin stripping, but its identity is currently unknown. Conclusions: We report, for the first time, the detection of circulating TMP protein adducts in subjects taking TMP-SMX. These data prove that TMP is reactive towards proteins, and is capable of hapten formation. Further work is needed to understand the relationship between TMP adduct formation and the development of IADRs, and whether these circulatory protein adducts could be used as biomarkers for IADR development.