The activities of drug inactive ingredients on biological targets

Abstract

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K _d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

Fig. 1.. Motivation for testing excipient activity, operational workflow, and selected in vitro concentration-response curves.

(A) The similarity between excipients (top) and FDA-approved drugs (bottom). Benzethonium chloride is itself FDA-approved as a topical antiseptic wash. (B) Workflow. More than 600 molecular excipients were screened computationally, and a list of potential protein targets was predicted for each one on the basis of its SEA E-value. A subset of high-ranking excipient-target pairs was tested in vitro. In a second set of experiments, commonly used excipients were experimentally tested against a panel of clinical toxicity targets; these tests were unrelated to the SEA predictions, although sometimes they overlapped. (C) Molecular structures of thimerosal and ethyl mercury. (D) Concentration-response curves of selected excipient-target pairs with activity ranging from low-nanomolar (propyl gallate inhibition of COMT) to mid-micromolar (tartrazine binding to dopamine D₁). Red curves represent a reference positive control; blue curves represent excipient binding: (a) propyl gallate and reference compound tolcapone binding to COMT; (b) tartrazine and reference compound (+)-butaclamol binding to DRD1; (c) diethyl phthalate and reference compound RO 20–1724 binding to PDE4D; (d) thimerosal and reference compound (+)-butaclamol binding to DRD3; (e) butylparaben and reference compound L670596 binding to TBXA2R; (f) benzethonium chloride and reference compound potriptyline binding to SLC6A2 (previously known). The D₃ binding curve for thimerosal is one representative of replicates in three separate laboratories. Additional dose-response curves are provided in fig. S1.

Fig. 2.. Time-concentration profiles of excipients administered to rats.

(A to D) Time-concentration curves of blood exposure in rats after i.v. (1 mg/kg) application of excipients and oral administration of propyl gallate (10 mg/kg) (A), D&C Red No. 6 (1 mg/kg) (B), cetylpyridinium chloride (1 mg/kg) (C), and FD&C Red No. 3 (1 mg/kg) (D). Insets are expanded views of the oral administration curves. Error bars denote SD from measurements in three rats.