Toxicity, pharmacokinetics and metabolism of a novel inhibitor of IL-6-induced STAT3 activation

Abstract

Purpose: The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) promotes gene transcription involved in cancer, and its activation by IL-6 is found in head and neck squamous cell carcinoma. Four triazolothiadizine STAT3 pathway inhibitors were evaluated to prioritize a single compound for in vivo examination.

Methods: Metabolic stability in mouse liver microsome incubation was used to evaluate four triazolothiadizine analogues, and UPCDC-10205 was administered to mice IV as single or multiple doses to evaluate toxicity. Single-dose pharmacokinetics (PK), bioavailability and metabolism were studied after IV 4 mg/kg, PO 4 mg/kg, or PO 30 mg/kg suspension in 1% carboxymethyl cellulose. Mice were euthanized between 5 min to 24 h after dosing, and plasma and tissues were analyzed by LC-MS. Non-compartmental PK parameters were determined.

Results: Of the four triazolothiadizine analogues evaluated, UPCDC-10205 was metabolically most stable. The maximum soluble dose of 4 mg/kg in 10% Solutol™ was not toxic to mice after single and multiple doses. PK analysis showed extensive tissue distribution and rapid plasma clearance. Bioavailability was ~5%. A direct glucuronide conjugate was identified as the major metabolite which was recapitulated in vitro.

Conclusions: Rapid clearance of UPCDC-10205 was thought to be the result of phase II metabolism despite its favorable stability in a phase I in vitro metabolic stability assay. The direct glucuronidation explains why microsomal stability (reflective of phase I metabolism) did not translate to in vivo metabolic stability. UPCDC-10205 did not demonstrate appropriate exposure to support efficacy studies in the current formulation.

Keywords: IL-6; LC-MS; Pharmacokinetics; Small molecule inhibitor of STAT3.

Figure 1

A) 669 series chemical structures. B) Mouse liver microsome incubation of UPCDC-10205 (□),UPCDC-10540 (○), UPCDC-10305 (Δ), and 864669 (◊).

Figure 2

Average mouse body weights (N=5; Error bars represent ±1 SD). A) Single dose toxicity study before and following a single IV injection of UPCDC-10205 at 4 mg/kg to male treated (▲), male vehicle (Δ), female treated (◊), and female vehicle (♦) mice. B) Multiple dose toxicity study, before, during and following QDx5 IV administration of UPCDC-10205 to female mice at 4 mg/kg (□), 2.7 (Δ), 1.3 (○) and vehicle (◊).

Figure 3

Concentration vs time results following administration of UPCDC-10205. Points represent the mean of the mice (N=3) and error bars represent ±1 SD. A) Plasma and tissues from IV 4 mg/kg UPCDC-10205. B) Plasma and tissues from PO 4 mg/kg UPCDC-10205. C) Plasma and tissues from PO 30 mg/kg UPCDC-10205 in 1% CMC; ◊=plasma, ■=liver, Δ=kidney, ●=lung, ▲=RBC, ◊=muscle, ○=brain. D) Plasma UPCDC-10205 concentration versus time from all three administration types. ◊= IV 4 mg/kg, ■= PO 4 mg/kg, Δ= PO 30 mg/kg in 1% CMC.

Figure 4

A) Chromatogram of the SRM scan (613 and 437 m/z channels) of mouse plasma from the PK study between 40 to 55 minutes. Line 1 is vehicle 437 m/z, line 2 is vehicle 613 m/z (5×10⁶ count offset), line 3 is treated 437 m/z (1.0×10⁷ count offset) and line 4 is treated 613 m/z (1.5×10⁷ count offset). B) 60 minute mouse liver microsome incubation with UPCDC-10205, alamethicin and UPDGA to produce direct N-glucuronide (613 m/z). Left axis shows quantification of UPCDC-10205 (□) represented by a percent of the starting amount. Right axis shows production of metabolite (○) represented as the ratio of analyte to internal standard values. C) Proposed metabolism of UPCDC-10205 to the conjugated N-glucuronide; note that there are 2 possible regioisomers for the glucuronide, one of which is shown.