Development and preclinical pharmacology of a novel dCK inhibitor, DI-87

Abstract

Background: Deoxycytidine kinase (dCK) is an essential enzyme for production of nucleotides via the salvage pathway; DI-87 is a novel dCK inhibitor in preclinical development for use in anticancer therapy. The current study utilizes PET imaging to evaluate PK-PD relationships and to determine optimal dosing of the drug.

Methods: NSG mice bearing CEM tumors had plasma and tumor PK assessed using mass spectrometry following oral administration of DI-87. dCK inhibition was assessed after a single dose of oral DI-87 followed by a [¹⁸F]CFA PET probe and PET imaging. Tumor growth inhibition was assessed by orally administering DI-87 with concurrent intraperitoneal thymidine.

Results: DI-87 had an in vitro EC₅₀ of 10.2 nM with low protein binding. Peak DI-87 concentrations were observed between 1-3 h and 3-9 h in plasma and tumor, respectively, with tumor concentrations less than one third of plasma. Full dCK inhibition, as evaluated by PET imaging, was observed as early as 3 h following 25 mg/kg dosing and was maintained for 12 h, with full recovery of enzyme activity after 36 h. When DI-87 was administered as repeated doses in combination with thymidine, full dCK inhibition was maintained at 12 h (25 mg/kg twice daily dose) and led to maximal tumor growth inhibition.

Conclusions: DI-87 is a promising new compound for use in combination therapy against tumors expressing dCK. Utilizing a [¹⁸F]CFA PET probe targeting the pathway of interest allowed for efficient and accurate identification of the optimal dose for growth inhibition.

Keywords: DI-87; Deoxycytidine kinase; PET scan; Pharmacodynamics; Pharmacokinetics; Preclinical.

Figure 1:

Schematic of population PK-PD model. DI-87 is an oral drug which is absorbed from the gut with an absorption constant (KA). It distributes between the plasma volume (Vp) and tumor volume (Vt) with an intercompartmental clearance Q and is eventually cleared from the plasma compartment (CL). Tumor DI-87 concentrations lead to drug effect (dCK inhibition or growth inhibition). DCK inhibition with modeled with a sigmoid Emax indirect response model while growth inhibition was modeled with an Emax indirect response model.

Figure 2:

Synthesis and in vitro activity of DI-87. (A) Structure of DI87. (B) Synthetic route of DI-87. (C) IC₅₀ values determined using ³H-dC uptake assay in CEM T-ALL cells to measure inhibition of dCK activity. (D) Dose response of DI-87 in CEM T-ALL cells treated with 10 nM Gemcitabine (n=4; mean±SD) for 72h determined using Cell Titer Glo. (E) Protein binding of DI-87 and (R)DI-82 assessed by comparing IC₅₀ of the compounds in presence of 25 and 50 mg/mL BSA (n=2; mean±SD).

Figure 3:

Plasma and tumor concentrations. Each data point represents the plasma and tumor concentrations from a single mouse (n=5 per time point). Plasma concentrations are higher than tumor concentrations and have an earlier peak Tumor sizes varied between experiments thus it was difficult to determine if the tumor concentrations were linear.

Figure 4:

dCK inhibition studies. A. dCK activity in tumors. DI-87 was administered as a single dose to mice and PET scans were completed following drug administration. dCK activity from representative mice over time is shown for 25 mg/kg, 10mg/kg, and 5 mg/kg. B. dCK activity as measured by PET scans. dCK inhibition was prolonged with higher doses of drug (25 mg/kg vs. 10 mg/kg). Minimal inhibition was seen at 5 mg/kg. C. Simulation of dCK activity for three doses of DI-87.

Figure 4:

dCK inhibition studies. A. dCK activity in tumors. DI-87 was administered as a single dose to mice and PET scans were completed following drug administration. dCK activity from representative mice over time is shown for 25 mg/kg, 10mg/kg, and 5 mg/kg. B. dCK activity as measured by PET scans. dCK inhibition was prolonged with higher doses of drug (25 mg/kg vs. 10 mg/kg). Minimal inhibition was seen at 5 mg/kg. C. Simulation of dCK activity for three doses of DI-87.

Figure 5:

A. Growth inhibition experiment. DI-87 was administered as multiple doses in combination with thymidine to mice. Near complete growth inhibition was seen with full DCK inhibition throughout the dosing interval (25 mg/kg BID). Data represent the mean and standard deviation of 5 mice. B. Growth inhibition model simulation. Solid lines represent simulations from final PK-PD model and symbols represent measured tumor size. Overall the model represents the data well.