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In Vitro Evaluation of Drug–Drug Interaction Potential of Doxecitine and Doxribtimine in Thymidine Kinase 2 Deficiency (TK2d) 

Biochemical/Metabolic and Therapeutics
  • Primary Categories:
    • Other Therapies
  • Secondary Categories:
    • Other Therapies
Introduction:
Doxecitine and doxribtimine powder for oral solution is a 1:1 mixture by weight of deoxycytidine (dC) and deoxythymidine (dT). It is being investigated as a treatment for patients with thymidine kinase 2 deficiency (TK2d), an ultra-rare genetic mitochondrial disease associated with progressive proximal myopathy and respiratory weakness. Here, we report findings on the in vitro plasma protein binding and potential interactions of doxecitine and doxribtimine with cytochrome P450 (CYP) enzymes and common drug transporters.

Methods:
Procedures followed guidelines on drug–drug interaction (DDI) investigations published by the European Medicines Agency (2013) and US Food and Drug Administration (FDA; 2017 and 2020). A 1:1 mixture of dC and dT by molar concentration was tested unless noted otherwise. Protein binding of [14C]-dC and [14C]-dT (1, 10 and 10 µM) in mouse, rat, dog, monkey and human plasma was determined using rapid equilibrium dialysis with liquid chromatography–tandem mass spectrometry (LC-MS/MS) and radiometric detection. Metabolism of [14C]-dC and [14C]-dT (0.5, 2.5 and 25 μM each) was studied separately in pooled human liver microsomes (HLM) analysed using LC-MS/MS and radiometric detection. Induction of CYP1A2, CYP2B6 and CYP3A4 mRNA was examined in cryopreserved primary human hepatocytes treated with dC and dT (0.05–50 000 μM each). Inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 by dC and dT (50–50 000 µM for CYP3A4/5, 0.015–15 μM for the others) was examined in pooled HLM using CYP-selective probe substrates and LC‑MS/MS analysis. Interaction (inhibition or substrate potential) of dC and dT with transporters was evaluated in transporter-expressing cells or membrane vesicles (for the bile salt export pump [BSEP]). Substrate potential of [3H]‑dC and [3H]‑dT for P‑glycoprotein (P-gp) and breast cancer resistance protein (BCRP) was tested individually in MDCK‑MDR1 and Caco-2 cells, respectively.

 

Results:
Plasma protein binding of dC and dT was relatively low (< 10%) and independent of concentration. In HLM, dC and dT were degraded in a time- and protein-dependent but not cofactor (NADPH)-dependent manner, suggesting no meaningful involvement of CYPs. There was little or no induction of CYP1A2, CYP2B6 or CYP3A4 mRNA (< 2-fold change) by dC or dT up to 50 000 µM. No direct inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19 or CYP2D6 was evident; inhibition of CYP3A4/5 was noted (IC50 ≥ 39 000 µM). No time- or metabolism-dependent inhibition of any CYP was observed. Doxecitine and doxribtimine did not inhibit the BSEP, organic anion transporter (OAT)1, OAT3, organic cation transporter (OCT)2, multidrug and toxic extrusion protein (MATE)1 or MATE2-K. Inhibition of organic anion transporter polypeptide (OATP)1B3, OATP1B1, OCT1, BCRP and P-gp was observed at IC50 values of 15.4, 20.8, 56.5, ~100 and > 100 mM, respectively. These values greatly exceed the human steady-state geometric mean Cmax values of dC (0.054 μM) and dT (0.112 μM) at the highest anticipated daily dose. Potential in vivo inhibition of P-gp, BCRP and OATP1B1/B3 was further evaluated using Igut/IC50 cut-off criteria (FDA 2020). Estimated Igut/IC50 ratios were below the cut‑off values for all 4 transporters, suggesting in vivo inhibition is unlikely. Doxecitine and doxribtimine was not a substrate for P-gp, OAT1, OAT3 or OCT2. dC was unlikely to be a substrate of BCRP, whereas dT was considered a possible substrate of BCRP. Fumitremorgin C, a reference BCRP inhibitor, had minimal or no effect on bidirectional efflux of dT (1 µM and 10 µM), indicating BCRP has a minor role, if any, in dT transport at these concentrations.

 

Conclusion:
Clinically relevant concentrations of doxecitine and doxribtimine are unlikely to cause or be subject to pharmacokinetic DDIs mediated by plasma protein binding displacement, CYP-mediated metabolism or activity at common drug transporters.



Study funded by UCB.

 

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