Modeling a plasma-esterase mediated prodrug conversion in mouse (PK-Sim) — unable to set initial enzyme concentration

[leap2006]

Title: Modeling a plasma-esterase mediated prodrug conversion in mouse (PK-Sim) — unable to set initial enzyme concentration

Hi all,

I am building a mouse PBPK model (PK-Sim, species: Mouse) for an ester prodrug that is reported to be rapidly and almost completely hydrolyzed by plasma esterases immediately upon reaching systemic circulation (reported half-life of conversion ≈ 6 min in humans; no parent compound is detectable in plasma). I am trying to replicate this mechanism in the mouse model.

What I have done so far:

• Created a custom Metabolizing Enzyme process ("esterase3") on the parent compound, linked to the metabolite, using "Intrinsic clearance – First Order" with a calibrated clearance value.
• Created a corresponding Expression Profile (Species: Mouse) for this enzyme.
• Under Localization, I selected only "Vascular endothelium membrane plasma-side" (unchecking all other localizations), since the conversion is described as occurring in plasma rather than intracellularly in a specific organ.

The problem:
With this localization, the "Relative expression"/initial concentration for the vascular system remains at 0 and does not appear editable, either directly in the Expression Profile (even with "Show initial concentration" checked — the compartment table stays empty) or after linking the profile to an Individual. As a result, the enzyme appears inactive in simulation: the parent compound shows no elimination at all (plateaus instead of declining after the dose), confirming the enzyme concentration is effectively zero. Screenshot attached showing the Expression Profile with "Vascular endothelium membrane plasma-side" checked and the empty initial-concentration table below.

My three questions:

1. How can I correctly set a non-zero initial/relative expression for a custom enzyme localized in "Vascular endothelium membrane plasma-side"? Is there a required step I am missing (e.g., does this localization only become editable after the profile is attached to a full Simulation rather than an Individual/Building Block alone)?

2. Has anyone previously modeled plasma-esterase-mediated hydrolysis in mouse using PK-Sim? If so, which gene/enzyme entry from the mouse gene expression database (Bgee-based) did you use as a proxy? I searched for "esterase" and found mostly intracellular carboxylesterases (Ces2 family, liver/intestine), but the literature points to a plasma-secreted murine esterase (Ces1c, historically "Es-1") for this type of ester hydrolysis — however, as expected, this gene shows liver (site of synthesis/secretion) rather than plasma expression in the database, which doesn't map cleanly onto the "vascular system" relative expression field.

3. Is there a simpler way to model this step given how fast and complete the conversion is? Since essentially 100% of the parent is converted to the active metabolite within minutes of reaching systemic circulation, and the parent is never detected in plasma in vivo, would it be more appropriate to bypass an explicit enzymatic process altogether (e.g., administering the active metabolite directly, or using a very fast non-saturable first-order "disappearance" process not tied to a specific enzyme localization) rather than trying to force a mechanistic vascular-endothelium enzyme expression that the UI does not seem to support well for custom (non-database) enzymes?

I am not a PBPK specialist and am working through this largely self-taught, so any pointers (even to existing example projects/forum threads covering an analogous ester-prodrug case) would be very much appreciated.

Thanks in advance!

[github-actions[bot]]

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[HenrikCordes]

Dear @leap2006 ,

as I understood your post, you are dealing with an unknown/uncharacterized enzyme that should do the metabolization process.
Why not just setting the values to 1 (or an arbitrary value) and simulate/test you hypothesis?
You can just declare relative expression to be 1.

Set up the compound to be metabolized:

Simulation:

Best,
Henrik

[leap2006]

Thanks for the quick reply!

To clarify where I'm currently stuck: with "Vascular endothelium membrane plasma-side" checked and "Show initial concentration" ticked, the compartment table at the bottom (Organ / Compartment / Parameter / Value / Normalized Expression) stays completely empty — no rows appear at all, so there is nothing for me to click into or overwrite with a value.

I'll rebuild the expression profile from scratch (delete and recreate it) and see if that resolves it — I suspect something may have gotten into an inconsistent state while I was testing different localization combinations. I'll report back with the result.

[leap2006]

In your working example, you have the Individual tab open alongside the Expression Profile — is that required for the table to populate, or was it just incidental? My table stays completely empty even with matching localization settings and an Individual already created in the project.

[HenrikCordes]

@leap2006
Yes, you have to add the expression profile to the individual:
You can check out the documentation for detailed explanation on how to use the software: https://docs.open-systems-pharmacology.org/working-with-pk-sim/pk-sim-documentation/pk-sim-creating-individuals

Expression profiles are individual building blocks and if they should be used, they have to be combined with individuals to have an impact in a simulation.

Best,
Henrik