# Stage 3 — New Hypotheses and Biological Rationale

**Pipeline stage:** 3 of 14
**Date:** 2 May 2026
**Author:** IQANOVA AI-QSP Master Pipeline v2.0

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## 3.1 Falsifiable mechanistic hypotheses

The v2.0 model architecture supports five falsifiable hypotheses about olipudase alfa pharmacology in ASMD. Each is expressed in the canonical form **"if [mechanism] then [measurable prediction] under [specific condition]"**, anchored to a published biological basis, and linked to the parameters whose GSA dominance (Stage 7) will determine whether the hypothesis is supported or rejected.

| # | Hypothesis | Biological basis | Key parameters (testable in GSA) | Falsifying experiment | Novelty |
|---|---|---|---|---|---|
| H1 | **ASM-activity gating** — if functional lysosomal ASM activity is the rate-limiting step for tissue burden resolution, then spleen, liver and lung burden percent change correlates more strongly with `ASM_active_fraction` (composite of `kcat_ASM`, `REA_baseline`, `Olipudase_lysosome`) than with the individual tissue resolution rate constants | Schuchman & Desnick 2017 (NPD enzymology); Wasserstein et al. 2019 (ASCEND adult Phase II); Diaz et al. 2021 (ASCEND-Peds Phase I/II) | `kcat_ASM`, `REA_baseline`, `k_tissue_to_lysosome`, `k_lysosome_degradation` | A patient subgroup with very low residual endogenous activity (REA) and intact paediatric pharmacology should show *slower* tissue resolution proportional to (REA + olipudase contribution) — measurable by serial spleen MN at 6, 12, 24 months | **Novel** in QSP form (mechanism known qualitatively but not previously linked to tissue-specific resolution rates in a fitted ODE model) |
| H2 | **ADA-positive sub-population** — if anti-drug antibodies neutralise functional lysosomal enzyme, then patients converting to high-titre ADA between months 6 and 12 should show a deceleration of tissue burden reduction beginning ~30–60 days after seroconversion, with the magnitude proportional to `ADA_neutralisation_potency × ADA_titre` | EMA Xenpozyme PI §4.4 (immunogenicity); Wasserstein et al. 2024 ASCEND-OLE 2y data | `ADA_neutralisation_potency`, `k_ADA_generation`, `k_ADA_clearance` | Compare ASCEND-OLE patients with confirmed neutralising ADA (NAb+) to NAb− on serial spleen MN trajectories from month 6 onward | **Novel** quantitative prediction (no previous QSP model has parameterised ADA dynamics for olipudase) |
| H3 | **Paediatric rapid-resolution advantage** — if paediatric tissue has higher functional ASM uptake (`k_tissue_to_lysosome`) and higher per-organ resolution capacity (`spleen_resolution_max_frac`, `liver_resolution_max_frac`), then ASCEND-Peds patients should reach 80% of their two-year tissue-burden response by month 6 (compared to month 9–12 in adults) | Schuchman & Desnick 2017 (paediatric tissue substrate clearance); Diaz et al. 2024 (ASCEND-Peds 2-year extension); Mistry et al. 2018 (paediatric ERT response in Gaucher type 1) | `k_tissue_to_lysosome`, `kcat_ASM`, `spleen_resolution_max_frac`, `liver_resolution_max_frac` | Bayesian meta-analysis of percent-change-from-baseline at month 6 vs month 12, paediatric vs adult, should show paediatric posterior mean ≥ 0.80 × Y2 mean by month 6 | **Confirmatory** at the qualitative level but **novel** at the rate-constant comparison level |
| H4 | **Lung is the rate-limiting compartment** — if pulmonary parenchymal sphingomyelin clearance is slower than splanchnic clearance (because lung interstitial macrophage population is smaller per-gram-tissue and turnover is slower), then DLCO percent change will lag spleen volume percent change by ≥ 3 months at any matched dose, and `k_lung_resolution` will rank in the top three GSA drivers for `DLCO_percent_change` | Wasserstein et al. 2022 (DLCO recovery dynamics); Bidari et al. 2023 (lung pathology in NPD-B autopsy series) | `k_lung_resolution`, `lung_resolution_max_frac`, `DLCO_kinetic_rate`, `DLCO_gain_max_pct` | Compare ratio (DLCO Δ% / spleen Δ%) at month 6 and month 12; should be < 0.5 at month 6 and > 0.7 at month 12 | **Novel** quantitative prediction |
| H5 | **Ceramide rebound dynamics** — if plasma ceramide is the sum of (a) acute SM-hydrolysis flux (transient increase) and (b) tissue-burden-coupled steady-state contribution (decreases with time), then plasma ceramide will show a transient peak around weeks 4–8 followed by net reduction by week 26, with the trough magnitude determined by `ceramide_burden_coupling` and `k_ceramide_clearance` | Wasserstein et al. 2019 (early plasma ceramide pharmacodynamics); Lipinski et al. 2020 (sphingolipid flux modelling) | `k_ceramide_generation_from_SM`, `ceramide_burden_coupling`, `k_ceramide_clearance` | Dense early sampling (every 4 weeks for first 6 months) of plasma ceramide should show a peak between week 4 and week 12 in both populations, with greater amplitude in paediatrics | **Novel** — v1.3 had no peak-then-trough behaviour; this is genuinely new mechanistic prediction |

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## 3.2 Mapping of hypotheses to v2.0 model components

| Hypothesis | SBML species touched | SBML rate rules touched | Assignment rules used |
|---|---|---|---|
| H1 | `Olipudase_lysosome`, `SM_spleen_burden`, `SM_liver_burden`, `SM_lung_burden` | All three burden ODEs | `ASM_active_fraction` |
| H2 | `ADA_titre`, `Olipudase_plasma`, `Olipudase_lysosome` | ADA ODE; Olipudase_plasma ODE | `Olipudase_effective` |
| H3 | All burden compartments; biomarkers | Burden ODEs (paediatric parameter set) | `spleen_volume_pct_change`, `liver_volume_pct_change` |
| H4 | `SM_lung_burden` | Lung burden ODE | `DLCO_percent_change` |
| H5 | `SM_lysosome`, `Ceramide_plasma` | Substrate and ceramide ODEs | `Ceramide_percent_change` |

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## 3.3 Pre-specified GSA dominance predictions (to be tested in Stage 7)

For each hypothesis, the GSA-dominant parameters predicted by mechanism are listed. If the Stage 7 GSA reveals different dominant parameters, the hypothesis is wholly or partly falsified.

| Hypothesis | Predicted GSA-dominant parameters | Predicted endpoint(s) |
|---|---|---|
| H1 | `kcat_ASM` (positive), `REA_baseline` (positive), `Km_SM` (negative) | spleen, liver, lung burden |
| H2 | `ADA_neutralisation_potency` (negative), `k_ADA_generation` (negative) | all burden + DLCO |
| H3 | `k_tissue_to_lysosome` (positive), `spleen_resolution_max_frac` (positive) | paediatric burden endpoints |
| H4 | `k_lung_resolution` (positive), `DLCO_kinetic_rate` (positive) | DLCO_percent_change |
| H5 | `ceramide_burden_coupling` (positive), `k_ceramide_clearance` (negative) | Ceramide_percent_change |

**Gate to Stage 4: OPEN.**
