GBA Activator criteria, Target profiles, Definition, GBA activity / GlcSph, RD-DDU GBA Activator, L444P Lyso-GL1, GBA GT start
GBA Activator criteria (continued)
Pk? Cbu target? → already kind of achieved, easier than potency. How to determine? → in vivo mice
On track?
Then Biology experiments:
Prospect
RD ? → no activation in cell free, ↑ activity in cell system (but at ~3 μM, so not very potent) (possible reason of discrepancy between cell free & cell system: chaperone effect) they don’t want to include activation in PE criteria, ↓ GlcSph, no activation in GBA KO cells (ie cell system)
AC inhibitor
20200428 Ceri meeting — Target profiles to meet the milestones (nGD)
Current profiles:
- Cell-free EC350: 0.5 - 1 μM
- GlcSph reduction MED in iPSC-neuron: 1 μM
- Cbu… by i.p.: ~4 μM / 300 mg/kg, i.p.
- Cbu… by p.o.: ~0.02 μM / 10 mg/kg, p.o.
- ADMET profiles: Under evaluation
| Cell-free EC350 | GlcSph reduction MED in iPSC-neuron | Cbu... by i.p. | Cbu... by p.o. | ADMET | |
|---|---|---|---|---|---|
| LG | ≤1 μM | ≤1 μM | ×10 | ×3 | |
| PE | ≤0.1 μM | ≤0.1 μM | ≥1 μM / 30 mg/kg, i.p. | 0.05 μM / 10 mg/kg, p.o. | No critical concern |
| CN | ≤0.03 μM | ≤0.03 μM | ×3 | ×2 | ≥0.1 μM / ≤10 mg/kg, p.o. Tolerable profiles |
We need to finalize target profiles of CN candidate by PE.
Correlation of GlcSph reduction in iPSC-neuron and in vivo (mouse liver or brain) should be investigated. CONFIDENTIAL
위 그림 내 해석: eg. CN 때는, 우리 물질이 i. [in vitro: GlcSph reduction in iPSC-neuron]에서, EC50 이 30 nM 이하이고, 또한, ii. [In vivo]에서 GlcSph reduction 이 50%이상이어야 함 (그리고 그 때의 약 Cmax 가 in vitro 의 농도와 equivalent 할 거라고 assume 하나봄), cf) target cell EC50s are 100 nM (PE) and 30 nM (CN) to achieve in vivo GBA activation at Cmax (PE) and GlcSph reduction (CN).
Definition (Below 20200529 LGE sDIB)
| Categories | Methods | Models / BMs | Objectives | PE target profile | CN (nGD) target profile |
|---|---|---|---|---|---|
| Cell-free potency | Enzyme assay | Recombinant GBA / synthetic substrate 4MU-β-D-glucopyranoside (4MUG) | • 1st screen • GBA-compound direct binding | EC350: 100 nM | EC350: 30 nM |
| In-cell potency | GlcSph reduction assay | iPSC-Neuron (L444P/L444P) / GlcSph | • Effectiveness in a target cell type: dopaminergic neuron • Chemotype Go/NoGo | EC50: 100 nM | EC50: 30 nM |
| In vivo target engagement | Enzyme assay | Wt mouse / 4MUG | • Acute effects after a single administration • PK/TE correlation | MED: 100 mpk 로 statistically significant GBA activity increase vs vehicle-treated mice (PE criterion) | MED: TBD* |
| In vivo pharmacodynamic | ↓ GlcSph | GBA mutant mouse (L444P/+ or others) / GlcSph (JS: Our GBA activator doesn't increase GBA ACTIVITY of D409V mice) | • Effects on proximal BM / Gaucher disease-related BM | - | 50% reduction, dose: TBD* |
*TBD based on in vitro enzyme activation/GlcSph reduction correlation, PK/TE correlation, and safety margin
GBA activity and GlcSph reduction
GBA activity
3.5-fold in the cell-free = ↓ 50% GlcSph (cerezyme 0.1 U/ml) in L444P iPSC-neuron = 130% GBA activity in ex vivo brain homogenate.
EC350 (N370S/4MUG, pH4.7) (% basal activity)
이 basal activity 는 WT 과 비교시 어느 수준?
Can we estimate how much the 350% of N370S correspond to the activity of WT GBA allele?: → No. The basal enzyme activities were different more than 100-fold between wt and N370S in this assay. We don’t have the explanation for this difference.
Cf) We assume that activation of recombinant GBA by 350% in our enzyme assay is the minimum effect we can observe in cell-based assays (일단, cell free 에서 했는데, 나중에 cell 에서 실험시
| LGE criteria | T-4391493 | CN | |
|---|---|---|---|
| cell-free enzyme assay | ≤1 μM | 0.91 μM | PD: 30 nM 로 GBA activity returning to the normal level 달성 |
| GD & PD 공통: 100 nM 으로 GBA activation by detectable level 달성 (?) |
GlcSph reduction
EC50 (% of that by Cerezyme at 0.1 U/ml, 100%면 cerezyme 과 동등한 정도로 줄임, 50%면 cerezyme 이 줄이는 것의 반밖에 못 줄임.) cerezyme 이 이 농도에서 어느 정도로 줄이는 걸까? 이 cerezyme 농도는 Clinical dose 에서의 plasma concentration 과 비교시 어떤 수준인가?
| LGE criteria | T-4391493 | CN | |
|---|---|---|---|
| iPSC-DaNs | ≤1 μM | 55% | GD: 30 nM 로 50% (or comparable to venglustat) 달성, |
The rationale of Cerezyme at 0.1 U/ml:
- The maximum dose is 1 U/kg/min for 1 h, once every weeks.
- Elimination half-life: 3.6 - 10.4 min (Wikipedia).
- Total blood volume is 7% of body weight.
- 1 U/70 ml × 10 min = 0.14 U/ml (70 ml 는 1kg=1L 의 7%) (Inazuka san 1 시간에 걸쳐 준 약이 다 준 이후 10 분동안 머무르니까 10min 을 곱했나? 이해 안감) 그럼 반감기가 1 달이면 1 달을 곱하나?
↓ GlcSph in vitro (by 50%) at 0.3 μM in iPSC DaNs.
↓ GlcSph in vivo (D409V/D409V 계획인데, 여기서 GBA activity activation 이 안 되서 다른 쥐 (D409V/WT, L444P/WT 등 고려 중)
| GlcSph in vitro (by 50%) at 0.3 μM in iPSC DaNs | LGE criterion | T-4391493 |
|---|---|---|
| 20 (D409V/D409V 계획인데 여기서 GBA activity activation 안 됨) | sym (mice ko) | |
| (D409V/WT, L444P/WT 등 고려 중) | 202 sep (mice study) |
RD-DDU GBA Activator
| LGE Criteria | D | Target | PE Date | target | CS | IN | D | |
|---|---|---|---|---|---|---|---|---|
| ↓ GlcSph in vivo (mouse liver) | No GBA activation in cell free assay The same binding site, but different chemotype | (202 0 Apr-Jun →) 2020 Sep | ↓ a-syn (mouse) |
20201013 Imaeda san: RD 거? → no activation in cell free, ↑ activity in cell system (but at ~3 μM, so not potent) (possible reason of discrepancy between cell free & cell system: chaperone effect) they don’t want to include activation in PE criteria, ↓ GlcSph, no activation in GBA KO cells (ie cell system)
below 20200619 Crosssite meeting: 그들것은 N370S 와 L444P 둘다에 유효한 듯, D409 는 No data
L444P Lyso-GL1 Substrate Results (CIT20-09, Exp 3), wt 없네?
Age at collection: 15.5 Weeks
Panel titles (Normalized): CIT20-09 (Exp 3) Spleen Lyso-GL1, Liver Lyso-GL1, Brain Lyso-GL1, Bone Marrow Lyso-GL1, Lung Lyso-GL1, Serum Lyso-GL1.
Group legend (Takeda):
- CR0005 106 13 (PO) — 11 Days Post Dose
- CR0005 167 34 (PO) — 11 Days Post Dose
- LT-291 (PO) — 11 Days Post Dose
- AOCA (IV) — 24 Hours Post Dose
- Venglustat (PO) — 11 Days Post Dose
GBA GT executive summary (start)
| LGE | PE Date | CN target | CS | IN | D | |
|---|---|---|---|---|---|---|
| Cassette expression in vitro (as plasmid) | • Cell lines - Non-neuronal | 20 20 Jul | Evaluation as AAV (tool capsid) → goal: Selection of 1 expression cassette Pilot 1 / Pilot 2 / Main study Mar 2021 2023/06 in vivo mouse study 2023/10 NHP preliminary safety NHP GLP tox/biodistribution (6m, standard tox) | NHP GLP toxicokinetics & biodistribution | ND |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Target profiles / Cbu rows | Cbu... by i.p. and Cbu... by p.o. | reads as written; the trailing ... indicates a clipped subscript. |
| GBA activity paragraph | (일단, cell free 에서 했는데, 나중에 cell 에서 실험시 | the sentence ends mid-clause. |