| In Vivo strategy overview | Validation | |||
|---|---|---|---|---|
| iPSC α-Syn | Microglia/Astrocytes/Neurons (Dr Lipton, Scripps) | Literature data at PE; Validation with internal compounds after PE | ||
| Rodent MOA | NLRP3 GoF mouse model (Dr Hoffman, UCSD) — Fully characterized; Validation with internal compounds before PE | α-Syn PFF rat model (Dr Sortwell, Michigan) | α-Syn PFF/iPSC microglia hum. mouse model (Dr Lipton, Scripps) | Currently characterizing NLRP3-IL-1β axis activation level |
| Primate & human | Primary Human Microglia Healthy vs PD patients (CRL) — Fully validated Internal compound data at PE | AAV-A53T SNCA / α-Syn PFF cyno model | PK/PD; RO/PD | |
(Takeda Pharmaceutical Company Limited footer logo)
| Atuka 1st | Atuka 2nd | sortwell | sortwell | |
|---|---|---|---|---|
| Construct |
aSyn AAV-aSyn : intranigral AAV1/2 human A53T α-syn AAV1/2 A53TaSyn 3694 bp (DNA cartoon: ITR / SAR / CBA promoter (green arrow) / HA tag / WPRE / alpha A53T synuclein / bGHpolyA / ITR) (Koprich et al, 2010, 2011 Mol. Neurodeg, PLoS-one) CBA promoter, for most CNS applications, provides ubiquitous expression in a wide variety of cells | aSyn PFF (Intrastriatal injection (3 sites, bilateral): α Syn monomer, α Syn PFF, naïve | aSyn AAV-aSyn Injection (2 bilateral): Intra-AAV asyn, Intras-AAV RFP, naïve groups !WT aSyn | |
| Animal | mouse | Rat: All animals will be F344 rats, 6-8 months of age at time of surgery 2 Fischer pff rat models: aging on-site until 6-8 months (early summer) | ||
| Compounds administered | the compounds are administered one day after the insult (prophylactically) for 2 months 63 days dosing. | |||
| Dosing |
098 (3 and 30 mpk, qd) 660 (30 mpk, qd) MCC950 (20 mpk, qd) | 098: 10 mpk 660: 10 & 30mpk, |
(20220202 M), NLRP3, ASC, Caspase-1 will be measured in brain, CSF and plasma samples from those animals. WE have made plans with Paul. [1 and 2 months after injection]
| |
| Tissue assays |
SN tissue will be assessed at Atuka by Stereology/IF = TH+ve cells, Total human a-SYN and pSYN Striatum tissue will be assessed at TSD by WB = TH, NLRP3 pathway, TSPO, pSYN and fragments of a-SYN (the latter very investigatory at the moment). CSF will be sent to TSD to be tested = TBD based on the available assays and what we think collectively makes more sense to test. | |||
Early readouts - September 2022
- cylinder test
- striatal dopamine and metabolite levels (via LC-MS/MS)
- dopamine transporter (DAT) (via autoradiography)
Later readouts - October/November 2022
- TH+ve neurons in SN
- Number of human alpha-syn expressing TH+ve neurons in SN
- Human a-syn (IF이라 고): total, phospho (antibody: EP1536Y), fragments (?)
- NLRP3, ASC, Casp-1,
CD11b, TSPO
| tudy elements | Due on full study commission (50%) | Due on delivery of UoP I (40%) | * Due after go/no decision (50% of addition) | Due on delivery of Draft Final Report (10% + 50% of additions) | Total |
|---|---|---|---|---|---|
| Five treatment groups; behaviour, striatal dopamine and DAT and the PK study | $90,485 | $72,388 | - | $18,097 | $180,970 |
| *Addition of TH/aSyn stereology (all 6 groups) | - | - | $51,678 | $51,678 | $103,356 |
| *Addition of pSer129/another marker stereology (all 6 groups) | $51,678 | $51,678 | $103,356 | ||
| *Addition of NLRP3 assessment (all 6 groups) | $17,226 | $17,226 | $34,452 | ||
| *Addition of ASC assessment (all 6 groups) | $17,226 | $17,226 | $34,452 | ||
| *Addition of CD11b assessment (all 6 groups) | $17,226 | $17,226 | $34,452 | ||
| *Addition of H&E (all 6 groups) | $6,251 | $6,251 | $12,502 |
- Based on the above data a second study to be initiated in fall 2022
| 098 | 3 (1st) | 10 (2nd) | 30 (1st) | |
| 660 | 10 (2nd) | 30 (1st & 2nd) | ||
| MCC950 | 20 |
| Katy 20220211 | (plasma availability) |
|---|---|
evaluate availability and feasibility of plasma assays for (in normal mouse plasma):
|
Sample availability: SN X, striatum O, CSF O, blood X Tail vein blood samples (plasma) to be collected monthly with terminal cardiac blood collected at 6 months, samples sent to Takeda (NLRP3/IL 1 β pathway) M: will add TSPO Rat: Order and evaluate available rat assays Measure plasma and CSF levels for Sortwell aSyn AAV-pff (?) rat models :
|
| Study to be performed in April - Analyzed data by September 2022 (officially CN) | |
Compounds
- Initial plan: TR06692993 AND/OR TR06693098 in addition to MCC-950
- 20220208: to One compound will be chosen from TR06692993 (→ 나중 note, de-prioritized IP issue) OR TR06693098 OR TR06698660 (CN슬라이드보면 이것도 FTO issue있다는데)
- 20220209 Satoshi proposal: one compound 만으로는 CS 성공가능성 위험, two COMPOUND 하자
- 20220210 M: In the end 2 compounds will be advancing in parallel to CN-CS
- 20220513: TR06693098 (30 mpk & 3mpk OD) & TR06698660 (30 mpk QD), MCC-950 (IP 20 mg/kg QOD would be suitable for MCC950 dosing 50 mg/kg po QD may be unsafe and IP 20 QOD has been adopted in several other reports) → no ability to define minimum efficacious dose from tis study to enable definition of safety margins in DSRE studies (ATUKA study would help to set the dose of 2wks tox study.
In vivo strategy details
- Initial plan: single dose
- 20220208: (To establish Efficacy-PD relationship and define the subsequent non-GLP safety studies after CN) Two doses (30 mg/kg and one more dose) will be tested
- 20220209 Satoshi proposal: (tentative) MED는 이 in vivo study 말고, PK/PD study 로 정하자
- 20220210 M: How to determine MED? Brain PK/PD assay plus time course studies OR AAV-A53T-αSyn mouse model ?
Cell numbers in AAV1/2 + pSyn mice
| All cells | neurons | TH+ | 3500 | TH+/paSyn+ | 500 (~15%) |
| TH+/paSyn- | 3000 | ||||
| TH- | |||||
| microglia | NLRP3+ | 3000 | NLRP3+/paSyn+ | 2.5% (~75) | |
| NLRP3+/paSyn- | 97.5% | ||||
| NLRP3- | |||||
| ODC | |||||
| astrocytes | |||||
| endothelial | |||||
NLRP3 증가 x, CASP-1 증가 X, microglia 수 (IBA1, TSPO) X,
What killed DA neurons?
NLRP3+/paSyn+: only 2.5%만 표현되었는데?
| 1m | 2m / 약효 | ||||
|---|---|---|---|---|---|
| model | 약효 | model | 약효 | ||
| NLRP3 | Microglia | i) Dying neuronal debris과 ii) (당연히) neuronal paSYN이 (iii) 다른 cell의 paSyn도 혹시) 더 많으므로 이것도 더 많을 가능성있어 볼가치O | (염증없는 2m때와 달리) 염증이 있다면 ↓가능성? | ||
| Casp-1 | microglia | i) 상동 ii) 2m 에서 아예 안 봤음. | |||
| asc | |||||
| Il-1 | |||||
| IL-18 | |||||
| TSPO | Microglia | (염증없는 2m때와 달리) 염증이 있다면 ↓가능성? | |||
| p-aSyn | Microglia | 모델상 그냥 당연 | (2m에서 Dose-dependent로) 줄이는경향 ㅇ → 볼가치O | ||
| p-aSyn | Neuron | 별로 영향없는 듯 | |||
| p-aSyn | oligo | 불명, (다수 cell type이니) 볼가치 | 불명, 볼가치 | 불명, 볼가치 | |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
top overview grid / column placement | the In Vivo strategy overview is a presentation-style figure with shaded rectangular boxes; column groups are pulled from the visible labels in body_r01_c01.jpg and body_r01_c02.jpg and may not exactly match the source slide layout. | low confidence on column-by-row placement. |
study elements cost table / row label | the header row reads tudy elements (likely a typo for Study elements due to clipping at the left edge); preserved verbatim. | low confidence on whether the leading S is clipped. |
Cell numbers / TH+/paSyn+ row | the 500 (~15%) value is read as ~15% of TH+ cells; preserved verbatim. | low confidence on whether the percent denotes paSyn+ fraction of TH+ cells or some other ratio. |
In vivo strategy per-marker per-cell sub-table / 1m vs 2m column placement | only the 1m / model / 약효 columns are clearly visible in the captured frame; the rightward 2m columns are mostly clipped and partly visible only as red Korean cells. | clipped at right edge; partial. |