Biomarkers measurements for planned in vivo studies
| Study | Striatum / (CORTEX) | Substantia Nigra | Plasma/CSF |
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ATUKA – mouse, AAV- aSyn, 9 weeks efficacy study MCC-950 vs 2 Takeda compounds ➤ start: April? ➤ end: September? |
All done at ATUKA
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All done at ATUKA
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Dr. Sortwell (MSU) – Rat aSyn-PFF model and AAV aSyn models – 1, 2, 6-month evaluation (no compounds tested) ➤ start: April? ➤ end: December? |
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Subchronic
| 1w dosing | 2w dosing |
|---|---|
| 30 mg/kg of TR098 treatment for 5 days did not reduce TSPO/NRLP3 protein expression |
In next study from Nov 7, the dosing period will be increased to 2 weeks to confirm the effect of lowering NLRP3and TSPO. ✓ Since the number of animals is statistically required to have 16 mice per group, and since some mice drop out during the study, only three groups(sham, vehicle, TR098)can be conducted, so only one dose of TR098 will be administered. The effect of 30 mg/kg of TR098 should be confirmed firstly. The results will be reported in mid Dec. -surgery (=sham): 노인 -surgery+bzATP (vehicle group): 노인+ PD -surgery+bzATP+약: 노인+ PD+ 약 The evaluation of TR660 in a sub-chronic mouse will be able to start after new year. The available API amount of TR660 will be checked (8 g available?) Several brain tissue samples of sub-chronic mice can be shipped to Ashley for ARG study. |
| MBM-based patient stratification approach was deprioritized by NS-TAU, and wasn't supported by patient brain tissue target validation result. |
Katy
Sharefolder: 2023
| NLRP3 | ASC/PYCARD | ASC speck | Caspase-1 protein (The Abs are directed to the P20, so probably measure so it will bind pro-form, p20 monomer, and tetramer. Unknown whether it binds to csp-1 in inflammasome.) | Casp-1 Activity : | Casp1 activity: probe | IL-1β | IL-18 | GSDMD | ||
|---|---|---|---|---|---|---|---|---|---|---|
| % homology w/human protein | 82 | 69-72 | 62 | Promega | 67-68 | 63-65 | ||||
| ELISA | ELISA | ELISA | MSD (ECL S-Plex assay ) | ELISA | ||||||
| Rat | Brain | Yes We have LBAs that can measure NLRP3 and ASC in brain homogenate | Yes, but incosistent | Yes ( | BLOD, Endog level unknown | Yes | ||||
| CSF | BLOD EL: unknown; good spike recovery Planned: pff 6 mo. CSF (If enough volume left) | BLOD .Normal endogenous level unknown | Yes: 200~300pg/ml (이게 꽤 높긴 한데 individual CSF) 좋은결과 Required volume: 10ul (1/20 의미?) Plan: 6m pff plasma | 안좋음 Some total activity in plasma, but little/no inhibition with caspase-1 inhibitor; | 안좋음 signal from recombinant caspase-1 is completely suppressed when spiked into plasma, (but not CSF) | Current V-plex: (~10 pg/ml) [Fischer rats] all BLOD, [pooled CSF from SD rats] (age unknown): just above the detectable level, so SD may be a bit higher than Fischer, or they were older. Required volume: NA Because Endog level is BLOD [S-plex]: : undetectable, even undiluted. We may be able to measure elevated levels, but we won't have a baseline comparison. (20ul CSF required) | Yes [Fischer rats] mostly BLQ at 5 mo., and higher at 10 mo. It was not significant, but it is highly significant if removing the outlier from the 5 mo (IL-18 Fischer Rat CSF inline scatter) group.Required volume:20ul, EL: 5m 60 pg/ml (tsd), 10m 89 pg/ml (TSD) Planned: limited spike recovery; 6 mo. pff CSF | |||
| Plasma | BLOD (LOD ~6 pg/ml) EL: unknown; good spike recovery Planned: pff6 mo. Plasma | BLOD (<15 pg/ml) Normal endogenous level unknown | Yes Yes: 200-350pg/ml 1/20 (5ul) Planned: 6 mo. pff CSF | V-plex: BLOD (<7pg/ml) [Fischer rats] all BLOD, Required volume: TBD, Endog level: 0.8-2pg/ml (Quanterix estimate) S-plex: endoge level: ~0.4-2.3 pg/ml (after adjusting for a minimum dilution factor of 2-fold) -S-plex: LOD 0.17 pg/ml, and LLOQ ~0.6 pg/ml -detectable but not strictly quantitative, as they are mostly between the LOD and the LLOQ. So we need to keep in mind that any comparison to baseline may be more of a relative measurement -Rat plasma spike recovery is low at all dilutions(~50%), so endogenous levels are possibly higher than measured. -Required volume: 20ul (as samples need to be diluted at least 2-fold to avoid matrix interference) | Yes [Fischer rats] (좋은결과) a significant elevation in plasma levels at 10 mo. vs. 5mo. (IL-18 Fischer Rat Plasma inline scatter) Required volume: 12ul, 85-406 pg/ml (manufacturer), EL: 5 m 392 pg/ml (TSD), 10m 621 pg/ml (TSD) Planned: limited spike recovery; 6 mo. pff plasma | |||||
| Brain | Yes | Yes | Yes | |||||||
| CSF O Just measurable @ 4 fold dilution 20-30 pg/ml (25ul, 3-4 mice) barely detectable, not in the quantitative range, and dilutional linearity in unknown could be run at risk | Just measurable @2 fold diln ; ~20 pg /ml (requires 40ul; 4-8 mice depending on age) barely detectable, not in the quantitative range, and dilutional linearity in unknown could be run at risk | Maybe, need further titration | 안좋음 mouse CSF has a very high level of contaminating blood caspase-1 that may confound an activity signal. | BLOD in a 2-fold dilution. Even if it could be detected undiluted, it would require at least 10 animals or more per data point (→ defeating the purpose). Required volume: NA Because Endog level is BLOD | Yes | Yes (~500 pg/ml) ¼ dilution (20ul required, 3-4 mice) : Spiked blood assays suggest that any signal in CSF is likely coming from systemic source (due to both the high plasma levels and the difficulty of drawing "clean" CSF from a mouse) Plan: 1. Measure it at risk, 2. Evaluate/develop enzymatic activity assay for mouse plasma (and possibly CSF) to mirror activity assay development for human CSF Not recommendable | Just measurable @4-fold diln; endogenous level: ~10 pg/ml LOQ: ~15 pg/ml, (TSD assay requires 100ul, so for one data point 25ul CSF is required.. With ~10ul CSF/mouse and considering pipetting dead volume 3-4 mice depending on age is required.. Plan: If it is decided this matrix will be selected for translation (time-frame?), we will look at developing our own 384-well assay in order to minimize CSF volume needed (potentially ~5ul/well). The signal is barely measurable, however it is most likely due to contaminating blood proteins (This has not been evaluated because the team decided to de-prioritize it.) | |||
| Detectable, but BLQ ~410 pg /ml : 2 fold dilution LOD~1.6pg/ml); limited spike recovery good NOMID gof >100X increase | BLOD (<7pg/ml) | Some total activity in plasma, but little/no inhibition with caspase-1 inhibitor | 안좋음 signal from recombinant caspase-1 is completely suppressed when spiked into plasma, (but not CSF) | Yes 1-2 ng/ml (1/10:10ul) 10ul 있으면 된다는 거 아니네.. 거네 | Yes (MSD S-plex: LOD ~0.1pg/ml) Endog level: ~0.2-0.8 pg/ml (TSD)? No S-plex for mouse IL-1b -detectable but not strictly quantitative, as ... Yes ~50-200 pg/ml 4-fold dilution NOMID gof >20X increase |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Katy table top header | ”Caspase-1 protein (The Abs are directed to the P20, so probably measure so it will bind pro-form, p20 monomer, and tetramer. Unknown whether it binds to csp-1 in inflammasome.)” | Long header text wraps inside the blue band; final phrase “in inflammasome” preserved as best-effort. |
| Rat / Plasma row, IL-18 inline chart | numeric annotations 5 mo / 10 mo | Inline IL-18 Fischer Rat Plasma chart data points (red and blue dots) are kept as evidence; per-point numeric values are not transcribed. |
| Brain row leftmost cell | ”BLOD, Endog level unknown” | Cell appears repeated under Brain row of Caspase-1 column; preserved as-seen. |
| Subchronic 2w dosing cell | ”노인+ PD+ 약” tail | Tail of the labelled-experiment list near the cell wrap is partly faded; preserved as legible. |
| Mouse Brain row, GSDMD column | ”~50-200 pg/ml 4-fold dilution NOMID gof >20X increase” | Numeric values straddle the cell border; preserved as best-effort. |
| Last row IL-18 entry | ”Yes (MSD S-plex: LOD ~0.1pg/ml) Endog level: ~0.2-0.8 pg/ml (TSD)?” | The “(TSD)?” annotation is partly cut off; preserved as legible. |