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The 5-fold increase of NLRP3 expression was confirmed in striatum of LPS injected rat models. IL-1b level was 1.5-fold upregulated of 0.5 mg/kg LPS rats but no increase was observer in 2 mg/kg LPS rats. The results of the 2 mg/kg group was reasonable because the final dosing of LPS was skipped. There was no TH loss in LPS models. ✓ The increase level of NLRP3 in LPS model was comparable to that in 6OHDA rat model. ✓ The remains of LPS rat samples (cortex, midbrain and hippocampus) will be shipped to Ashley for ARG study. If the striatum sections are needed, Masato can collect it again. It will take 3 weeks to deliver the samples. |
mg/kg). There was no big difference of Kp of TR06647850 (Tissue/plasma) between 0.01 mg/kg and 1 mg/kg in both control and LPS model rats, suggesing The SB of TR06647850 to the brain may be weak in this LPS model rats. There was no big difference of Kp (Tissue/plasma) and fold change of striatum between 0.05 mg/kg and 1 mg/kg of TR06683014 in both control and LPS model rats, (although In TR06683015, Kp and fold change of striatum were slightly enhanced at both 0.05 mg/kg and 1 mg/kg of LPS model rats. The specific binding of TR06683014 and TR06683015 to the brain may be weak in this LPS model rats. | |
| Interpretation |
After obtaining the Kp results of TR850 at lower doses (ie 0.01 mpk) in 6OHDA rats (will be generated next week), team will discuss the necessity of additional Kp study of TR850 at lower doses in LPS rats. Just in case, adhoc meeting will be scheduled. ✓ The sensitivity of TR850 at lower dose are checking in TSHO by API6500 (the same machine as TSD) this week. ✓ Since LPS rats have not undergone brain surgery, it would be desirable to see brain uptake. | |
| Next steps |
To evaluate TR06683014 and TR06683015 with improved BBB permeability and lipophlicity were improved using LPS rats. This study will be proceeded by Bozo After checking the results of TR014 and TR015 in LPS rat model, the evaluation of both compounds would be discussed with 6OHDA rat model. |
LPS (bain injection) Rat
| 1st | 2nd (20220505) | |
|---|---|---|
| Tissue collection | 3 d, 5 d | |
| NLRP3 Level? | Paul Rolzin: | |
| tracer | [3H]TR-126 | |
| Blocker | ||
| Result (microglia) |
[3H]PBR28 binding increased on the ipsilateral side of injection Binding increased in 3-day LPS rats compared to 5-day LPS | |
| Result (NLRP3) | .[3H]TR-126 (80nM) showed high SB in SF9 insect cells over-expressing NLRP3, but did not show increased binding on the side of LPS injection. |
Other models
| NOMID mice | PS19 mice | |
|---|---|---|
| NLRP3 level |
results will be generated in Aug it will take 6-7 months to receive the mice |
analyzing NLRP3 expression (2, 5, 8, 11 months old mice) by August Shonan has a license for PS19 mice, and Toni is working to use this mice in TSD |
ARG Postmortem-AD
| 1st | 2nd | |
|---|---|---|
| Tracer | [3H]TR-126 | |
| Blocker | ||
| Result (microglia) | ↑ 2nM [3H]PBR28 (vs HC) | |
| Result (NLRP3) |
- Some specific binding of 30nM [3H]MCC950 (60% and 75% Inhibition observed at 30nM [3H]MCC950 in Tris and Ion buffer, respectively) - AD vs HC 비교는 무의미 - Tracer는 MCC950만 사용. |
[PD & AD: ] -At 13nM (~ Ki), no usable binding observed in control or diseased tissue -At 80nM: Specific binding window increased using [3H]TR-126 compared to [3H] MCC950 at ~80nM in human tissue, but Comparison not included herein [AD] - Tracer (30-190nM) SB window was not improved using [3H]TR-126 (vs [3H] MCC950) -Suggests binding to available NLRP3 site is lower affinity and easily washed away -NSB driving observed signal ↓ ↓ [Centrifugation assay, homogenate, with increased protein amounts (5 & 10mg)] - No SB observed with both [3H]TR-126 & [3H]MCC-950 (suggest right-shifted affinity, low Bmax?; high NSB in human tissues, Higher affinity radioligand necessary) |
PET activities (with start/end date)
[Study lists_NLRP3 PET.xlsx](Study lists_NLRP3 PET.xlsx)
Scenario
| Problem | Human brain ARG | In vitro | Non-imaging triaging (LC-MS) | Animal PET study | Risk | |
|---|---|---|---|---|---|---|
| 1st | NA | O | 다음 둘 다 확립필요: i) normal 에서 signal, ii) disease vs HC signal window More than 1 nM is criteria for Bmax, and more than 10 would be the ideal target value of Bmax/Kd ratio in human PD/AD brain. |
Do we need disease model? le window 확립해야 하나? Hdac6 는 normal animal 만 했는데 team can use the in vivo animal model to triage the compounds (More than 2-fold would be desirable as SB window in vivo LC-MS study) |
My disease NHP model → NHP 는 아뇌, blocking (window 없으니, 의미 소실되네) Js: below should be the same here the brain uptake and elimination profile will be tested in NHP PET scan without blockable binding (?). we can move forward to PET scan with 6-OHDA rats. | As below should be the same here the brain uptake and elimination profile will be tested in NHP PET scan without blockable binding then, human PET will be performed at risk |
| worst e1 | O | PET tracer candidate will be selected in vitro human brain tissue, and | X | |||
| worst e2 | 6OHDA-induced ARG 불능 | O | PET tracer candidate will be selected in vitro human brain tissue, (and 6-OHDA model would not be appropriate to evaluate the Bmax of NLRP3) | But if, SB is confirmed in 6-OHDA rats by in vivo LC-MS/MS study, we can move forward to PET scan with 6-OHDA rats. | ||
| worst e2 | In vitro-in vivo disconnect | X | ||||
|
- Measure TR06647850 in 6-OHDA side vs naïve side to determine if there is enrichment of compound - What can it tell us? Blocking study possible with this animal model? | ||||||
|
- Enrichment study: principle, only Tsho DMPK? TCAL DMPK 가 어떻게 받아들였나? - Worst cast scenario: how to select by in vitro human brain tissue - 6OHDA Model: 두 선 확립 못 하니, 이걸로 - 6OHDA-induced ARG 불능 이어도 → LCMS Blocking study 가나? LPS model 도? 둘 다 blocking study? | ||||||
[Thaw cycle]
ARG: freeze thaw 없이 하는 방법 없겠지.
Homogenate: i) binding : freeze thaw 유무 비교 분명 가능한데 (Paul 이 이미 WB때 이렇게 함), ii) WB again
이거 freeze thaw 유무 비교 할까?
| NLRP3 stability | ||
|---|---|---|
| Snap freezing → homogenation | Ok | |
| Fresh → homogenation → freezing | ↓ (but only in 6OHDA model) |
PK
| Free fraction | T1/2 | Repeat doseing |
|---|---|---|
| T1/2 of 600 mg/kg group in rat 3-day tox was quite longer and higher Kp of liver. At therapeutic dose, rat T1/2 was around 8-12h. | there was no difference of exposure between Day1 and Day3 (no accumulation). | |
| The free fraction of TR06693098 in the plasma and the brain was 0.035/0.045 | T1/2 was longer than 12h. |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Scenario animal PET cell | My disease NHP model | The leading My likely is No; OCR-confused N/M; preserved as visible. |