20240722_184826

	analysis	Image development		Tomimatsu: just using Image J

Cf) microARG:
- radiolabeled substrate taken up by individual prokaryotic cells → visualized by means of a radiation-sensitive silver halide emulsion covering the radiolabeled organisms → subsequently processed by standard photographic procedures

Example		Method	Readout
Ashley's AD postmortem: [11C]NCGG401 (Ogata, 2022 #2023)		-tracer 농도는 변화 [³H]MCC950 10-100 nM, - blocker 농도는 고정 (MCC950 10 µM) Tracer 와 blocker (10 µM NCGG401) 모두 농도 고정
(Au - Griem-Krey, 2019 #1506)	Jove protocol. mouse 포함.	Fig4) total binding: as fmol/mg tissue equivalents (TE) for the radioligands [3H]GHB (30 nM). Non-specific binding was not detected in the presence of 1 mM GHB. Data is presented as mean ± SD of four biological replicates each performed in three technical replicates.
AC Immune's aSyn PET	Human brain section (p14, showing n of 2)	한번은 radio-labelled 처리 & 다른 한번은 self-blocking 해놓고 (with ACI-3710 5 µM),	Ki
SPAL	Mouse (good의 p9 fig3-2중 맨 아래) & human Patient (good의 p9 fig3-2중 위로부터 세개, 조각인듯) (n=1?)	Self-blocking
(Kroth et al. 2019, PMID 31264169)	Human brain section (frozen 18 µm): fig4 (Brakk I, III, V당 n=1인듯). Entorhinal cortex / hippocampus, frontal cortex 의 각 조각에서 본 듯, fig5: only amygdala (n=1인듯)	non-radioactive test compounds were used in excess (10 µM, 이렇게 완전히 막아놓고 나오는 signal 보는 것이니 'non-specific binding') and mixed with the ¹⁸F-labeled tracer (0.8-2.4 kBq/Ml,	Tracer 의 affinity (specific binding)를 보려는 목적이니 labeled tracer 를 여러 dose 로 quantification 없는 듯
	Micro-ARG (Frozen tissue from entorhinal cortex brain region of an AD donor)	-
위 Bmax에 (Hostetler, 2016 #889)	NHP Brain (n=4), human (n=3) 은 region별 (cortex, thal, Hippo etc)	-
(Hall, 1998 #1259)	Human brain section (whole hemisphere)	-	DA & HT receptor subtypes as well as of their transporters
(Pan, 2016 #1260)	Human postmortem AD brain hippocampus sections (7 µm). 확실히 AD 2명, Control 2명	- Chunks of frozen tissue were dissected
(Martin-Cora, 2004 #1488)	Human postmortem frontal cortex (a, b), hippocampus (c, d), mesencephalum (e, f), and thalamus: fig 4 (coronal sections)	상세한 정량화 table 3!, By caudate nucleu, putamen, GP, SN 등 Region별로 다 했음 human)	Saturation curve (rat)
(Strebl, 2017 #475)	Bavarostat: rat brain	Sagittal slices of rat brain tissue were exposed to [¹⁸F]Bavarostat in the presence of 10 µM Bavarostat or Tubastatin A Results: the binding of [¹⁸F]Bavarostat was reduced by almost 40% in the cerebellum, relative to corpus callosum, a region of high nonspecific binding. Tubastatin A, an HDAC6-selective compound, reduced the amount of bound radioactivity to a comparable level as Bavarostat. These data indicate that the binding to brain tissue occurs with selectivity for HDAC6.
(Perry, 1999 #1619)	Human postmortem sections (hippocampus, cerebral cortex)	cf) Autoradiography with [³H]EB in human brain yielded excellent images, with low to nearly undetectable nonspecific binding (Fig 3C). 그래서 위 그림에서 nonspecific binding 고려햇군. Quantified results in the table 1 (caudate nucleus only, without putamen)
(Farkas, 2012 #1694)	human striatum	-	midbrain
(Bannon, 2002 #1695)	Human midbrain	-	정량화 x

Selectivity

‘in vivo SB’ (tracer 후보의) (by pretreatment with unlabeled Bavarostat)
Target occupancy (약후보의)

SB 얻는 방법 두가지 (2017 Zhang):

i) Competitin

ii) Target KO: mimicking complete TO. i)보다 덜좋은 방법이라는 듯, target receptor 가 아니라 protein 전체가 없기 때문인가?

Zhang 2017: a ligand candidate in a non-radiolabeled “cold” form at a low tracer dose (typically <10 µg/kg) was injected into test animals.

LC-MS cold method 의 좋은 review: (Barth 2014): i) radiometabolite 관련 ii) anatomic delineation 어려움 iii) false negative iv) (Paul) For LC/MS studies due to detection limits you are somewhere around 0.5-1 mg/kg, which is a significantly higher dose than those for PET study dose.. However for those targets with low expression, you can essentially block all the target and so the majority of the material present is non-specific and so you will be unable to differentiate the specific binding.

↑ ↑ ↑ NSB, 그래서 (blocker 가 target 을 block 해서 생기는) SB 의 비중 (SB/Total)이 너무 작아짐, ie SB:NSB 비율이 너무 작음.

↑ ↑ ↑ total binding,

Specific binding: (우측그림의 왼쪽) Validation: Comparing WT and KO mice (ligand 후보의 specific binding) →

TO: (우측 그림의 우측) drug (412)의 dose 를 변화시키면서 (ligand 후보 molecule의 dose (Zhang 2017에서는 typically <10 g/kg라던데 우측은 훨씬 많이 썼네) 는 고정), 412와 competition시키면서

ligand 후보 (465)가 아니라 약후보 (412)의 TO를 보는 것임.

LC-MS Non-imaging triaging (마치면 BTV)	Mice
	Cold (readout: drug conc)
	20210120 Paul: $150K for any rodent work
in vivo Rodent		Hot, -PK: Brain uptake & retention -Specific binding (sometimes not necessary, but IP6K did this because selective binding in non-imaging study was unclear) - tracer doses typically used 0.1 - 10 ng injected i.v. to a rat (Barth 2014), - Example: 우리 HDAC6는 이거 안 한 듯.

PET probe discovery for HDAC6 (Cont’d)

T-4035187 (OnT: TBOS1)

IDAC6 IC₅₀: 4.9 nM
HDAC1 IC₅₀: 4000 - 10000 - 12000 nM

T-4055455 (OnT: Shenyang)

IDAC6 IC₅₀: 2.7 nM
HDAC1 IC₅₀: 350 - 3000 - 4400 nM
IDT vs HDAC6-KO

Validation in KO mouse / Competition with T-412 (panels)

LC-MS-based target occupancy study indicates that T-4035187 and T-4055455 are useful for target engagement study of HDAC6 inhibitor
Validation study in NHP with radiolabeled molecules is underway

Which was better to evaluate specific binding: Kp.hippocampus or Hippocampus concentration

(Slide overlay panels showing four occupancy/concentration scatter plots with red callout boxes)

This can represent NULL occupancy of 412. 위에 그림으로 부터 추측 가능 (4그림 중에 첫번째). 즉 412에 의해 막히지 않은 condition.
This can represent full occupancy of 412. 4그림 중 마지막 (즉 KO이라거나, R 465가 매우 적은데다가 가까이 412에 의해 fully 막혀있게 되는 거랑 같다는 의미). 그런거지 같음 받은 의미라는…
412의 dose @ 변화시킨 거와 4번 째에 실린 occupancy 를 잘다라가 양상으로 같지를 결정.
Concentrations in plasma matched those in hippocampus, caused Kp.hippocampus.
Kp.hippocampus values correlate with specific binding among study batches: (WT/Vehicle, HDAC6-KO):
13.3 ± 5 [CV 64%, n=12], 3.61 ± 0.88 [CV 28%, n=4]
Hippocampus concentrations corresponding to null occupancy were stable among study batches: (Mean ± SD): 6.13 ± 0.24 [CV 4%, n=12], 1.85 ± 0.24 [CV 13%, n=4]

→ Hippocampus concentration could be better to estimate target occupancy.

Non-imaging methods to triage selected compounds and assess specific target binding

LC-MS quantification of “cold-tracer” tissue distribution
- A novel, efficient paradigm for identifying PET tracers

Step 1: IV administration of non-radiolabeled compound

Step 2: Tissue dissection and sample preparation at desired time-points

Step 3: LC-MS quantification of compound concentration

Step 4: Tissue distribution and time course analysis

LC-MS allows for the quantification of a compound’s regional distribution without needing to prepare a PET tracer precursor or develop radiolabeling methods. It can be used to assess specific binding, time-course activity as well as metabolite profile and translates well into NHP

A LCMS/MS evaluation in rat of the dopamine D2/D3 tracer FLB-457 (n = 4) over 60 min (ACS Chem Neurosci (2014) 5: 1154; rat regions Cerebellum / Striatum / Frontal Cortex)

Flow/criteria of PET tracer development (cold study)

Blue: Criteria

Step 1

IC₅₀: <10 nM (Bmax/Kd (>10))
MDR1 ratio: <3
LOGD: 1-3
fu. brain: >1% free (>5% preferable)
Mouse IV: rapid elimination
Labeling position: ¹¹C, ¹⁸F

Target expression / distribution analysis (rodent, monkey, human / WB, mRNA)

Step 2 — Specific uptake

IV screening for specific binding in rodents (LC/MS/MS)
1. Differential-distribution (target/reference region)
  1. reference region O. PDE10A
2. Dose-dependently change of Kp 2. reference region X. CHQ4H
Metabolite identification in vitro: No/little undesirable metabolites
Monkey IV (cassette): rapid elimination

Step 3

↑ Cold — Competitiveness: Pretreatment study by drug candidate (LC/MS/MS) → Competitive
Selectivity: LC/MS/MS study in KO mouse → Negligible off-target binding
↓ Hot (Collaboration): Radiolabeling, PET imaging in NHP or Rodent

Bavarostat: (Strebl, 2017 #475) fig3

(Slide reproducing Step 1–4 mouse-to-LC-MS workflow for Bavarostat)

Traditional vs Novel Testing Scheme for PET Tracer Discovery

"Traditional" Testing Scheme for PET Tracer Discovery Radioligand approach	Novel Testing Scheme for PET Tracer Discovery LC-MS approach
Compound Synthesis → In vitro Binding Assay (Affinity & Selectivity) → Prepare radioligand [usually ³H or ¹⁴C] → Autoradiography Study (Non-specific Binding) → Prepare PET ligand [usually ¹¹C or ¹⁸F] → PET Experiment (Brain Penetration / Clearance / Formation of radioactive Metabolites) ~4 weeks	Compound Synthesis → In vitro Binding Assay → LC-MS Tracer Evaluation (Non-... / Brain... / Clearance...) → Prepare PET ligand [usually ¹¹C or ¹⁸F] → PET Experiment (Form... radioactive Metab...) ~2 days

Uncertain Spans

location	transcription	uncertainty
Top reference table column structure	(Example / [tissue] / Method / Readout columns)	The reference table starts on `20240722_184823`; the leftmost narrow columns of this row block are mostly empty on this capture, so the column structure is reconstructed from the visible header tokens and from the previous photo.
`T-4035187` and `T-4055455` HDAC1 IC50 values	`4000 - 10000 - 12000 nM`, `350 - 3000 - 4400 nM`	The slide shows three IC50 values per compound separated by hyphens; the text is small and the middle token is partially blurred.
‘Which was better’ callout boxes	(red Korean annotation boxes overlaid on scatter plots)	Several Korean callout fragments inside the embedded slide are partially occluded by the chart traces; preserved as evidence and the legible portions transcribed inline.
Step 2 reference region rows	`1. reference region O. PDE10A`, `2. reference region X. CHQ4H`	The ‘CHQ4H’ token renders ambiguously between `CH04H` and `CHQ4H`; preserved as printed.
Bottom ‘Bavarostat: fig3’ workflow	(Step 1–4 cartoon mouse→tissue→LC-MS→bar chart)	Bottom of the slide is cut at the `Step 4` panel; the remaining text continues on `20240722_184829`.

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