20240722_184819

Target validation workflow

In vitro assays

☐ Affinity/selectivity for target
☐ BBB permeability (TP, permeability)
☐ Low NSB (log D, fup/fub, in vitro MetID)

In vivo assays

☐ Determination of SB by LC-MS/MS
- ▶ KO animal (if available)
- ▶ IV study
  1. Differential-distribution (target/reference region)
  2. Dose-dependently change of Kp
- ▶ Blocking study (TO evaluation)
☐ Low NSB (in vivo MetID)

Target validation

Bmax
- ▶ expression level
- ▶ species difference

Radiochemistry development → Preclinical PET study

SB; specific binding NSB; non-specific binding TO; target occupancy

Assay flow for 1st set of PET tracer candidates

1st step — All compounds — Kd / Structure / MDR1 at 10 uM / PAMPA
→ 12 compounds
2nd step — MDR1 at low conc. / Metabolite identification — Prioritization
- ⇩ Dose dependent of Kp
- ⇩ Replacement
→ PET in NHP

if no plausible candidate was found, go to strategy for next screening (next page).

if number of candidates is more than 12, Reference study

fb
f_u,brain
logD
CL, Vd (rodents)
CL, Vd (monkey)

PET in NHP — 200万円/scan

33 | 0000 | DDMMYY Takeda Pharmaceutical Company Limited

Tentative flow for GCSi — Flow of PET tracer development (preclinical)

Koike-san’s slide

Step 1	vitro / rodent PK / target expression		For GCSi PET
	IC₅₀ <10 nM, Bmax/Kd >4 (10 preferable) Labeling position ¹¹C, ¹⁸F LogD 1~3	MDR ratio (1 μM) < 3 Mouse IV (cassette) rapid clearance Selectivity fu (%) >1% free (> 5% preferable)	For GCSi PET
Target expression / distribution analysis (rodent, monkey, human / WB, mRNA)
Step 2	Rodent LC/MS/MS		PK / metabolite
	IV screening for specific binding in rodents target/reference region > 2 dose-dependent change of specific binding ratio		Monkey IV (cassette) rapid clearance Metabolite Identification active metabolite, stability of labeling position
Step 3	Rodent LC/MS/MS competition		KO mice LC/MS/MS GCS KO is not available.
	Pretreatment study by drug candidate competitiveness		IV screening for specific binding in KO mouse no specific uptake to target region
Cold tracer / Hot tracer
Step 4	Radiolabeling, NHP imaging
	Radiolabeling feasibility Target specific uptake of radio labeled tracer Blockage of tracer binding by pretreatment of drug candidate

0 | PET tracer | Mar., 2016 | Confidential Takeda Pharmaceutical Company Limited

ELOW Good!

Work Flow for PET Tracer Discovery

Start Here — HTS Library Screen
- Selective compound series identified?
  - No → (loop back)
  - Yes → Compound design and synthesis
    - Affinity Maturation
      - Yes → In vitro evaluation of target density: Rodent, NHP and human
Lead Candidates Identified?
- No → (loop back)
- Yes → Optimize characteristics through rational PET tracer design
  - Tractable Target: (B_max >1 nM)
    - No → Terminate PET effort
    - Yes → Initiate in vivo PET Imaging process
Competition binding assay; IC >10% B_max → Non-imaging triaging of candidates
Potential PET tracer identified?
- No → Evaluate Published PET Tracer
- Yes → Synthesize 5-10 mg of precursor and 1 mg of standard
  - Establish reliable and reproducible radiosynthesis?
    - No → PET DT agrees to terminate PET effort
    - Yes → Specific signal blocked by drug candidate?
      - No → PET DT agrees to terminate PET effort
      - Yes → Distribution matches target profile?
        
        No → PET DT agrees to terminate PET effort
        
        Yes → Preclinical in vivo study: CNS-NHP, IO/GI-relevant animal model
        
        Establish target occupancy/plasma relationship
        
        Yes → Clinically translate PET tracer

Swim-lane / responsibility annotations: 3 — TML, Biology discuss models; DMPK to perform; TML, Biology discuss in vitro experiments; Chemist to perform; 2 — TML-Chemist Biology; Imaging group to perform; Chemist; Imaging group lead; NEUROSCIENCE.

* Initiate in vivo PET Imaging process

Now, there is no non-imaging method step

PET Ligand Development Work Flow — Reviewed by PET Steering Committee (PETSC)

Doing a formal Bmax assessment as an feasibility step early (and formalizing oversight of responsible leaders and decision-making minutes) is the only change from Takeda’s current process.

Project Specific Translation Development Team (TDT)
- Is a PET tracer required?
  - No → PET DT agrees to terminate PET effort
  - Yes → TDT to present rationale and impact of PET tracer to PET Steering Committee (PETSC)
    - Is novel PET tracer required?
      - No → Evaluate Published PET Tracer
      - Yes → In vitro PET tractability assessment across species — currently need to outsource
        
        Is it a tractable Target? (B_max >1 nM)*
        
        No → PET DT agrees to terminate PET effort
        
        Yes → PETSC to formulate discovery strategy and present overview to TDT for execution
        
        Does TDT support PET discovery strategy and timelines?
        
        No → (continues onto next photo)
        
        Yes → Research Team optimizes …

Goal: Imaging to coordinate clinical translation of PET tracer

PET DT validated PET tracer preclinically as fit-for-purpose?
- Yes → Imaging to coordinate clinical translation of PET tracer
- No → PET DT agrees to terminate PET effort

Uncertain Spans

location	transcription	uncertainty
Slide A title	(no visible title; flowchart starts at `In vitro assays / In vivo assays`)	Slide A is partial — its title bar is above the page top and not photographed; the visible body is transcribed but the slide page-number footer is also missing.
Slide D, swim-lane label `3`	`3 — TML, Biology discuss models; DMPK to perform`	The leading red-circled `3` is part of the slide’s numbered swim-lane indicator; whether the number reflects step ordering (`Step 3`) or a footnote ID is not certain from this photo alone.
Slide D, `Establish target occupancy/plasma relationship` arrow	continues to `Clinically translate PET tracer`	The flowchart edge from `Establish target occupancy/plasma relationship` to `Clinically translate PET tracer` is drawn but its `Yes` / branch label position is partially obscured.
Slide E, lower flowchart	`Research Team optimizes ...`	The text inside the `Research Team optimizes ...` box and the next decision diamond are clipped at the bottom of the photo and continue onto `20240722_184823`.

JS PD KB

탐색기