Questions

How many data points are needed to get Emax model?

[PET]

Antibody radiolabelling

purpose	to characterise the interaction of an antibody with its specific epitope.
procedure	Radioiodination with Iodine-125 → immunoscintigraphic image It is therefore essential that the antibody retains the ability to bind to its epitope throughout the labelling procedure.

Parameters of PET ligand

parameter	description	Criteria	HDAC6
		General (Van de Bittner et al. 2014, PMID)
MW		<500 Da (Nerella 2022 #2523) (Nerella 2022 #2523) number of hydrogen bond donors is <1
in vitro Bmax study		1. Potent activity (Ki <5 nM), 2. High Fu brain (> 5%), 3. High specific activity (>67.6 Ci/mmol)
in vivo PET study		2. Ki, 2. Fub, 3. MDR1(A>B*/ER) : - (<6)    4. Good PK (brain uptake and elimination)
	Occupy the same binding site for the clinical drug Candidate to allow competitive blocking and target occupancy
	촬영현장근처에서 재빨리 Rapid radiolabeling 할 수 있는 functional group 보유해야 한다.
	Pharmacological effect를 유발 안 해야 할걸,	PET can be conducted under conditions in which the ligand occupies <5% of the target and has no pharmacologically relevant activity ('tracer conditions').
potency	Subnanomolar, But dose is too small to actually show its pharmacological effect. 그래서 이것은 그냥 potency의 marker인가 보다.		IC50 < 20 nM
Selective		>100-fold selectivity, >30-100x selective (pfizer), >50 (2014 Honer) selective ligand lacking significant off-target binding (>100-fold selective over other targets) is desirable,	> 50x (PET SC)
Metabolite		No brain-permeable radioactive metabolite (as PET detector measures total radioactivity without distinction of origin)
	Reversible binding
PK	↑ Metabolism (clearance): 이래야 i) ↓ non-displaceable signal (↓ free portion 이라서겠지?), ii) we can complete scanning not too long. PET tracer의 条件 Radiosynthesis considerations Radiolabeling position (choice of radiolabeled parent compound or structural analog) Radiolabeling yield (typically >10% at end of synthesis) Specific activity requirements (typically > 500 Ci/mmol (> 18.5 TBq/mmol)) In vitro characteristics Binding selectivity/specificity (> 100-fold for target site(s)) Binding affinity (typically < 1 nM for neuroreceptor sites) Lipophilicity (logP typically in range of 1–3) In vivo characteristics Brain uptake (>0.10% ID-kg/g at early times post-injection) Binding selectivity/specificity (should be demonstrated in vivo with blocking or displacement studies) Pharmacokinetics of specific binding (should be reversible and the binding rate should be much less than the brain uptake rate (k_on < K_1)) Pharmacokinetics of non-specific binding (off-rate should be rapid; brain clearance t_1/2 <30 min for 11C- (20.3 min half-life) and 18F-labeled (109.8 min half-life) agents Metabolism (absence of radiolabeled metabolites in brain) Protein binding (should be reversible) Toxicology Injected drug masses are typically <10 μg Limited toxicology packages are required by the FDA for new agents Radiation dosimetry Limits dependent on regulatory approval route Human measurements usually required for IND Annual Reports in Medicinal Chemistry 40, 49 (2005).	Brain clearance t1/2<30 min for 11C-(20.3 min haf-life) and 18F-labeled (109.8 min half-life) agents (Annual Reports in Medicinal Chemistry eBook ISBN: 9780080458175) → (Mitsuhiro Nishihara) but We Not usually calculate the brain t1/2 directly and Not set the criteria in the cold study. The clearance rate of novel cold ligand candidates can be predicted in vitro from co-incubation experiments with liver microsomes, hepatocytes or assessed in vivo from pharmacokinetic studies. Maki ] we want 10 <   < 80 mL/min/kg the GW2580 PET ligand may require more optimization to move forward to clinical development as it showed slow elimination in the brain (ie 90-min PET data acquisition is required to quantify the tracer retention)
Brain uptake
Lipophilicity	if ↑ lipophilicity → ↑ NSB (ie brain fats (and proteins)) If ↓ lipophilicity → ↓ BBB penetrance	logD (Distribution-coefficient) refers the the water:octanol partition coefficient at a specific pH, normally pH 7.4. 1. 1≤LogD7.4≤3, only maters. Paul: for Bmax, practically Free fraction doesn't matter, high affinity with logP [Merck] <3.5 is preferred.	LogD < 2.5
fu,b = Brain free fraction = brain unbound fraction	fu,b: unbound free fraction in brain? Brain free fraction of functional tracers (from Friden paper) [38 functional tracers bar chart; on-figure annotations: 38 functional tracers / Among 38 functional tracers / fub of 36 tracers were <0.1 (10%) / fub of 22 tracers were <0.05 (5%) / There is no suitable criteria for fub / 30/38 tracers <10% / 22/36 tracers <5%] Friden et al. EJMMM Research 2014, 4:62	Eg. Fu,b 10%: mean that '10% of the total concentration is unbound free fraction 2017 Zhang) fu,b >5% (중요, 이래야 ↓NSB, 이게 높단 건 albumin등에 이미 많이 binding하고 있단느 의미이미로), 2014 Honer) fu,b>15% (preferable)	fu,b >0.05 (ie 5%) 이게 Bmax 시작에 필요함.
fu,p	fu,p: unbound free fraction in plasma?	However, a high fP is not a prerequisite, there are several successful tracers with an fP <10%. 2014 Honer: fu,p >10%	fu,p >5%,
Kp:  BBB penetrance의 지표임	Total Brain/Plasma Ratio	Brain/Plasma Ratio 그래서 이건 단위가 없구나(%로 표현되지 않음) brain에서 binding 여부 무관한 듯 (ie free radioligand 도 포함하는 듯) Kp>1 (matthews 2012), 참고: 그러나 Bmax측정용 ligand 는 BBB penetrance 필요없다, one-to-one interaction 만 있으면 된다 (ligand to target protein) (Honer 2014) An initial brain uptake of >5% of the injected dose at 2 min after tracer injection (for rodent biodistribution studies) or a peak SUV of >2 for brain tissue in PET studies is considered desirable [14].	Kp>0.5    (출처: Takeda slide)
MPO	(Multi-parameter Optimization	GOOD 설명 in 20150625 Zhang ACS (pdf)
MDR	[Permeability assay schematic; on-figure labels: Compounds / Apical side (A) / Basolateral side (B) / Chemical / Human MDR1 expressing cell / Semi-permeable membrane / Permeability of drug from A to B (absorption direction) / from B to A (excretion direction) are evaluated / Efflux ratio (ER) can be determined / ER = BtoA / AtoB / Criteria Efflux ratio(ER): Unlikely ER < 2 / Likely 2≤]	MDR1 gene encodes for P glycoprotein (P-gp), -"AtoB" (A>B) indicates the permeability of drug from (A)apical side to (B) basolateral side. (ie 'absorption', we'd want this ↑) -ER (Efflux ratio) is calculated by BtoA/AtoB. (we'd want this ↓) *TR06647850, "MDR (AtoB/ER):19/34" means that A to B is 19 nm/sec, and ER is 34 (more than 6, TR850 is MDR substrate). Permeability Efflux MDR1(A>B*/ER) A>B (의미: A to B) ER 희망 ↑ ↓ (의미: not MDR substrate) criteria ? 3 too low, 30 ok <6
Bmax	-Represents: maximum specific binding, the total number of binding sites -unit of Bmax: the same units as Y (cpm, sites/cell, or fmol/mg protein). -Bmax: The maximum amount of drug (usually expressed in picomoles per mg protein) which can bind specifically to the receptors in a membrane preparation. If one drug molecule binds to each receptor, it also indicates the concentration of receptors in the tissue Yamatake: Desired profile for in vitro Bmax study Potent activity (Ki <5 nM) : High Fu brain (> 5%) : High specific activity (>67.6 Ci/mmol) (2017 Zhang) Bmax > 1nM 이 'viable target' 이라고 씀. (Bmax먼저 알아놓고, Bmax/Kd>10 인 molecule 을 찾음이 효율적 Bmax assessment in rats [Cbound/Cunbound vs Cunbound (nM) plot with WT_fitted / KO_fitted curves and WT_obs / KO_obs points; Specific binding (SB) / off-target binding / Non-specific binding annotated arrows] Brain slice from WT/HDAC6 KO rats / +[³H]T-3789533 at 0.1–1000 nM / Incubated for 4h at 37C / Measured radioactivity in brain slice (Cbound)/ buffer (Cunbound) / Determined Bmax/Kd/k values by Eqs. HDAC6 KO:   C_bound / C_unbound = Bmax_KO / (Kd_KO + C_unbound) + k_KO WT:   C_bound / C_unbound = Bmax_SB / (Kd_SB + C_unbound) + Bmax_KO / (Kd_KO + C_unbound) + k_KO Parameter Unit Value estimated 95% CI BmaxSB nM 3.1 0.0–10.6 KdSB nM 1.2 0.0–4.5 BmaxKO nM 33.5 0–133.8 KdKO nM 22.4 0–84.5 kKO — 2.7 1.9–3.5 Takeda Pharmaceutical Company Limited - Takeda slide: Bmax > 1 nM (minimal) Summary of Bmax and Kd value Species Region SRC Mertis Binding assay IC50, 60min (nM) Kd (nM) Tracer T-3789533 Bmax (nM) Kd (nM) Tracer T-3888058 Bmax* (nM) Rat Striatum - - - 15.61 ND Cerebrum - 1.2 3.1 Monkey Striatum - - - 16.39 1.7 Caudate 41.38 2.9 Human Striatum 2.1 - - 14.38 0.5 Thalamus 2.99 2.7 *Bmax was calculated using 20 mg protein/g brain tissue. Points to be cared 1) Bmax values should be carefully dealt because different tracers were used between rats and monkey/human assays. 2) Bmax in rats were estimated using WT/KO rats, meanwhile those in monkey and human were estimated only by WT monkeys and Healthy subjects, respectively. Off-target and non-specific binding should be taken into account Bmax in monkey/human. There seemed to be little species difference in Bmax. Takeda Pharmaceutical Company Limited	MCC-950 h/r/m: 12/16/17 nM 14% TR06683014 h/r/m: 3.9/-/16 nM 10% h/r/m: 5.9/-/16 nM

Bmax & Kd는 homogenate에서 당연히 얻을 수 있고, ARG에서도 saturation analysis를 하면 얻을 수 있다 (증거: (Au - Griem-Krey, 2019 #1506, fig6) (그런데 tracer를 많이 쓰니 비용 문제 발생)

[Unit] If one has historical data on Bmax, for example, from literature sources, which are expressed relative to protein concentrations, it is possible to convert this to molarity. The conversion for brain is that there is 50mg of protein per gram of tissue. Using this, a binding concentration of

Uncertain Spans

location	transcription	uncertainty
Parameters of PET ligand, third row criteria	(Nerella 2022 #2523) number of hydrogen bond donors is <1	Word table cell text reads <1; whether the constraint is <1 (a single donor maximum) or part of a longer constraint that wraps onto the cut next-line is unclear.
Bmax row, parameter table	Bmax_KO 33.5 0–133.8 and Kd_KO 22.4 0–84.5	The 95% CI for KO parameters is read as 0–133.8 and 0–84.5; the upper bound digits (133.8, 84.5) are at the edge of slide-screenshot resolution.
Summary of Bmax and Kd value, Rat row	Bmax (nM) 3.1 for Cerebrum	The Cerebrum Bmax cell shows 3.1 highlighted with a red rectangle in the slide; whether the red rectangle is a callout or part of the data is unclear from this photo alone.
Summary of Bmax and Kd value, second header group label	Mertis	The right-hand header band reads Mertis (Takeda internal compound code or slide-deck label); the exact spelling is small and partially clipped.
Trailing paragraph	last cell ends with binding concentration of	The next clause 100 fmol/mg protein in the brain converts to 5 nM. ... is partially visible only in the page status bar and continues onto 20240722_184809.