Bmax assessment in rats
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.
Saturation binding curve (figure, evidence-only): Cbound/Cunbound vs Cunbound (nM, log scale 0.01–10000) with WT_fitted, KO_fitted lines and WT_obs, KO_obs points. Legend swatches: Specific binding (red), off-target binding (gray), Non-specific binding (light gray).
Equations:
- HDAC6 KO:
Cbound / Cunbound = Bmax_KO / (Kd_KO + Cunbound) + k_KO - WT:
Cbound / Cunbound = Bmax_SB / (Kd_SB + Cunbound) + Bmax_KO / (Kd_KO + Cunbound) + k_KO
| Parameter | Unit | Value estimated | 95% CI |
|---|---|---|---|
| Bmax_SB | nM | 3.1 | 0.0–10.6 |
| Kd_SB | nM | 1.2 | 0.0–4.5 |
| Bmax_KO | nM | 33.5 | 0–133.8 |
| Kd_KO | nM | 22.4 | 0–84.5 |
| k_KO | — | 2.7 | 1.9–3.5 |
(Takeda Pharmaceutical Company Limited)
- Takeda slide: Bmax > 1 nM (minimal)
- (Honer 2014) the target with the lowest Bmax imaged in a robust manner with PET is the extrastriatal D2 receptor pool which has a Bmax of 1 nM (10 fmol/mg protein)
- 아래는 ‘(important) 201904 Paul McQudade Takeda PET Tracer Discovery Strategy_iPark 3rd April 2019’
- Bmax & Kd 는 homogenate에서 당연히 얻을 수 있고, ARG 에서도 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 100 fmol/mg protein in the brain converts to 5 nM. This particular conversion factor is only valid for brain.
Summary of Bmax and Kd value
(table reproduced from a Takeda slide, with two highlighted column groups labelled [3H]T-3789533 and MeNz — Bmax* annotated *Bmax was calculated using 20 mg protein/g brain tissue.)
| Species | Region | Binding assay IC50, 60min (nM) | Kd (nM) Tracer T-3789533 | Bmax (nM) | Kd (nM) Tracer T-3789533 | Bmax* (nM) |
|---|---|---|---|---|---|---|
| Rat | Striatum | — | — | — | 15.61 | ND |
| Cerebrum | — | 1.2 | 3.1 | 16.39 | 1.7 | |
| Monkey | Striatum | — | — | — | 41.38 | 2.9 |
| Caudate | — | — | — | 14.38 | 0.5 | |
| Human | Striatum | 2.1 | — | — | 2.99 | 2.7 |
| Thalamus | — | — | — | — | — |
Points to be used:
- Bmax values should be carefully dealt because different tracers were used between rats and monkey/human assays
- 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.
| Bmax (nM) | PET Tracer Tractability | ||||
|---|---|---|---|---|---|
| Rodent | NHP | Human | |||
| Target 1 | <1 | <1 | <1 | Low — Target tractable. Anticipate smaller specific PET signal in human as compared to preclinical species | |
| Target 2 | 100 | 110 | 105 | High | |
| Target 3 | 200 | 220 | 25 | High | |
Kd (흔히 Ki로 대치됨)
| Definition | Notes |
|---|---|
equilibrium dissociation constant, Binding affinity, Binding strength. The smaller the KD value, the greater the binding affinity of the ligand for its target., where [A], [B], and [AxBy] are the equilibrium concentrations of A, B, and the complex AxBy, respectively.K_D = [A]^x [B]^y / [A_x B_y]Kd: 50% 가 free 이고, 50% 가 binding 됐을때의 tracer 의 농도임. 3H-MCC950 data 보면 Ki (11nM) 와 Kd (92.63nM) 값이 다르네. |
(2014 er) Kd<10 nM (common criteria); Cf) Kd of CKD-504 is 5 nM. NLRP3: Ki <5nM 20220326 AD Immune: Capotosti reported that the tracer's dissociation constant was between 8 to 30 nM for α-syn aggregates in tissue slices and brain homogenates from different α-synopathies. This seemed shockingly weak to Mathis, who said most researchers agree the sweet spot for tracer affinity is 1 nM or lower. −1/KD: first order kinetic rate constants for radioligand binding and release from the target, kon/koff, Bmax & Kd 는 homogenate에서 당연히 얻을 수 있고, ARG 에서도 saturation analysis를 하면 얻을 수 있다 (증거: (Au – Griem-Krey, 2019 #1506, fig6))(단지 tracer를 많이 쓰니 비용 문제 발생) Kd는 대략 Ki, IC50 값과 유사함 (but 아닐 수도 있음) |
Homogenate binding and ARG produce similar estimates for Kd, (whereas Bmax may be underestimated in homogenate binding).
| Autopsy, striatum | Homogenate | Kd | |
|---|---|---|---|
| (Dean, 1997 #1991) | human | 3.4 (unit?) | |
| Makoto Fushimi: | In vitro (겠지) | Kd can be quantitiated by SPR, (not by NMR, MS) | |
| (Olsson 2004) [18F]FLB-457 | in vitro | 20 pM | |
| in vivo (healthy human) | 0.27–0.43 nM (그런데 in vitro 값보다 Kd 가 증가하는데 해당 논문에 이유 설명) Bmax 는 유사 | ||
| 그런데 KD is normally considered to have a single, usually unknown, value in vivo. (Matthews et al. 2012, PMID 21838787) | |||
Bmax/Kd binding potential
| Definition | Notes |
|---|---|
|
Binding potential, affinity, 이것은 다른 공식에 의해서, 다음의 의미로 된다. : the maximum bound to free ratio. That is, the maximum ratio of radiotracer bound in the target tissue to the free radiotracer in the circulation : is proportional to SB divided by the free concentration of the radioligand. because Bmax and KD are expressed in the same units (e.g., nM), BP is unitless. |
(Matthews et al. 2012, PMID 21838787) Practical range for the binding potential, is between 0.5–15. Values less than 0.5 or greater than 15 suggest that a candidate radioligand may suffer from either undesirably high variability or low precision, respectively. If <0.5 → ↑ variability of radioligand If >15 → ↓ precision - >10 (이래야 TO study 가능함), but not a definite criteria, 아래 그림 중 위는 (Patel and Gibson 2008, PMID 19026942), et al. 2014, PMID) (2014 Honer) A Bmax:Kd of >10 (ideal), whereas a ratio of 3 is often regarded to be the minimum, (Takeda) Minimum acceptance: Bmax/Kd ≥ 1 |
In vitro affinity / Bmax and Kd
(Patel & Gibson 2008 Table 1 reproduction — figure, evidence-only)
Generalised summary of the relationship between Bmax/Kd ratio and size of in vivo specific signal, taken from mammalian CNS data obtained from the literature.
| Target | Radiotracer | Bmax (nM) | Kd (nM) | Bmax/Kd | In vivo Sp/Ns signal | References |
|---|---|---|---|---|---|---|
| DAT | [11C]cocaine | 150* | 130* | 1.2 | 1 | [13–17] |
| DAT | [18F]β-CFT | 180* | 11* | 16 | 4 | [18–20] |
| D₂ | [11C]raclopride | 19* | 3.5* | 5.4 | 4 | [21–24] |
| D₂ | [11C]N-methylspiperone | 19* | 0.1* | 190 | 8 | [25,26] |
| CB1 | [18F]MK9470 | 14* | 0.3* | 47 | 3 | [27,28] |
| m-AChR | [123I]/[11C]QNB | 150* | 0.2* | 750 | >3b | [29–32] |
| m-GluR5 | [18F]FPEB | 50* | 0.2* | 250 | 7 | [33,34] |
| m-GluR5 | [18F]PycPEB | 50* | 16* | 3 | 0 | [33,34] |
S. Patel, R. Gibson / Nuclear Medicine and Biology 35 (2008) 805–815
Pie-chart pair (figure, evidence-only): Functional 35 tracers — 94 % green / 6 % red; Non-functional 12 tracers — 50 % green / 50 % red; with legend Bmax/Kd ■ X > 10 (green) ■ X ≤ 10 (red), and caption Bmax/Kd > 10 is desirable, but not definitive criteria. Source: Friden et al. EJNMMI Research 2014, 4:50.
Test-retest variability of PET tracer
| within-subject variability, prerequisite for longitudinal studies (eg. occupancy study), 어떤 cross sectional study에서는 안 하기도 했네. | >5–10% | (Ogden et al. 2007, PMID 16736050) (Hirvonen et al. 2008, PMID 18183028) | 5-HT transporter | HV | Two serial scans within the same day | ~15% |
| DAT | HV | in NHP ≤ 20 % |
Dosimetry
The total number of PET measurements is limited by the radiation absorbed dose received by the subjects, = the absorbed dose of individual radioligand
Safety
- single dose IV micro dosing,
- Structure alerts/Gentox may be tolerated
- Dose: safety window at multiple of projected clinical dose (ug scale)
- GLP toxicity study is required for exploratory IND submission (?),
- acute intra venous (IV) dosing in a single species, typically rats, plus 14-day observation (Wagner & Langer 2011).
Specific activity (Ci/mmol)
the amount of radiolabeled mass in a sample, often expressed as Ci/mmol or Ci/mg. For example, 33P-gamma-ATP in the image below has a specific activity of 3000 Ci/mmol (on the calibration date). These units indicate that there are 3000 Curies per millimole of ATP — specific activity (>67.6 Ci/mmol)
Outcome parameters (in PET study)
| Parameter | Definition | Notes |
|---|---|---|
| %ID | (SB와 상관없이 Brain uptake 의 적도이군) Note: good review for in vivo parameters: {Nerella, 2022 #2211} (Peak tracer uptake) |
the ratio of the activity in a tissue (given in kBq/cc or mCi/cc) divided by the decay-corrected activity injected into the animal (given in kBq or mCi). This ratio is defined as the percent injected dose per cubic centimeter (or milliliter) in tissue (%ID/cc or %ID/mL). Commonly, a density of 1 g/cc in tissue is assumed and therefore the uptake is often defined as %ID/g. [Van de Bittner] PET tracers with a %ID/cc above 0.1 % in rat or 0.01 % in NHP within 5 min of injection have suitable BBB penetration for CNS PET imaging studies — ≥ 1.0 |
| SUV (standardized uptake value): static PET data analysis gives SUV? |
(Peak tracer uptake) Weight corrected (Percentage of injected radiotracer per unit volume (or weight) of tissue)%ID × 100 / subject weightSUV = c_img / c_inj
|
Definition: [Tissue radioactivity concentration (Bq/mL) / Administered dose (Bq)] / Body weight (g). free radioligand 도 포함하나? SUV is the ratio of the image-derived radioactivity concentration cimg and the whole body concentration of the injected radioactivity cinj. An SUV value of 1 corresponds to the concentration of radiotracer that would result from uniform distribution of the injected dose throughout the entire body. (Honer 2014) a peak SUV >2 for brain tissue in PET studies is considered desirable {Pike, 2009 #2535} table 1: peak SUV values of several CNS PET tracers |
| VT (VOLUMES OF DISTRIBUTION) |
total distribution volume (VT)C_T / C_P (SB와 상관없이 Brain uptake 의 적도이군)when a reference region doesn't exists: VT is the typical outcome measure |
VT is a ratio at equilibrium of the radioligand concentration in tissue to that in plasma and can be expressed in terms of kinetic rate parameters as VT = K1/k2 (1 + k3/k4) where K1 and k2 are influx and efflux rates for radiotracer passage across BBB and k3 and k4 describe the radioligand transfer between the free and non-specific compartments and the specific binding compartment 단점: SB, NSB and free radioligand 미구분 (Suridjan 2019) As a rule of thumb, very low VT measured in the course of a PET experiment (< 0.2) indicates very low levels of brain entry, if any, that may not be confidently distinguished from experimental noise, intravascular radioactivity, or scatter from extra-cranial radioactivity |
Js: SB 확인 방법들
- ARG with a blocker
- Eg
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Summary of Bmax and Kd value table | column header [3H]T-3789533 for the first Kd/Bmax group and MeNz for the second group | The two column-group headers in the source slide are small graphic labels; transcribed as best interpretation but the second (MeNz / methyl-Nz / similar) is not perfectly legible. |
| Bmax (nM) Targets table, Target 1 PET Tracer Tractability cell | ”Low — Target tractable. Anticipate smaller specific PET signal in human as compared to preclinical species” | The Low and High ratings in the right column align with three target rows, but the row-to-rating mapping is small print and the assignment of Low to Target 1 versus Target 2/3 is inferred from horizontal position. |
| Patel & Gibson Table 1, Bmax and Kd values | values like 150, 180, 19, 14, 50 with superscript footnote markers *, b, etc. | Footnote superscripts are tiny; numerals and footnotes preserved as best read. |
| Test-retest variability row, last cell | in NHP ≤ 20 % | Visible cell shows in NHP ≤20% partially merged into the SUV row above; the row alignment for this cell is not perfectly crisp. |
| Dosimetry row | the absorbed dose of individual radioligand | The trailing fragment is small print at the row boundary; transcribed as visible. |
| Outcome parameters → SUV → Notes | [Tissue radioactivity concentration (Bq/mL) / Administered dose (Bq)] / Body weight (g). | The exact bracket nesting and division order in the original equation is small; transcribed as the most likely interpretation. |