Safety BM tail, Kim 2018 carmofur GBA-KO results, Figure 7 / Figure 8, GalSph / psychosine summary, Krabbe questions, GALC enzyme summary

Safety BM tail

• TSR suggested liver pathology would be sensitive indicator of ACi on target toxicity
• Safety margin will be estimated with front-loaded schedule
• aSyn leading to the slowing of disease progression. AC inhibition might lead to ceramide increase
• We need to assess safety in the increase of GalCer and Ceramide, although current data does not indicate a ceramide increase as there are multiple pathways to process.

Target validation and correction — Kim 2018 #1185 carmofur GBA-KO

	Pre-Tx	Post-Tx
Protein levels of acid ceramidase		↑ (~20%)
GlcSph		↓
Cer		↑
GlcCer		↑ (No effect on C14-GluCer)

Figure 7 caption (lipid species panels):

Carmofur treatment reverts reduced ceramide levels and increases GluSph levels in GCase-deficient cells. Wild-type and GCase-deficient cells were treated with either DMSO or 7.5 μM carmofur for ~20 h. After drug treatments, total lipids were extracted and measured as described in the Materials and Methods section. Lipid analysis results were expressed as lipid levels of picomoles (pmol)/three million cells. Levels of GluSph, GluCer species and dihydroceramide (A), and ceramide species (B) are shown. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test, *p<0.001, **p<0.01,

↓ GalCer (AC 적으니 당연), ↑ mature (50 kDa) GalCer 중 C18:1 만 증가시킴

Figure 8 (panel labels): C18-GalCer, C18:1-GalCer, C24-GalCer, C24:1-GalCer

GalCer (=, 80 kDa) immature, mature (50 kDa) GalCer

Figure 8 caption (GalCer / GALC):

Galactosylceramide (GalCer) levels are reduced in and mature GALC levels are elevated in GCase-deficient cells. (A) Reduced galactosylceramide (GalCer) levels in GCase-deficient cells. Wild-type and GCase-deficient cells were treated with either DMSO or 7.5 μM carmofur for ~20 h. After drug treatments, total lipids were extracted and measured as described in the Materials and Methods section. Lipid analysis results were expressed as lipid levels of picomoles (pmol)/three million cells. Levels of galactosylceramide species. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test *P<0.001 (B) Mature GALC levels are elevated in GCase-deficient cells. Total cell lysates from wild-type and GCase-deficient cells were subjected to immunoblot analysis using antibody against GALC, GCase or tubulin. Immunoblot data is shown in the left. Band intensities of the mature form of galactosylceramidase (m-GALC) and the immature form of galactosylceramidase (i-GALC) were normalized to tubulin, and compared with wild-type cells. Graph represents mean ± S.E.M. n=3, two-tailed unpaired t-test, **P<0.01; compared with wild-type cells. n.s = not significant. (C) Levels of Sph and Sph-1-P. For each group, three independent samples were analyzed. Data represent mean ± S.E.M. Two-tailed unpaired t-test, **P<0.01, *P<0.05.

Bands: i-GALC (immature) → ↑, m-GALC (mature) → ↑, GCase (controls), Tubulin (loading)

GalSph (psychosine) summary

• ↑ aSyn
• ↑ LC3B, ↑ p62, ↑ ubiquitin (는 안 봤네)
• ↑ LAMP2

(Li, 2019 #1189) Carmofur, Twi: GALC mice (Krabbe disease model), Twi/FDH: GALC, ASAH mice — Human fibroblasts Krabbe.

Human Krabbe → ↓ psychosine, ↓ aSyn, ↓ LC3B / p62 / ubiquitin / LAMP2 → 넷 다 줄임.

Krabbe diseases mouse model Brain: WT vs Carmofur Twi vs WT-DMSO comparison. Relative Psychosine bar plot.

Reference: \\mytakeda.sharepoint.com@SSL\DavWWWRoot\sites\InterACT-RAD-Pipeline\NS DU ACi\Shared Documents\Meetings\20221012 Jaewon and Kentaro

Krabbe / open questions

Kentaro Krabbe’s disease → no psychosine in late onset Krabbe, genotype-phenotype correlation, acid

Psychosine: existing data, detectible? PPMI data (longitudinal?)

AC increase in PD? Effect size calculation from the paper (Marshall?)

GALC KO → ↑ aSyn?

Galc variant: LOF vs GOF (Senkevich paper: ↑ galc level & activity, we’d want ↓, as seen in Krabbe’s dis)

GlcSph is under LLOQ in Parkinson’s disease patients while is under LLOQ in Parkinson’s disease patients while…

20211006: GlcSph was successfully measured in CSF of individual HVs as well as monkey CSF even with reduced volume of 200 μL, (LLOQ: 0.1 pg/mL), cf) Sanofi: 5 pg/mL.

GBAL444P/L444P iPSC-DAn results

Parameter	Pre-Tx	Post-Tx
GlcSph	↓	Restored (↑)
Ceramide (16:0, 18:0, 24:1, 26:0)		No change
GlcCer (16:0, 18:0, 24:1)		No change
Sph		No change

Patient segmentation questions

• How many GALC-PD. Severe GALC-PD?
• Patient segmentation: only by genotyping
• Psychosine → aSyn truncation?
• DTBZ → [18F]AV-133

GALC enzyme summary

field	content
frequency	(left blank)
Coded protein (enzyme)	galactosylceramidase
level	(left blank)
activity	(left blank)
Substrates	galactosylceramides
Product	Galactosylsphingosine (sphingosine)