Krabbe psychosine continued, KD subtypes, Pipeline of KD, MOA for PD, Monthly Report (AC Inhibitor), Safety / Target validation

Krabbe psychosine continued

뇌의 전영역에서 (late-onset KD 도 WM, Caudate 에선 증가로 매우 evident).

In vivo

• Infantile onset
• the late infantile/juvenile (1-16 years) and adult (>16 years)

The latter type includes a spectrum of late infantile-onset (7 months to 2 years), juvenile-onset (3-8 years) and adult onset (>9 years) onset KD (Xu, Sakai, Tanike, Inui, & Ozono, 2006)

usually die before the age of 2-3 years.

a slower rate than infantile patients with some adults living until the sixth decade with mild symptoms.

The disease is generally fatal 2-7 years after the symptoms

Pipeline of KD

https://www.businesswire.com/news/home/20230117005486/en

MOA for PD

↓ GALC activity → ↑ psychosine → ↑ aSyn

	post mortem (Marshall, 2018 #2200)
GALC activity	fig 1 (M, N, O, P) WM 에서만 ↑
↑ psychosine	(Marshall, 2018 #2200) fig 1 (e, f, g, h, Q) GM 에서만 증가
severe GALC mutation PD patient 에서 psychosine 더 높음
Niigata: ↑ psychosine	Amygdala: d=0.72 (76% of control would be below average person in PD group), Occipital: d=0.01

(Marshall, 2018 #2200) correlation between psychosine and GALC activity is small (r²=0.13)

isoluble truncated aSyn and oligomer were observed after GalSph treatment (in-house data). aSyn truncation may be caused by abnormality of lysosomal proteases (J. Biol. Chem. (2020) 295(30) 10224-10244)

(Marshall, 2018 #2200) In vitro, psychosine accelerates aggregation of a-syn [8] by direct binding to the carboxy-terminus and facilitation of an aggregation-prone protein conformation (Abdelkarim, …)

In house) GALC KD or AC OE → ↑ aSyn (insoluble) oligomer; severe GALC mutation PD patient 에서 psychosine 더 높음 (d=0.53)

(Smith, 2014 #2199), (Abdelkarim, 2018 #2201) genetic correction of GALC deficiency completely prevents a-syn aggregation. Genetic KO of a-syn reduces, but does not completely prevent, neurological signs in a mouse model of KD.

Monthly Report (AC Inhibitor) 202101

GlcSph reduction in iPSC-DaNs (L444P/L444P), 14 days treatment.

Concentration (nM) ratio (n=) groups visible:

• Sanofi GCSi
• Takeda ACi
• rhGBA
• Venglustat
• T-4343016
• T-4178815
• T-4889033 (BT GA4 LAMA4/ALP)
• T-1xxx0C8
• Cerezyme (U/mL)

p-α-Syn (induced by PFF) reduction in mouse primary neurons:

• Y-axis: Normalized Phospho-α-Syn DxA by Nuclei number
• Series: DMSO, 10 μM T-4378886, 10 μM T-3269966
• X-axis: mPFF concentration (ng/mL) 0.1 to 100

Ceramide, GlcCer and Sph levels was not changed by ACi.

202102 Pipeline preclinical

(Caputo, 2020 #1196) (Di Martino, 2020 #1197) — SH-SY5Y compound: 4d (their lead compound, IC50 = 166 nM, Figure 2 and Table S1) as a lead compound with good oral bioavailability, excellent brain penetration, and target engagement in two animal models of neuropathic GD and KD.

Safety

• carmofur has been known to induce leukoencephalopathy, characterized by progressive damage to white matter in the brain with stroke-like symptoms. [8],[9],[10]
• Carmofur: A clinical trial for small hepatocellular carcinoma was stopped prematurely because 56% of the treated patients had unacceptable side effects.
• genetic loss of ACDase activity → Farber disease: Systemic nodules and inflammation, neurologic deterioration, and hepatosplenomegaly with ceramide accumulation
• Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) (1)
• SMA-PME: Epileptic disorder with lower motor neuron dysfunction related to ceramide metabolism (2)

Safety BM

• Carmofur: A clinical trial for small hepatocellular carcinoma was stopped prematurely because 56% of the treated patients had unacceptable side effects.
• genetic loss of ACDase activity → Farber disease: Systemic nodules and inflammation, neurologic deterioration, and hepatosplenomegaly with ceramide accumulation
• → SMA-PME: Epileptic disorder with lower motor neuron dysfunction related to ceramide metabolism (2)
• Monitor ceramide level in vitro / in vivo
• Representative inhibitors did not show effect on ceramide level in GBA KO HAP1 cells
• 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

Where? (Kim, #1185) carmofur GBA KO cells (HEK293-FT cells)

	Pre-Tx	Post-Tx
↑ (~20%) Protein levels of acid ceramidase
↓ GlcSph
↑ Cer
↑ GlcCer
C14-GluCer	A → No effect	B
GluSph	15-…	C16-Cer C18-Cer

Figure 7. 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 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 ± SEM. Two-tailed unpaired t-test, *p<0.001, **p<0.01,

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

↑ AC activity, ↑ Cer, ↑ sphingosine, = SM, ↑ HexCer

Uncertain Spans

location	transcription	uncertainty
Monthly Report compound list	T-1xxx0C8	reads as written; the partially-clipped compound code is preserved verbatim.