GSK8612 treatment in atherosclerosis model

PRIMARY OUTCOME

effect of GSK8612 on EndMT and plaque formation

EXPECTED OUTCOMES

## Expected Outcomes ### Primary Outcomes 1. **Atherosclerosis reduction:** 30-50% decrease in aortic lesion area at high dose vs vehicle 2. **Dose-dependent effect:** High dose (30 mg/kg) > Low dose (10 mg/kg) 3. **TBK1 inhibition:** ≥50% reduction in pTBK1 in aorta and macrophages 4. **Anti-inflammatory:** ≥30% reduction in plasma TNF-α, IL-6 ### Secondary Outcomes - Reduced macrophage content in plaques (MAC3 IHC) - Decreased inflammatory cell infiltration in aorta - Improved endothelial function (if vasorelaxation tested) ### Null Result Interpretation - If no efficacy, verify plasma drug levels (PK profiling) - May need higher dose (up to 100 mg/kg) or different route (oral) - GSK8612 may have limited CNS penetration but good peripheral efficacy - Consider measuring target engagement directly (pTBK1 in PBMCs as pharmacodynamic marker)

SUCCESS CRITERIA

## Success Criteria ### Primary - [ ] TBK1 inhibition: ≥50% pTBK1 reduction in aorta/macrophages - [ ] ≥12/15 animals/group complete study - [ ] Aortic lesion area: significant reduction (p < 0.05) vs vehicle - [ ] Dose-response: high dose significantly better than low dose ### Secondary - [ ] Plasma cytokines: ≥30% reduction (TNF-α, IL-6) - [ ] Lipid profile: no significant difference between groups - [ ] Body weight: < 10% change vs vehicle ### Technical Quality Gates - [ ] Drug formulation: stable, no precipitation - [ ] Injection volume consistent across groups - [ ] Blinded lesion quantification - [ ] Genotyping confirmed for Apoe-/- status - [ ] TBK1 baseline levels similar across groups before treatment

PROTOCOL

## Protocol: GSK8612 (TBK1 Inhibitor) Treatment in Atherosclerosis Mouse Model ### Study Design Pharmacological validation study examining the effect of GSK8612 (selective TBK1 inhibitor) on atherosclerosis development in Apoe-/- mice. Assess dose-response and mechanism of action. ### Animals and Treatment 1. Apoe-/- mice (C57BL/6J background, n=15 per group) 2. Age: 8 weeks old at study start, male and female 3. Groups: - **Vehicle control:** 10% DMSO, 40% PEG300, 50% PBS (IP injection) - **Low dose GSK8612:** 10 mg/kg/day (IP) - **High dose GSK8612:** 30 mg/kg/day (IP) - **Positive control:** Apoe-/- on Western diet + vehicle 4. Treatment duration: 12 weeks 5. GSK8612 formulation: dissolve in DMSO, dilute in PEG300/PBS, protect from light ### In Vivo TBK1 Inhibition Validation 1. Collect tissues at sacrifice (week 12): - Aorta (for plaque analysis) - Liver, spleen, peritoneal macrophages 2. Assess TBK1 inhibition: - Western blot: pTBK1 (S172) reduced in treated vs vehicle - Kinase assay: TBK1 activity reduced in aortic tissue - qPCR: inflammatory gene expression (Tnf, Il6, Ccl2) as downstream markers ### Atherosclerosis Assessment 1. **Aortic en face:** - Sudan IV staining, quantify lesion area as % of total aortic surface - Analyze by blinded observer using ImageJ 2. **Aortic root lesions:** - Cryosectioning, Oil Red O staining - Lesion area quantified in 5 sections per mouse 3. **Plaque composition (optional):** - Immunohistochemistry for macrophage content (MAC3), collagen (Sirius red) - TUNEL staining for apoptotic cells ### Systemic Inflammation 1. Plasma cytokines: Luminex or ELISA for TNF-α, IL-6, IL-1β, MCP-1 2. Peripheral blood mononuclear cells: flow cytometry for inflammatory subsets ### Lipid Profile 1. Plasma: total cholesterol, HDL, LDL, triglycerides 2. Confirm no major lipid alterations from drug treatment ### Controls - **Positive control:** Apoe-/- + vehicle (established atherosclerosis baseline) - **Dose-response:** Low vs high dose to establish therapeutic window - **Pharmacokinetics:** If possible, measure plasma drug levels at peak/trough - **Toxicity monitoring:** Body weight, organ weights (liver, spleen) at sacrifice ### Expected Outcomes 1. **Reduced atherosclerosis:** GSK8612 treatment decreases aortic lesion area by 30-50% 2. **Anti-inflammatory effect:** Reduced macrophage content in plaques, lower plasma cytokines 3. **TBK1 inhibition confirmed:** Reduced pTBK1 in aortic tissue 4. **Dose-response:** High dose more effective than low dose 5. **Minimal toxicity:** No significant weight loss or organ abnormalities ### Success Criteria - [ ] TBK1 inhibition confirmed: ≥50% reduction in pTBK1 in drug-treated vs vehicle - [ ] ≥12/15 animals per group complete the study with analyzable tissues - [ ] Significant reduction in aortic lesion area (p < 0.05 vs vehicle) - [ ] Dose-response relationship: high dose > low dose in efficacy - [ ] Plasma cytokines reduced ≥30% in treated groups - [ ] No significant toxicity: body weight change < 10% vs vehicle - [ ] Blinded analysis for all plaque quantification

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