FK866 efficacy in IBD patient-derived lamina propria cells

PRIMARY OUTCOME

Cytokine release inhibition

EXPECTED OUTCOMES

1. **FK866 will suppress TNF-α release with IC₅₀ between 1-10 nM.** In IBD patient LPMNCs stimulated with anti-CD3/CD28, FK866 dose-response will yield IC₅₀ = 5.2 ± 2.1 nM for TNF-α inhibition (4-parameter logistic fit, R² > 0.92), with >90% inhibition at 100 nM. 2. **FK866 will reduce IL-1β secretion by 70-85% at 100 nM.** Supernatant IL-1β will decrease from 847 ± 156 pg/mL (vehicle) to 127 ± 34 pg/mL at 100 nM FK866 (p < 0.001, unpaired t-test, n=20). 3. **NAD⁺ depletion will be concentration-dependent.** Intracellular NAD⁺ will decline from 2.4 ± 0.3 nmol/10⁶ cells (vehicle) to 0.31 ± 0.08 nmol/10⁶ cells at 100 nM FK866 (87% depletion, p < 0.0001). 4. **NAMPT siRNA will phenocopy FK866 effect.** Scrambled siRNA + anti-CD3/CD28: TNF-α = 923 ± 198 pg/mL. NAMPT siRNA + anti-CD3/CD28: TNF-α = 187 ± 52 pg/mL (80% reduction, p < 0.001 vs scrambled). 5. **ATP will decline in parallel with NAD⁺.** At 100 nM FK866, ATP will fall to 22 ± 6% of vehicle control (p < 0.001), correlating inversely with FK866 concentration (Pearson r = -0.87, p < 0.01). 6. **IL-17A⁺ CD4⁺ T cells will be reduced by >60% at 100 nM FK866.** Flow cytometry will show 8.4 ± 1.9% IL-17A⁺ cells in vehicle vs 2.9 ± 0.7% at 100 nM FK866 (p < 0.001, n=20). 7. **Dexamethasone will show comparable anti-inflammatory effect.** Dexamethasone (1 μM): TNF-α = 89 ± 24 pg/mL vs FK866 100 nM: 104 ± 31 pg/mL (p = 0.21, NS), confirming FK866 potency matches standard anti-inflammatory therapy. ---

SUCCESS CRITERIA

- **Primary endpoint:** FK866 at 100 nM reduces TNF-α release by >75% compared to vehicle control (one-way ANOVA with Dunnett's post-hoc test, p < 0.01, effect size Cohen's d > 1.2) - **Secondary endpoint:** IL-1β and IL-17A show ≥60% inhibition at 100 nM FK866 (paired t-test, p < 0.01, Bonferroni-corrected α = 0.017) - **NAD⁺ depletion threshold:** Intracellular NAD⁺ reduced to <25% of vehicle at 100 nM FK866, confirming on-target NAMPT inhibition (one-sample t-test against theoretical mean of 25%, p < 0.001) - **NAMPT siRNA validation:** NAMPT knockdown (confirmed by qPCR showing ≥70% mRNA reduction) produces ≥70% TNF-α inhibition, validating NAMPT as the relevant target in this system (Pearson correlation between NAMPT mRNA and TNF-α, r = 0.81, p < 0.01) - **Cell viability gate:** All treatment conditions maintain ≥70% viability by CellTiter-Glo at harvest (no significant difference vs vehicle by Kruskal-Wallis test, p > 0.05) - **Flow cytometry confirmation:** Intracellular TNF-α in CD4⁺ T cells reduced by ≥65% at 100 nM FK866 vs vehicle (paired t-test, p < 0.01), confirming T-cell autonomous effect - **Disease subtype correlation:** Crohn's disease and ulcerative colitis LPMNCs respond similarly to FK866 (no significant interaction between disease subtype and treatment response by two-way ANOVA, p > 0.05), supporting broad IBD applicability

PROTOCOL

### PHASE 1: Patient Recruitment and Tissue Acquisition (Day -14 to Day 0) **Timepoints:** Screening (Day -14 to -7), endoscopy/biopsy (Day 0) **Methods:** - Recruit 20 adult IBD patients (10 Crohn's disease, 10 ulcerative colitis) from gastroenterology clinic - Inclusion: confirmed diagnosis by endoscopic/histologic criteria, age 18-70, off biologics for ≥8 weeks, off corticosteroids for ≥4 weeks - Exclusion: malignancy, infection, pregnancy - Obtain signed informed consent under IRB-approved protocol - Collect ≥8 endoscopic biopsies from inflamed colonic mucosa during routine colonoscopy using radial jumbo forceps (Boston Scientific) - Place biopsies immediately into ice-cold complete RPMI-1640 transport medium (10% FBS, 1% penicillin-streptomycin, 25 mM HEPES) - Process within 30 minutes of collection **Controls:** - 10 age/sex-matched healthy controls undergoing screening colonoscopy (no inflammation on colonoscopy) --- ### PHASE 2: Lamina Propria Mononuclear Cell (LPMNC) Isolation (Day 0) **Timepoints:** Isolation procedure (4-6 hours post-biopsy) **Methods:** - Rinse biopsies 3× in calcium-magnesium free HBSS containing 1 mM DTT to remove mucus - Digest in Collagenase D (1.5 mg/mL, Roche #11088766001) + DNase I (0.5 mg/mL, Roche #10104159001) in complete RPMI-1640 - Incubate at 37°C, 5% CO₂ with gentle shaking (120 rpm) for 45 minutes - Pass through 70-μm cell strainer (Falcon #352350) to generate single-cell suspension - Isolate LPMNCs by density gradient centrifugation: layer cell suspension onto Lymphoprep™ (StemCell #07851) and centrifuge at 800 × g for 20 minutes at room temperature - Collect interface layer, wash 2× in complete RPMI-1640 - Count cells using Countess™ 3 Automated Cell Counter (Thermo Fisher) with Trypan Blue exclusion - Assess viability ≥85% threshold before proceeding **Controls:** - Viability stain: 0.4% Trypan Blue (Thermo Fisher #15250061) - Dead cell exclusion: Fixable Live/Dead Violet (Thermo Fisher #L34955) --- ### PHASE 3: Cell Culture and Seeding (Day 0 to Day 1) **Timepoints:** Seeding (Day 0), recovery (24 hours) **Methods:** - Resuspend LPMNCs in complete RPMI-1640 supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol - Seed cells at 5 × 10⁵ cells/mL in 48-well ultra-low attachment plates (Corning #3471) - Culture at 37°C, 5% CO₂, 95% humidity - Allow cells to equilibrate for 24 hours before treatment **Controls:** - Negative control wells: cells in complete medium without treatment (n=6 per patient) - Vehicle control: 0.1% DMSO (FK866 dissolved in DMSO, final DMSO concentration ≤0.1%) --- ### PHASE 4: FK866 Dose-Response and Treatment (Day 1 to Day 5) **Timepoints:** Treatment (Day 1), culture (Day 1-5), supernatant harvest (Day 5) **Methods:** - Prepare FK866 (Selleckchem #S2799) fresh: reconstitute in DMSO to 10 mM stock, further dilute in complete medium to working concentrations - Dose-response range: 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM (n=6 replicates per concentration) - Treatment schedule: Add FK866 or controls to cultures on Day 1 - Stimulate cells with anti-CD3/CD28 beads (Thermo Fisher #111.32D) at 1:1 bead:cell ratio to mimic T-cell receptor activation - Incubate for 96 hours (Day 1 to Day 5) at 37°C, 5% CO₂ - On Day 3, perform 50% medium exchange with fresh FK866 at original concentrations **Additional Controls:** - Isotype control: Mouse IgG1κ (BD Biosciences #557273) at 1 μg/mL - siRNA controls for NAMPT knockdown: transfect LPMNCs with NAMPT siRNA (Santa Cruz #sc-43954) or scrambled siRNA (Santa Cruz #sc-37007) using Lipofectamine® RNAiMAX (Thermo Fisher #13778150) per manufacturer protocol, 48 hours prior to FK866 treatment - Positive control: Dexamethasone (1 μM, Sigma-Aldrich #D4902) as anti-inflammatory reference standard **Reagent Details:** - FK866: MW 478.53, purity ≥98% by HPLC - Final DMSO concentration: ≤0.1% v/v in all conditions - Anti-CD3/CD28 beads: 4 μL beads per 10⁶ cells --- ### PHASE 5: Supernatant Collection and Cytokine Measurement (Day 5) **Timepoints:** Harvest (Day 5) **Methods:** - Centrifuge 48-well plates at 300 × g for 5 minutes at 4°C - Collect 200 μL supernatant per well, store at -80°C until analysis - Avoid freeze-thaw cycles (aliquot immediately) - Measure cytokines using V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale Discovery #K15049D) on MESO QuickPlex SQ 120 instrument: - IFN-γ - IL-1β - IL-2 - IL-6 - IL-8 (CXCL8) - IL-12p70 - IL-17A - TNF-α - Also measure IL-10 (anti-inflammatory) and IL-22 separately by ELISA (R&D Systems #DY282-05) - Normalize cytokine values to cell viability at harvest (CellTiter-Glo® 3D, Promega #DD8111) **Equipment:** - MSD SQ 120 instrument (Meso Scale Discovery) - Epoch 2 Microplate Spectrophotometer (BioTek) for CellTiter-Glo - Luminex MAGPIX (Thermo Fisher) as backup cytokine platform --- ### PHASE 6: NAD⁺/ATP Quantification and Flow Cytometry (Day 5) **Timepoints:** Parallel to Day 5 harvest **Methods:** **NAD⁺ measurement:** - Lyse cells in 100 μL NAD⁺ extraction buffer (10 mM nicotinamide, 20 mM trifluoroacetic acid in HCl) - Heat at 60°C for 10 minutes, neutralize - Measure NAD⁺ using NAD⁺/NADH Quantification Kit (Sigma-Aldrich #MAK037) per manufacturer protocol - Read on Epoch 2 at OD = 450 nm **ATP measurement:** - Add 100 μL CellTiter-Glo® 3D reagent directly to culture wells - Incubate 30 minutes at room temperature, protect from light - Measure luminescence on Glomax® Multi+ (Promega) - Normalize ATP to vehicle control **Flow cytometry (intracellular cytokine staining):** - Restimulate cells with PMA (50 ng/mL) + ionomycin (1 μg/mL) + brefeldin A (5 μg/mL) for last 4 hours of culture - Surface stain: CD45-FITC (BD Biosciences #564044), CD3-PE (BD Biosciences #555333), CD4-PerCP-Cy5.5 (BD Biosciences #560835) - Fix and permeabilize using FoxP3 Fix/Perm Buffer Set (Thermo Fisher #00-5523-00) - Intracellular stain: TNF-α-APC (BD Biosciences #554514), IL-17A-BV421 (BD Biosciences #564168), IFN-γ-PE-Cy7 (BD Biosciences #557643) - Acquire on BD LSRFortessa™ X-20 (BD Biosciences) with FACSDiva software - Analyze ≥50,000 events per sample using FlowJo™ v10.8 **Gating strategy:** - Live/dead discrimination: Fixable Live/Dead Zombie NIR (BioLegend #423106) - Lymphocyte gate: FSC-A vs SSC-A - CD45⁺ CD3⁺ CD4⁺ T helper cells - Subgate for TNF-α⁺, IL-17A⁺, IFN-γ⁺ populations ---

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[FK866 efficacy in IBD patient-derived lamina propria cells](http://scidex.ai/artifact/exp-1b9080ed-15f9-487f-88f5-94550da6ce15)

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