FK866 efficacy in IBD patient-derived lamina propria cells

Clinical Score: 0.950 Price: $0.50 Inflammatory bowel disease Human IBD patient-derived LPMNCs Status: proposed

What This Experiment Tests

Clinical experiment designed to assess clinical efficacy targeting NAMPT in Human IBD patient-derived LPMNCs. Primary outcome: Cytokine release inhibition

Description

Clinical validation study using lamina propria mononuclear cells (LPMNCs) isolated from patients with inflammatory bowel disease (IBD) to test the therapeutic efficacy of FK866 compared to standard treatments including dexamethasone and infliximab. This experiment provided direct clinical relevance by demonstrating that FK866 effectively suppressed cytokine release from patient-derived immune cells. The study showed that FK866 was as effective as or superior to established IBD therapies in controlling inflammatory responses in human tissue, providing strong translational evidence for the potential clinical application of NAMPT inhibition in IBD treatment.

TARGET GENE

NAMPT (Score: 0.543)

MODEL SYSTEM

Human IBD patient-derived LPMNCs

ESTIMATED COST

TIMELINE

0 months

PATHWAY

NAD metabolism, inflammatory cytokine production

SOURCE

extracted_from_pmid_28877980

PRIMARY OUTCOME

Cytokine release inhibition

Scoring Dimensions

📖 Wiki Pages

NAMPT Genegene NAMPT Proteinprotein Treatmentsindex

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 minut

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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

Expected Outcomes

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.

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).

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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)

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Related Hypotheses (2)

TREM2 R47H Variant-Driven Metabolic Dysfunction as the Primary Trigger for Failed DAM Transition0.858

Metabolic NAD+ Salvage Pathway Enhancement Through NAMPT Overexpression0.887

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