FOXO4

Research Overview

FOXO4-DRI (Proxofim) is a first-in-class senolytic peptide designed to selectively eliminate senescent cells — damaged, non-dividing "zombie" cells that accumulate with age and secrete harmful inflammatory factors known as the Senescence-Associated Secretory Phenotype (SASP). The peptide was developed by Peter L.J. de Keizer and colleagues at the Erasmus University Medical Center Rotterdam and first described in a landmark 2017 publication in Cell.[1]

FOXO4-DRI is derived from the Forkhead domain of the human FOXO4 transcription factor — specifically the region that interacts with p53. Its name reflects two key structural modifications: "D" denotes the use of D-amino acids (mirror images of natural L-amino acids), and "Retro-Inverso" means the amino acid sequence is reversed. Together, these modifications produce a peptide that mimics the 3D surface of the original protein while being highly resistant to enzymatic degradation by proteases. The peptide is further fused to the HIV-TAT protein transduction domain to enable rapid cellular uptake within 2–4 hours.[1][2]

The fundamental insight behind FOXO4-DRI is that senescent cells resist apoptosis by upregulating FOXO4, which binds and sequesters p53 within PML nuclear bodies, preventing p53 from executing its normal pro-apoptotic functions. FOXO4-DRI acts as a competitive decoy, outcompeting endogenous FOXO4 for p53 binding and liberating p53 to translocate to the mitochondria and trigger caspase-dependent apoptosis. Because non-senescent cells express minimal FOXO4 and do not depend on this survival mechanism, they are spared — achieving remarkable selectivity.[1][3]

Preclinical research has demonstrated FOXO4-DRI's therapeutic potential across a broad spectrum of age-related conditions including chemotherapy-induced toxicity, renal function decline, male hypogonadism (testosterone deficiency), vascular aging, pulmonary fibrosis, osteoarthritis, liver fibrosis, and therapy-resistant cancers. The peptide has been shown to restore fur density, physical activity, and kidney function in both fast-aging and naturally aged mice at a standard dose of 5 mg/kg.[1][4][5]

The biotechnology company Cleara Biotech B.V., founded by de Keizer, is advancing optimized 4th-generation variants (CL04183/CL04177) with enhanced binding affinity and improved pharmacokinetic profiles toward clinical development. A structural milestone was reached in 2025 when Bourgeois et al. solved the NMR structure of the FOXO4-DRI/p53 complex, identifying the p53 Transactivation Domain 2 (TAD2) as the specific binding site and demonstrating that p53 phosphorylation at Ser46 and Thr55 enhances binding affinity.[2][3]

Mechanism of Action

FOXO4-DRI functions as a competitive peptide antagonist that disrupts the critical survival interaction between FOXO4 and p53 in senescent cells, triggering a process called Targeted Apoptosis of Senescent Cells (TASC).[1]

Primary Target & Binding Characteristics

Property	Detail	Evidence
Primary Target	FOXO4-p53 protein-protein interaction interface	Baar et al. (2017)[1]
Specific Binding Site on p53	Transactivation Domain 2 (TAD2) of p53	Bourgeois et al. (2025) NMR structure[2]
Binding Dynamics	Both FOXO4-DRI and p53 TAD2 are intrinsically disordered; fold synergistically upon binding to form a transiently folded complex	Bourgeois et al. (2025)[2]
Affinity Modulation	Phosphorylation of p53 at Ser46 and Thr55 significantly enhances FOXO4-DRI binding affinity	Bourgeois et al. (2025)[2]
HIV-TAT Contribution	Cationic HIV-TAT residues contribute additional contacts with p53 TAD2, stabilizing the interaction	Bourgeois et al. (2025)[2]
Cell Penetration	Intracellular uptake within 2–4 hours; detectable for ≥72 hours	Baar et al. (2017)[1]

The Senescence Lock (Pre-Treatment State)

In senescent cells, FOXO4 is upregulated and physically interacts with p53 within the nucleus, specifically localizing to PML bodies (Promyelocytic Leukemia bodies) and DNA-SCARS (DNA Segments with Chromatin Alterations Reinforcing Senescence). This binding sequesters p53 in the nucleus, preventing it from initiating pro-apoptotic functions — effectively keeping the senescent cell in a suspended "zombie" state.[1][3]

Downstream Signaling Cascade (TASC Pathway)

Step	Event	Molecular Detail
1. Competitive Inhibition	FOXO4-DRI competes with endogenous FOXO4 for p53 binding	Binds p53 TAD2 with high affinity, displacing endogenous FOXO4[1][2]
2. Nuclear Exclusion	Liberated p53 is excluded from the nucleus	p53 released from PML bodies / DNA-SCARS[1]
3. Mitochondrial Translocation	Active, mono-ubiquitinated p53 translocates to mitochondria	Transcription-independent apoptosis pathway[1]
4. BAX/BAK Activation	p53 interacts with BAX and BAK (pro-apoptotic Bcl-2 family members)	Mitochondrial outer membrane permeabilization (MOMP)[1][5]
5. Cytochrome C Release	BAX/BAK pores release Cytochrome C into the cytosol	Initiates the intrinsic apoptosis cascade[1]
6. Caspase Activation	Cytochrome C triggers cleavage of Caspase-3 and Caspase-7	Terminal effector caspases execute apoptosis[1][5]
7. Selective Senolysis	Senescent cell undergoes intrinsic apoptosis and is eliminated	Non-senescent cells unaffected (low FOXO4, no dependency on FOXO4-p53 axis)[1]

Cellular & Tissue-Level Effects

Effect	Detail	Evidence
Senolysis	Selective elimination of senescent fibroblasts (IMR90), chondrocytes, and Leydig cells; 11.73-fold selectivity over non-senescent cells	Baar et al. (2017); Huang et al. (2021)[1][6]
SASP Suppression	Downregulation of IL-6, IL-1β, TNF-α, TGF-β, and other pro-inflammatory cytokines	Zhang et al. (2020); Hu et al. (2026)[4][5]
Marker Modulation	Decreased p16 and p21 expression; increased Ki-67 (proliferation marker) and Lamin B1 (nuclear envelope marker)	Hu et al. (2026)[5]
Renal Restoration	Normalized plasma urea and creatinine; reduced tubular senescence markers	Baar et al. (2017)[1]
Testosterone Restoration	Selective apoptosis of senescent Leydig cells; improved testicular microenvironment; restored testosterone secretion	Zhang et al. (2020)[4]
Vascular Health	Reduced aortic wall thickness, reduced ROS levels, improved endothelial-dependent vasodilation, reduced Pulse Wave Velocity	Hu et al. (2026)[5]

Selectivity vs. Related Compounds

Comparison	FOXO4-DRI	Comparator
vs. ABT-737 / Navitoclax (BCL-2 inhibitors)	No thrombocytopenia; targets p53/FOXO4 axis specifically	Causes low platelet counts by affecting non-senescent cells[1]
vs. Dasatinib + Quercetin	Peptide-based; single-target mechanism (FOXO4-p53); DRI stability	Small molecule combination; multi-target kinase inhibition
vs. CL04183 (4th-generation)	Original compound; shorter half-life; narrower therapeutic window at high doses	Enhanced binding affinity; improved liver enzyme stability; broader therapeutic window[2]
vs. Endogenous FOXO4	Antagonist: competes for p53 to trigger apoptosis; protease-resistant DRI form	Agonist of senescence maintenance: sequesters p53 to keep senescent cells alive[1]

FOXO Family Specificity: The peptide design focused on a region of FOXO4 that differs from FOXO1 and FOXO3 to minimize cross-reactivity with these essential transcription factors. Cross-species conservation of the binding domain allows direct translational studies between mice and humans.[1]

Research Applications

FOXO4-DRI is utilized in preclinical research to study the effects of clearing senescent cells (senolysis) across 9+ research domains:

1. General Aging & Frailty — FOXO4-DRI restores tissue homeostasis in naturally aged mice (104–130 weeks). Benefits include improved fur density, increased physical activity (running wheel activity), improved responsiveness, and reduced p16-driven bioluminescence (senescence burden). Treatment of Xpd^TTD/TTD fast-aging mice yielded fur insulation restoration (approaching wildtype levels) and increased running from 1.37 km/day to near-wildtype levels.[1]
2. Chemotherapy-Induced Toxicity — In Doxorubicin-treated C57BL/6J mice, FOXO4-DRI (5 mg/kg i.v., 3 doses) neutralized chemotherapy-induced liver toxicity (normalized plasma AST), prevented body weight loss, and reduced IL-6 and FOXO4 foci in liver tissue.[1]
3. Renal Function Decline — In both fast-aging and naturally aged mice, FOXO4-DRI normalizes plasma urea and creatinine levels, restoring kidney filtering capacity and reducing tubular senescence markers. Significant reductions observed 30 days after 3 i.p. injections.[1]
4. Male Hypogonadism / Testosterone Deficiency — In aged male mice (20–24 months), FOXO4-DRI selectively induces apoptosis in senescent Leydig cells, reducing SASP factors (IL-1β, IL-6, TGF-β), improving the testicular microenvironment, and significantly restoring serum testosterone levels (p<0.05).[4]
5. Cardiovascular Aging & Endothelial Dysfunction — FOXO4-DRI reduces reactive oxygen species (ROS) in the aorta, suppresses vascular aging markers (p16, p21), thins the aortic wall, lowers Pulse Wave Velocity (improved elasticity), and improves endothelial-dependent vasodilation in both naturally aged and D-galactose progeroid mice.[5]
6. Pulmonary Fibrosis — FOXO4-DRI ameliorates bleomycin-induced pulmonary fibrosis by targeting senescent myofibroblasts, downregulating extracellular matrix receptor interaction pathways, attenuating collagen deposition, and increasing Type 2 alveolar epithelial cells (AEC2).[8][9]
7. Osteoarthritis & Cartilage Regeneration — In expanded human chondrocytes, FOXO4-DRI (25 µM, 5 days) selectively removed >50% of senescent cells (PDL9), reduced SA-β-gal to <5%, and decreased expression of SASP factors (IL-6, IL-8) in engineered cartilage tissue.[6]
8. Cancer & Metastasis — FOXO4-DRI is being explored against therapy-resistant and metastatic cancers (triple-negative breast cancer, metastatic colon cancer) by targeting "scarred" cancer cells that share features with senescent cells, and for radiosensitizing non-small cell lung cancer.[10][11]
9. Liver Fibrosis — FOXO4-DRI and optimized variants (CL04183) counter the CD44-high dedifferentiation state in hepatocytes driven by senescence, restoring liver function markers in fibrosis models.[10]

Preclinical Research Summary

Key Preclinical Animal Studies

Study	Model	Key Findings	Ref
Baar et al. (2017) Cell	C57BL/6J mice — Doxorubicin chemotoxicity; 5 mg/kg i.v., 3 doses over 5 days	Neutralized plasma AST elevation (liver toxicity); prevented body weight loss; reduced IL-6 and FOXO4 foci in liver tissue	[1]
Baar et al. (2017) Cell	XpdTTD/TTD fast-aging mice; 5 mg/kg, initiated at ~26 weeks	Fur density restored (infrared imaging); running activity increased from 1.37 km/day toward wildtype 9.37 km/day; plasma urea normalized	[1]
Baar et al. (2017) Cell	Naturally aged C57BL/6J (p16::3MR), 104–130 wks; 5 mg/kg i.p., 3 doses over 5 days; observed 30 days	Reduced plasma urea and creatinine (restored renal function); reduced p16-driven bioluminescence; improved responsiveness to stimuli	[1]
Zhang et al. (2020) Aging	Naturally aged male C57BL/6 (20–24 mo); 5 mg/kg i.p., 3 doses every other day; analyzed 30 days post-treatment	Serum testosterone significantly increased (p<0.05); decreased p53, p21, p16 in testes; reduced IL-1β, IL-6, TGF-β; no change in body/testis weight	[4]
Hu et al. (2026) Front. Bioeng. Biotech.	Naturally aged mice (17 mo); 5 mg/kg i.p., every 2 days for 1 month	Aortic wall significantly thinner (p<0.05); lower PWV (improved elasticity); downregulated P21/P16; upregulated Ki-67/Lamin B; decreased IL-1β, IL-6, CXCL15, TNF-α	[5]
Hu et al. (2026) Front. Bioeng. Biotech.	D-galactose progeroid mice (200 mg/kg/day D-gal for 8 wks); 5 mg/kg i.p., every 2 days for 4 weeks	Reduced aortic wall thickness; reduced ROS (DHE staining); improved blood flow; confirmed p53/Bcl-2/Caspase-3 pathway activation	[5]
Han et al. (2022) J. Cell. Mol. Med.	Bleomycin-induced pulmonary fibrosis mouse model	Decreased senescent cells; attenuated BLM-induced collagen deposition; increased AEC2 percentage; decreased myofibroblasts	[8]
Toxicity Data Cleara/Patent	C57BL/6J mice; MTD 2x/week for 4 weeks; acute toxicity up to 100 mg/kg single dose (BALB/c)	At 5 mg/kg: well tolerated, no obvious side effects. At MTD: decreased body weight, elevated platelet counts, elevated ALP/ALT/AST. Acute 100 mg/kg: no mortality or observable toxicity within 24h	[1]

In Vitro / Human Cell Studies

Study	Cell Type	Key Results	Ref
Baar et al. (2017)	Human IMR90 fibroblasts (IR/Doxorubicin-induced senescence)	Potent, selective reduction in senescent cell viability; 11.73-fold selectivity vs non-senescent cells; p53 mitochondrial translocation; caspase-3/7 activation; non-senescent cells unaffected	[1]
Huang et al. (2021)	Human chondrocytes (PDL9 senescent vs PDL3 non-senescent)	25 µM for 5 days: removed >50% senescent cells; SA-β-gal <5% remaining; decreased p16, p21, p53; reduced IL-6, IL-8 SASP; non-senescent PDL3 cells unaffected	[6]
Zhang et al. (2020)	Human testicular tissue (observational immunofluorescence)	FOXO4 localized to Leydig cell nuclei in elderly men (≥65 yrs) but cytoplasmic in young men (<30 yrs); validates FOXO4 as a human aging target; correlated with reduced steroidogenic enzyme expression	[4]
Bourgeois et al. (2025)	Solution NMR structural analysis (p53-FOXO4-DRI complex)	Identified p53 TAD2 as specific binding site; both peptide and p53 TAD2 fold synergistically upon binding; phospho-Ser46/Thr55 enhances affinity; HIV-TAT contributes stabilizing contacts	[2]

Clinical / Human Trial Status

There are currently no completed or published human clinical trials for FOXO4-DRI. The compound remains in the preclinical stage. Cleara Biotech describes itself as a "preclinical-stage company" and is preparing the lead candidate CL04183 for Investigational New Drug (IND) applications. Some private wellness clinics offer FOXO4-DRI off-label; these are not registered clinical trials and the substance is not FDA-approved for human use.[1]

Dosage Summary

Setting	Dose	Route / Schedule	Notes
In Vitro (standard)	25 µM	Cell culture; 5 days exposure	Most common effective concentration[1][6]
In Vitro (range)	6.25–50 µM	Cell culture	Dose-dependent senolytic effect[1]
In Vivo (standard)	5 mg/kg	i.p. or i.v.; 3 doses every other day, or every 2 days for 1 month	Used across all major efficacy studies[1][4][5]
Acute Toxicity	Up to 100 mg/kg	Single i.v. injection (BALB/c mice)	No mortality or observable toxicity within 24h
Human (clinical)	None established	No clinical trials conducted	Off-label clinics report 100–400 mcg/kg (not validated)

Safety Profile

Parameter	At Therapeutic Dose (5 mg/kg)	At High Dose (MTD)
Body Weight	No significant change	Decreased total body weight
Platelet Counts	No thrombocytopenia (unlike BCL-2 inhibitors)	Elevated platelet counts
Liver Enzymes	Normal ALT, AST levels	Elevated ALP, ALT, AST (liver toxicity)
Kidney Function	Normal BUN, creatinine	Not separately reported
Long-term Tolerability	Over 10 months, 3x/week: no obvious side effects	Narrower therapeutic window[1]
In Vitro (human cells)	Non-senescent fibroblasts and chondrocytes consistently unaffected at senolytic doses[1][6]