Ezrin (Hep-receptor in the α domain, amino acids 308–372). No IC50, Ki, EC50, or DC50 values reported. [1]
B-cell receptor (BCR) complex; ezrin-actin cytoskeleton. [1]
Antiviral against various viruses such as HIV, HCV, herpes viruses, HPV, and influenza viruses
HEP-1 enhances antibody titers generated by hepatitis B vaccination and is effective against infections by various viruses including HIV, HCV, herpes viruses, HPV, and influenza viruses. As an immunomodulator, it enhances immune responses to vaccination and viral infection.

In human peripheral white blood cells stimulated with PHA (3 μg/mL, 24h, 37°C), GEPON (Rupe312, active sequence of RepG3) at concentrations of 1, 3, 10, 30, 100 ng/mL inhibited IL-6, IL-1β, IL-8, and TNFα expression. At 100 ng/mL, IL-6 expression was reduced by 95% relative to control (zero peptide). IL-2 expression was unaffected. [1]
In mouse DSS-induced colitis model, HEP1 (Gepon) and RepG3 (GEKKRRETVEREGG) reduced IL-6 mRNA expression in colon tissue in the presence or absence of LPS. RepG3 showed twice the anti-inflammatory activity of HEP1. [1]
In NIH/3T3 fibroblasts, GEPON induced differentiation, activated ERK1/2-dependent signalling, and increased cell motility by 25% in scratch test. GEPON also induced FAK phosphorylation. [2]
In vitro culture of bone marrow Ly6C+ monocytes from DSS-colitis mice treated with GEPON (50 μg/mL, 14h, 37°C, 5% CO2) significantly decreased mRNA expression of IL-1β (p=0.034) and IL-6 (p=0.022). [2]

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In vitro, HEP-1 demonstrates antiviral activity against multiple viruses including HIV, HCV, herpes viruses, HPV, and influenza viruses. Its mechanism involves immunomodulation and enhancement of immune responses, which contributes to its antiviral efficacy.

In a DSS-induced murine colitis model (C57BL/6 mice, 2 cycles of 2.5% DSS, 5 days on/5 days off, i.p. GEPON 2.25 mg/kg every 48h from day 1 to day 19), GEPON significantly attenuated body weight loss (DSS group lost 14%; GEPON group lost <6%, p<0.0001), reduced disease activity index (DAI from 5-6 to 2-3), lowered mortality, and prevented colon shortening (DSS: 7.0±0.6 cm; GEPON: 8.5±0.6 cm, p=0.008; healthy: 9.6±0.5 cm). GEPON reduced Il1b, Il6, and Nos2 mRNA in colon tissue by 2.8-, 3.4-, and 3.2-fold respectively. No significant change in Tgfb1, Il10, or Arg1. [2]
GEPON reduced colon CD45+ leukocyte infiltration from 1.2×10⁶ to 0.7×10⁶ cells/colon (p=0.009), reduced Ly6G+ granulocytes from 5.6×10⁴ to 2.5×10⁴ (p=0.012), and Ly6C+ monocytes from 9.3×10⁴ to 4.5×10⁴ (p=0.008). No significant change in macrophages, eosinophils, CD4+ or CD8+ T cells. Monocyte:macrophage ratio reduced 2-fold (p=0.003). [2]
In sorted colonic Ly6C+ monocytes from DSS mice, GEPON treatment reduced Tnf mRNA 4.1-fold (p=0.004), Nos2 mRNA 4.5-fold (p=0.009), Arg1 mRNA 2.5-fold (p=0.035). In Ly6G+ granulocytes, Tnf mRNA reduced 3.6-fold (p=0.017), Nos2 mRNA 4.3-fold (p=0.048). No effect on macrophages. [2]
In mice immunised with sheep erythrocytes (SE) or egg albumin (EA), HEP1 (Gepon) (i.p. 0.1 μg/mouse, 1h before immunisation) increased IgM antibody-forming cells in spleen 2.5-fold (from 600 to 1500 per spleen). Oral HEP1 pre-treatment (0.5 mg/mouse/day for 20 days) increased serum haemagglutinin and haemolysin titres 2- to 3-fold after booster immunisation. [1]
In mice with lethal herpes virus infection (HSV-1, 1000 LD50), pre-treatment with HEP1 (100 ng/mouse i.p. at 48h and 24h before infection) extended survival to 10.1 days, compared to 1.25 days in untreated controls and 6 days with Ridostin (100 μg/mouse). [1]
In a hepatitis B vaccination study in children (Kombiotech vaccine, 4 injections), sublingual HEP1 (2 mg/2 mL before each injection) increased average anti-HBs titre from 90.4 to 181.1 mIU/mL, and doubled the percentage of patients in the 100–1000 mIU/mL range and quadrupled those >1000 mIU/mL. [1]

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In vivo, HEP-1 is an orally administered peptide that enhances antibody titers generated by hepatitis B vaccination. This indicates that the peptide is bioavailable when administered orally and can modulate immune responses in living organisms.

For non-cellular in vitro assays, HEP-1 may be evaluated for its binding to immune cell receptors or its effects on cytokine production. As a peptide derived from ezrin, it may interact with proteins involved in immune signaling pathways. Binding affinity studies using surface plasmon resonance or ELISA could be employed.

Human peripheral white blood cells were isolated and stimulated with phytohaemagglutinin (PHA) at 3 μg/mL for 24 hours at 37°C. Various concentrations of GEPON (Rupe312) at 1, 3, 10, 30, 100 ng/mL were added to the culture medium. After incubation, cytokine concentrations (IL-6, IL-1β, IL-8, TNFα, IL-2) were measured in picograms per mL and expressed relative to the zero-peptide control (set to 100). A 95% reduction of IL-6 expression was observed with 100 ng/mL peptide. [1]
Bone marrow Ly6C+ monocytes and Ly6G+ granulocytes were isolated from naive and DSS-colitis mice (day 6). Cells were sorted by FACS and plated in DMEM culture medium with FBS in 96-well plates at densities of 50,000 Ly6C+ or 150,000 Ly6G+ cells per well in triplicate. GEPON was added at 50 μg/mL. Cells were incubated for 14 hours at 37°C and 5% CO2, then harvested for mRNA analysis by real-time PCR. GEPON significantly decreased IL-1β and IL-6 mRNA in Ly6C+ monocytes (p=0.034 and p=0.022 respectively). [2]
NIH/3T3 fibroblasts were cultured and treated with GEPON to assess differentiation and motility. A scratch test was performed, and GEPON treatment resulted in a 25% increase in cell motility. ERK1/2-dependent intracellular signalling cascade activation and FAK phosphorylation were observed. [2]

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For in vitro cellular assays, HEP-1 is tested in immune cell cultures to assess its immunomodulatory effects. Cells are treated with the peptide, and cytokine production, cell proliferation, and activation markers are measured. Antiviral activity is assessed in virus-infected cell cultures by measuring viral replication and cytopathic effects.

For DSS colitis model: Eight- to ten-week-old male C57BL/6 mice (21-23 g) were given 2.5% DSS polymer in drinking water for two 5-day cycles separated by 5 days of plain water, followed by 4 more days of water. GEPON (2.25 mg/kg body weight) was dissolved in water and administered intraperitoneally (i.p.) at a volume of 50 μL every 48 hours from day 1 to day 19. Control groups received water only or DSS with vehicle (water i.p.). Mice were sacrificed on day 19; colons were collected for length measurement, FACS, and PCR analysis. [2]
For mouse immunisation studies: Mice (CBA × C57Bl) F1 were given HEP1 orally at 0.5 mg/0.2 mL per mouse per day for 20 days prior to immunisation. Immunisation was with 20 million sheep erythrocytes (SE) i.p. on day 1, followed by a booster of 20 million SE on day 21. Serum haemagglutinin and haemolysin titres were measured weekly. Alternatively, mice received i.p. injection of HEP1 (0.01 to 10 μg per mouse) 1 hour before i.p. immunisation with 5×10⁶ SE cells. After 4 days, spleens were collected to count antibody-forming cells. [1]
For lethal herpes infection model: B/P white mice (10-12 g) received i.p. injections of HEP1 (1, 10, or 100 ng per mouse in 0.2 mL saline) at 48h and 24h before i.p. injection of herpes simplex virus type 1 (HSV-1 strain L2) at 1000 LD50 (titre 3.5 Log/0.2 mL). Mice were observed for 14 days. Positive control received Ridostin (100 μg/mouse), negative control received no adjuvant. [1]
For hepatitis B vaccination study in children: Children with malignant tumours received Kombiotech hepatitis B vaccine (KHBV) injections at monthly intervals (3 doses) plus a fourth dose 6 months after start. In the HEP1 group (n=15), 2 mg/2 mL HEP1 solution was given sublingually, held for 2 minutes then swallowed before each of the four vaccine injections. Control group (n=16) received vaccine without HEP1. Anti-HBs titres were measured. [1]

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For in vivo animal studies, HEP-1 is administered orally to animal models to assess its immunomodulatory and antiviral effects. Efficacy is evaluated by measuring antibody titers following vaccination, viral load reduction, and survival rates in infection models.

HEP-1 is an orally administered peptide, indicating that it has favorable oral bioavailability. Its pharmacokinetic properties include absorption from the gastrointestinal tract and distribution to target tissues. As a peptide, it may be subject to proteolytic degradation, but its oral activity suggests sufficient stability.

No adverse reactions, no side effects, no allergic reactions, no hypersensitivity, and no contra-indications were observed in children receiving intra-nasal GEPON (2 mg in 2 mL water, 5 drops per nostril twice daily for 5 days). No aggravation of atopic dermatitis was detected. No adverse drug interactions were reported. [1]

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Toxicological data for HEP-1 are limited. As a peptide derived from a human protein, it is expected to have low immunogenicity and toxicity. Comprehensive safety evaluations would be required for therapeutic development. The compound has a molecular weight of 1817.9979 and a molecular formula of C₇₄H₁₃₂N₂₆O₂₇.

[1]. Ezrin Peptide Therapy from HIV to COVID: Inhibition of Inflammation and Amplification of Adaptive Anti-Viral Immunity. Int J Mol Sci. 2021 Oct 28;22(21):11688.
[2]. Pharmaceutical grade synthetic peptide Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu ameliorates DSS-induced murine colitis by reducing the number and pro-inflammatory activity of colon tissue-infiltrating Ly6G+ granulocytes and Ly6C+ monocytes. Peptides. 2020 Oct;132:170364.

GEPON (HEP1) is registered for human use in the Russian Federation since 2001. It is an immunomodulator for topical and internal use. It is effective against opportunistic infections in HIV patients, chronic recurrent vaginal candidiasis, acute and chronic enterocolitis, dysbacteriosis, gastric and duodenal ulcers, and inflammatory bowel disease. It has been used as an inhaled spray for mild-to-moderate COVID. [1]
Mechanism hypothesis: Ezrin peptides bind to a “receptor” conformation of ezrin on cell surfaces (lymphocytes, epithelial cells, fibroblasts, monocytes, macrophages), inducing allosteric opening of active ezrin, which organises signalling complexes (Ras>Raf>MEK>ERK, PI3K>AKT) and enhances B-cell activation and antibody production. Suppression of IL-6 may occur via CREB activation competing for CBP/p300 co-factors with NFκB. [1]
GEPON is also known as HEP1 (Human Ezrin Peptide One) and its active pharmaceutical ingredient is TEKKRRETVEREKE. A derivative RepG3 (GEKKRRETVEREGG) has twice the anti-inflammatory activity in DSS colitis model. [1]

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HEP-1 Acetate (Human ezrin peptide (324-337)) is an orally administered peptide with anti-HIV activity. It enhances antibody titers generated by hepatitis B vaccination and has antiviral, anti-inflammatory, and immunomodulatory activities against various viruses including HIV, HCV, herpes viruses, HPV, and influenza viruses.

C74H132N26O27

1817.99789619446

1816.975

174641-44-2

25078222

Thr-Glu-Lys-Lys-Arg-Arg-Glu-Thr-Val-Glu-Arg-Glu-Lys-Glu

TEKKRRETVEREKE

White to off-white solid at room temperature

-20.3

31

34

69

127

3730

16

O=C([C@H](CCCCN)NC([C@H](CCCCN)NC([C@H](CCC(=O)O)NC([C@H]([C@@H](C)O)N)=O)=O)=O)N[C@H](C(N[C@H](C(N[C@H](C(N[C@@H]([C@@H](C)O)C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(=O)O)CCC(=O)O)=O)CCCCN)=O)CCC(=O)O)=O)CCC/N=C(\N)/N)=O)CCC(=O)O)=O)C(C)C)=O)=O)CCC(=O)O)=O)CCC/N=C(\N)/N)=O)CCC/N=C(\N)/N

HTQONPPEZULJKY-XRBNWYQFSA-N

InChI=1S/C74H132N26O27/c1-36(2)56(69(124)97-47(22-27-52(107)108)66(121)93-44(19-13-35-87-74(83)84)61(116)94-45(20-25-50(103)104)64(119)90-41(16-7-10-32-77)63(118)98-49(71(126)127)24-29-54(111)112)99-70(125)57(38(4)102)100-67(122)48(23-28-53(109)110)95-62(117)43(18-12-34-86-73(81)82)92-60(115)42(17-11-33-85-72(79)80)91-59(114)39(14-5-8-30-75)88-58(113)40(15-6-9-31-76)89-65(120)46(21-26-51(105)106)96-68(123)55(78)37(3)101/h36-49,55-57,101-102H,5-35,75-78H2,1-4H3,(H,88,113)(H,89,120)(H,90,119)(H,91,114)(H,92,115)(H,93,121)(H,94,116)(H,95,117)(H,96,123)(H,97,124)(H,98,118)(H,99,125)(H,100,122)(H,103,104)(H,105,106)(H,107,108)(H,109,110)(H,111,112)(H,126,127)(H4,79,80,85)(H4,81,82,86)(H4,83,84,87)/t37-,38-,39+,40+,41+,42+,43+,44+,45+,46+,47+,48+,49+,55+,56+,57+/m1/s1

(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-4-carboxybutanoyl]amino]hexanoyl]amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-4-carboxybutanoyl]amino]-3-hydroxybutanoyl]amino]-3-methylbutanoyl]amino]-4-carboxybutanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-4-carboxybutanoyl]amino]hexanoyl]amino]pentanedioic acid

HEP-1; gepon; Human ezrin peptide (324-337); 174641-44-2; orb1942551; SCHEMBL30942637;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~ 50 mg/mL (27.5 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)