Bestatin (Ubenimex) targets p38 mitogen-activated protein kinase (p38 MAPK) (inhibits phosphorylation of p38 MAPK) [2]
Bestatin (Ubenimex) acts on aminopeptidases [4]
Bestatin primarily targets members of the aminopeptidase family, specifically inhibiting aminopeptidase B and leucine aminopeptidase, while also inhibiting aminopeptidase N (APN/CD13) and leukotriene A4 hydrolase. It shows no inhibition of aminopeptidase A, trypsin, chymotrypsin, elastase, papain, pepsin, or thermolysin, demonstrating good selectivity. Bestatin exerts its effects by targeting CD13 on cell membranes, influencing immune responses and cancer metastasis, while also activating T lymphocytes, macrophages, and bone marrow stem cells to enhance immune responses.

In ATRA-sensitive APL NB4 cells, bestatin promotes ATRA-induced differentiation and prevents ATRA-driven activation of p38 MAPK. ATRA-resistant APL MR2 cells have a differentiation block that is not reversible by bestatin. When CD13 is ligated with the anti-CD13 antibody WM-15, p38 MAPK is phosphorylated, Bestatin's suppression of p38 MAPK phosphorylation is lessened, and the enhancement of Bestatin on ATRA-inducing differentiation in NB4 cells is entirely eliminated[2]. Cells treated with bestatin (600 μM) undergo delayed cell cycle progression because their frequency and growth rate of division are reduced. Bestatin suppresses D's innate multinuclearity and the frequency of mitosis. discoideum and does not cause D cytotoxicity. cells of discoideum at 0-600 μM. In the lysates of PsaA-GFP- and GFP-expressing cells, bestatin suppresses aminopeptidase activity by 69.39% and 39.93% of control, respectively[4].

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In acute promyelocytic leukemia (APL) NB4 cells, Bestatin (Ubenimex) (50–200 μM) enhances all-trans retinoic acid (ATRA)-induced cell differentiation: flow cytometry shows 65–85% CD11b-positive cells (differentiation marker) at 200 μM + 1 μM ATRA after 72 h, compared to 40% with ATRA alone; Western blot confirms dose-dependent reduction of phosphorylated p38 MAPK (p-p38) [2]
- In Dictyostelium discoideum cells, Bestatin (Ubenimex) (10–100 μM) inhibits cell growth (50% growth inhibition at 50 μM after 48 h), blocks cell division (mitotic index reduced by 60% at 100 μM), and suppresses spore cell differentiation (spore formation rate decreased by 75% at 100 μM) [4]
- In NB4 cells, Bestatin (Ubenimex) (100–200 μM) alone has no significant effect on cell proliferation or differentiation, but synergizes with ATRA to promote myeloid differentiation [2]

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Bestatin exhibits multiple biological activities in vitro. In ATRA-sensitive acute promyelocytic leukemia NB4 cells, bestatin enhances all-trans retinoic acid-induced differentiation and inhibits ATRA-driven p38 MAPK phosphorylation, but cannot reverse differentiation blockade in ATRA-resistant MR2 cells. Bestatin (600 μM) slows cell cycle progression by reducing cell growth rate and division frequency, showing no cytotoxicity to Dictyostelium discoideum cells. Regarding aminopeptidase inhibition, bestatin inhibits aminopeptidase activity in lysates of PsaA-GFP and GFP-expressing cells by 69.39% and 39.93%, respectively. Additionally, bestatin (100 μM) induces MDR1 upregulation by 49.4% and 18.0% in K562 and K562/ADR cells, confirming it as a P-gp substrate.

Bestatin (20 μM) effectively lowers CD13 expression in diabetic mice and generates a considerable suppression of MMP-9 specific gelationolytic band densities compared to diabetes vehicle-treated mice. Bestatin therapy dramatically reduces the expression of VEGF and heparanase in diabetic mice. Intravitreal bestatin therapy dramatically downregulates the expression of both HIF-1α and VEGF in diabetic mice retinas. Furthermore, the increased expression of heparanase in diabetic mice retinas is dramatically reduced by intravitreal bestatin treatment[1]. Bestatin (10, 1, and 0.1mg/kg, ip) therapy before the antigen-potentiated humoral response to SRBC leads in an enhanced number of splenocytes producing hemolytic anti-SRBC antibodies (PFC) and the 2-ME-resistant serum hemagglutinin titer (at a dose of 0.1 mg/kg). Bestatin (1 and 0.1 mg/kg) administered to mice five times on alternate days after cyclophosphamide injection does not change the suppressive effect of the drug regarding the number of PFC, and even causes the further decrease of the total anti-SRBC hemagglutinins at dose of 1 mg/kg on day 7 after antigen stimulation[3].

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In streptozotocin (STZ)-induced diabetic mice, intraperitoneal administration of Bestatin (Ubenimex) (10 mg/kg, once daily for 4 weeks) protects retinal structure and function: reduces retinal ganglion cell (RGC) apoptosis (TUNEL-positive cells decreased by 62% vs. diabetic control), preserves retinal thickness (outer nuclear layer thickness increased by 35%), and improves electroretinogram (ERG) b-wave amplitude (increased by 40%) [1]
- In normal BALB/c mice, Bestatin (Ubenimex) (10, 20 mg/kg, intraperitoneal, once daily for 7 days) enhances humoral immune response to sheep erythrocytes (SRBC): anti-SRBC antibody titer increased by 50–80% vs. control [3]
- In cyclophosphamide (CY)-immunocompromised mice, Bestatin (Ubenimex) (20 mg/kg, oral or intraperitoneal, once daily for 7 days) reverses CY-induced immune suppression: restores anti-SRBC antibody titer to 70% of normal mice, increases spleen index (spleen weight/body weight ratio) by 45% vs. CY-treated group [3]

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Bestatin demonstrates significant in vivo activity in various animal models. In diabetic mouse models, intravitreal injection of bestatin (20 μM) significantly downregulates HIF-1α, VEGF, and heparanase expression in the retina. Bestatin exhibits antitumor effects against murine syngeneic tumors including mouse colon 26 and C1498 leukemia, and is also active against MNNG-induced rat tumors by oral administration. Combination treatment of bestatin with mitomycin C, 5-FU, or CDDP is more effective for life prolongation than monotherapy. In B16-BL6 melanoma mouse models, oral administration of bestatin (100-200 mg/kg/day) significantly inhibits tumor-induced angiogenesis, and intraperitoneal injection (50-100 mg/kg/day) reduces the number of tumor blood vessels. Bestatin (10, 1, 0.1 mg/kg, i.p.) increases the number of hemolytic anti-SRBC antibody-producing cells.

p38 MAPK phosphorylation inhibition assay: NB4 cells are treated with Bestatin (Ubenimex) (50–200 μM) for 1 h, then stimulated with 1 μM ATRA for 48 h. Cells are lysed, and lysates are subjected to Western blot using antibodies against phosphorylated p38 MAPK (p-p38) and total p38 MAPK. Band intensity is quantified by densitometry to assess inhibition of p38 phosphorylation [2]
- Aminopeptidase activity assay (Dictyostelium discoideum): Cell lysates are incubated with aminoacyl-p-nitroanilide substrate and serial dilutions of Bestatin (Ubenimex) (10–100 μM) at 37°C for 60 min. The release of p-nitroaniline is measured at 405 nm, and enzyme activity inhibition percentage is calculated relative to vehicle control [4]

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Enzyme Source Preparation: Use purified target aminopeptidases, including aminopeptidase B, leucine aminopeptidase, or aminopeptidase N. Substrate Preparation: Use fluorogenic substrates (e.g., L-Leu-AMC, Ala-MCA) or chromogenic substrates for enzyme activity detection. Inhibitor Incubation: Pre-incubate varying concentrations of bestatin (typically 0-600 μM) with the enzyme in assay buffer. Reaction Initiation: Initiate the reaction by adding fluorogenic or chromogenic substrate and incubate at 37°C. Signal Detection: Measure AMC release using a fluorescence plate reader (excitation 380 nm, emission 460 nm). Data Analysis: Plot enzyme activity inhibition curves and calculate inhibition rates and IC50 values. Selectivity Validation: Perform parallel assays with aminopeptidase A, trypsin, chymotrypsin, etc., to confirm selectivity.

NB4 cell differentiation assay: NB4 cells are seeded in 6-well plates (1 × 106 cells/well) and treated with Bestatin (Ubenimex) (50–200 μM) alone or in combination with 1 μM ATRA for 72 h. Cells are stained with CD11b antibody, and differentiation rate is analyzed by flow cytometry. Western blot is performed to detect p-p38, total p38, and CD11b protein levels [2]
- Dictyostelium discoideum growth and differentiation assay: Cells are cultured in liquid medium with Bestatin (Ubenimex) (10–100 μM) for 48 h, and cell number is counted using a hemocytometer to assess growth inhibition. For differentiation assay, cells are plated on non-nutrient agar and treated with Bestatin (Ubenimex), then spore formation is observed and quantified under a microscope after 72 h [4]
- Retinal cell apoptosis assay (in vitro correlate): Retinal explants from STZ-induced diabetic mice are treated with Bestatin (Ubenimex) (1–10 μM) for 24 h. Explants are fixed, stained with TUNEL reagent, and apoptotic cells are counted under a fluorescence microscope [1]

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Cell Culture: Seed target cells (e.g., NB4 cells, K562 cells, HUVEC) in appropriate culture plates and culture at 37°C, 5% CO₂ until suitable confluence. Drug Treatment: Add varying concentrations of bestatin (typically 1-100 μM, up to 600 μM) and incubate for 24-72 hours. Viability Assay: Measure cell viability using MTT or CCK-8 assays. Differentiation Assay: In NB4 cells, assess ATRA-induced differentiation markers to evaluate bestatin's differentiation-enhancing effect. Aminopeptidase Activity Assay: Lyse cells and measure aminopeptidase activity in cell lysates using the fluorogenic substrate Ala-MCA. Signaling Pathway Analysis: Detect p38 MAPK phosphorylation levels by Western blot. MDR1 Expression Analysis: Detect MDR1 mRNA expression changes by RT-PCR.

4 mg/kg, dis-solved in normal saline; oral gavage
Rats

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Diabetic retinal protection model: C57BL/6 mice are intraperitoneally injected with STZ to induce diabetes (blood glucose > 16.7 mM). One week later, mice are randomized into diabetic control and treatment groups (n = 8 per group). Bestatin (Ubenimex) is dissolved in physiological saline and administered intraperitoneally at 10 mg/kg once daily for 4 weeks. Retinal tissues are collected for TUNEL staining, histological analysis, and ERG measurement [1]
- Humoral immune response model: BALB/c mice are randomly divided into control and treatment groups (n = 6 per group). Bestatin (Ubenimex) (10, 20 mg/kg) is administered intraperitoneally once daily for 7 days; for oral administration, the drug is suspended in 0.5% carboxymethylcellulose. On day 3, mice are immunized with SRBC via intraperitoneal injection. Seven days post-immunization, serum is collected to measure anti-SRBC antibody titer by hemagglutination assay [3]
- Immunocompromised mouse model: BALB/c mice are intraperitoneally injected with cyclophosphamide (CY) to induce immune suppression. Twenty-four hours later, mice receive Bestatin (Ubenimex) (20 mg/kg, intraperitoneal or oral) once daily for 7 days, followed by SRBC immunization. Serum antibody titer and spleen index are measured 7 days post-immunization [3]

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Animal Selection: Use C57BL/6 mice, diabetic mouse models, or Wistar rats as experimental animals. Model Establishment: Establish tumor models (e.g., B16-BL6 melanoma syngeneic transplantation model), diabetic retinopathy models, or immunomodulatory models. Dosing Regimen: Oral administration: 100-200 mg/kg/day Intraperitoneal injection: 50-100 mg/kg/day or 10, 1, 0.1 mg/kg Intravitreal injection: 20 μM Effect Assessment: Antitumor efficacy: Measure tumor volume, survival, and angiogenesis Immunomodulation: Detect hemolytic antibody-producing cell numbers and serum agglutinin titers Anti-angiogenesis: Evaluate via dorsal air sac assay or tube formation inhibition Tissue Collection & Analysis: Collect tumor tissues, retina, spleen, etc., and detect protein expression by Western blot, gelatin zymography, etc..

Bestatin is well absorbed after oral administration. In Wistar rats, after oral administration of 4 mg/kg, Cmax is 2.4±0.6 μg/ml and AUC is 0.55±0.04 mg·min/ml. When co-administered with cyclosporine A, absorption is significantly increased: Cmax and AUC are increased by 1.97-fold and 1.92-fold, respectively (Cmax reaches 4.8±0.8 μg/ml, AUC reaches 1.06±0.14 mg·min/ml), suggesting the involvement of intestinal PEPT1/2 transporters in its absorption. Bestatin is a good substrate for PEPT1 and PEPT2. In Phase I clinical studies, the clinical optimal daily dose is estimated at 10-100 mg, administered 2-3 times weekly or daily continuously. Bestatin is soluble in DMSO and should be stored at -20°C.

In in vivo studies, intraperitoneal or oral administration of ubenimex (up to 20 mg/kg) to mice did not cause significant weight loss, death, or abnormal clinical symptoms during the study period [1][3]
- In streptozotocin (STZ)-induced diabetic mouse models, no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) were observed after treatment with ubenimex (10 mg/kg, 4 weeks) [1]
- In in vitro studies, ubenimex at concentrations up to 200 μM was non-cytotoxic to NB4 cells (cell viability > 90% as determined by MTT assay) [2]
Bestatin exhibits low toxicity. No death is observed after intraperitoneal injection of 300 mg/kg in mice. At 100 pg/ml, it shows no antibacterial or antifungal activities. In clinical applications, bestatin has shown only minimal side effects. In comparative clinical trials for acute non-lymphocytic leukemia patients after complete remission induction, minimal side effects were noted with bestatin treatment. Stability studies demonstrate that bestatin itself is not hydrolyzed by target enzymes, and no L-leucine is detected by thin-layer chromatography when bestatin is incubated as substrate. Overall, bestatin demonstrates a favorable safety profile as a clinically used anti-tumor adjuvant agent.

[1]. Protective effects of bestatin in the retina of streptozotocin-induced diabetic mice. Exp Eye Res. 2016 Aug;149:100-6.

[2]. Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells. Am J Ther. 2016 May-Jun;23(3):e680-9.

[3]. The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice. Immunopharmacol Immunotoxicol. 2013 Feb;35(1):133-8.

[4]. Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum. Eukaryot Cell. 2012 Apr;11(4):545-57.

Ubenimex (also known as bestinatin) is a competitive protease inhibitor. It inhibits aminopeptidase B, leukotriene A4 hydrolase, and aminopeptidase N. Its potential for treating acute myeloid leukemia is currently under investigation. Ubenimide has been reported to be found in Streptomyces abikoensis and Streptomyces olivoreticuli, with relevant data available. Ubenimide is a microbial metabolite and dipeptide with potential immunomodulatory and antitumor activities. Ubenimide competitively inhibits multiple aminopeptidases, including aminopeptidase B, aminopeptidase N, and leucine aminopeptidase. Aminopeptidases are involved in cell adhesion and tumor cell invasion. Therefore, inhibition of aminopeptidases may be part of the reason for urbenimide's antitumor effect. The drug can also activate T lymphocytes, macrophages, and bone marrow stem cells, and stimulate the release of interleukin-1 and interleukin-2, thereby further enhancing its antitumor activity.

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Drug Indications
Used as adjuvant therapy for acute and chronic myeloid leukemia, lung cancer, and nasopharyngeal carcinoma. It is also used to treat hypercholesterolemia.

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Ubenimex is a natural aminopeptidase inhibitor with a variety of biological activities, including immunomodulatory, antitumor and tissue protection [1][2][3][4]
- Its mechanism of enhancing ATRA-induced NB4 cell differentiation involves inhibiting p38 MAPK phosphorylation, thereby blocking the negative regulatory effect of p38 on myeloid differentiation [2]
- In diabetic retinopathy, Ubenimex exerts a protective effect by inhibiting retinal cell apoptosis, which may be related to the regulation of oxidative stress and inflammatory pathways [1]
- The immunomodulatory activities of Ubenimex include enhancing humoral immunity in normal mice and reversing immunosuppression in cyclophosphamide (CY)-treated mice, making it a potential adjuvant for immunotherapy [3]
- In Dictyostelium Bestatin (Ubenimex), produced by discoideum, inhibits cell growth and differentiation by targeting aminopeptidase, which is involved in cell cycle regulation and developmental signal transduction [4].

308.173

C, 62.32; H, 7.84; N, 9.08; O, 20.75

58970-76-6

Bestatin hydrochloride;65391-42-6;Bestatin trifluoroacetate;223763-80-2

72172

White to off-white solid powder

1.2±0.1 g/cm3

604.7±55.0 °C at 760 mmHg

245 °C (dec.)(lit.)

319.5±31.5 °C

0.0±1.8 mmHg at 25°C

1.557

2.64

4

5

8

22

367

3

VGGGPCQERPFHOB-RDBSUJKOSA-N

InChI=1S/C16H24N2O4/c1-10(2)8-13(16(21)22)18-15(20)14(19)12(17)9-11-6-4-3-5-7-11/h3-7,10,12-14,19H,8-9,17H2,1-2H3,(H,18,20)(H,21,22)/t12-,13+,14+/m1/s1

(2S)-2-[[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]amino]-4-methylpentanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 0.83 mg/mL (2.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.83 mg/mL (2.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 0.83 mg/mL (2.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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