Cenupatide (UPARANT) targets the urokinase-type plasminogen activator receptor (uPAR) pathway and formyl peptide receptors (FPRs), specifically FPR1, FPR2, and FPR3. The compound inhibits uPAR binding to FPRs and also affects the αvβ3 integrin/Rac-1 pathway.
IC50, Ki, EC50, or DC50 values were not reported in the provided literature. [1][2][3]

In human retinal endothelial cells, UPARANT prevented VEGF-induced permeability and inhibited angiogenic activity. The peptide inhibited FPR-mediated regulation of transcription factors coupled to inflammation, including NF-κB and CREB. UPARANT downregulated the expression of uPAR and its membrane partners FPR1 and FPR2 at both transcript and protein levels in retinal tissues from SDT rats. In STZ rats, however, no effects of UPARANT on transcript or protein levels of uPAR or FPRs could be observed. [2]
In mouse models of rubeosis iridis, UPARANT reduced FPR1 overexpression at the transcript level, while no alteration in transcript levels of FPR2 or FPR3 was observed. Immunofluorescence analysis revealed that FPR1 was strongly colocalized with iris vasculature, while FPR2 and FPR3 showed less intense colocalization signals. UPARANT reduced the phosphorylation of CREB and NF-κB, master regulators of pro-inflammatory responses. At the molecular level, UPARANT downregulated transcript levels of genes associated with inflammation and extracellular matrix degradation, including PAI-1, uPA, uPAR, IL-1β, IL-6, TGFα, CCL2, and CXCR4, without affecting hypoxia response genes (PGK1 and EPO) or canonical angiogenesis genes (VEGF, PLGF, and their receptors). Protein expression analysis showed that UPARANT reduced IL-6 and MMP2 levels. [3]
In kidney extracts from STZ-induced diabetic rats, UPARANT (8 mg/kg) reduced uPA levels and activity without affecting uPAR levels. The compound inhibited FPR2 expression in isolated glomeruli, suggesting that the drug acts downstream of uPAR by blocking its binding to FPRs. UPARANT recovered the increased activity of the αvβ3 integrin/Rac-1 pathway, reducing αvβ3 integrin levels, β3 integrin phosphorylation, and Rac-1 activity. UPARANT also reduced renal levels of ECM components including fibronectin, collagen I, and collagen IV, and increased plasmin and MMP-2/MMP-9 levels and activity in kidney extracts. [1]

In the spontaneously diabetic Torii (SDT) rat model of type 2 diabetic retinopathy, preventive administration of UPARANT (7 mg/kg, subcutaneous, 3 times per week for 19 weeks) initiated 7 weeks after diabetes onset prevented electroretinogram dysfunction, including preservation of a-wave and b-wave amplitudes and retinal sensitivity (k). UPARANT prevented blood-retinal barrier leakage as demonstrated by reduced fluorescein leakage and Evans blue dye extravasation, prevented upregulation of VEGF and FGF-2, preserved tight junction proteins (claudin-1, claudin-5, ZO-1), and reduced gliosis (GFAP) and retinal cell death (caspase-3). In SDT rats, UPARANT reduced the upregulation of uPAR, FPR1, and FPR2 at transcript and protein levels by 1.7- to 1.9-fold, without affecting FPR3. UPARANT also reduced the phosphorylation of STAT3 (Tyr705) and NF-κB p65 (Ser276) by 3.4-fold and 1.6-fold, respectively, and downregulated inflammatory cytokines TNF-α, IL-1β, and IL-6 at both transcript and protein levels. [2]
In the STZ-induced diabetic rat model of diabetic nephropathy, subcutaneous administration of UPARANT at 8 mg/kg (daily for 5 days) significantly improved renal parameters including reduced urine output (1.7-fold), urine albumin (1.8-fold), urine creatinine (1.3-fold), albumin-to-creatinine ratio (2.3-fold), plasma creatinine (1.4-fold), creatinine clearance (1.3-fold), and blood urea nitrogen (1.7-fold). UPARANT restored vascular permeability by increasing ZO-1 (1.7-fold) and occludin (1.8-fold) levels, decreasing VEGF expression (1.8-fold) and plasma extravasation (1.4-fold). The compound increased AQP2 expression in the medulla (2.8-fold). UPARANT reduced inflammatory markers including iNOS, ICAM-1, phosphorylated NF-κB p65 (Ser276), phosphorylated CREB (Ser133), and HIF-1α. The compound also reduced renal fibrosis as evidenced by decreased ECM components (fibronectin, collagen I, collagen IV) and reduced Masson's trichrome staining of fibrotic areas. Histological evaluation demonstrated that UPARANT attenuated glomerular hypertrophy and mesangial expansion, and transmission electron microscopy revealed recovery from thickening of the glomerular basement membrane and podocyte foot process effacement. UPARANT at 1 mg/kg was ineffective on most parameters. [1]
In the mouse puncture-induced rubeosis iridis model, intravitreal administration of UPARANT (7.6 g/L, 1 μL per injection on days 4, 8, and 12) reduced iris vascular density from approximately 20% above control to control levels by day 8, with effects sustained through day 15. Microvasculature analysis showed significant reduction in total vasculature, sprouts, and vascular branching. Subcutaneous administration of UPARANT (15.2 mg/kg, daily loading dose for 5 days) also effectively counteracted iris vascular response, with no statistical difference between induced eyes and fellow controls after the last injection. UPARANT downregulated transcripts of FPR1, PAI-1, uPA, uPAR, IL-1β, IL-6, CCL2, and CXCR4 to control levels. [3]

uPA activity was measured using a colorimetric assay with a commercial uPA Activity Assay Kit. Kidney homogenates or plasma samples were processed according to the manufacturer's instructions, and uPA activity was determined by colorimetric detection. In STZ rats, uPA activity in kidney extracts was increased by approximately 2.5-fold, and in plasma by 1.5-fold compared to controls. UPARANT at 8 mg/kg reduced uPA activity in kidney extracts by 1.4-fold and in plasma by 1.3-fold. [1]
Plasmin activity was assayed using Chromozym PL, a plasmin-specific chromogenic substrate. Samples were incubated with the substrate and plasmin activity was determined by measuring chromogenic product formation. In STZ rats, plasmin activity was decreased in kidney extracts by 2.1-fold and in plasma by 1.3-fold compared to controls. UPARANT at 8 mg/kg increased plasmin activity in kidney extracts by 1.7-fold and in plasma by 1.3-fold. [1]
MMP-2/MMP-9 activity was assessed using a fluorogenic assay with a collagen-like fluorogenic substrate. Cleavage of the substrate by MMP-2/MMP-9 resulted in increased fluorescence measured at excitation 320 nm and emission 405 nm. In STZ rats, MMP-2/MMP-9 activity in kidney extracts was decreased by 2.7-fold, while in plasma it was increased by 2.2-fold compared to controls. UPARANT at 8 mg/kg increased MMP-2/MMP-9 activity in kidney extracts by 1.9-fold without affecting plasma MMP activity. [1]
Rac-1 activity was measured using a quantitative ELISA assay that recognizes the active GTP-bound form of Rac-1. Protein extracts were processed according to the manufacturer's protocol, and active Rac-1 was determined by ELISA. In STZ rats, Rac-1 activity increased by 2.1-fold, and UPARANT at 8 mg/kg reduced Rac-1 activity by 1.2-fold. [1]

For quantitative real-time PCR analysis, total RNA was extracted from retinal tissues or isolated glomeruli using a commercial RNA isolation kit. First-strand cDNA was generated from 1 μg of total RNA using reverse transcription. qPCR amplification was performed with SYBR Green supermix on a real-time PCR detection system. Target genes were assayed concurrently with housekeeping genes Rpl13a and Hprt. Samples were compared using the relative threshold cycle (Ct method), and fold changes were determined relative to controls after normalization to housekeeping genes. All reactions were performed in triplicate. FPR primer sequences included: FPR1 forward 5'-GTTTCCGCATGAAACGCACT-3', reverse 5'-CATGACCAGGCTGACGATGT-3'; FPR2 forward 5'-GCTTCACAATGCCCATGTCC-3', reverse 5'-ACTCGTAAAGGGACGACTGGA-3'; FPR3 forward 5'-TCCCTTTCAACTGGTTGCCC-3', reverse 5'-GCCAATGAGTTGGTTGGCATA-3'. [1][2][3]
For Western blot analysis, tissues were homogenized in RIPA buffer or appropriate lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Protein concentration was determined using the Micro BCA method or Bradford method. Equal amounts of proteins (typically 30 μg or 15 μg) were separated by SDS-PAGE and transferred onto nitrocellulose or PVDF membranes. Membranes were probed with primary antibodies against targets including uPA, uPAR, FPRs, αvβ3 integrin, phosphorylated β3 integrin (Tyr773), Rac-1, plasminogen, plasmin, MMP-2, MMP-9, fibronectin, collagen I, collagen IV, ZO-1, occludin, VEGF, iNOS, ICAM-1, NF-κB p65 (total and phosphorylated at Ser276), CREB (total and phosphorylated at Ser133), HIF-1α, AQP2, GFAP, caspase-3, STAT3 (total and phosphorylated at Tyr705), and β-actin as loading control. After incubation with HRP-conjugated secondary antibodies, bands were visualized using enhanced chemiluminescence substrate and quantified by densitometry using image analysis software. [1][2][3]
For ELISA analysis, protein levels of VEGF, FGF-2, TNF-α, IL-1β, IL-6, uPA, soluble uPAR, plasminogen, and MMPs were quantified using commercially available ELISA kits. Protein extracts or plasma samples were processed according to manufacturers' instructions. ELISA plates were evaluated spectrophotometrically, and data were expressed as nanograms or picograms of target per milligram of protein. All experiments were performed in duplicate. [1][2]
For immunofluorescence analysis, tissues were fixed, cryopreserved, and sectioned. Sections were incubated with primary antibodies directed to target proteins followed by fluorophore-conjugated secondary antibodies. Confocal images were obtained using a confocal laser-scanning microscope. For retinal vasculature analysis, whole mounts were immunolabeled with CD31 antibody and analyzed by fluorescence microscopy. [1][2][3]

For the STZ-induced diabetic nephropathy model, male Sprague-Dawley rats (150-200 g) received a single intraperitoneal injection of 65 mg/kg streptozotocin in citrate buffer (0.1 mol/L citric acid and 0.2 mol/L sodium phosphate, pH 4.5). Animals with plasma glucose >350 mg/dL were considered diabetic. Four weeks after diabetes induction, UPARANT succinate was dissolved in PBS and administered subcutaneously at 1 mg/kg or 8 mg/kg daily for 5 days. Rats were kept individually in metabolic cages for 24 hours to collect urine for measurement of urine output. Systolic blood pressure was measured by tail-cuff blood pressure system. Rats were killed with 65 mg/kg pentobarbital. Blood was collected in EDTA tubes for plasma isolation, and kidneys were removed for protein extraction, immunohistochemistry, or electron microscopy. [1]

For the SDT rat diabetic retinopathy preventive study, male SDT rats were used. Age-matched SD rats served as nondiabetic controls. Treatments were initiated 7 weeks after diabetes onset (corresponding to 13 weeks before significant ERG dysfunction). UPARANT at 7 mg/kg was administered subcutaneously 3 times per week for 19 weeks. Blood glucose was measured by tail sampling. Body weight and blood glucose were recorded. ERG monitoring was performed longitudinally. [2]

For the STZ rat diabetic retinopathy study, SD rats received a single intraperitoneal injection of 65 mg/kg STZ. Treatments were initiated 4 weeks after diabetes onset. UPARANT at 8 mg/kg was administered subcutaneously daily for 5 days. [2]

For the puncture-induced rubeosis iridis model, 12.5-day-old BALB/c mice of either sex were subjected to two self-sealing uveal punctures with a 30 G beveled needle immediately posterior to the limbus on both eyes. Punctures were repeated every 4 days until experimental day 12. For intravitreal administration, UPARANT at 7.6 g/L (1 μL) was injected on experimental days 4, 8, and 12. For subcutaneous administration, UPARANT at 15.2 mg/kg was administered daily from experimental day 4 to day 8 (5 days loading dose). Mice were anesthetized with 4% isoflurane in room air, treated with 1% tetracaine hydrochloride solution, and rehydrated with subcutaneous injection of sterile saline solution. On experimental day 15, mice were euthanized and eyes were dissected for molecular analysis or immunofluorescence. In vivo iris vasculature was photographed using an objective-adapted camera prior to each procedure. [3]

Following subcutaneous administration of UPARANT (20 mg/kg), the drug rapidly appeared in plasma, being quantifiable at 0.25 hours post-dose. Cmax was reached at 2.3 hours, and plasma concentrations declined following a monophasic profile. UPARANT was still detectable at 24 hours, though values were below the lower limit of quantification. The elimination half-life (t1/2) was 2.2 hours in the elimination phase. No differences in pharmacokinetic parameters were found between control and STZ rats. In the kidney of control rats at 24 hours post-dosing, UPARANT concentration was 45.9 ± 3.6 μg/g, which was 2.2-fold higher than in the liver (21.1 ± 2.4 μg/g). In STZ rats, the liver concentration of UPARANT (20.4 ± 7.5 μg/g) did not differ from controls, while the kidney concentration was slightly lower (42.4 ± 4.5 μg/g) compared to controls. Additional pharmacokinetic parameters were evaluated using a two-phase model equation. [1]

In the STZ rat model, subcutaneous administration of UPARANT at the doses used (up to 8 mg/kg daily for 5 days) did not affect body weight, kidney weight/body weight ratio, or blood glucose levels, indicating no significant systemic toxicity at these doses. No histopathologic alterations of the liver and kidney were observed, suggesting the subcutaneous delivery route is safe for the most important organs for detoxification processes. No differences in systolic blood pressure were observed among experimental groups. [1][2]

[1]. Inhibiting the urokinase-type plasminogen activator receptor system recovers STZ-induced diabetic nephropathy. J Cell Mol Med. 2019 Feb;23(2):1034-1049.

[2]. Diabetic Retinopathy in the Spontaneously Diabetic Torii Rat: Pathogenetic Mechanisms and Preventive Efficacy of Inhibiting the Urokinase-Type Plasminogen Activator Receptor System. J Diabetes Res. 2017;2017:2904150.

[3]. UPARANT is an effective antiangiogenic agent in a mouse model of rubeosis iridis. J Mol Med (Berl) . 2019 Sep;97(9):1273-1283.

Cenupatide (UPARANT) was designed to compete with N-formyl-Met-Leu-Phe peptide for binding to FPRs. The compound was originally developed as a uPAR-derived peptide inhibitor of VEGF-driven angiogenesis. In addition to its antiangiogenic effects, UPARANT has significant anti-inflammatory activity both in vitro and in vivo. The compound has been shown to protect the retina from pathologic changes induced by diabetic retinopathy in animal models and to improve diabetic kidney lesions in STZ-induced diabetic rats. The drug was evaluated for therapeutic effects on diabetic nephropathy and for preventive effects on diabetic retinopathy in the spontaneously diabetic Torii rat model. UPARANT acts on multiple pathways involved in diabetic complication pathogenesis, including the uPAR/FPR system, αvβ3 integrin/Rac-1 pathway, and inflammatory pathways involving NF-κB, CREB, and STAT3. The drug constitutes a promising multitarget strategy for treating diabetic complications. UPARANT is also effective in reducing VEGF-independent neovascularization in the puncture-induced rubeosis iridis model, suggesting potential utility for patients who do not respond to anti-VEGF treatments. The compound was developed with funding from Kaleyde Pharmaceuticals AG, and certain authors are holders of the UPARANT patent. [1][2][3]

C28H47N11O5

617.743484735489

617.376

C, 54.44; H, 7.67; N, 24.94; O, 12.95

1006388-38-0

1006388-38-0;unknown (TFA);

134694272

Ac-Arg-{Aib}-Arg-{aMePhe}-NH2
Ac-Arg-Aib-Arg-aMePhe-NH2
N-acetyl-L-arginyl-alpha-methyl-alanyl-L-arginyl-alpha-methyl-L-phenylalaninamide

RXRX
Ac-R-{Aib}-R-{aMePhe}-NH2

White to off-white solid at room temperature

-2.3

9

7

18

44

1060

3

CC(=O)N[C@@H](CCCN=C(N)N)C(=O)NC(C)(C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@](C)(CC1=CC=CC=C1)C(=O)N

JDNFBDNDFFJJIB-JVAKCPTJSA-N

InChI=1S/C28H47N11O5/c1-17(40)36-19(12-8-14-34-25(30)31)21(41)38-27(2,3)24(44)37-20(13-9-15-35-26(32)33)22(42)39-28(4,23(29)43)16-18-10-6-5-7-11-18/h5-7,10-11,19-20H,8-9,12-16H2,1-4H3,(H2,29,43)(H,36,40)(H,37,44)(H,38,41)(H,39,42)(H4,30,31,34)(H4,32,33,35)/t19-,20-,28-/m0/s1

(S)-2-acetamido-N-(1-(((S)-1-(((S)-1-amino-2-methyl-1-oxo-3-phenylpropan-2-yl)amino)-5-guanidino-1-oxopentan-2-yl)amino)-2-methyl-1-oxopropan-2-yl)-5-guanidinopentanamide

Cenupatide; UPARANT; Cenupatide; 1006388-38-0; Cenupatide [INN]; A92EH9EGL4; UNII-A92EH9EGL4; Ac-Arg-Aib-Arg-α(Me)Phe-NH2)

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: ≥ 100 mg/mL (161.9 mM)
DMSO: ~100 mg/mL (161.9 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.)