CD88 (C5a receptor) – Potent antagonist (inhibits C5a-induced neutrophil myeloperoxidase release with IC50 of 22 nM; inhibits C5a-induced chemotaxis with IC50 of 75 nM) [2]
Mas-related gene X2 (MrgX2) – Low-affinity agonist (induces degranulation at concentrations ≥30 nM) [1]

- In human LAD2 mast cells, PMX-53 (1 μM) induced degranulation reaching approximately 60% (β-hexosaminidase release) in a dose-dependent manner; PMX-53S (scrambled linear peptide) also induced degranulation but with lower magnitude; PMX-53C (Trp-Arg replaced with Ala-dArg) did not induce degranulation even at 1 μM [1]
- In LAD2 mast cells, PMX-53 (100 nM and 1 μM) induced Ca²⁺ mobilization; PMX-53C (100 nM) was inactive and 1 μM induced only small variable response [1]
- In HMC-1 cells, PMX-53 (10 nM) almost completely inhibited C5a (10 nM)-induced Ca²⁺ mobilization but had no effect on C3a (10 nM)-induced response; PMX-53C and PMX-53S had no effect on either C5a or C3a-induced responses [1]
- In RBL-2H3 cells stably expressing CD88, PMX-53 (10 nM) inhibited C5a (1 nM)-induced degranulation [1]
- In LAD2 cells, G protein antagonist 2 (GPA-2, 1 μM, 30 min) substantially inhibited PMX-53-induced degranulation; pertussis toxin (100 ng/mL, 16 h) also inhibited PMX-53-mediated degranulation [1]
- PMX-53 (≥30 nM) induced degranulation in LAD2 mast cells, CD34⁺ cell-derived primary human mast cells, and RBL-2H3 cells stably expressing MrgX2, but did not activate RBL-2H3 cells expressing MrgX1 [1]
- In RBL-2H3 cells expressing MrgX2, PMX-53 (1 μM) caused sustained Ca²⁺ mobilization similar in magnitude and duration to that observed in LAD2 cells and CD34⁺-derived mast cells; PMX-53C had no effect [1]
- Replacement of Trp with Ala and Arg with dArg (PMX-53C) abolished the ability of PMX-53 to inhibit C5a-induced Ca²⁺ mobilization in HMC-1 cells and to cause degranulation in RBL-2H3 cells expressing MrgX2 [1]

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With IC50 values of 22 nM and 75 nM, respectively, PMX-53 is a strong CD88 antagonist that blocks C5a-induced neutrophil myeloperoxidase release and chemotaxis [1]. PMX-53 (10 nM) prevents HMC-1 cells from being excited by C5a, but at concentrations greater than 30 nM, it promotes the degranulation of CD34+ cell-derived mast cells, MrgX2-expressing RBL cells, and LAD2 mast cells. Replacing Trp with Ala and Arg and PMX-53 with dArg prevented MrgX2-expressing RBL-2H3 cells from degranulating and prevented C5a-induced Ca2+ mobilization in HMC-1 cells [1].

- In rats, local pretreatment with PMX-53 (60 or 180 μg per paw, intraplantar, 30 min before stimulus) inhibited zymosan (30 μg per paw)-induced mechanical hypernociception; effect was sustained for 6 h after zymosan injection and decreased after 24 h. Therapeutic treatment with PMX-53 (60 μg per paw, given 3 days after zymosan) significantly reduced ongoing zymosan-induced hypernociception [2]
- PMX-53 (60 μg per paw) inhibited hypernociception induced by zymosan-activated serum (1:300 dilution) and recombinant C5a (40 ng) [2]
- PMX-53 (60 μg per paw) inhibited LPS (0.5 μg per paw)- and carrageenan (100 μg per paw)-induced mechanical hypernociception [2]
- PMX-53 (60 μg per paw) pretreatment reduced antigen (ovalbumin, 25 μg per paw) challenge-induced hypernociception in previously immunized rats [2]
- PMX-53 (60 μg per paw) did not alter PGE₂ (100 ng)- or dopamine (3 μg)-induced hypernociception [2]
- PMX-53 (60 μg per paw) reduced neutrophil migration induced by zymosan, zymosan-activated serum, and C5a, but not that induced by carrageenan or LPS, as measured by myeloperoxidase activity [2]
- In mice, systemic PMX-53 (0.3, 1, or 3 mg/kg, s.c., 30 min before stimulus) reduced zymosan (30 μg intra-articular)-induced articular hypernociception in a dose-dependent manner [2]
- PMX-53 (3 mg/kg, s.c., 30 min before stimulus) reduced zymosan-induced neutrophil migration to the tibiotarsal joint (measured by MPO activity) and reduced TNF-α (100 pg intra-articular)-induced articular hypernociception [2]
- In ApoE⁻/⁻ mice (fed normal chow), chronic treatment with PMX-53 (3 mg/kg s.c. 3 times/week plus approximately 1 mg/kg/day orally in drinking water, from 5 to 30 weeks of age) reduced brachiocephalic artery lesion size (intimal area and intima-to-media ratio reduced by ~40%, P<0.05) and reduced plaque lipid content (P<0.05) [5]
- PMX-53 treatment in ApoE⁻/⁻ mice did not affect body weight or serum cholesterol levels [5]
- In ApoE⁻/⁻ mice, PMX-53 treatment resulted in a ~40% reduction in mean lipid-rich lesion area in ascending aorta by en face staining, but this did not reach statistical significance due to high variation among untreated controls [5]

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Zymosin E2, which is the direct acting mediator of hypernociceptors, is not inhibited by PMX-53 (0.3–3 mg/kg; subcutaneous injection; once; Wistar staining) treatment. On the other hand, hypernociception caused by lever, carrageenan, lipopolysaccharide (LPS), reinforcement, and dopamine is inhibited by PMX-53 (60–180 μg per paw). After the warehouse powder (3 mg/kg) was administered, pharmacokinetic study revealed that PMX-53 (3D53) started to accumulate in the bones in 5 minutes and reached its maximum blood concentration of about 0.3 μM in 20 minutes. Here, the recession phase is divided in half, lasting roughly 70 minutes[3]. With a Kd of 30 nM, a non-chained variant of PMX-53 (3D53) binds to isolated mouse neutrophils (mouse C5a binding Kd).

- Degranulation assay: LAD2 cells (5×10³ cells/well) or CD34⁺-derived mast cells were seeded into 96-well plates overnight in the presence of human IgE (1 μg/mL). Cells were washed and incubated with different concentrations of peptides for 30 min. β-hexosaminidase release was measured by incubating 20 μL of supernatant with 20 μL of 1 mM p-nitrophenyl-N-acetyl-β-D-glucosamine for 1.5 h at 37°C. Reaction was stopped with 250 μL of 0.1 M Na₂CO₃/NaHCO₃ buffer, and absorbance was measured at 405 nm [1]
- Calcium mobilization assay: Cells (0.2×10⁶ human mast cells or 10⁶ RBL-2H3 cells) were loaded with 1 μM indo-1 acetoxymethyl ester in the presence of 1 μM Pluronic acid F-127 for 30 min at room temperature. Cells were washed and resuspended in HEPES-buffered saline. Ca²⁺ mobilization was measured in a spectrophotometer with excitation wavelength of 355 nm and emission wavelength of 410 nm [1]
- RBL-2H3 cells stably expressing MrgX1 or MrgX2 were generated by nucleofection with plasmids encoding HA-tagged receptors, cultured in presence of G418 (1 mg/mL), and sorted using anti-HA antibody [1]
- RT-PCR for MrgX1 and MrgX2: Total RNA from mast cells was extracted using TRIzol, treated with DNase I, reverse transcribed to cDNA, and amplified with specific primers. Human β-actin primers were used as internal control [1]

- Rat mechanical hypernociception test: Adult male Wistar rats (180-200 g) received intraplantar injections (100 μL) of drugs. Mechanical hypernociception was tested using constant-pressure rat-paw test (20 mmHg applied to plantar surface). Latency to freezing reaction was measured before (zero time) and after stimulus administration. Hypernociception intensity was quantified as reduction in reaction time [2]
- Rat dosing for hypernociception studies: PMX-53 (60 or 180 μg per paw, i.p.l.) was given 30 min before zymosan (30 μg), LPS (0.5 μg), carrageenan (100 μg), or ovalbumin (25 μg in immunized rats). For therapeutic treatment, PMX-53 (60 μg per paw) was injected on day 3 after zymosan. For ZAS and C5a studies, PMX-53 (60 μg per paw) was given 30 min before ZAS (1:300 dilution) or C5a (40 ng) [2]
- Neutrophil depletion: Rats received vinblastine sulfate (0.8 mg/kg, i.v.) 72 h before intraplantar injection of stimuli [2]
- Mouse articular hypernociception model: Male C57BL/6 mice (20-25 g) were lightly anesthetized, and zymosan (30 μg in 5 μL) or TNF-α (100 pg in 5 μL) was injected into the right tibiotarsal joint. Mechanical hypernociception was measured using an electronic pressure meter with a modified large tip (4.15 mm²) applied to plantar surface to induce dorsal flexion of the tibiotarsal joint. PMX-53 (0.3, 1, or 3 mg/kg, s.c.) or vehicle (saline) was given 30 min before stimulus [2]
- MPO assay: Tissues were homogenized in EDTA/NaCl buffer (pH 4.7), centrifuged at 3000g for 15 min at 4°C. Pellets were resuspended in 0.5% hexadecyltrimethyl ammonium bromide buffer (pH 5.4), frozen and thawed three times, centrifuged at 3000g for 15 min at 4°C. Supernatant was used for MPO assay with 1.6 mM tetramethylbenzidine, 80 mM NaPO₄, 0.5 mM hydrogen peroxide. Reaction terminated with 4 M H₂SO₄, absorbance read at 450 nm [2]
- Cytokine measurement by ELISA: Paw skin was homogenized in buffer with protease inhibitors. Microtiter plates were coated with sheep anti-rat TNF-α or IL-1β antibodies overnight at 4°C. Standards and samples were added for 2 h at room temperature, then biotinylated polyclonal antibodies (1:500) were added for 1 h, followed by avidin-horseradish peroxidase (1:5000) for 30 min. OPD substrate was added for 15 min, reaction stopped with H₂SO₄, absorbance read at 490 nm [2]
- ApoE⁻/⁻ mouse atherosclerosis model: Homozygous female ApoE⁻/⁻ mice (on C57BL/6J background) were fed normal chow ad libitum. PMX-53 was prepared in autoclaved water (6 mg/L) for oral dosing and in sterile 5% glucose solution (3 mg/mL) for subcutaneous dosing. Mice were treated from 5 to 30 weeks of age with PMX-53 in drinking water (approximate daily oral dose of 1 mg/kg/day) plus triweekly subcutaneous injections of PMX-53 (3 mg/kg). Controls received vehicle (5% glucose in sterile water) injections and autoclaved drinking water [5]
- Histological analysis of brachiocephalic arteries: Arteries were perfusion-fixed with 4% buffered formaldehyde, embedded in cryoprotectant mounting medium, frozen, and cut into 10-μm transverse sections. Sections were stained with Miller's Elastin Van Gieson stain, Gabe's aldehyde-fuchsin stain, Picrosirius red (collagen), and Oil-red-O (lipids). Morphometric analyses were performed using ImageJ software. Areas enclosed by external elastic lamina, internal elastic lamina, and lumen were measured to derive total vessel area, medial area, and intima-to-media ratio [5]
- En face staining of ascending aorta: Aortic root and ascending aorta were opened longitudinally, stained with Oil-red-O, mounted on wax, and lesion area (stained red) was expressed as percentage of total vessel area [5]
- Immunohistochemistry on brachiocephalic arteries: Sections were blocked with 5% goat serum in PBS for 30 min, incubated with primary antibodies (anti-CD68 for macrophages, anti-von Willebrand factor for endothelial cells, anti-α-SM actin for smooth muscle cells, anti-CD88, anti-C5L2) overnight at 4°C, then with Alexa Fluor-conjugated secondary antibodies (1:500) for visualization. Negative controls had primary antibodies replaced by irrelevant IgG [5]
- Flow cytometry on aortic cells: Aortas from 25-week-old ApoE⁻/⁻ mice were enzymatically dissociated with Liberase Blendzyme TL for 30 min at 37°C. Single-cell suspensions were stained with anti-mouse CD88-Alexa Fluor 647 and anti-mouse CD31-PE or anti-mouse F4/80-PE for 30 min on ice, blocked with anti-CD16/CD32 antibody, and analyzed on a flow cytometer [5]

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Animal/Disease Models: Adult male Wistar rat (body weight 180-200 g ) injection of zymosan [2]
Doses: 0.3 mg/kg is 0.3 nM) and inhibits C5a-induced chemotaxis in mice, with an IC50 value of 0.5 nM [3]. , 1 mg/kg or 3 mg/kg.
Doses: subcutaneous injection; primary
Experimental Results:inhibits excessive nociception caused by zymosan-activated serum and C5a.

PMX-53 is noted to be orally active in a number of animal models of inflammatory disease and has exhibited safety and tolerability in phase I and IIa clinical trials [5]

- PMX-53 has exhibited safety and tolerability in phase I and IIa clinical trials [5]
- No LD50, hepatotoxicity, nephrotoxicity, drug-drug interactions, or plasma protein binding data were described for PMX-53 in these papers [1][2][5]

[1]. PMX-53 as a dual CD88 antagonist and an agonist for Mas-related gene 2 (MrgX2) in human mast cells. Mol Pharmacol. 2011 Jun;79(6):1005-13.

[2]. Role of complement C5a in mechanical inflammatory hypernociception: potential use of C5a receptor antagonists to control inflammatory pain. Br J Pharmacol. 2008 Mar;153(5):1043-53.

[3]. Synthetic small-molecule complement inhibitors. Curr Opin Investig Drugs. 2004 Nov;5(11):1164-73.

[4]. Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a. J Med Chem. 1999 Jun 3;42(11):1965-74.

[5]. Complement C5a inhibition reduces atherosclerosis in ApoE-/- mice. FASEB J. 2011 Jul;25(7):2447-55.

[6]. Complement c5a receptor facilitates cancer metastasis by altering T-cell responses in the metastatic niche. Cancer Res. 2014 Jul 1;74(13):3454-65.

- PMX-53 is a cyclic hexapeptide (Ac-Phe-[Orn-Pro-dCha-Trp-Arg]) based on the terminal amino acid sequence of C5a, modeled on the C-terminal region of native human C5a [1][2][5]
- PMX-53 is a potent, small-molecule peptidic receptor antagonist highly selective for CD88 [5]
- PMX-53 inhibits C5a-induced neutrophil myeloperoxidase release and chemotaxis with IC50 values of 22 and 75 nM, respectively [2]
- PMX-53 is effective in numerous inflammatory disease models in mice and rats including rheumatoid arthritis, inflammatory bowel disease, ischemia-reperfusion injuries, and neurodegeneration [2][5]
- PMX-53 has been shown to be effective in reducing C5a-mediated inflammatory response and the development of pathology in several inflammatory diseases [5]
- PMX-53 is currently undergoing clinical trials for treatment of osteoarthritis [1]
- PMX-53 functions as a dual-action molecule: high-affinity antagonist of CD88 but low-affinity agonist of MrgX2. At low concentrations, PMX-53 blocks inflammation by acting as a CD88 antagonist; at higher concentrations (≥30 nM), it can promote innate immunity by mimicking actions of defensins on mast cell activation via MrgX2 [1]
- PMX-53 requires Trp and Arg residues for its activity as both a CD88 antagonist and a MrgX2 agonist [1]
- Murine mast cells, which do not express MrgX2, are unresponsive to activation by PMX-53, indicating specificity for human MrgX2 [1]

C47H65N11O7

896.088510274887

895.506

C, 63.00; H, 7.31; N, 17.19; O, 12.50

219639-75-5

852629-88-0

6918468

Ac-Phe-{Orn}-Pro-{dCha}-Trp-Arg (Lactam bridge: Orn2-Arg6)

Ac-F-{Orn}-P-{dCha}-WR (Lactam bridge: Orn2-Arg6)
Ac-Phe-Orn(1)-Pro-D-Cha-Trp-Arg-(1)

White to off-white solid powder

1.4±0.1 g/cm3

1.684

0.81

9

8

13

65

1680

6

CC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@H]2CCCNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](NC(=O)[C@@H]3CCCN3C2=O)CC4CCCCC4)CC5=CNC6=CC=CC=C65)CCCN=C(N)N

YOKBGCTZYPOSQM-HPSWDUTRSA-N

InChI=1S/C47H65N11O7/c1-29(59)53-37(25-30-13-4-2-5-14-30)42(61)55-36-20-11-22-50-41(60)35(19-10-23-51-47(48)49)54-44(63)39(27-32-28-52-34-18-9-8-17-33(32)34)56-43(62)38(26-31-15-6-3-7-16-31)57-45(64)40-21-12-24-58(40)46(36)65/h2,4-5,8-9,13-14,17-18,28,31,35-40,52H,3,6-7,10-12,15-16,19-27H2,1H3,(H,50,60)(H,53,59)(H,54,63)(H,55,61)(H,56,62)(H,57,64)(H4,48,49,51)/t35-,36-,37-,38+,39-,40-/m0/s1

(S)-N-((3R,6S,9S,15S,20aS)-6-((1H-indol-3-yl)methyl)-3-(cyclohexylmethyl)-9-(3-guanidinopropyl)-1,4,7,10,16-pentaoxoicosahydropyrrolo[1,2-a][1,4,7,10,13]pentaazacyclooctadecin-15-yl)-2-acetamido-3-phenylpropanamide

PMX 53; PMX 53; PMX53; FBK4LNR4Q7; Ac-Phe-cyclo(Orn-Pro-D-Cha-Trp-Arg); AcPhe(ornithine-Pro-cyclohexylamine-Trp-Arg; AcPhe(ornithine-Pro-cyclohexylamine-Trp-Arg)

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~200 mg/mL (~223.19 mM)
H2O : ~2.5 mg/mL (~2.79 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.32 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.08 mg/mL (2.32 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 20.8 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: ≥ 2.08 mg/mL (2.32 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 20.8 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.)