Cell experiment:

The mouse mammary tumor cell lines 4T1 are plated at a density 5.0×104 cells/ml/well of 96-multiwell plates in complete medium. After 24 h of incubation, the medium is changed with serum starve medium to synchronize the cell cycle and growth as to ensure that the cells reaches its plateau phase. After 24 h of serum-starving, cells are treated with 5 μL/well of 0.1 M of agonist, EP54, antagonist, PMX 205 and the positive control is treated with Tamoxifen drug. 5 μL/well of the 12 mM ck solution is added 5 h before reading is taken and about 50 μL/well of the SDS-HCL solution is added and mixed thoroughly using pipette 3 h before reading is taken. The MTT assay is assessed at different time point; 0, 24, 48 and 72 h at wavelength 570 nm respectively by using ELISA plate reader Infinite M200[1].

Animal experiment:

Rats[2]Transgenic SOD1G93A rats expressing human mutant G93A SOD1 (NTac:SD-Tg SOD1G93A L26H) are used. Three experimental groups are chosen: untreated, PMX 205 treats from day 28 onward, and PMX 205 treats from day 70 onward. Animals are administered PMX 205 via drinking water (1 mg/kg/day) , from day 28 or day 70 onward; controls receive water only[2].Mice[3]Tg2576 mice are treated (starting at or after the initiation of plaque pathology) for 2-3 mo with PMX 205 given in the drinking water only (10-20 μg/mL, equivalent to ~3-6 mg/kg/day) or both in drinking water (10-20 μg/mL) and s.c. (1 mg/kg) twice weekly throughout the treatment period. Untreated transgenic animals of same age are used as controls. Nontransgenic littermates are similarly treated or not treated with the drug. 3×Tg mice are also treated with PMX 205 in the drinking water. Due to the low pathology of the males, only female mice of this strain are used for these studies[3].

PMX205 is a cyclic hexapeptide (c[Arg-Trp-D-Cha-Pro-Orn]-Hca), which is an antagonist of the C5a complement receptor. [1]

Human C5a, a complement-derived anaphylatoxin, is a potent mediator of human leukocyte chemotaxis. C5a in particular is a potent inflammatory mediator and signalling through CD88 mediates chemotaxis of leukocytes, vascular permeability could be increased, mast cells release inflammatory mediator and smooth muscle cell contracts and other activities, but all the activities contribute to inflammation and potent tissue damage. C5a-mediated leukocyte chemotaxis release from cytochalasin B-treated cells closely paralleled uptake of the ligand, clearly indicating that it is a receptor-C5a interaction that leads to stimulation of these cellular responses. [2]

PMX205 have been shown to be effective in causing a significant reduction in fibrillar amyloid deposits and activated glia in two mouse models of Alzheimer’s disease. PMX205 -treated rats exhibited reduced striatal lesion size, apoptosis, neutrophil infiltration, and hemorrhage.[3]

PMX205 is used as anti-inflammatory drug. PMX205 significantly prevented DSS-induced colon inflammation, and dealed with PMX205, subjected rats had lower pro-inflammatory. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 did not affect C5a’s levels. The positive effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation. Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis.[3]

References:
[1] Delisle Milton RC, Milton SC, Chamberlin AR.  Improving the Fmoc Solid Phase Synthesis of the Cyclic Hexapeptide Complement C5a Antagonist, PMX205. Int J Pept Res Ther. 2011 Dec;17(4):337-342.
[2] Chenoweth DE, Hugli TE.  Demonstration of specific C5a receptor on intact human polymorphonuclear leukocytes. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3943-7.
[3] Jain U, Woodruff TM, Stadnyk AW.  The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10. Br J Pharmacol. 2013 Jan;168(2):488-501.