MIP-1α-CCR5 (IC50 = 3.3 nM); RANTES-CCR5 (IC50 = 5.2 nM); MIP-1β-CCR5 (IC50 = 7.2 nM); HIV-1 (Ba-L) (IC50 = 1.1 nM (IC50 in PM-1 cells)

A powerful selective CCR5 antagonist, maraviroc (UK-427857) effectively inhibits the human immunodeficiency virus type 1 (HIV-1). With IC50s ranging from 7 to 30 nM for MIP-1β, MIP-1α, and RANTES, maraviroc suppresses events that follow chemokine-induced intracellular calcium redistribution. In a 5-day antiviral assay employing isolated multiple (Pooled), Maraviroc (UK-427857) is active (IC90) against HIV-1 Ba-L, a laboratory-engineered R5 strain, at low nanomolar doses [1]. The assay assessed IC90 on donor PBMC (3.1 nM), IC90 on single-donor PBMC (1.8 nM), or IC90 on PM-1 cells (1.1 nM).

Following intravenous injection, clearance values were moderate to high in dogs and rats (74 and 21 mL/min/kg, respectively). In both species, maraviroc likewise exhibits a moderate volume of distribution (4.3 to 6.5 L/kg). Maraviroc has a half-life of 0.9 hours in rats and 2.3 hours in dogs. Dogs received 2 mg/kg orally, and 1.5 hours later, Cmax (256 ng/mL) was reached. 40% of the substance is bioavailable after administration. Roughly thirty percent of the oral dose is absorbed from the gut in rats, according to research on concentrations found in the portal vein following oral administration [1]. Maraviroc specifically decreases the recruitment of leukocytes harboring CCR5 in DSS/TNBS colitis and metastatic models, thereby mitigating the development of intestinal inflammation[2].

Inhibition of chemokine binding to CCR5. [1]
Binding of 125I-labeled MIP-1α, MIP-1β, and RANTES to CCR5 was measured essentially as described previously using intact HEK-293 cells stably expressing the receptor or membrane preparations thereof. Briefly, cells were resuspended in binding buffer (50 mM HEPES containing 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin [BSA] and adjusted to pH 7.4) to a density of 2 × 106 cells/ml. For membrane preparations, phosphate-buffered saline (PBS)-washed cells were resuspended in lysis buffer (20 mM HEPES, 1 mM CaCl2, 1 tablet COMPLETE per 50 ml, pH 7.4) prior to homogenization in a Polytron hand-held homogenizer, ultracentrifugation (40,000 × g for 30 min), and resuspension in binding buffer to a protein concentration of 0.25 mg/ml (12.5 μg of membrane protein was used in each well of a 96-well plate). 125I-radiolabeled MIP-1α, MIP-1β, and RANTES were prepared and diluted in binding buffer to a final concentration of 400 pM in the assay. Appropriatemaraviroc dilutions were added to each well to a final volume of 100 μl, the assay plates incubated for 1 h, and the contents filtered through preblocked and washed Unifilter plates which were counted following overnight drying.

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Inhibition of soluble recombinant HIV-1 gp120 (Ba-L strain) binding to CCR5. [1]
This assay was performed as described previously. Briefly, HEK-293 cell aliquots (100 μl at 1 × 106 cells/ml) were plated into poly-d-lysine-coated plates and incubated at 37°C overnight. A 1:1 mix of soluble recombinant human CD4 (sCD4) (diluted to 4.5 nM in culture medium) and HIV-1 gp120 was incubated at room temperature for 15 min prior to its addition to PBS-washed cells in the presence of dilutions of maraviroc to enable IC50 determination. The assay plates were incubated at 37°C for 1 h and washed. Eu3+-labeled anti-gp120 antibody (1/500 dilution in assay buffer) was added to each well (50 μl) and incubated for 1 h. The plate was washed three times with wash buffer prior to the addition of enhancement solution (200 μl/well) and measurement of Eu3+ fluorescence (Victor2 multilabel counter; “Europium” protocol). Nonspecific binding was taken as the fluorescence measured for gp120 incubated with cells in the absence of preincubation with sCD4.

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Inhibition of CCR5 signaling: calcium flux. [1]
Maraviroc-dependent inhibition of CCR5-mediated signaling was investigated by measuring chemokine-dependent intracellular calcium redistribution (flux) by fluorescence assay using a calcium-sensitive dye, essentially as previously reported. Briefly, CCR5-stable transfected HEK-293 cells were washed in PBS and then incubated at 37°C for 1 h in cell culture medium containing fluo-3 dye (5 μg/ml; Molecular Probes). The dye-loaded cells were washed in PBS and resuspended in flux buffer (10 mM HEPES buffer, pH 7.4, containing 1.6 mM CaCl2 and 1 bottle of Hanks' balanced salts powder) to 5 × 105 cells/ml for the assay. The cell suspension (160 μl) was divided into aliquots, placed in each well of a black-walled clear-base 96-well plate, and centrifuged (400 × g) for 5 min. Dilutions of maraviroc and solutions of chemokines were divided into aliquots and placed in separate 96-well plates to enable their sequential addition to the HEK-293 cells and subsequent measurement of intracellular calcium redistribution effects in a fluorescent laser imaging plate reader (FLIPR). The FLIPR added maraviroc dilutions (20 μl) after 30 s, followed 4 min later by the addition of the RANTES chemokine (20 μl) to a final concentration of 20 nM in situ. Fluorescence (488-nm and 530-nm excitation and emission wavelengths, respectively) was measured over 8 min to investigate the direct effects of maraviroc on cell signaling and inhibitory effects of maraviroc on chemokine-mediated signaling.

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Inhibition of CCR5 internalization. [1]
The effects of maraviroc on CCR5 internalization were measured by flow cytometry, using a FACSCalibur instrument. Aliquots of 300.19 cells (100 μl at 5 × 106/ml) were incubated for 45 min at 37°C with maraviroc, RANTES, or SDF-1α (all 100 nM in situ) to enable CCR5 internalization. The samples were washed twice, resuspended in 0.5% BSA-PBS (40 μl), and incubated for 45 min at 4°C with 10 μl 2D7 (anti-human CCR5 mouse monoclonal antibody) or isotype control antibody (mouse IgG2a). The samples were washed (0.5% BSA-PBS) and incubated with phycoerythrin-goat antimouse secondary antibody (75 μl) at 4°C for 45 min. The samples were washed and fixed with 1% (vol/vol) formaldehyde-PBS (1000 μl), and expression of CCR5 was evaluated using excitation/emission wavelengths of 488 nm and 530 nm, respectively.

Drug susceptibility assays were performed in 24-well tissue culture plates. Duplicate eight-point dilution series of maraviroc were prepared in DMSO and medium to yield a final DMSO concentration of 0.1% (vol/vol) in the assay. PHA-stimulated PBMC or PM-1 cells were infected with virus for 1 h at 37°C. Cells were subsequently washed once, and 3.6 × 105 PBMC or 2.0 × 105 PM-1 cells were added to each well of assay plates containing diluted compound. Plates were incubated for 5 days (lab-adapted strains) or 7 days (primary isolates) at 37°C in a humidified 5% CO2 (vol/vol) atmosphere. Control compounds, saquinavir (an HIV-1 protease inhibitor) and RANTES, were included in all assays.

Pharmacokinetic studies with rats and dogs. [1]
Preclinical pharmacokinetic studies were carried out with maravirocfollowing a single intravenous and oral administration to both male Sprague-Dawley rats (1 mg/kg of body weight given intravenously [i.v.] and 10 mg/kg given orally [p.o.]; n = 2) and male beagle dogs (0.5 mg/kg i.v. and 2 mg/kg p.o; n = 4). Plasma samples were taken for up to 24 h postdose, and the concentrations of unchanged maraviroc were determined using a specific high-performance liquid chromatography-tandem mass spectrum assay.

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In this study, researchers aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5(-/-) mice or adoptive transfer of splenic naïve CD4(+) T-cells from wild type or CCR5(-/-) mice into RAG-1(-/-). CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4(+) and CD11b(+) leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maravirocattenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs.

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Dissolved in phosphate-buffered saline, sterile-filtered and adjusted to a final concentration of 4 mg/mL (7.8 mM). A 3.4% gel preparation of hydroxyl-ethyl cellulose (HEC) is added to achieve a final concentration of 5 mM maraviroc in 2.2% HEC gel; ~64 μg; A 25 μL volume of the gel formulation is carefully applied in to the vaginal vault of mice.

Humanized BALB/c-Rag2 / γc / and BALB/c-Rag1 / γc / (RAG-hu) mice

[1]. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother. 2005 Nov;49(11):472.

[2]. Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis. Sci Rep. 2016 Aug 5;6:30802.

[3]. Effect of maraviroc on HIV-disease progression-related biomarkers. Antimicrob Agents Chemother. 2012 Nov;56(11):5858-64.

[4]. NRSF and CCR5 Established Neuron-glia Communication during Acute and Chronic Stresses. Journal of Drug Metabolism & Toxicology. January 10, 2016.

Maraviroc is a monocarboxylic acid amide obtained by formal condensation of the carboxy group of 4,4-difluorocyclohexanecarboxylic acid and the primary amino group of (1S)-3-[(3-exo)-3-(3-isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropylamine. An antiretroviral drug, it prevents the interaction of HIV-1 gp120 and chemokine receptor 5 (CCR5) necessary for CCR5-tropic HIV-1 to enter cells. It has a role as an antiviral drug, a chemokine receptor 5 antagonist and a HIV fusion inhibitor. It is an azabicycloalkane, an organofluorine compound, a member of triazoles and a monocarboxylic acid amide.
Maraviroc is a CCR5 Co-receptor Antagonist. The mechanism of action of maraviroc is as a Chemokine Co-receptor 5 Antagonist.
A cyclohexane and triazole derivative that acts as an antagonist of the CCR5 RECEPTOR. It prevents infection by HIV-1 virus strains which use CCR5 as a co-receptor for membrane fusion and cellular entry.
See also: Maraviroc (annotation moved to).

C29H41F2N5O

513.67

513.327

C, 67.81; H, 8.05; F, 7.40; N, 13.63; O, 3.11

376348-65-1

Maraviroc-d6;1033699-22-7

3002977

White to off-white solid powder

1.3±0.1 g/cm3

79-81ºC

1.628

3.6

1

6

8

37

751

3

CC1=NN=C(N1C2C[C@H]3CC[C@@H](C2)N3CC[C@@H](C4=CC=CC=C4)NC(=O)C5CCC(CC5)(F)F)C(C)C

GSNHKUDZZFZSJB-HLMSNRGBSA-N

InChI=1S/C29H41F2N5O/c1-19(2)27-34-33-20(3)36(27)25-17-23-9-10-24(18-25)35(23)16-13-26(21-7-5-4-6-8-21)32-28(37)22-11-14-29(30,31)15-12-22/h4-8,19,22-26H,9-18H2,1-3H3,(H,32,37)/t23-,24+,25?,26-/m0/s1

4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.87 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 25.0 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: ≥ 2.5 mg/mL (4.87 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 25.0 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: ≥ 2.5 mg/mL (4.87 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.87 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 2.5 mg/mL (4.87 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 6: ≥ 0.5 mg/mL (0.97 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 7: 2% DMSO +Corn oil : 10 mg/mL

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