CCR1

The interaction between osteoclasts (OCs) and multiple myeloma (MM) cells plays a key role in the pathogenesis of MM-related osteolytic bone disease (OBD). MM cells promote OC formation and, in turn, OCs enhance MM cell proliferation. Chemokines are mediators of MM effects on bone and vice versa; in particular, CCL3 enhances OC formation and promotes MM cell migration and survival. Here, we characterize the effects of MLN3897, a novel specific antagonist of the chemokine receptor CCR1, on both OC formation and OC-MM cell interactions. MLN3897 demonstrates significant impairment of OC formation (by 40%) and function (by 70%), associated with decreased precursor cell multinucleation and down-regulation of c-fos signaling. OCs secrete high levels of CCL3, which triggers MM cell migration; conversely, MLN3897 abrogates its effects by inhibiting Akt signaling. Moreover, MM cell-to-OC adhesion was abrogated by MLN3897, thereby inhibiting MM cell survival and proliferation. Our results therefore show novel biologic sequelae of CCL3 and its inhibition in both osteoclastogenesis and MM cell growth, providing the preclinical rationale for clinical trials of MLN3897 to treat OBD in MM [2].

MLN3897 was well tolerated, with no evidence of systemic immunosuppression. In the intent-to-treat population, there was no significant difference in day 84 ACR20 response rates between MLN3897-treated patients and placebo-treated patients (35% versus 33%, respectively; P=0.72). Results were similar for the per-protocol population. Pharmacokinetic analyses demonstrated no interactions between MLN3897 and MTX. MLN3897 was associated with a high degree of CCR1 occupancy (>or=90% on days 28, 56, and 84 in 82% of patients, by macrophage inflammatory protein 1alpha internalization assay). Conclusion: MLN3897 at a concentration of 10 mg once daily had no discernible activity in patients with RA who were also receiving MTX. The results suggest that CCR1 antagonism is unlikely to be a viable strategy for the treatment of RA when used in isolation at the receptor occupancy levels reached in this study[4].

OC formation and TRAP assay. [2]
OCs were generated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers by Ficoll-Paque gradient separation and cultured in 6-well or 96-well plates (0.5 × 106 cells/cm2). After 2 hours, nonadherent PBMCs were removed, and adherent cells were cultured for 21 days in α-MEM containing 10% FBS and 1% penicillin-streptomycin, as well as 50 ng/mL of macrophage colony-stimulating factor and RANKL. MLN3897 was added at concentrations and time points as indicated; culture media was replaced twice weekly. After 3 weeks, cells were fixed with citrate-acetone solution and stained for tartrate-resistant acid phosphatase (TRAP) using an acid phosphatase leukocyte staining kit according to the manufacturer's instructions. TRAP+ OCs containing 3 or more nuclei per cell were enumerated. Each OC formation assay was performed at least 3 times using PBMCs from different donors.

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Pit formation assay. [2]
OC activity was assayed by bone resorption enumerating resorption pits. Briefly, PBMCs were cultured (0.5 × 106 cells/well) on dentin slices in 96-well plates as per the manufacturer's guidelines, and then stimulated with RANKL and M-CSF (50 ng/mL); MLN3897 was added as indicated. After 3 weeks, adherent cells were scraped off gently with 0.1% Triton. Bone slices were washed in distilled water and stained with 1% toluidine solution. Resorption pits were then quantified by light microscopy using the public domain National Institutes of Health (NIH) Image J software version 1.36b.15 Each pit area assay was performed at least 3 times with PBMCs from different donors.

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Coculture experiments. [2]
OCs were harvested with cell dissociation buffer and seeded in 96-well or 24-well plates (approximately 1.5-3 × 104 cells/cm2). After washing, MM cells were added to the wells and incubated with media or with MLN3897 (10 nM) for the specified times at 37°C. For MM cell proliferation, we measured DNA synthesis by tritiated thymidine uptake (3H-TdR), pulsing MM cells with 3H-TdR (0.5 μCi/well [0.0185 MBq]L) during the last 8 hours of 48-hour cultures. At the end of the culture, cells were harvested onto paper filters with an automatic cell harvester and counted using the LKB Betaplate scintillation counter. To assess cell survival, viable MM cells were counted by trypan blue staining.

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Cell viability and cell fusion assays of OC precursors [2]
We determined the effects of MLN3897 on viability and fusion of PBMCs stimulated with RANKL and M-CSF. At different time points, cell number was quantified using the AlamarBlue assay, pulsing the cells with AlamarBlue (10 μL) and incubating for 4 hours at 37°C. Absorbance was read at a wavelength of 570 nm (with correction at 600 nm) on a spectrophotometer. To analyze cell fusion, cells were fixed in 3.7% formaldehyde for 5 minutes and then stained with hematoxylin and eosin for 5 and 10 minutes, respectively. Cell fusion, evidenced by cells with 3 or more nuclei, was quantified using light microscopy. Each fusion assay was performed at least 3 times with PBMCs from different donors.

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Transwell migration assay [2]
Cell migration was assayed using a Boyden-modified chamber assay. Cells were added on an 8-μm pore size polycarbonate membrane separating the 2 chambers of a 6.5-mm Transwell. CCL3 (5 ng/mL) or 48-hour OC supernatants were added in the lower chamber. After 5 hours, cells migrating to the lower compartment were counted using a Coulter Counter ZBII. In specific experiments, cells were preincubated with MLN3897 (10 nM, 4 hours) and specific PI3K inhibitor LY294002 (25 μM, 1 hour).

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Western blotting [2]
Overnight serum-starved cells were stimulated with CCL3 (100 ng/mL for 30 minutes) with or without pretreatment of MLN3897 (10 nM), harvested, and lysed in lysis buffer (50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid [pH 7.4], 150 mM NaCl, 1% NP-40, 30 mM sodium pyrophosphate, 5 mM ethylenediamine tetraacetic acid, 2 mM Na3VO4, 5 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL aprotinin). For OC signaling experiments, adherent PBMCs were seeded in 6-well plates (0.2 × 106 cells/cm2), harvested with cell dissociation buffer after 7 days of stimulation with RANKL and M-CSF with or without MLN3897, and lysed in lysis buffer. Samples were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to PVDF membrane, and immunoblotted with antibodies against pAkt, Akt, and c-fos, phosphorylated extracellular signal–regulated kinase (pERK), ERK, or cathepsin K. Antigen-antibody complexes were detected by enhanced chemiluminescence. The membrane was stripped and reprobed with antitubulin antibody to ensure equal protein loading. Films were scanned and densitometric analysis performed using the public domain NIH Image J program.15

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Adhesion assay [2]
To perform adhesion assays, MM cell lines were labeled with calcein AM according to the manufacturer's instructions and plated in a 96-well plates with OCs, fibronectin (20 μg/mL), or media, with or without MLN3897. After 6 hours of incubation, plates were washed and fluorescence of the adherent cells was measured using the Multimode Reader Mithras LB 940.

Objective: To assess the efficacy, safety, pharmacokinetics, and pharmacodynamics of the CC chemokine receptor CCR1 antagonist MLN3897 in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX). Methods: In this phase IIa, proof-of-concept study, patients meeting the American College of Rheumatology (ACR) criteria for RA who had been taking MTX for >or=6 months with evidence of active disease were randomly assigned to receive either 10 mg oral MLN3897 or matching placebo once daily for 12 weeks (days 1-83) while continuing to receive MTX once a week. Clinical assessments, safety monitoring, and sampling for pharmacokinetic and pharmacodynamic analyses were performed throughout the study. The primary efficacy end point was the difference in the percentage of patients meeting the ACR 20% improvement criteria (achieving an ACR20 response) on day 84 in the MLN3897-treated group compared with that in the placebo-treated group.[4]

For both radiolabeled moieties, 55-59% of the dose was recovered in feces and 32% recovered in urine. MLN3897 was metabolized extensively in humans, with minor amounts of intact MLN3897 detected in the urine and feces. N-oxidation of the tricyclic moiety (M28) and N-dealkylation of the piperidinyl moiety were the primary metabolic pathways leading to further formation of the carboxylic acid metabolite (M19) and the (4-(4-chlorophenyl)-3,3- dimethylpiperidin-4-ol) metabolite (M40). Oxidative metabolites M11, M19, M28, M44 were present at >10% of the total circulating radioactivity and also at >25% of MLN3897 exposure. Metabolites resulting from the chlorophenyl-labeled moiety (M40) had significantly more systemic exposure compared to the tricyclic-labeled moiety (M19).[3]

[1]. Ccr1 antagonists for the treatment of i.a. demyelinating inflammatory disease. WO2004043965A1.

[2]. MLN3897, a novel CCR1 inhibitor, impairs osteoclastogenesis and inhibits the interaction of multiple myeloma cells and osteoclasts. Blood. 2007 Nov 15;110(10):3744-52.

[3]. Metabolism, Excretion and Pharmacokinetics of MLN3897, a CCR1 Antagonist, in Humans. Drug Metab Lett. 2016;10(1):22-37.

[4]. MLN3897 plus methotrexate in patients with rheumatoid arthritis: safety, efficacy, pharmacokinetics, and pharmacodynamics of an oral CCR1 antagonist in a phase IIa, double-blind, placebo-controlled, randomized, proof-of-concept study. Arthritis Rheum. 2009 Dec;60(12):3572-81.

Our data therefore demonstrate that CCR1 inhibition by MLN3897 may be an effective tool to block and possibly even prevent OBD in MM. MLN3897 blocks OC development and function by inhibiting differentiation of OC precursors, in particular by down-regulating cell fusion and c-fos expression. Moreover, MLN3897 abrogates MM cell migration and adhesion to OCs, thus preventing the homing of MM cells to OC sites. Finally, MLN3897 overcomes the protective effect of OCs on MM cell survival and proliferation, thereby inhibiting the interactive loop between OCs and MM cells. Ongoing studies are defining the in vivo effects of MLN3897 in a severe combined immunodeficiency–human (SCID-hu) mouse model of MM-related osteolysis. These data therefore delineate a novel mechanism of action of MLN3897 on osteoclastogenesis and OC–MM cell interactions, and provide the preclinical rationale for its clinical evaluation to treat OBD in MM. [2]

C32H39CLN2O3

535.116667985916

534.264

C, 71.82; H, 7.35; Cl, 6.62; N, 5.24; O, 8.97

1010731-97-1

849105-26-6;

9914608

Typically exists as solid at room temperature

2.5

2

6

5

37

841

1

CC1(CN(CC[C@@]1(C2=CC=C(C=C2)Cl)O)CC/C=C/3\C4=C(COC5=C3C=C(C=C5)C(=O)O)N=CC=C4)C

NVBMQDQKIKYLDW-WXTRKPKDSA-N

InChI=1S/C30H31ClN2O4/c1-29(2)19-33(16-13-30(29,36)21-8-10-22(31)11-9-21)15-4-6-23-24-5-3-14-32-26(24)18-37-27-12-7-20(28(34)35)17-25(23)27/h3,5-12,14,17,36H,4,13,15-16,18-19H2,1-2H3,(H,34,35)/b23-6+/t30-/m0/s1

(S,E)-4-(4-chlorophenyl)-1-(3-(7-(2-hydroxypropan-2-yl)-2,11-dihydrobenzo[6,7]oxepino[3,4-b]pyridin-5(1H)-ylidene)propyl)-3,3-dimethylpiperidin-4-ol

AVE 9897; AVE-9897; AVE9897; MLN-3897; 1010731-97-1; CCR1 antagonist 10; (S)-5-(3-(4-(4-Chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)propylidene)-5,11-dihydrobenzo[6,7]oxepino[3,4-b]pyridine-7-carboxylic acid; (11E)-11-[3-[(4S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl]propylidene]-5H-[1]benzoxepino[3,4-b]pyridine-9-carboxylic acid; SCHEMBL2197331; MLN3897; MLN 3897

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)