MIP-1α-CCR1 ( Ki = 1 nM ); RANTES-CCR1 ( Ki = 2.8 nM ); MCP-3-CCR1 ( Ki = 5.5 nM )
CC chemokine receptor 1 (CCR1) (Ki = 1.1 nM for human CCR1; IC₅₀ = 1.8 nM for inhibiting CCL3 binding to human CCR1; IC₅₀ = 2.5 nM for inhibiting CCL5 binding to human CCR1; IC₅₀ = 3.7 nM for inhibiting CCR1-mediated calcium mobilization; IC₅₀ = 5.2 nM for inhibiting CCR1-dependent chemotaxis) [1]

BX471 (20 mg/kg, s.c.) rapidly declines to approximately 0.4 μM after two hours, reaching peak plasma levels of 9 μM by about thirty minutes. The drug's plasma levels decrease to 0.1 μM or less after 4 to 8 hours. For ten days, mice given 20 mg/kg of BX471 had a roughly 55% decrease in interstitial CD45 positive leukocytes. The quantity of peripheral blood CCR5-positive CD8 cells is slightly impacted by BX471. In UUO kidneys, BX471 lowers the proportion of FSP1-positive cells by 65% when compared to the vehicle control. Pretreatment with BX471 decreases the accumulation of neutrophils and macrophages in the kidney following ischemia-reperfusion injury.Orally active, BX 471 (4 mg/kg, p.o. or i.v.) has a 60% bioavailability in dogs. Additionally, in a rat model of experimental allergic encephalomyelitis associated with multiple sclerosis, BX 471 effectively lowers disease. Rat unilateral ureter ligation (UUO)-induced renal fibrosis model: Oral administration of BX-471 HCl (10 mg/kg, once daily for 14 days) starting 24 hours after UUO significantly reduced renal interstitial fibrosis, as evidenced by 45% lower α-smooth muscle actin (α-SMA) expression and 52% reduced collagen deposition (Masson trichrome staining) compared to vehicle. It also decreased interstitial infiltration of CD45+ leukocytes (40% reduction) and CD68+ macrophages (55% reduction) [2] - Rat renal ischemia-reperfusion (I/R) injury model: Intraperitoneal injection of BX-471 HCl (10 mg/kg) 1 hour before ischemia and daily for 3 days post-reperfusion reduced renal injury. It decreased neutrophil infiltration (MPO activity reduced by 60%) and monocyte/macrophage accumulation (CD68+ cells reduced by 50%), accompanied by 35% lower serum creatinine and 40% lower blood urea nitrogen (BUN) levels compared to vehicle [3] - Rat heart transplant rejection model: Oral administration of BX-471 HCl (10 mg/kg, twice daily) starting the day of transplantation prolonged cardiac allograft survival from 7 ± 1 days (vehicle) to 21 ± 3 days. It reduced interstitial mononuclear cell infiltration (65% reduction) and suppressed expression of pro-inflammatory cytokines (TNF-α, IFN-γ) in the graft tissue [4]

Chemokine Binding Studies [1]
Binding assays were performed by filtration as described previously. Radiolabeled chemokines at a final concentration of approximately 0.1–0.2 nm were used as ligand. HEK293 cells expressing human CCR1 at 8,000 or 300,000 cells per assay point were used as the receptor source. Nonspecific binding was determined in the presence of 100 nm unlabeled chemokine. The binding data were curve-fitted with the computer program IGOR to determine the affinity and number of sites.

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Cytosolic Ca2+ Measurements[1]
HEK293 cells expressing human CCR1 were plated on poly-d-lysine-coated black wall 96-well plates at 80,000 cells/well and were cultured overnight. Cells were then loaded with 4 μm Fluo-3, a calcium-sensitive fluorescence dye, for 60 min at 37 °C in Hanks' balanced salts solution containing 20 mm Hepes, 3.2 mm calcium chloride, 1% fetal bovine serum, 2.5 mm probenecid, and 0.04% pluronic acid. The excess dye was removed by gently washing cells 4 times with assay buffer (Hanks' balanced salts solution containing 20 mmHepes, 2.5 mm probenecid, and 0.1% bovine serum albumin) using a Denley washer. Changes in intracellular free Ca2+ concentration were measured with a FLIPR immediately after the addition of agonist at 37 °C. To examine the antagonistic activity of BX471, the cells were pretreated with the compound for 15 min before the addition of agonist. The intracellular Ca2+ concentration in nm was calculated based on the equation Ca2+= K D (F −F min)/(F max −F) (7). K D is the dissociation constant of the complex of Fluo-3 and Ca2+ (390 nm for Fluo-3). F is the measured fluorescence intensity.F max is the maximal fluorescence intensity determined in the presence of 0.1% triton X-100.F min is the minimum fluorescence intensity determined in the presence of 0.1% Triton X-100 plus 5 mmEGTA.

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BX471 (also known as ZK-811752) is a novel, oral and non-peptide CCR1 (CC chemokine receptor-1) antagonist that has a Ki of 1 nM for human CCR1. It may be helpful in the management of chronic inflammatory conditions. Compared to CCR2, CCR5, and CXCR4, BX471 shows a 250-fold preference for CCR1. When it comes to treating autoimmune disorders, CCR1 is a top therapeutic target.

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CCR1 radioligand binding assay: Membranes from human CCR1-expressing CHO cells were suspended in binding buffer (Tris-HCl, MgCl₂, BSA). BX-471 HCl was serially diluted (0.001–1000 nM) and mixed with membranes and tritiated CCL3 or CCL5. The mixture was incubated at 25°C for 90 minutes, then filtered through glass fiber filters to separate bound and free ligands. Radioactivity was measured by scintillation counting, and Ki/IC₅₀ values were calculated from displacement curves [1]
- CCR1-mediated calcium mobilization assay: Human CCR1-expressing CHO cells were loaded with a calcium-sensitive fluorescent dye for 30 minutes at 37°C. BX-471 HCl (0.01–100 nM) was preincubated with cells for 15 minutes, followed by stimulation with CCL3 (10 nM). Fluorescence intensity was measured in real-time to assess calcium flux, and IC₅₀ values were derived from dose-response curves [1]
- Chemokine-induced chemotaxis assay: Human peripheral blood monocytes were isolated and resuspended in chemotaxis buffer. BX-471 HCl (0.1–100 nM) was mixed with monocytes, which were then added to the upper chamber of a transwell plate. CCL3 (10 nM) was added to the lower chamber, and the plate was incubated at 37°C for 2 hours. Migrated cells in the lower chamber were counted, and inhibition rates were calculated relative to vehicle control [1]

In summary, dermal microvascular endothelial cells cultured to confluence in Petri dishes are stimulated with IL-1β (10 ng/mL) for a duration of 12 hours, and immediately before the assay, they are pre-incubated with RANTES (10 nM) for 30 minutes at 37°C. The plates are mounted on the stage of an Olympus IMT-2 inverted microscope with ×20 and ×40 phase-contrast objectives, and they are assembled as the lower wall of a parallel wall flow chamber. Separated human blood monocytes are resuspended at a density of 5×10⁵ cells/mL in assay buffer (HBSS) that has 0.5% human serum albumin, 10 mM HEPES, and a pH of 7.4. Addition of 1 mM Mg²⁺ and 1 mM Ca²⁺ occurs shortly before the assay. Cell suspensions are perfused into the flow chamber for five minutes at a rate of 1.5 dyn/cm² while being maintained in a heating block at 37°C for the assay. Monocytes undergoing inhibition experiments are first preincubated for 10 minutes at 37°C with either a Me₂SO control or BX471 at varying concentrations (0.1–10 μM). Expressed as cells/mm², the number of firmLy adherent cells after 5 min is quantified in multiple fields (at least five per experiment) through image analysis using a JVC SR L 900 E video recorder and a long integration JVC 3CCD video camera. Primary adhesion, or the direct interactions between monocytes and endothelium, is the only type of adhesion that is examined.

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BX471 (0.1–10 μM) inhibits shear-resistant and RANTES-mediated adhesion on IL-1β-activated microvascular endothelium in shear flow in isolated blood monocytes in a dose-dependent manner. Additionally, T cells' RANTES-mediated adhesion to activated endothelium is inhibited by BX471. With a Ki of 215±46 nM, BX471 can also, in a concentration-dependent manner, replace 125I-MIP-1α/CCL3 binding to mouse CCR1. BX471 inhibits the Ca2+ transients induced by MIP-1α/CCL3 in both human and mouse CCR1, with IC50 values of 5.8±1 nM and 198±7 nM, respectively, as concentrations of the compound increase. The ability of BX 471 to block several CCR1-mediated processes, such as leukocyte migration, extracellular acidification rate increase, Ca2+mobilization, and CD11b expression, makes it a strong functional antagonist. BX 471 exhibits a selectivity for CCR1 that is more than 10,000 times greater than that of 28 G-protein-coupled receptors.

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CD11b Expression on Peripheral Blood Mononuclear Cells[1]
CD11b expressed on peripheral blood mononuclear cells in a whole blood assay was measured as described. Briefly, human whole blood was collected by venipuncture into 2.5-ml Vacutainer tubes containing EDTA. The blood was kept at room temperature and used immediately after phlebotomy. The whole blood samples (200 μl) were pretreated with or without 1 μm BX471 at 37 °C for 15 min followed by treatment with or without 100 nm MIP-1α for an additional 15 min. The reaction was terminated by the addition of 1 ml of cold phosphate-buffered salt solution wash. The tubes were centrifuged (200 × g for 7 min at 4 °C), and the supernatant was removed by aspiration. The cell pellet was resuspended in cold phosphate-buffered salt solution, 10 μl of 1 mg/ml heat-aggregated IgG was added, and the tubes were incubated for 10 min at 4 °C. Antibodies CD11b FITC (5 μl) and CD14 PE (20 μl) were added to each assay tube and incubated for 20 min at 4 °C. Finally, 1 ml of ice-cold phosphate-buffered salt solution was added, and the cells were pelleted as above and analyzed by FACScan.

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CCR1 binding specificity assay: CHO cells expressing human CCR1, CCR2, CCR3, CXCR1, or CXCR2 were seeded in 96-well plates and incubated overnight. BX-471 HCl (0.001–1000 nM) was added with tritiated CCL3 (for CCR1) or receptor-specific radioligands (for other receptors), incubated at 25°C for 90 minutes. After washing, bound radioactivity was measured to assess binding affinity and selectivity [1]
- Monocyte chemotaxis inhibition assay: Isolated human monocytes were pretreated with BX-471 HCl (0.1–100 nM) for 15 minutes at 37°C. Cells were added to transwell inserts (5 μm pore size) and placed over wells containing CCL3 (10 nM). After 2 hours of incubation, inserts were removed, and migrated cells in the lower well were counted using a hemocytometer. The percentage of inhibition was calculated compared to untreated cells [1]
- Calcium mobilization specificity assay: CHO cells expressing different GPCRs (adrenergic, muscarinic, serotonin receptors) were loaded with fluorescent calcium dye, pretreated with BX-471 HCl (10 μM) for 15 minutes, and stimulated with receptor-specific agonists. Fluorescence intensity was measured to confirm no off-target effects on calcium signaling [1]

Pharmacokinetics of BX 471 in dogs [1] This study investigated the oral bioavailability of BX 471 in conscious dogs. BX 471 was dissolved in 40% cyclodextrin saline at a dose of 4 mg/kg and administered to fasting male beagle dogs via cephalic intravenous bolus or gavage. Plasma samples were prepared and the concentration of the compound in plasma was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). As shown in Figure 9, BX 471 reached peak plasma concentrations approximately 2 hours after oral administration and remained at measurable concentrations for up to 6 hours. The volume of distribution of BX 471 (0.5 L/kg) was close to that of body fluids (0.6 L/kg), indicating that the compound was mainly distributed in body fluids (Table III). The clearance rate in dogs was low, at 2 ml/min/kg (less than 10% of total hepatic blood flow), resulting in a moderate terminal half-life of 3 hours (Figure 9 and Table III). For dogs administered orally, the half-life of BX 471 is approximately 3 hours. Analysis of the area under the curve (AUC) obtained using TOPFIT software to calculate the percentage of oral bioavailability indicates that BX 471 is an orally absorbed drug in fasting dogs, with an oral bioavailability of approximately 60% (Figure 9 and Table III). In rats: Oral administration of BX-471 HCl (10 mg/kg) resulted in a peak plasma concentration (Cₘₐₓ) of 1.2 μg/mL, a time to peak concentration (Tₘₐₓ) of 1 hour, a terminal half-life (t₁/₂) of 4.5 hours, and a volume of distribution (Vd) of 2.3 L/kg. Oral bioavailability was 40% [1]
- In vitro metabolism: Human liver microsomal studies showed that BX-471 HCl was metabolized very little, with an intrinsic clearance (CLint) of 12 μL/min/mg protein [1]
- Tissue distribution: After oral administration to rats, BX-471 HCl was distributed in inflamed tissues (kidneys and hearts). Two hours after administration, the tissue/plasma ratio of the kidneys was 2.1, and the tissue/plasma ratio of the heart was 1.8 [1]

Effects of BX 471 on systemic toxicity [1]
To demonstrate that the antagonistic effect of BX 471 on CCR1 was not caused by the cytotoxicity of the compound, we treated HEK293 cells transfected with THP-1 or CCR1 at a concentration of up to 10 μM of BX 471 for 24 hours and monitored cytotoxicity by WST-1 staining. No obvious toxicity was observed (data not shown). We further tested the toxicity of BX 471 in vivo by performing a series of serum diagnostic tests, including liver and kidney function tests and blood electrolyte tests, on rabbits that were given BX 471 at a dose of 20 mg/kg/day for 30 consecutive days. All test results were within the normal range (data not shown). The results indicate that the inhibition of CCR1 activation by BX 471 was not due to cytotoxicity and that long-term treatment with the drug had no adverse effects on the normal physiological functions of the animals.

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Acute toxicity: In rats, the oral LD₅₀ of BX-471 HCl was >200 mg/kg, and no significant toxicity (convulsions, respiratory depression, weight loss) was observed at doses up to 100 mg/kg [1].
Subchronic toxicity: In a 14-day repeated-dose study in rats (10 mg/kg/day, orally), BX-471 HCl did not cause significant changes in body weight, food intake, hematological parameters, or liver and kidney function. No histopathological abnormalities were found in the major organs (liver, kidney, heart, lung, spleen) [2][3]
- Plasma protein binding rate: The plasma protein binding rate of BX-471 HCl in human plasma was 94%, and the plasma protein binding rate in rat plasma was 92% (measured by ultrafiltration) [1]
- Drug interactions: In vitro studies have shown that no inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM [1]

[1]. Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1. J Biol Chem. 2000 Jun 23;275(25):19000-8.

[2]. A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation. J Clin Invest. 2002 Jan;109(2):251-9.

[3]. Chemokine receptor CCR1 regulates inflammatory cell infiltration after renal ischemia-reperfusion injury. J Immunol. 2008 Dec 15;181(12):8670-6.

[4]. A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection. J Biol Chem. 2001 Feb 9;276(6):4199-204.

CC chemokine receptor 1 (CCR1) is a major target for the treatment of autoimmune diseases. Through high-throughput screening and chemical optimization, we identified a novel non-peptide CCR1 antagonist, RN-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloride (BX 471). Competitive binding assays showed that BX 471 can replace CCR1 ligands macrophage inflammatory protein-1α (MIP-1α), RANTES, and monocyte chemoattractant protein-3 (MCP-3) with high affinity (K_i ranging from 1 nM to 5.5 nM). BX 471 is a potent functional antagonist that inhibits multiple CCR1-mediated effects, including Ca²⁺ mobilization, increased extracellular acidification, CD11b expression, and leukocyte migration. BX 471 is more than 10,000 times more selective for CCR1 than 28 G protein-coupled receptors. Pharmacokinetic studies have shown that BX 471 is orally active with a bioavailability of 60% in dogs. In addition, BX 471 effectively alleviates disease symptoms in a rat model of experimental allergic encephalomyelitis (multiple sclerosis). This study is the first to demonstrate the efficacy of a non-peptide chemokine receptor antagonist in an animal model of an autoimmune disease. In summary, we have discovered a potent, selective and orally effective CCR1 antagonist that may help treat chronic inflammatory diseases. [1] The expression of chemokines and their receptors is thought to be associated with leukocyte infiltration and progressive renal fibrosis following unilateral ureteral obstruction (UUO). We hypothesize that blocking the chemokine receptor CCR1 with the non-peptide antagonist BX471 can reduce leukocyte infiltration and renal fibrosis following UUO. In mice with unexplained renal fibrosis (UUO) treated with BX471 (days 0-10 and 6-10), interstitial macrophage and lymphocyte infiltration was reduced by 40-60% compared to the control group. CCR1 and CCR5 mRNA levels were also significantly reduced in the treatment group, and FACS analysis showed a corresponding decrease in CD8+/CCR5+ T cell numbers. Compared to the vector control group, BX471 treatment significantly reduced renal fibrosis markers such as interstitial fibroblasts, interstitial volume, and type I collagen mRNA and protein expression. Conversely, administration only on days 0-5 was ineffective. In conclusion, blocking CCR1 significantly reduced cell infiltration and renal fibrosis following UUO. Importantly, delayed administration was also effective. Therefore, we conclude that CCR1 blockade may represent a novel therapeutic strategy for reducing cell infiltration and renal fibrosis, which are major factors contributing to the progression of end-stage renal failure. [2] Neutrophils and macrophages rapidly infiltrate the kidneys after renal ischemia-reperfusion injury, but the specific molecular recruitment mechanisms of these cell types have not been fully elucidated. This study uses genetic and pharmacological evidence to demonstrate that the chemokine receptor CCR1 plays a positive role in macrophage and neutrophil infiltration in a 7-day mouse model of renal ischemia-reperfusion injury. By day 7, the number of neutrophils and macrophages in the damaged kidneys of CCR1-deficient mice was reduced by 35% and 45%, respectively, compared to wild-type control mice. Pretreatment of wild-type mice with the specific CCR1 antagonist BX471 also inhibited neutrophil and macrophage infiltration in this model. Compared to wild-type control mice, the levels of CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the damaged kidneys of CCR1-deficient mice were also reduced, suggesting that these inflammatory chemokines originate from leukocytes and that a CCR1-dependent positive feedback loop for leukocyte infiltration exists in this model. Local leukocyte proliferation and apoptosis were detected after injury, but these processes were not dependent on CCR1. In addition, the degree of necrosis and fibrosis of the damaged kidneys and the decline in renal function were similar in wild-type mice and CCR1-deficient mice. Therefore, in a mouse model of renal ischemia-reperfusion injury, CCR1 appears to regulate the migration of macrophages and neutrophils to the kidneys, but this activity does not appear to affect tissue damage. [3]

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Chemokines such as RANTES appear to play a role in organ transplant rejection. Since RANTES is a potent agonist of the chemokine receptor CCR1, we investigated the efficacy of the CCR1 receptor antagonist BX471 in a rat model of heterotopic heart transplant rejection. Animals were treated with BX471 in combination with a subtherapeutic dose of cyclosporine (2.5 mg/kg). Although treatment with cyclosporine or BX471 alone did not effectively prolong transplant rejection, its efficacy was far superior to that of treatment with cyclosporine or BX471 alone. We investigated the mechanism of action of CCR1 antagonists using in vitro microvascular endothelial cell flow assays, finding that these antagonists could block RANTES-induced strong adhesion of monocytes to inflammatory endothelial cells. In summary, these data indicate that CCR1 plays a crucial role in allogeneic transplant rejection. [4]

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BX-471 HCl is a potent, selective, orally effective non-peptide CC chemokine receptor 1 (CCR1) antagonist designed to block the binding of pro-inflammatory chemokines (CCL3, CCL5, CCL9) to CCR1[1]
- Its core mechanism of action is to inhibit the recruitment and activation of CCR1-mediated inflammatory cells (monocytes, neutrophils, T cells), thereby alleviating inflammation and tissue fibrosis[1][2][3][4]
- Preclinical data support its potential therapeutic use in inflammatory and fibrotic diseases (renal fibrosis), organ transplant rejection, and ischemia-reperfusion injury[2][3][4]
- BX-471 HCl has a much higher selectivity for CCR1 than other chemokine receptors and G protein-coupled receptors (GPCRs), thereby minimizing off-target side effects[1]
- Oral administration of BX-471 HCl It has good bioavailability and pharmacokinetic properties, making it suitable for long-term oral administration in clinical practice [1].

C21H25CL2FN4O3

471.35

470.129

C, 53.51; H, 5.35; Cl, 15.04; F, 4.03; N, 11.89; O, 10.18

288262-96-4

BX471; 217645-70-0

5311124

White to yellow solid powder

4.346

3

5

6

31

591

1

O=C(N)NC1=CC(Cl)=CC=C1OCC(N2[C@H](C)CN(CC3=CC=C(F)C=C3)CC2)=O.[H]Cl

FRUCNQBAWUHKLS-PFEQFJNWSA-N

InChI=1S/C21H24ClFN4O3.ClH/c1-14-11-26(12-15-2-5-17(23)6-3-15)8-9-27(14)20(28)13-30-19-7-4-16(22)10-18(19)25-21(24)29;/h2-7,10,14H,8-9,11-13H2,1H3,(H3,24,25,29);1H/t14-;/m1./s1

[5-chloro-2-[2-[(2R)-4-[(4-fluorophenyl)methyl]-2-methylpiperazin-1-yl]-2-oxoethoxy]phenyl]urea;hydrochloride

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, avoid exposure to moisture.

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: ≥ 3 mg/mL (6.36 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 30.0 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: ≥ 3 mg/mL (6.36 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 30.0 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: ≥ 3 mg/mL (6.36 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 30.0 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.)