Integrin CD11b/CD18

A particular agonist of leukocyte surface integrin CD11b/CD18 (αMβ2; CR3; Mac-1) is leukadherin-1. Monokine-stimulated natural killer (NK) cells secrete less interferon (IFN)-γ, tumor necrosis factor (TNF), and macrophage inflammatory protein (MIP)-1β when pretreated with leukadherin-1. Pretreatment with leukadherin-1 also decreases the release of TNF, IL-1β, and IL-6 by TLR-2 and TLR-7/8 activated monocytes[3].

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Leukadherin-1 is a specific small-molecule CR3 agonist that has shown therapeutic promise in animal models of vascular injury and inflammation. We show that Leukadherin-1 pretreatment reduces secretion of interferon (IFN)-γ, tumour necrosis factor (TNF) and macrophage inflammatory protein (MIP)-1β by monokine-stimulated NK cells. It was associated with a reduction in phosphorylated signal transducer and activator of transcription (pSTAT)-5 following interleukin (IL)-12 + IL-15 stimulation (P < 0·02) and increased IL-10 secretion following IL-12 + IL-18 stimulation (P < 0·001). Leukadherin-1 pretreatment also reduces secretion of IL-1β, IL-6 and TNF by Toll-like receptor (TLR)-2 and TLR-7/8-stimulated monocytes (P < 0·01 for all). The R77H variant did not affect NK cell response to Leukadherin-1 using ex-vivo cells from homozygous donors; nor did the variant influence CR3 expression by these cell types, in contrast to a recent report. These data extend our understanding of CR3 biology by demonstrating that activation potently modifies innate immune inflammatory signalling, including a previously undocumented role in NK cell function. We discuss the potential relevance of this to the pathogenesis of SLE. Leukadherin-1 appears to mediate its anti-inflammatory effect irrespective of the SLE-risk genotype of CR3, providing further evidence to support its evaluation of Leukadherin-1 as a potential therapeutic for autoimmune disease[3].

In an experimental model of bronchopulmonary dysplasia (BPD), leukadherin-1 (1 mg/kg; ip; twice daily for 14 days) is helpful in preventing hyperoxia-induced neonatal lung injury[1].
Administration of Leukadherin-1 (LA1) is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) and received twice-daily intraperitoneal injection of LA1 or placebo for 14 days. Hyperoxia exposure in the presence of the placebo resulted in a drastic increase in the influx of neutrophils and macrophages into the alveolar airspaces. This increased leukocyte influx was accompanied by decreased alveolarization and angiogenesis and increased pulmonary vascular remodeling and pulmonary hypertension (PH), the pathological hallmarks of BPD. However, administration of LA1 decreased macrophage infiltration in the lungs during hyperoxia. Furthermore, treatment with LA1 improved alveolarization and angiogenesis and decreased pulmonary vascular remodeling and PH. These data indicate that leukocyte recruitment plays an important role in the experimental model of BPD induced by hyperoxia. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.[1]

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Here, researchers used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins (such as Leukadherin-1 (LA1)). These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.[2]
Administration of LA1 30 min before injection with thioglycolate significantly reduced the amount of neutrophil accumulation by 40% (P < 0.05) compared to that in mice pretreated with vehicle, whereas LA2 reduced neutrophil accumulation by 65% (P < 0.0001), and LA3 reduced it by 55%[2].

Leukadherin-1, also known as LA1, is a novel and specific agonist of Complement receptor 3 (CR3) and the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Complement receptor 3 (CR3, CD11b/CD18) is a multi-functional receptor expressed predominantly on myeloid and natural killer (NK) cells. Leukadherin-1 (LA1) does not modulate signal transducer and activator of transcription (STAT)-4 phosphorylation. Leukadherin-1 modulates TLR-2 and TLR-7/8-induced monocyte cytokine secretion. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.

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αA domain ligand-binding assay [2]
MaxiSorp 96-well plates were coated overnight with fibrinogen (1 μg per well) in 10 mM phosphate-buffered saline (PBS, pH 7.4) and blocked with 1% BSA in PBS. Binding of purified, GST-tagged αA domain (50 μl per well of a 5 μg/ml solution) to immobilized fibrinogen was performed in TBS-based assay buffer (TBS containing 0.1% BSA, 1 mM MgCl2, 1 mM CaCl2, and 0.05% Tween 20) (TBS-Ca/Mg buffer) for 1 hour at room temperature. The αA domain was also added to uncoated wells on the plate to estimate the maximum amount of protein that could be captured and detected in each well for data normalization. Unbound αA domain was removed by washing the wells twice with TBS-Ca/Mg buffer. Subsequently, the amount of bound protein was determined by incubation with horseradish peroxidase–conjugated antibody against GST (GE, 1:2000 dilution) for 1 hour. Unbound antibody was removed by washing the wells twice with TBS-Ca/Mg buffer. Detection of bound protein was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate kit according to the manufacturer’s protocol. Absorbance was read with a SpectraMax M5 spectrophotometer). Absorbance values were normalized such that the mean absorbance from the input αA domain wells was set at 100%, and the results are presented as the percentage of the total input amounts of the wild-type αA domain. Assays were performed in triplicate wells, and the data shown are from one of at least three independent experiments.

Cell stimulation [3]
Supernatant cytokines are quantified after stimulation and culture for 18 h (monocytes) or 24 h (NK cells). Except for bead-based stimulation, all experiments are conducted using 100 µL cells in a 96-well plate format. NK cell stimuli are added as follows: (1) Syk inhibitor (1 μM), (2) Leukadherin-1 or dimethylsulphoxide (DMSO) (vector control) (7.5 μM). Shown to induce∼82% of maximum response with negligible off-target effect, (3) anti-CD210 or isotype control (5 µg/mL), (4) 30-45 min after Leukadherin-1 NK cells are stimulated with combinations of IL-12 (10 ng/mL), IL-15 (30 ng/mL) or IL-18 (10 ng/mL): either IL-12 + IL-15 or IL-12 + IL-18. Monocytes are stimulated using pam3csk4 (TLR-2 agonist, 300 ng/mL) or R848 (TLR-7/8 agonist, 2 µg/mL). Supernatants are stored at −80ºC for .

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Phagocytosis assay with complement iC3b-coated sheep erythrocytes (EiC3bs) [2]
Sheep erythrocytes coated with complement iC3b were prepared and used in the phagocytosis assay as described previously. Coated erythrocytes (EiC3bs) were diluted to a concentration of 1.5 × 107 to 6 × 107 cells/ml. K562 CD11b/CD18 cells were washed twice in TBS and resuspended to 1 × 106/ml, of which 40 μl (4 × 104 cells) was incubated in suspension with EiC3bs (1.2 × 106) in a total volume of 100 μl at 37°C for 25 min in the presence of 1 mM each of CaCl2 and MgCl2 (in the absence or presence of 50 to 100 μM Leukadherin-1 (LA1), LA2, or LA3), in 1 mM MnCl2, or in 10 mM EDTA. Binding was detected by visually analyzing the formation of rosettes [the binding of multiple erythrocytes (EiC3bs) to individual K562 cells] by phase-contrast microscopy, as has been described previously. For scoring, only those K562 cells that were bound to ≥3 EiC3bs were scored as positive, and >200 cells were examined in multiple fields under each condition. Binding results, showing the percentages of all cells showing rosettes in a field, are reported as histograms representing the mean ± SEM of triplicate experiments; the data shown are from one of at least three independent experiments.

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Cell viability assays [2]
Cell viability assays were performed with commercially available reagents and kits. Briefly, 1 × 104 K562 CD11b/CD18 cells or wild-type B6 neutrophils were incubated in each well of a 96-well plate with increasing amounts of the indicated compounds, and the number of viable cellswas determined with the MTS reagent, according to the manufacturers’ instructions, after 4 hours (neutrophils) or 24 hours (K562 cells) of incubation. A SpectraMaxM5 spectrophotometer was used to read the assay plates. Data are representative of at least two independent experiments.

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Western blotting analysis [2]
K562 CD11b/CD18 cells were incubated with Leukadherin-1 (LA1), LA2, or LA3 (15 μM) or fibrinogen (200 μg) in serum-free medium for 1 hour at 37°C. Cell lysates were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane by means of established protocols. Membranes were incubated with a 1:1000 dilution of an antibody against phosphorylated extracellular signal–regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), stripped with Reblot mild stripping solution, and then incubated, first with an antibody against total ERK1/2 and then with an antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and developed according to the manufacturer’s instructions. Data presented are representative of at least three independent experiments.

Animal/Disease Models: Newborn Sprague Dawley rat pups[1]
Doses: 1 mg/kg
Route of Administration: Ip; twice (two times) daily for 14 days
Experimental Results: Beneficial on preventing the lung inflammatory response, improved alveolarization and vascular development, and decreased pulmonary vascular remodeling and PH in a hyperoxia-induced experimental model of BPD.

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Animal Model and Experimental Protocol [1]
Newborn Sprague Dawley rat pups (n = 12 per group) were randomized on Postnatal Day 2 into four groups: normoxia (21% O2) plus placebo, normoxia plus Leukadherin-1 (LA1), hyperoxia (85% O2) plus placebo, and hyperoxia plus Leukadherin-1 (LA1). Rat pups received Leukadherin-1 (LA1) (1 mg/kg, mixed in 2% DMSO) or placebo (2% DMSO) (equal volume) twice daily by intraperitoneal injection for 14 days. Pups were killed on Postnatal Day 15 for analyses.

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In vivo peritonitis model [2]
Thioglycolate-induced peritonitis in 8- to 10-week-old wild-type B6 and CD11b−/− B6 mice was performed as previously described. Leukadherins were administered 30 min before intraperitoneal injection with 3% thioglycolate. Leukadherin-1 (LA1) and LA2 (200 μl of a 50 μM solution in saline) were administered intravenously, whereas LA3 was administered intraperitoneally (500 μl of a 50 μM solution in saline). To evaluate peritoneal neutrophil recruitment, we euthanized mice at 4, 12, or 24 hours after thioglycolate injection; the peritoneal lavage was collected, and the number of emigrated neutrophils was quantified by flow cytometric analysis for cells expressing both GR-1 and Mac-1 on the surface, as described previously

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Anti-GBM nephritis [2]
Experimental anti-GBM nephritis was induced in wild-type B6 mice (n = 4 mice per group) by intraperitoneal injection of sheep antibody against rabbit GBM (0.5 ml per 20 g body weight per day for 2 consecutive days) as previously described. One group of animals was treated with leukadherins by daily intraperitoneal injection of Leukadherin-1 (LA1) (500 μl of a 50 μM solution in saline) starting at 2 hours before induction of nephritis and continuing until the end of the experiment. A group of animals was treated with a known antagonist, the blocking antibody M1/70, as described previously (76). Briefly, M1/70 (100 μg per injection) in a saline solution was injected intraperitoneally every other day starting at 2 hours before induction of nephritis and continuing until the end of the experiment. Urine collections were performed every 24 hours and analyzed for evidence of proteinuria on SDS-PAGE gels with BSA standards, as described previously. The amounts of creatinine were determined with a creatinine assay kit according to the manufacturer’s instructions. Mice were killed on days 0, 3, and 7, and renal biopsies were obtained from each animal. Tissue sections were fixed with 4% paraformaldehyde and were used for histochemical analyses and leukocyte enumeration.

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Evaluation of neutrophil sequestration [2]
To evaluate whether leukadherins induced tissue sequestration of neutrophils, we fixed various organs from wild-type B6 mice (n = 3 mice per group) with formalin and stained them with hematoxylin and eosin (H&E). The numbers of neutrophils in untreated and Leukadherin-1 (LA1)-treated animals were quantified from four random fields for each specimen at either 40× or 1000× magnification in a blinded fashion. Bone marrow was isolated as described previously. Briefly, mice were euthanized, and the femurs and tibia from both hind legs were removed and freed of soft tissue. The extreme ends of the bones were cut off, and a solution of RPMI 1640 containing 10% FBS was forced through the bone with a 27-gauge syringe needle. Cell clumps were dispersed, passed through an 80-μm filter, and collected by centrifugation. Red blood cells (RBCs) were removed by hypotonic lysis buffer followed by two washes with HBSS buffer containing 0.1% BSA. Neutrophil numbers were determined by flow cytometry as described earlier.

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Intravital microscopy [2]
Mice were given an intrascrotal injection of TNF-α (500 ng) in 0.25 ml of saline 3 hours before cremaster muscle exteriorization. Some animals also received intravenous injections of the blocking mAb M1/70 (30 μg per mouse) in 0.1 ml of saline and intraperitoneal injections of Leukadherin-1 (LA1) (100 μM) or DMSO in 0.5 ml of saline 30 min before injection with TNF-α. Mice were anesthetized with an intraperitoneal injection of ketamine (125 mg/kg), xylazine (12.5 mg/kg), and atropine sulfate (0.025 mg/kg) and placed on a 38°C heating pad. After tracheal intubation and cannulation of one carotid artery, the cremaster was exteriorized, pinned to the stage, and superfused with thermocontrolled bicarbonate-buffered saline (131.9 mM NaCl, 18 mM NaHCO3, 4.7 mM KCl, 2.0 mM CaCl2, and 1.2 mM MgCl2) equilibrated with 5% CO2 in N2. Cremaster muscles were illuminated with stroboscopic flash epi-illumination (DPS-1, Rapp OptoElectronic) and halogen transillumination. Microscopic observations were made on postcapillary venules with a diameter of between 20 and 40 mm by means of an intravital microscope with a saline immersion objective (SW 40/0.75). A charge-coupled device (CCD) camera (model SIT66, DAGE-MTI) was used for recording. In a limited analysis, cells adjacent to the venules were counted to determine the number of transmigrated neutrophils. The surface area (S) was calculated for each vessel as S = πdIν, where d is the diameter of the vessel and Iν is the length of the vessel. Adherent leukocytes were defined as those cells that were stationary for more than 30 s.

[1]. Efficacy of Leukadherin-1 in the Prevention of Hyperoxia-Induced Lung Injury in Neonatal Rats. Am J Respir Cell Mol Biol. 2015;53(6):793-801.

[2]. Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease. Sci Signal. 2011;4(189):ra57.

[3]. The complement receptor 3 (CD11b/CD18) agonist Leukadherin-1 suppresses human innate inflammatory signalling. Clin Exp Immunol. 2016;185(3):361-371.

Lung inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. The challenge in BPD management is the lack of effective and safe antiinflammatory agents. Leukadherin-1 (LA1) is a novel agonist of the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Its functional significance in preventing hyperoxia-induced neonatal lung injury is unknown. We tested the hypothesis that administration of LA1 is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) and received twice-daily intraperitoneal injection of LA1 or placebo for 14 days. Hyperoxia exposure in the presence of the placebo resulted in a drastic increase in the influx of neutrophils and macrophages into the alveolar airspaces. This increased leukocyte influx was accompanied by decreased alveolarization and angiogenesis and increased pulmonary vascular remodeling and pulmonary hypertension (PH), the pathological hallmarks of BPD. However, administration of LA1 decreased macrophage infiltration in the lungs during hyperoxia. Furthermore, treatment with LA1 improved alveolarization and angiogenesis and decreased pulmonary vascular remodeling and PH. These data indicate that leukocyte recruitment plays an important role in the experimental model of BPD induced by hyperoxia. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.[1]

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The integrin CD11b/CD18 (also known as Mac-1), which is a heterodimer of the α(M) (CD11b) and β(2) (CD18) subunits, is critical for leukocyte adhesion and migration and for immune functions. Blocking integrin-mediated leukocyte adhesion, although beneficial in experimental models, has had limited success in treating inflammatory diseases in humans. Here, we used an alternative strategy of inhibiting leukocyte recruitment by activating CD11b/CD18 with small-molecule agonists, which we term leukadherins. These compounds increased the extent of CD11b/CD18-dependent cell adhesion of transfected cells and of primary human and mouse neutrophils, which resulted in decreased chemotaxis and transendothelial migration. Leukadherins also decreased leukocyte recruitment and reduced arterial narrowing after injury in rats. Moreover, compared to a known integrin antagonist, leukadherins better preserved kidney function in a mouse model of experimental nephritis. Leukadherins inhibited leukocyte recruitment by increasing leukocyte adhesion to the inflamed endothelium, which was reversed with a blocking antibody. Thus, we propose that pharmacological activation of CD11b/CD18 offers an alternative therapeutic approach for inflammatory diseases.[2]

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Our results demonstrate that CR3 is a negative regulator of this inflammatory loop and that Leukadherin‐1 is capable of marked NK cell cytokine down‐regulation. Furthermore Leukadherin‐1 is equally capable of down‐regulating signalling even in cells that are homozygous for the genetically encoded under‐functioning variant of CR3, presumably because the mechanism of CR3 signal transduction is different following engagement with this small molecule compound compared with natural ligand. We did not demonstrate any significant effect of Leukadherin‐1 in modulating cytokine production following engagement of CD16 on NK cells. Whether this would also apply to other CD16‐mediated functions, such as antibody‐dependent cellular cytotoxicity (ADCC), would require further investigation. In addition to documenting ex‐vivo effects of Leukadherin‐1 we have, in part, explained the mechanism in NK cells by demonstrating an inhibition of IL‐15‐induced phosphorylation of the transcription factor STAT‐5. Our data suggest that Leukadherin‐1 interferes with events that take place upstream of, or during, STAT‐5 phosphorylation – such as Janus kinase (JAK) phosphorylation or JAK/STAT association – rather than through a mechanism of STAT dephosphorylation. Activation of JAK‐1 and JAK‐3 associated with the IL‐2Rβ and γc‐chains, respectively, has been shown to be crucial for STAT‐5 tyrosine phosphorylation in response to IL‐15. A CR3‐mediated effect on JAKs has been shown recently in human monocytes/macrophages, in which CR3 activation with anti‐CD18 or anti‐CD11b antibodies blocks the phosphorylation of IL‐13 receptor‐associated JAK‐2 and tyrosine kinase‐2 (TYK‐2) kinases, which further inhibits the downstream tyrosine and serine phosphorylation of STAT‐1, STAT‐3 and STAT‐6. Further work is needed to determine the upstream mechanism through which the Leukadherin‐1‐mediated inhibition of STAT‐5 phosphorylation occurs in NK cells.[3]

C22H15NO4S2

421.49

421.044

C, 62.69; H, 3.59; N, 3.32; O, 15.18; S, 15.21

344897-95-6

(Z)-Leukadherin-1;2055362-72-4;ADH-503;2055362-74-6

5342077

Light yellow to yellow solid powder

1.5±0.1 g/cm3

634.7±65.0 °C at 760 mmHg

337.6±34.3 °C

0.0±2.0 mmHg at 25°C

1.756

4.88

1

6

5

29

680

0

S1C(N(C(/C/1=C(\[H])/C1=C([H])C([H])=C(C2C([H])=C([H])C(C(=O)O[H])=C([H])C=2[H])O1)=O)C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=S

AEZGRQSLKVNPCI-UNOMPAQXSA-N

InChI=1S/C22H15NO4S2/c24-20-19(29-22(28)23(20)13-14-4-2-1-3-5-14)12-17-10-11-18(27-17)15-6-8-16(9-7-15)21(25)26/h1-12H,13H2,(H,25,26)/b19-12-

4-[5-[[4-Oxo-3-(phenylmethyl)-2-thioxo-5-thiazolidinylidene]methyl]-2-furanyl]-benzoic acid

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)

DMSO: ~5 mg/mL ( 11.86 mM）
Water: Insoluble
Ethanol: Insoluble

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.)