BTK (Ki = 0.91 nM); BTK C481R (Ki = 1.3 nM); BTK C481S (Ki = 1.6 nM); BTK T474M (Ki = 3.4 nM); BTK T474I (Ki = 12.6 nM)

GDC-0853 only inhibits 3 out of 286 off-target kinases in a broad panel of human kinase biochemical assays when tested at 1 μM. The selectivity for Btk is >100-fold against each of these three off-targets, according to the calculated IC50 values: Bmx (153-fold), Fgr (168-fold), and Src (131-fold). GDC-0853 inhibits the signaling of monocyte FcγR and B-cell BCR. The average residence time of GDC-0853 with Btk in the in vitro biochemical Btk enzyme assay is 18.3 ± 2.8 hours. GDC-0853 blocks cellular autophosphorylation of WT Btk and the C481S mutant[1]. GDC-0853 treatment of CLL (chronic lymphocytic leukemia) cells in vitro prior to BCR stimulation results in decreased BTK phosphorylation and decreased activation of downstream targets such as PLCγ2, AKT, and ERK. GDC-0853 decreases activation, hinders migration, and inhibits NF-κB-dependent transcription. GDC-0853 does not influence T-cell receptor activation and does not inhibit EGFR or ITK in the cellular system[3].

GDC-0853 has a moderate clearance of 27.4 mL/min/kg and an excellent bioavailability (F=65%) in rats administered 0.2 mg/kg via intraperitoneal injection or 1 mg/kg PO. The plasma half-life (t_1/2) is 2.2 hours, the volume of distribution (Vd) is 5.42 L/kg, and the plasma clearance is 27.4 mL/min/kg. GDC-0853 shows positive PK characteristics in dogs as well. In canine toxicology research, achieving adequate exposures is further made possible by the 3.8-hour half-life (Cl_p 10.9 mL/min/kg, V_d 2.96 L/kg) and high oral bioavailability (85%). Both rats and dogs tolerate GDC-0853 well, and it has an overall good safety profile. GDC-0853 is helpful in the treatment of autoimmune diseases mediated by B-cells or myeloid cells, including rheumatoid arthritis. GDC-0853 has shown excellent tolerance in two studies: a single ascending dose (SAD) trial (0.5 mg to 600 mg) and a multiple ascending dose (MAD) study (250 mg BID to 500 mg QD) lasting 14 days. Both studies have shown no dose-limiting toxicities and no severe adverse events. GDC-0853 exhibits dose-proportional, linear pharmacokinetics and is well absorbed [1]. GDC-0853 and other structurally diverse BTK inhibitors administered for 7 days or more in Sprague-Dawley (SD) rats result in pancreatic lesions that include pigment-laden macrophages, inflammation, fibrosis, and multifocal islet-centered hemorrhage, along with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. At far higher exposures, similar results are not seen in mice or dogs[2].

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Preclinical Efficacy in Rheumatoid Arthritis and Lupus[1]
To evaluate the in vivo efficacy of GDC-0853 (29), we tested it in a B and myeloid cell-dependent inflammatory arthritis model. Female Lewis rats with developing collagen-induced arthritis (30) were dosed orally for 16 days with 29 at a range of doses once (0.06, 0.25, 1, 4, and 16 mg/kg QD) (Figure 8A) or twice (0.125, 0.5, and 2 mg/kg BID) daily (Figure 8B). 29 dose-dependently reduced ankle thickness following QD (Figure 8A) and BID (Figure 8B) dosing regimens. Furthermore, 29 showed a dose responsive beneficial effect on a panel of ankle histopathology parameters (inflammation, pannus, cartilage damage, bone resorption; data not shown). We also assessed the ankle diameter expressed as area under the curve (AUC) as an efficacy parameter. Ankle diameter AUC was significantly reduced toward normal for rats treated QD with 16 mg/kg (99% reduction), 4 mg/kg (89%), 1 mg/kg (71%), and 0.25 mg/kg 29 (26%) or BID with 2 mg/kg (96%), 0.5 mg/kg (79%), and 0.125 mg/kg 29 (50%) as compared with the respective disease controls. The QD 16 mg/kg dose and the BID 2 mg/kg dose of 29 showed comparable (to slightly improved) efficacy relative to dexamethasone. In naı̈ve rats treated with vehicle, the ankle diameters did not change over the course of the study and ankle diameters from normal rats were significantly smaller (P < 0.05) than those of the rats with CIA that were treated with vehicle.

Btk assays. [1]
Btk kinase activity and inhibition was assessed following the previously published peptide phosphorylation assay1 and using wild type and mutant Btk enzymes expressed and purified at Genentech. The Btk proteins were used as obtained from the purification and no special measures were taken to activate them. Inhibition constant (Ki) values were calculated from inhibitor titration data as follows. Btk fractional activity (vi /vo) was plotted against test article concentration and the data were fit using Genedata Screenersoftware (Genedata; Basel, Switzerland) to the tight-binding inhibition equation2 to calculate the apparent inhibition constant, Ki app.

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Kinase selectivity.[1]
Btk inhibitor kinase selectivity is evaluated at a concentration of 1 µM in a panel of up to 287 recombinant human kinase activity and binding tests, such as lipid kinases, serine/threonine kinases, and cytoplasmic and receptor tyrosine kinases. Whereas the binding assays tracked the displacement of ATP sitebinding probes, the kinase activity assays quantify peptide phosphorylation or ADP production. For every kinase, the ATP concentrations used in the activity assays are usually within two times the experimentally determined apparent Michaelis constant (Km^app) value, while the competitive binding tracer concentrations used in the binding assays are usually within three times the experimentally determined dissociation constant (Kd) values. For every kinase, inhibitors are tested in duplicate, and the mean percentage of inhibition is reported. The same assays are used for 10-point inhibitor titrations to identify the inhibitor concentrations that cause 50% inhibition (IC50) for kinases that are inhibited by nearly or more than 80% at the test concentration.

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Kinase assay[3]
A total of 160 ng human recombinant wild-type and C481S BTK were incubated with DMSO or 1 µM GDC-0853 for 30 minutes. Recombinant protein was then combined with 50 µM adenosine triphosphate and 5 µg poly (4:1, Glu:Tyr) peptide for 30 minutes at room temperature in 1× reaction buffer to allow for phosphorylation of the peptide substrate. ADP-glo kinase reagent and kinase detection reagent were then used to quench and quantify the reaction, respectively. Luminescence was measured using a DTX880 plate reader.

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Liver Microsome Metabolic Stability Assays [1]
Metabolic stability of test compounds was evaluated in pooled donor human, mouse, and rat liver microsomes. The final incubations contained: 1 µM of test compound, 1 mM NADPH, and 0.5 mg/mL microsomal protein in 0.1 M potassium phosphate buffer (pH 7.4). Following a 5-minute pre-incubation period, the enzymatic reactions were initiated by the addition of NADPH and test compound to the microsomes diluted in phosphate buffered saline. The mixtures were incubated at 37 °C for 0, 20, 40, and 60 min and the resultant compound concentrations were assessed by LC-MS/MS. Intrinsic clearance based upon microsomal stability data was determined using a substrate depletion method and scaled to hepatic clearance using the well-stirred model.

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Hepatocyte Metabolic Stability Assays [1]
Metabolic stability assays of test compounds were carried out using cryopreserved pooled donor mouse, rat, dog, cynomolgus monkey, and human hepatocytes. Membrane integrity of the cells was assessed by trypan blue exclusion. Test compounds (1 µM with 0.1% DMSO) were incubated with cells (0.5 million cells/mL) at 37 °C in a 95% air/5% CO2 atmosphere for 0, 20, 40, or 60 min. Concentrations of test compounds in hepatocyte incubations were determined by LC-MS/MS. Intrinsic clearance was determined using a substrate depletion method and scaled to hepatic clearance using the well-stirred model (vide supra).

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GDC-0853 Residence Time Assay [1]
Btk enzyme (10 nM) was incubated for 2 h at room temperature with GDC-0853 (11 nM) or DMSO vehicle in 50 mM HEPES buffer (pH 7.5). After this incubation, the Btk samples were diluted 200-fold into assay mixture containing ATP and peptide substrate1 and the levels of unreacted substrate peptide and phosphorylated peptide product were monitored approximately every 2.5 min for 8.5 h. The progress curve for product formed by Btk that had been pre-incubated with GDC-0853 was fit to an equation that describes the recovery of activity4 :

Cellular Btk phosphorylation. [1]
The effect of GDC-0853 and ibrutinib on cellular wild type Btk and Btk C481S mutant phosphorylation on Y223 was assessed in transiently transfected HEK293T cells as previously described.

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B cell and monocyte assays [1]
Human B cells or monocytes were isolated from peripheral blood mononuclear cells (PBMCs) by FicollPaque PLUS separation and negatively selected by magnetic cell sorting following manufacturer’s instructions. Human B cells were stimulated with 10 or 25 µg/mL goat anti-IgM-F(ab’)2 or 10 µg/mL CD40L, and proliferation was measured by [ 3H]thymidine incorporation. Human monocytes were incubated with 40 µg/mL immobilized HSA/antiHSA ICs. TNFα production by FcγR-activation was measured by ELISA.

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Cells treated with dimethyl sulfoxide (DMSO) or GDC-0853 were similarly pelleted and then resuspended in 10% fetal bovine serum RPMI-1640 medium containing DMSO or GDC-0853. Experiments that occurred over several days included daily addition of drug and medium replacement.[3]

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NK cell–mediated ADCC[3]
Effector NK cells were isolated from Leukopaks obtained through the American Red Cross and incubated with target CLL cells loaded with radioactive Cr51 at an effector to target ratio of 25:1. Following treatment of purified NK cells with DMSO, 1 µM GDC-0853, or 1 µM ibrutinib for 1 hour, CLL cells were incubated with trastuzumab, alemtuzumab, rituximab, ofatumumab, or obinutuzumab at a concentration of 10 µg/mL and cocultured with NK cells to allow for lysis. After 4 hours of coculture, supernatant was collected and measured for radiation using a PerkinElmer Wizard2 γ counter. β decay measurements were scaled according to a no-NK cell coculture group with baseline CLL lysis and a detergent-treated CLL group with complete lysis.

Sprague-Dawley, Wistar-Han and Fischer-344 rats (6 to 12 weeks old)
5 or 10 mL/kg
p.o.

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Rat whole blood pBtk assay [1]
Sprague-Dawley rats were euthanized using CO2 asphyxiation. Blood was collected in heparin tubes by cardiac puncture. Rat whole blood was incubated with a titration of GDC-0853 (starting at 6 µM followed by 3-fold dilution for a 11-point dilution curve) for 4 h at 37 ºC. Blood was treated with an equal volume of MSD lysis buffer containing protease and phosphatase inhibitors. Thirty-five µL of lysate was added to MSD plates coated with 100 ng/well of total anti-BTK antibody and incubated for 2 h with shaking at room temperature. Wells were washed three times with TBST buffer and incubated with 12 µg/mL of anti-rabbit pBTK antibody detection antibody for 2 h at room temperature with constant shaking. Wells were washed and then incubated with 1 µg/mL of sulfo-tag anti-rabbit antibody for 45 min at room temperature with constant shaking. After incubation, wells were finally washed with S34 TBST and pBTK levels were detected by adding 150 µL of MSD Reading buffer in each well and read on a MSD Sector Imager 6000. IC50 values were calculated using Prism software.

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Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.[3]

Once-daily oral administration of 1, 4, and 16 mg/kg GDC-0853 (29) maintained plasma drug concentrations above the whole-blood pBtk inhibitory efficacy (IC50 = 9 nM, IC70 = 27 nM, IC90 = 135 nM as determined by inhibition of Btk Y223 autophosphorylation) for at least 12 hours over a 24-hour dosing cycle. Twice-daily oral administration of 0.125, 0.5, and 2 mg/kg (Figure 9B) also maintained plasma drug concentrations above the IC50, IC70, and IC90 levels for at least 6 hours over a 12-hour dosing cycle. Once-daily (QD) doses of 1 mg/kg and twice-daily (BID) doses of 0.5 mg/kg maintained plasma concentrations above the whole-blood pBtk IC70 (27 nM) for at least 12 hours over a 24-hour period. This exposure-efficacy relationship indicates that the concentration of compound 29 in plasma needs to exceed the IC70 for 12 hours to achieve efficacy, defined as a reduction of approximately 75% in ankle swelling. The once-daily (QD) 4 mg/kg dose group showed higher target coverage, exceeding the IC90 within 24 hours and further improving efficacy. The inhibitory effect of the non-covalent Btk inhibitor was also evaluated in an SLE mouse model. (14) In summary, the significant efficacy of our non-covalent Btk inhibitor in vivo, along with the excellent preclinical pharmacology, pharmacokinetics, and in vitro safety of compound 29, gives us confidence to advance the tolerability studies of this molecule. [1]

In studies designed to assess the safety of this molecule, GDC-0853 (29) demonstrated good tolerability and overall good safety in both rats and dogs. In the most sensitive preclinical animal model, dogs, no adverse event eliminator (NOAEL) was observed at doses more than 80 times higher than the target effective exposure, i.e., exceeding the IC70 concentration (from human whole blood CD69 assay) within 12 hours. In Sprague-Dawley rats, GDC-0853 (29) and other structurally different Btk inhibitors have been shown to be associated with islet-centered pancreatic lesions at clinically relevant doses. After a comprehensive review of strain and species sensitivity differences, Btk knockout (KO) mice, and XLA-mutant human case reports, we conclude that GDC-0853-associated pancreatic lesions in Sprague-Dawley rats are a result of the rodent-specific, strain-specific, and targeted action of Btk inhibitors, and are not related to humans. Histological evaluation of the pancreas in untreated Btk KO Sprague-Dawley rats supported this conclusion, showing that the pancreatic pathology was identical to that of GDC-0853. Given the good safety profile of GDC-0853 and the fact that the observed pancreatic toxicity was mouse-specific, we selected GDC-0853 (29) as our preferred candidate for clinical development. [1]

[1]. J Med Chem . 2018 Mar 22;61(6):2227-2245.

[2]. J Pharmacol Exp Ther . 2017 Jan;360(1):226-238.

[3]. Blood . 2018 Sep 6;132(10):1039-1049.

Fenbubrutinib is being investigated in the clinical trial NCT03174041 (GDC-0853, a study of drug interactions with midazolam, itraconazole, rosuvastatin, and simvastatin). Fenbubrutinib is an oral Bruton's tyrosine kinase (BTK) inhibitor with potential antitumor activity. After administration, fenbubrutinib inhibits BTK activity and blocks activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and activation of BTK-mediated downstream survival pathways, thereby inhibiting the growth of BTK-overexpressing malignant B cells. BTK is a member of the Src-associated BTK/Tec family of cytoplasmic tyrosine kinases and is overexpressed in B-cell malignancies; it plays an important role in B lymphocyte development, activation, signal transduction, proliferation, and survival. Bruton's tyrosine kinase (BTK) is a non-receptor cytoplasmic tyrosine kinase involved in the activation of B cells and myeloid cells, located downstream of the B-cell receptor and Fcγ receptor, respectively. Preclinical studies have shown that inhibiting Btk activity may provide a potential treatment for autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. This article reports the discovery and preclinical characteristics of a highly potent, selective, non-covalent Btk inhibitor that is currently in clinical development. GDC-0853 (29) inhibits disease components mediated by B cells and myeloid cells and exhibits dose-dependent activity in a rat model of inflammatory arthritis. In preclinical and ongoing Phase II studies in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria, the drug has demonstrated highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles. Based on its high potency, selectivity, long target dwell time, and non-covalent mode of inhibition, compound 29 is expected to be a top Btk inhibitor for the treatment of a variety of immune diseases. [1]

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The clinical success of ibrutinib has demonstrated that Bruton's tyrosine kinase (BTK) inhibition is an effective strategy for the treatment of hematologic malignancies, including chronic lymphocytic leukemia (CLL). Although ibrutinib can achieve durable remission in patients, acquired resistance can still occur, most commonly due to mutations at the C481 site of BTK in the ibrutinib binding site. This article characterizes a novel BTK inhibitor, GDC-0853, and evaluates its preclinical efficacy in ibrutinib-naïve and ibrutinib-resistant patients with chronic lymphocytic leukemia (CLL). GDC-0853 differs from previously reported BTK inhibitors in that it does not rely on a covalent reaction with C481 to stabilize its occupancy at the BTK adenosine triphosphate (ATP) binding site. Similar to ibrutinib, GDC-0853 effectively reduces B-cell receptor signaling, cell viability, NF-κB-dependent transcription, activation, and migration in untreated CLL cells. We found that GDC-0853 inhibited the most common ibrutinib-resistant BTK mutant (C481S) in both biochemical enzyme activity assays and in stably transfected 293T cell lines, and maintained cytotoxicity in CLL cells from patients carrying the C481S BTK mutation. In addition, GDC-0853 did not inhibit endothelial growth factor receptor (EGFR) or ITK, which are two alternative targets of ibrutinib that may cause some adverse reactions and may reduce the efficacy of ibrutinib in combination with antibodies. Our results using GDC-0853 suggest that non-covalently selective BTK inhibitors may be effective for chronic lymphocytic leukemia (CLL), whether as monotherapy or in combination with therapeutic antibodies, especially for patients who have developed resistance to ibrutinib. [3] In conclusion, we have identified a potential best-in-class BTK inhibitor, GDC-0853 (29), which is currently under clinical investigation for a variety of immune diseases. It exhibits high efficacy and is the most selective BTK inhibitor reported to date. Its favorable preclinical pharmacokinetic profile suggests potential for once-daily oral administration. Furthermore, the efficacy data reported in this paper indicate its potential application in treating rheumatoid arthritis and other B-cell or myeloid cell-mediated autoimmune diseases. These findings, combined with its excellent tolerability and safety profile demonstrated in various animal studies, have prompted us to advance clinical investigations of compound 29 in the field of autoimmune diseases. In single-dose escalation (SAD) studies (0.5 mg to 600 mg) and multiple-dose escalation (MAD) studies (250 mg twice daily to 500 mg once daily) in healthy volunteers, GDC-0853 was well-tolerated, with no serious adverse events, safety signals, or dose-limiting toxicities observed. Moreover, compound 29 is well absorbed and exhibits linear dose-proportional pharmacokinetic characteristics. We evaluated its target binding (CD63, CD69, pBTK) and found that the pharmacodynamic markers were completely inhibited within 24 hours. Based on satisfactory Phase I clinical trial results, compound 29 has entered Phase II clinical trials for rheumatoid arthritis, lupus, and chronic urticaria. Further details and clinical results will be released in due course. [1]

C37H45CLN8O4

701.26

700.325

2128304-54-9

1434048-34-6;2128304-54-9 (HCl);2128304-53-8 (mesylate);2128304-55-0 (sulfate);

154723936

Typically exists as solid at room temperature

1.59

3

9

7

50

1340

1

C[C@H]1CN(CCN1C2=CN=C(C=C2)NC3=CC(=CN(C3=O)C)C4=C(C(=NC=C4)N5CCN6C7=C(CC(C7)(C)C)C=C6C5=O)CO)C8COC8.Cl

UFNQEETXQCTBNF-BQAIUKQQSA-N

InChI=1S/C37H44N8O4.ClH/c1-23-18-42(27-21-49-22-27)9-10-43(23)26-5-6-33(39-17-26)40-30-13-25(19-41(4)35(30)47)28-7-8-38-34(29(28)20-46)45-12-11-44-31(36(45)48)14-24-15-37(2,3)16-32(24)44;/h5-8,13-14,17,19,23,27,46H,9-12,15-16,18,20-22H2,1-4H3,(H,39,40);1H/t23-;/m0./s1

(S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one hydrochloride

RG-7845 HCl; GDC-0853 HCl; RG7845 HCl; GDC 0853; RG 7845 hydrochloride; GDC0853 HCl

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.)