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]

Oral dosing with GDC-0853 (29) at 1, 4, and 16 mg/kg QD maintained plasma concentrations above the rat whole blood pBtk potency, as determined by inhibition of Btk Y223 autophosphorylation (IC50 = 9 nM, IC70 = 27 nM, IC90 = 135 nM), for a minimum of 12 h of the 24-h dosing period. BID dosing at 0.125, 0.5, and 2 mg/kg (Figure 9B) also maintained plasma concentrations above the IC50, IC70, and IC90 levels for a minimum of 6 h of the 12-h dosing period. Doses of 1 mg/kg QD and 0.5 mg/kg BID were associated with plasma concentrations that, at a minimum, exceeded the whole blood pBtk IC70 (27 nM) for approximately 12 h in a 24-h period. This exposure-efficacy relationship suggests that plasma concentrations of 29 in excess of the IC70 for 12 h were required to achieve efficacy, defined as a reduction of ∼75% of ankle swelling. Increased target coverage, as seen at the 4 mg/kg QD dose covered the IC90 for 24 h and offered further efficacy improvement. The efficacy of Btk inhibition by noncovalent inhibitors has also been assessed in mouse models of SLE. (14) Taken together, these impressive in vivo efficacy results with our noncovalent Btk inhibitors, combined with the excellent preclinical pharmacologic, pharmacokinetic and in vitro safety profile of 29, gave us confidence to progress this molecule into tolerability studies.[1]

In studies designed to assess the safety of the molecule, GDC-0853 (29) was well tolerated in both rats and dogs and displayed an overall favorable safety profile. The no observed adverse effect level (NOAEL) in dogs, the most sensitive preclinical species, was >80-fold higher than the targeted efficacious exposure, i.e., exceeding for 12 h the IC70 concentration (from the human whole blood CD69 assay). In Sprague–Dawley rats,GDC-0853 (29) and other structurally distinct Btk inhibitors have been shown to be associated with islet-centric pancreatic lesions at clinically relevant doses. After a thorough investigation involving evaluation of strain and species sensitivity differences, Btk knockout (KO) mice, and literature reports of humans with XLA mutations, we concluded that the GDC-0853-related pancreas findings in the Sprague–Dawley strain were the result of a rat-specific, strain-variable, on-target effect of Btk inhibition that is not relevant for humans. These conclusions have been supported by a histologic evaluation of the pancreas of untreated Btk KO Sprague–Dawley rats, that demonstrated the presence of identical pancreatic pathology. With a favorable safety profile and evidence that the observed pancreatic toxicity was a rat-specific phenomenon, we selected GDC-0853 (29) as our lead 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.

Fenebrutinib is under investigation in clinical trial NCT03174041 (A Drug-Drug Interaction Study Between GDC-0853 and Midazolam, Itraconazole, Rosuvastatin, and Simvastatin).
Fenebrutinib is an orally available inhibitor of Bruton's tyrosine kinase (BTK) with potential antineoplastic activity. Upon administration, fenebrutinib inhibits the activity of BTK and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and BTK-mediated activation of downstream survival pathways, which leads to the inhibition of the growth of malignant B-cells that overexpress BTK. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed in B-cell malignancies; it plays an important role in B-lymphocyte development, activation, signaling, proliferation and survival.

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Bruton's tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.[1]

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The clinical success of ibrutinib validates Bruton tyrosine kinase (BTK) inhibition as an effective strategy for treating hematologic malignancies, including chronic lymphocytic leukemia (CLL). Despite ibrutinib's ability to produce durable remissions in patients, acquired resistance can develop, mostly commonly by mutation of C481 of BTK in the ibrutinib binding site. Here, we characterize a novel BTK inhibitor, GDC-0853, to evaluate its preclinical efficacy in ibrutinib-naive and ibrutinib-resistant CLL. GDC-0853 is unique among reported BTK inhibitors in that it does not rely upon covalent reaction with C481 to stabilize its occupancy within BTK's adenosine triphosphate binding site. As with ibrutinib, GDC-0853 potently reduces B-cell receptor signaling, viability, NF-κB-dependent transcription, activation, and migration in treatment naïve CLL cells. We found that GDC-0853 also inhibits the most commonly reported ibrutinib-resistant BTK mutant (C481S) both in a biochemical enzyme activity assay and in a stably transfected 293T cell line and maintains cytotoxicity against patient CLL cells harboring C481S BTK mutations. Additionally, GDC-0853 does not inhibit endothelial growth factor receptor or ITK, 2 alternative targets of ibrutinib that are likely responsible for some adverse events and may reduce the efficacy of ibrutinib-antibody combinations, respectively. Our results using GDC-0853 indicate that noncovalent, selective BTK inhibition may be effective in CLL either as monotherapy or in combination with therapeutic antibodies, especially among the emerging population of patients with acquired resistance to ibrutinib therapy.[3]

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In summary, we have discovered a potential best-in-class Btk inhibitor, GDC-0853 (29), that is currently in clinical investigation for several immune disorders. It is highly potent and is the most selective Btk inhibitor reported to date. Its preclinical pharmacokinetic characteristics are favorable, indicating the potential for QD oral dosing. In addition, the supporting efficacy data reported here suggest that it could have utility in treating rheumatoid arthritis and other B-cell or myeloid cell mediated autoimmune diseases. These findings, combined with a very desirable tolerability and safety profile in multiple species, encouraged us to progress 29 into clinical studies in autoimmune diseases. In a single ascending dose (SAD) study (0.5 mg to 600 mg) and multiple ascending dose (MAD) study for 14 days (250 mg of BID to 500 mg of QD) in healthy volunteers, GDC-0853 was very well tolerated with no severe adverse events, no safety signals, and no dose limiting toxicities. Additionally, 29 was well absorbed and had linear, dose-proportional PK. Target engagement (CD63, CD69, pBTK) was assessed and complete suppression of PD markers was maintained over 24 h. With favorable Phase 1 results, 29 entered Phase 2 clinical studies in rheumatoid arthritis, lupus, and chronic urticaria. Further details and clinical results will be reported 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.)