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Fenebrutinib (formerly RG-7845; GDC-0853) is a novel, potent, selective, orally bioavailable, and noncovalent (reversible) bruton's tyrosine kinase (BTK) inhibitor (Ki = 0.91 nM) with anticancer and anti-inflammatory activity. Treatment for systemic lupus erythematosus and rheumatoid arthritis is being developed. Administering GDC-0853 and other structurally diverse BTK inhibitors for at least seven days resulted in pancreatic lesions in Sprague-Dawley (SD) rats. These lesions included multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages, surrounded by lobular exocrine acinar cell atrophy, degeneration, and inflammation. At significantly higher exposures, no comparable results were seen in mice or dogs. Between four and seven daily doses of GDC-0853, peri-islet vasculature hemorrhage appeared. Histologically, this hemorrhage resembled changes that age-related SD rats naturally experience. This implies that GDC-0853 may make a background finding in juvenile animals worse. In the wake of a glucose challenge, there was dysregulation of glucose homeostasis; however, this was not linked to the onset or severity of pancreatic lesions and only happened after 28 days of administration. Other widely used blood biomarkers that evaluate exocrine and endocrine pancreatic function did not change. Neither magnetic resonance imaging nor computed tomography were able to easily identify these lesions. According to these findings, pancreatic lesions in rats are probably a class effect of BTK inhibitors, which could make an islet-centered pathology worse and make it less likely that humans will experience it. One of the cytoplasmic tyrosine kinases in the Tec family that is involved in B-cell and myeloid cell signaling is bruton's tyrosine kinase (BTK).
Physicochemical Properties
| Molecular Formula | C37H44N8O4 |
| Molecular Weight | 664.7965 |
| Exact Mass | 664.348 |
| Elemental Analysis | C, 66.85; H, 6.67; N, 16.86; O, 9.63 |
| CAS # | 1434048-34-6 |
| Related CAS # | 1434048-34-6;2128304-54-9 (HCl);2128304-53-8 (mesylate);2128304-55-0 (sulfate); |
| PubChem CID | 86567195 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 936.3±65.0 °C at 760 mmHg |
| Flash Point | 520.0±34.3 °C |
| Vapour Pressure | 0.0±0.3 mmHg at 25°C |
| Index of Refraction | 1.725 |
| LogP | 1.59 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 49 |
| Complexity | 1340 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | O1C([H])([H])C([H])(C1([H])[H])N1C([H])([H])C([H])([H])N(C2=C([H])N=C(C([H])=C2[H])N([H])C2C(N(C([H])([H])[H])C([H])=C(C=2[H])C2C([H])=C([H])N=C(C=2C([H])([H])O[H])N2C(C3=C([H])C4=C(C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C4([H])[H])N3C([H])([H])C2([H])[H])=O)=O)[C@@]([H])(C([H])([H])[H])C1([H])[H] |
| InChi Key | WNEODWDFDXWOLU-QHCPKHFHSA-N |
| InChi Code | InChI=1S/C37H44N8O4/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)/t23-/m0/s1 |
| Chemical Name | 10-[3-(hydroxymethyl)-4-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl]amino]-6-oxopyridin-3-yl]pyridin-2-yl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-9-one |
| Synonyms | RG-7845; GDC-0853; RG7845; GDC 0853; RG 7845; GDC0853; Fenebrutinib [INN]; (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; |
| HS Tariff Code | 2934.99.9001 |
| Storage |
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 (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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]. |
| ln Vitro |
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]. Fenebrutinib potently inhibits anti-IgM-induced CD69 expression on B cells in human whole blood with an IC₅₀ of 8.0 ± 6.0 nM. It inhibits anti-IgM-induced Btk Y223 autophosphorylation in human whole blood with an IC₅₀ of 11 ± 4.2 nM. In isolated primary human B cells, it inhibits anti-IgM-induced Btk Y223 phosphorylation with an IC₅₀ of 3.1 ± 0.7 nM. It inhibits anti-IgM and CD40L-induced B-cell proliferation with IC₅₀ values of 1.2 nM and 1.4 nM, respectively. It inhibits FcγRIII-triggered TNFα production in human monocytes with an IC₅₀ of 1.3 nM. It exhibits a long target residence time with Btk of 18.3 ± 2.8 h as determined in a jump-dilution enzyme assay. |
| ln Vivo |
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 (t1/2) 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 (Clp 10.9 mL/min/kg, Vd 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]. 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. Fenebrutinib dose-dependently reduces ankle swelling in a rat collagen-induced arthritis model when administered orally once daily (0.06–16 mg/kg) or twice daily (0.125–2 mg/kg). Plasma concentrations above the rat whole blood pBtk IC₇₀ (27 nM) for 12 h were associated with significant efficacy (∼75% reduction in ankle swelling). |
| Enzyme Assay |
Btk assays. [1] \nBtk 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. \nKinase selectivity.[1] \nBtk 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 (Kmapp) 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. \n\nKinase assay[3] \nA 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. \n\nLiver Microsome Metabolic Stability Assays [1] \nMetabolic 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. \n\nHepatocyte Metabolic Stability Assays [1] \nMetabolic 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). \n\n\nGDC-0853 Residence Time Assay [1] \nBtk 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 : Btk enzyme inhibition was assessed by monitoring Btk-catalyzed phosphorylation of a synthetic peptide substrate. Inhibitor Ki values were determined in at least triplicate. A jump-dilution assay was used to measure dissociation kinetics. Btk preincubated with Fenebrutinib was diluted into assay mixture, and delayed recovery of activity indicated slow dissociation. Data were fitted to a first-order dissociation model to calculate residence time. |
| Cell Assay |
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. 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. 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] 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. CD69 expression on CD19⁺ B cells in human whole blood was measured after stimulation with anti-IgM. Cells were treated with compound and stimulated overnight, followed by flow cytometry analysis. Btk Y223 phosphorylation in human whole blood was assessed using anti-phospho-Btk antibody after anti-IgM stimulation. In isolated primary human B cells, anti-IgM-induced Btk phosphorylation and proliferation were measured using phospho-specific antibodies and proliferation assays. In human monocytes, FcγRIII-triggered TNFα production was measured after stimulation. |
| Animal Protocol |
Sprague-Dawley, Wistar-Han and Fischer-344 rats (6 to 12 weeks old) 5 or 10 mL/kg p.o. 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. 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] In the rat collagen-induced arthritis model, female Lewis rats were immunized with type II collagen and orally dosed with Fenebrutinib once daily (0.06, 0.25, 1, 4, 16 mg/kg) or twice daily (0.125, 0.5, 2 mg/kg) for 16 days. Ankle diameter was measured daily. Pharmacokinetic studies in rats and dogs were conducted via cassette dosing. Rats received 0.2 mg/kg IV and 1.0 mg/kg PO; dogs received 0.2 mg/kg IV and 0.5 mg/kg PO. Blood samples were collected for plasma concentration analysis. |
| ADME/Pharmacokinetics |
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 rats, Fenebrutinib showed moderate clearance (27.4 mL/min/kg), volume of distribution of 5.42 L/kg, half-life of 2.2 h, and oral bioavailability of 65%. In dogs, clearance was 10.9 mL/min/kg, volume of distribution was 2.96 L/kg, half-life was 3.8 h, and oral bioavailability was 85%. Human liver microsome-predicted hepatic clearance was 7.2–17 mL/min/kg for related analogs. |
| Toxicity/Toxicokinetics |
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] Fenebrutinib showed no cytotoxicity in primary human hepatocytes (IC₅₀ > 300 μM). It was negative in Ames and micronucleus tests, indicating low genotoxicity risk. It showed low hERG channel inhibition (IC₅₀ > 30 μM). In Sprague–Dawley rats, pancreatic lesions were observed but were considered a rat-specific on-target effect not relevant to humans. In dogs, no observed adverse effect level was >80-fold above efficacious exposure. |
| References |
[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. |
| Additional Infomation |
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. Drug Indication Treatment of multiple sclerosis Fenebrutinib is a noncovalent Btk inhibitor with a binding mode orthogonal to covalent inhibitors like ibrutinib, allowing retention of activity against Btk C481S mutation. It is highly selective for Btk over other kinases, inhibiting only 3 of 286 off-target kinases at 1 μM. It entered Phase 2 clinical trials for rheumatoid arthritis, systemic lupus erythematosus, and chronic spontaneous urticaria. |
Solubility Data
| Solubility (In Vitro) | DMSO: ≥ 23 mg/mL (~34.6 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (1.50 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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. Solubility in Formulation 2: ≥ 1 mg/mL (1.50 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 10.0 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. Solubility in Formulation 3: ≥ 1 mg/mL (1.50 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5042 mL | 7.5211 mL | 15.0421 mL | |
| 5 mM | 0.3008 mL | 1.5042 mL | 3.0084 mL | |
| 10 mM | 0.1504 mL | 0.7521 mL | 1.5042 mL |