BTK

Cys481 may play a role in how Elsubrutinib inhibits BTK, as Elsubrutinib inhibits BTK (C481S) with an IC50 of 2.6 μM, indicating a significant loss in potency upon exchanging the targeted thiol nucleophile with an alcohol. Elsubrutinib prevents BTK-dependent cellular activation and BTK enzyme activity irreversibly. Elsubrutinib blocks the release of histamine from basophils stimulated by IgE and IL-6 from monocytes stimulated by IgG, which use the Fce and Fcc receptors, respectively. Elsubrutinib inhibits IgM-mediated B cell proliferation, which is dependent on signaling through the BCR. Elsubrutinib also prevents TNF from being released from PBMCs stimulated by CpG-DNA, which is signaled through TLR9. However, it has no effect on TLRs that do not use ITAM motifs, such as TLR4 (which is stimulated with LPS) or TLR7/8 (which is stimulated with R848). Elsubrutinib significantly affects the proliferation of B cells mediated by IgM[1].

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Elsubrutinib (ABBV-105): An irreversible, selective and potent inhibitor of BTK [1]
ABBV-105 was optimized to irreversibly inhibit BTK using an acrylamide moiety as an electrophile to covalently modify the active site cysteine 481. A similar strategy has been shown to successfully provide durable BTK inhibition and a favorable selectivity profile against the majority of protein kinases, which lack an active site cysteine [24]. ABBV-105 inhibits activity of the catalytic domain of BTK in a time-dependent manner, decreasing BTK enzymatic activity with longer pre-incubation of BTK with drug (data not shown). As ABBV-105 has an irreversible interaction (see below), this IC50 is not an equilibrium estimate of binding affinity, but rather a surrogate for a reaction rate constant. Here we define the IC50 based on the degree of inhibition observed under the defined condition of a one hour enzymatic assay without pre-incubating drug and enzyme, and the resulting IC50 of ABBV-105 for BTK catalytic domain is 0.18 μM (Figure 1(A), Table 1). Mutating Cys481 to serine results in a BTK catalytic domain with a similar specific activity and ATP KM as the wild-type construct (not shown, manuscript in preparation). ABBV-105 inhibits BTK (C481S) with an IC50 of 2.6 μM, indicating a significant loss in potency upon exchanging the targeted thiol nucleophile with an alcohol, suggesting Cys481 is important in the manner in which ABBV-105 inhibits BTK.

The kinome selectivity of a covalent kinase inhibitor like Elsubrutinib (ABBV-105) needs to be addressed in terms of two distinct aspects: its potential covalent reactivity toward the ten other protein kinases with a cysteine in the analogous position (a time-dependent phenomenon) and its binding affinity toward the remainder of the kinome without an active site nucleophile (an equilibrium measurement). We generated activity assays for each of these 10 other kinases with an active site cysteine, maintaining the same time parameters as for the BTK method, and generated IC50 values for ABBV-105 with each. Selectivity ratios for ABBV-105 (relative to BTK) range from 33 to >280 for these ten kinases (Figure 1(B)). This experiment also revealed that for many kinases, ABBV-105’s selectivity was superior to a previously described BTK covalent inhibitor CC-292 also run in our panel. Relative to CC-292, ABBV-105 showed improvements in selectivity within the TEC family with increased ITK, ETK/BMX, TEC, and TXK selectivity ratios.

Elsubrutinib (ABBV-105) was also tested in the DiscoverX KINOMEscan® panel consisting of 456 kinases, which uses a ligand competition method. We evaluated kinome selectivity at a concentration that achieves 80% inhibition, the threshold we used in our PK/PD modeling to determine the efficacious exposure. This was found to occur at 0.015 μM ABBV-105 in a dose-response experiment in the DiscoverX BTK assay. Kinome profiling at 0.015 μM ABBV-105 found that only significant inhibition on BTK (Supplemental Table 1). The lower BTK biochemical IC50 in the DiscoverX panel (3.1 nM, not shown) is likely due to a longer incubation time than used to generate Figure 1(A), highlighting the importance of controlling for the time variable in comparing biochemical IC50 estimates for a time-dependent inhibitor.

To confirm that Elsubrutinib (ABBV-105) interacts irreversibly with BTK, we evaluated the dissociation kinetics of ABBV-105. ABBV-105, at the IC90 was mixed and incubated with BTK for 30 minutes to allow for complete association. This mixture was then diluted 400-fold to less than the IC10, and the enzymatic activity was measured by detection of the phosphorylated product by TR-FRET. The rate of recovery of kinase activity was measured relative to BTK that had not been exposed to inhibitor. Pre-treatment of BTK with ABBV-105 results in a lack of any measurable BTK activity once the compound is diluted, suggesting that the inhibitor is irreversibly bound to BTK for greater than 24 hours (Figure 1(C)).

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Evaluation of Elsubrutinib (ABBV-105) in multiple cellular signaling pathways [1]
As BTK is downstream of ITAM-coupled signaling receptors in several different immunological cell types, we evaluated ABBV-105 in cell-based assays to assess the specificity and mechanism of action of ABBV-105 (Figure 1(D); Table 1). ABBV-105 inhibited histamine release from IgE-stimulated basophils and IL-6 release from IgG-stimulated monocytes, which utilize Fcε and Fcγ receptors respectively. ABBV-105 inhibited IgM-mediated B cell proliferation, which is dependent on signaling through the BCR. ABBV-105 also inhibited TNF-release from CpG-DNA stimulated PBMCs, which signals through TLR9, although it did not inhibit the function of TLRs that do not use ITAM motifs, namely, TNF release from PBMCs stimulated either through TLR4 (with LPS) or through TLR7/8 (with R848).

Antibody responses to NP-Ficoll and NP-KLH are inhibited by elsubrutinib (10 mg/kg; po), but not to NP-LPS or Prevnar-13[1]. At 10 mg/kg QD and BID doses, elsubrutinib (0.1~10 mg/kg; po) effectively delays the start of proteinuria, prolongs survival, and inhibits paw edema throughout the course of the disease; lower doses do not appreciably inhibit these endpoints[1]. Paw volume gains are shown to be exposure-dependently inhibited by elsubrutinib. Elsubrutinib substantially reduces bone volume loss in a dose-dependent manner that is in line with the anti-inflammatory effects that have been noted[1].

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Evaluation of Elsubrutinib (ABBV-105) in thymus-independent and thymus-dependent antibody responses [1]
XLA patients demonstrate a lack of specific antibody production and an increased risk of bacterial infections. Similarly, BTK deficient mice fail to elicit normal antibody responses to both thymus-independent and thymus-dependent antigens. Since ABBV-105 has significant impacts on IgM-mediated B cell proliferation in vitro there is a possibility that clinical use of a BTK inhibitor could reduce or eliminate the ability of patients to mount antibody responses to antigenic challenges. To better understand the effect of ABBV-105 on antibody responses to different antigens, we examined the responses to the thymus-independent antigens NP-LPS and NP-Ficoll, the pneumococcal vaccine Prevnar, or to the thymus-dependent antigen NP-KLH.

We evaluated the anti-NP IgM and IgG3 responses in mice on day 7 following immunization with the thymus-independent antigens NP-LPS and NP-Ficoll. Mice were treated with Elsubrutinib (ABBV-105) 10mg/kg QD or BID starting one day prior to immunization. ABBV-105 did not significantly inhibit the anti-NP IgM or IgG3 response to NP-LPS (Figure 2(A)). In contrast, when mice were immunized with NP-Ficoll, the anti-NP IgM and IgG3 responses were both significantly inhibited (Figure 2(B)). BID dosing of ABBV-105 did not provide a significant increase in response compared to QD dosing. As a consequence of the covalent and irreversible nature of the binding of ABBV-105 to BTK, target engagement can be quantitated in vivo by measuring BTK occupancy using a biotinylated probe derived from a related covalent BTK inhibitor. Splenic BTK occupancy was measured 24 hours after the last dose of ABBV-105 for mice dosed QD and 12 hours for mice dosed BID. Splenic occupancy was similar between mice immunized with NP-LPS (10 mg/kg QD = 64.3% ± 8.1%; BID = 77.8% ± 1.5%) or NP-Ficoll (10 mg/kg QD = 53.1% ± 8.1%; BID = 65.6% ± 3.0%).

To evaluate the potential effect of Elsubrutinib (ABBV-105) on responses to vaccines, we evaluated the antibody response to the pneumococcal vaccine Prevnar. Treatment with a dose response of ABBV-105 began on the day of immunization with Prevnar and IgM and IgG antibody responses to the 13 polysaccharides contained in the vaccine were measured 14 days later. ABBV-105 did not significantly inhibit either the anti-pneumococcal IgM or IgG response, whereas the positive control cyclophosphamide completely abrogated the antibody response (Figure 2(C)). Splenic BTK occupancy was measured 24 hours after the last dose of ABBV-105 and demonstrated a dose dependent increase in occupancy (10 mg/kg = 46.8% ± 3.4%; 3 mg/kg = 40.2% ± 3.2%; 1 mg/kg = 34.3% ± 4.1%).

To further understand the effect of BTK inhibition on antibody responses, we evaluated the primary and secondary anti-NP IgM and IgG1 responses to the thymus-dependent antigen NP-KLH. Elsubrutinib (ABBV-105) did not impact anti-NP IgM responses prior to the boost on day 35 (Figure 2(D)). However, following the boost on day 35, ABBV-105 treatment significantly reduced anti-NP IgM antibodies, and this occurred both in mice that were treated from the beginning of the study as well as in those where treatment began just prior to the boost. No significant difference was observed between QD and BID treatment with ABBV-105. ABBV-105 did not significantly impact anti-NP IgG1 responses on days 7, 14, or 21 (Figure 2(E)). Treatment with 10 mg/kg ABBV-105 significantly inhibited the anti-NP IgG1 response on day 35, but the other treatment groups were not significantly different from the vehicle control. Splenic BTK occupancy was measured two hours after the last dose of ABBV-105 and demonstrated comparable occupancy across all groups (10 mg/kg QDD1–42 = 99.0% ± 0.2%; 10 mg/kg BIDD1–42 = 97.2% ± 0.5%; 10 mg/kg QDD34–42 = 98.9% ± 0.2%; 10 mg/kg BIDD34–42 = 97.7% ± 0.6%).

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Efficacy of Elsubrutinib (ABBV-105) in CIA and the relationship to BTK occupancy [1]
In RA, pathogenic antibodies activate downstream inflammatory processes in part through FcγR-mediated signaling. Since we had previously demonstrated that ABBV-105 inhibits IgG-stimulated cytokine production (Figure 1(D)), we next wanted to know if inhibition of BTK was sufficient to inhibit inflammation in a rodent model of arthritis known to have a significant contribution from FcγR signaling. Therefore, we evaluated ABBV-105 in a rat CIA model with therapeutic treatment, beginning at the first signs of inflammation.

Daily, oral treatment of rats with Elsubrutinib (ABBV-105) resulted in dose-dependent inhibition of paw swelling throughout the course of disease (Figure 3(A); n = 9 per group). Whole blood samples were collected from three animals per group to measure drug concentration at the end of the study and the area under the curve (AUC) drug concentration values ± SEM were used to evaluate the exposure response-relationship. Paw swelling data from two independent CIA experiments were used for the pharmacokinetic/pharmacodynamics (PKPD) modeling. We used a direct Emax model with drug concentration AUC selected as the exposure parameter for PKPD evaluation. ABBV-105 demonstrates exposure-dependent inhibition of increases in paw volume (Figure 3(B)). The exposures that provide 50% and 80% inhibition of paw swelling on the last day were calculated (AUC50, 0–24 = 4.5 ± 1.9 ng × hour/mL; AUC80, 0–24 = 19 ± 8 ng × hour/mL).

Within the rat CIA model, inflammatory processes result in bone destruction in the ankle joint mediated by osteoclast activity. In addition, studies in mice have demonstrated that BTK plays a significant role in osteoclastogenesis. To assess the disease modifying effect of Elsubrutinib (ABBV-105) on bone, we analyzed ankles by micro-computed tomography (μCT) at the termination of the studies. Bone erosions can be quantified by three-dimensional evaluation of the ankle joint to calculate total bone volume. Compared to vehicle control-treated animals, ABBV-105 significantly inhibited bone volume loss in a dose dependent manner consistent with the observed anti-inflammatory effects (Figure 3(C)).

To understand the association between target engagement and efficacy in the CIA model, we evaluated splenic BTK occupancy to assess the relationship between it and inhibition of paw swelling. Following administration, there was a rapid clearance of Elsubrutinib (ABBV-105) in plasma while occupancy of BTK by ABBV-105 in homogenized spleen samples is maintained for an extended period of time (Figure 3(D)). BTK occupancy increased in a dose-dependent manner, reaching a maximum at two hours post-dose and decreasing over time (Figure 3(E)). At 2 and 12 hours post-dose, there is a strong, positive correlation between BTK occupancy and inhibition of paw swelling (Figure 3(F)).

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Efficacy of Elsubrutinib (ABBV-105) in an IFNα accelerated lupus nephritis model [1]
Through its role in BCR signaling, BTK plays a key role in the development of pathogenic autoantibodies. This has been demonstrated in murine lupus models as demonstrated by studies that crossed the BTK deficient xid strain onto the lupus prone MRL/lpr background resulting in a reduction of autoantibody production. In addition, BTK inhibitors have demonstrated efficacy across both the MRL/lpr and NZB/W models of lupus nephritis. We therefore evaluated ABBV-105 in an IFN-α-accelerated model of lupus in NZB/W F1 mice. Typically, female mice of the F1 generation of NZB x NZW crosses develop proteinuria leading to increased mortality by 12 months of age. Administration of an IFN-α expressing adenovirus to pre-diseased mice can lead to a rapid onset of proteinuria and mortality, thereby creating a shorter model to characterize novel therapeutics.

We evaluated Elsubrutinib (ABBV-105) treatment in the IFN-α accelerated model, initiating QD or BID treatment seven days after the injection of the IFN-α adenovirus, but prior to the onset of proteinuria. ABBV-105 significantly prevented the onset of proteinuria and prolonged survival at the 10 mg/kg QD and BID doses, while lower doses did not significantly inhibit these endpoints (Figure 4(A,B); n = 20 per group). BID dosing with ABBV-105 did not provide any greater effect on survival or proteinuria than QD dosing. We also evaluated the effect of ABBV-105 on the production of anti-dsDNA autoantibodies on day 14 and 28 following IFN-α adenovirus administration. Both the 10 mg/kg QD and BID doses of ABBV-105 significantly reduced plasma levels of anti-dsDNA IgG antibodies on day 28 (Figure 4(C)). The anti-dsDNA IgG antibody levels were not significantly different between the 10 mg/kg BID and QD doses.

Evaluation of BTK enzyme occupancy [1]
Assessment of BTK covalent occupancy by Elsubrutinib (ABBV-105) was similar to published work. Briefly, snap frozen spleens were homogenized using a Dounce tissue homogenizer in the presence of protease inhibitors. Spleen homogenate was mixed with a covalent occupancy probe and incubated on a plate shaker two hours at room temperature. Samples were transferred to a streptavidin-coated 96-well plate and incubated on a plate shaker for one hour at room temperature. After washing, BTK was detected using rabbit anti-mouse BTK (1:1000) followed by goat anti-rabbit HRP-conjugated antibody (1:5000). Percent BTK occupancy was calculated as a proportion of free BTK in a vehicle-treated spleen compared to a compound-treated spleen.

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Biochemical assays [1]
Human BTK (aa 393–659) was expressed in SF9 cells with an N-terminal His6 tag. Activity was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) based detection of phosphorylated peptide (Biotin-(Ahx)-GAEEEIYAAFFA-COOH). Final concentrations: 9 nM enzyme (BTK), 0.2 μM peptide, 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4, and 0.01 mM ATP. Reaction was quenched at 60 minutes by with 100 mM EDTA, followed by 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K, and 0.6 μg/mL SAX. TR-FRET counts were measured after 60 minutes on a Rubystar (BMG). Reversibility of BTK Binding was measured by incubating 400 nM BTK with inhibitors at their IC90 for 30 minutes. Activity was measured as above after a 400-fold dilution into reaction buffer. Additional kinases assays were measured as above at ATP equivalent to its KM.

IgE-mediated basophil degranulation [1]
Heparinized human whole blood was incubated with inhibitor for 30 minutes at 37 °C, stimulated with anti-IgE (Beckman Coulter, Brea, CA), and incubated for 30 minutes at 37 °C. After holding on ice for 10 minutes, samples centrifuged for 10 minutes at 1000 rpm at 4 °C. Supernatants were analyzed for histamine by Homogeneous Time Resolved Fluorescence (HTRF).

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IgM-mediated human B-cell proliferation [1]
Primary human B cells were thawed, re-suspended in media (RPMI supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Penicillin-Streptomycin), and plated at 1 × 105 cells/well in 100 μL. Inhibitor in media/1% DMSO was added to cells and incubated for 30 minutes. Cells were then stimulated with anti-human IgM, incubated for 48 hours, pulsed with 1 μCi/25μL/well [CH3-3H] thymidine, and incubated 16–18 hours. Cells were harvested using the UniFilter-96 Cell Harvester. Samples were washed with 1× PBS, dried, and counted TopCount NXT HTS (PerkinElmer, Waltham, MA) after addition of 50 μL/well scintillant.

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IgG-mediated macrophage IL-6 production [1]
Primary human monocytes were added to inhibitor as 37 °C for 30 minutes (2 × 105 cells/well, in RPMI with 10% FBS, 1% Penicillin-Streptomycin, 1% L-glutamine, and 0.4% DMSO). Cells were added to plates coated with 40 ng/μL human serum IgG, incubated overnight, centrifuged, and supernatants were collected to evaluate IL-6.

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TLR-stimulated human PBMC TNF production [1]
Primary human PBMCs were thawed, re-suspended in media (RPMI supplemented with 2% FBS and 1% Penicillin-Streptomycin), plated at 2 × 105 cells/well, and incubated with inhibitor for 30 minutes. Cells were stimulated with either CpG (2.5 μM), LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO), or R848 (3 μg/ml). After 24 hours, plates were centrifuged, and supernatant was collected for TNFα.

Animal/Disease Models: Female C57/BL6 mice[1]
Doses: 10 mg/kg
Route of Administration: Po
Experimental Results: Inhibited antibody responses to NP-Ficoll and NP-KLH, but not to NP-LPS or Prevnar-13.

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Animal/Disease Models: Lewis rats[1]
Doses: 0.1~10 mg/kg
Route of Administration: Po
Experimental Results: Resulted in dose-dependent inhibition of paw swelling throughout the course of disease.

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Animal/Disease Models: NZBWF1 mice[1]
Doses: 0.1~10 mg/kg
Route of Administration: Po
Experimental Results: Dramatically prevented the onset of proteinuria and prolonged survival at the 10 mg/kg QD and BID doses , while lower doses did not Dramatically inhibit these endpoints.

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Immunization of mice with thymus-dependent and -independent antigens [1]
To assess antibody responses to thymus independent antigens, female C57/BL6 were immunized IP with either 20 μg NP-LPS or NP-Ficoll (Biosearch Technologies, Petaluma, CA) in PBS. Mice were dosed orally with Elsubrutinib (ABBV-105) once a day (QD) or twice a day (BID) beginning one day prior to immunization. After seven days, mice were sacrificed and plasma was collected for anti-NP antibody analysis.

To assess antibody responses to Prevnar-13 vaccine, female C57/BL6 mice were immunized IP with 1 μg Prevnar-13 in PBS. Mice were dosed orally QD with Elsubrutinib (ABBV-105) for 14 days or IP three times per week with cyclophosphamide beginning on the day of immunization. Plasma was collected on day 14 to evaluate antibody titers.

For evaluation of the antibody response to the thymus dependent antigen, female C57/BL6 mice were immunized IP with 100 μg NP(23)-KLH in PBS with Imject Alum Thermo Scientific, IL). After 35 days, mice were boosted with 100 μg NP(23)-KLH in PBS. Mice were dosed orally QD or BID with 10mpk Elsubrutinib (ABBV-105). Treatment started on day –1 continuing through day 42 or started on day 35 prior to boost continuing through day 42. Mice were bled, and plasma collected on days 7, 14, 21, 35, and 42.

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Induction of collagen-induced arthritis [1]
Lewis rats were immunized with 600 µg bovine type II collagen in incomplete Freund’s adjuvant (IFA) intradermally on day 0 on the base of tail, left flank, and right flank and boosted with collagen in IFA near the same locations on day 6. Paw volume was measured using a micro-controlled volume meter plethysmograph with left and right paw volumes averaged for analysis. Paw volume baseline was assessed on day 8 (baseline), 11, 13, 15, and 18. Rats were dosed orally QD for seven days beginning on day 11 post-immunization with Elsubrutinib (ABBV-105). Data are presented as a pooled analysis of two independent experiments. On day 18, plasma was collected to measure drug concentrations from three animals per group at 0.25, 0.5, 1, 2, 4, 6, 12, and 24 hours post-dose. For BTK occupancy analysis spleens were collected and frozen in liquid nitrogen from three animals per group at 2, 12, and 24 hours post-dose.

[1]. ABBV-105, a selective and irreversible inhibitor of Bruton's tyrosine kinase, is efficacious in multiple preclinical models of inflammation [published correction appears in Mod Rheumatol. 2019 May;29(3):v]. Mod Rheumatol. 2019;29(3):510-52.

[3]. Primary carboxamides as Btk inhibitors. Patent WO2014210255Al.

Elsubrutinib is under investigation in clinical trial NCT04451772 (A Study of the Safety of Oral Elsubrutinib Capsules and Oral Upadacitinib Tablets Given Alone or in Combination (ABBV-599) for Adult Participants With Moderately to Severely Active Systemic Lupus Erythematosus to Assess Change in Disease State).
ELSUBRUTINIB is a small molecule drug with a maximum clinical trial phase of II (across all indications) and has 2 investigational indications.

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Objectives: Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease.
Methods: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker.
Results: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy.
Conclusion: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.[1]

C17H19N3O2

297.351663827896

297.147

C, 68.67; H, 6.44; N, 14.13; O, 10.76

1643570-23-3

Elsubrutinib;1643570-24-4

117773886

White to off-white solid powder

1.6

2

2

3

22

465

1

N1C2=C(C([C@H]3CCCN(C(=O)C=C)C3)=CC=C2C(N)=O)C=C1

UNHZLHSLZZWMNP-NSHDSACASA-N

InChI=1S/C17H19N3O2/c1-2-15(21)20-9-3-4-11(10-20)12-5-6-14(17(18)22)16-13(12)7-8-19-16/h2,5-8,11,19H,1,3-4,9-10H2,(H2,18,22)/t11-/m0/s1

4-[(3R)-1-prop-2-enoylpiperidin-3-yl]-1H-indole-7-carboxamide

(R)-Elsubrutinib; 1643570-23-3; SCHEMBL16337373; (R)-ABBV-105;

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: 25 mg/mL (84.08 mM)

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.)