BTK; Ikaros (IKZF1); Aiolos (IKZF3)

With an EC50 value of <30 nM, NX-2127 inhibits the proliferation of BTK-C481S mutant TMD8 cells[1]. Primary human T cells produce more IL-2 when exposed to NX-2127 [1].

Cynomolgus monkeys treated with NX-2127 (1 mg/kg; oral; once daily for 14 days) showed effective BTK degradation [1]. Oral dosing of NX-2127 causes BTK in plasma to degrade to less than 10% of baseline levels in circulating and splenic B cells, with exposure occurring in a dose-proportionate manner [1]. In mouse WT TMD8 and C481S mutant xenograft models, NX-2127 results in greater tumor growth inhibition (TGI) [1].

Assessment of BTK degradation by homologous time-resolved fluorescence (HTRF) Cells were incubated with compounds for 4 hours, and BTK levels were determined using a Cisbio Total-BTK HTRF kit (63ADK064PEG) according to manufacturer’s protocol. HTRF ratio was calculated using the equation below: [3]

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In addition to potent BTK degradation, NX-2127 possesses IMiD-like properties through the design of the CRBN binding harness that catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1). This activity is associated with increased T cell activation and anti-tumor effects of the IMiD drugs lenalidomide and pomalidomide. In primary human T cells, NX-2127 catalyzes the degradation of Aiolos and Ikaros with of 25 nM and 54 nM, respectively, potencies which are similar to those of lenalidomide (20 nM and 343 nM, respectively). Corresponding with such degradation, NX-2127 stimulates T cell activation as measured by increased IL-2 production in primary human T Cells in a manner similar to lenalidomide and pomalidomide. The dual activity of BTK degradation combined with immunomodulation of NX-2127 supports its development for the treatment of B-cell malignancies. [1]

Assessment of Aiolos and Ikaros degradation in human PBMCs [3]
Human peripheral blood mononuclear cells (PBMCs) were treated with 2000-0.00512 nM compound for 24 hours at 37 °C, and then cells were fixed and permeabilized using a Foxp3/Transcription Factor Fixation/Permeabilization Kit. Cells were stained with antibodies against Aiolos, Ikaros, CD20, and CD3. Additional samples were stained with Aiolos and Ikaros isotype control antibodies as staining controls. Flow cytometry was performed on an Attune NxT Acoustic Focusing Flow Cytometer and data were analyzed using FlowJo (v10.5.3) and GraphPad Prism. T cells (CD3+ CD20-) were gated, and the geometric mean fluorescence intensity (MFI) of Aiolos and Ikaros protein was calculated.

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Quantification of IL-2 secretion by activated T cells [3]
For T cell activation, primary human T cells were isolated from Leukopaks by immunomagnetic negative selection according to manufacturer’s protocol. T cells were pre-treated with compound for one hour and then stimulated for 24 hours with plate bound anti-CD3 antibody (1 µg OKT3/well) and 5 µg/mL soluble antiCD28 (clone 28.2) in the presence of compound. Total IL-2 in the cell-free supernatant was evaluated by ELISA

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Proteomics Assay [3]
Human PBMCs (2 million cells per sample) were treated with DMSO or degrader at a concentration of 50 nM for 4h. Cells were tested for viability and washed with PBS prior to storage at -80° C. Proteomic sample preparation was performed using PreOmics iST 96x kit. Cells were subsequently lysed before proceeding to proteolytic digestion and clean-up. Upon completion of sample processing, data acquisition was performed by liquid chromatography tandem mass spectrometry (LCMS/MS) via data independent analysis (DIA). Acquired DIA spectra were processed using Spectronaut (17.1.221229.55965 using library-free S75 proprietary directDIA algorithm and the default BGS factory settings. Search results (precursors and proteins) with Q valve cutoff of 1% were exported to a Nurix developed application for statistical analysis and visualization.

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NX-2127 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. NX-2127 degrades 50% of cellular BTK (DC50) at < 5 nM across multiple cancer cell lines and in human PBMCs. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 15 nM after 72 hours). Importantly, NX-2127 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-2127 EC50 values of < 30 nM versus > 1 μM for ibrutinib)[1].

Pharmacodynamic in vivo studies [3]
For pharmacodynamic assessment, 8–9-week-old female BALB/cJ mice or CD-1 mice were orally dosed with compound or vehicle. Mice were bled via tail vein or cardiac puncture at the indicated time points. Red blood cells were lysed and remaining cells were stained with live/dead dye, then fixed, permeabilized using FoxP3/Transcription Factor Staining Buffer Set and stained with cell surface markers CD45, TCR β chain, CD3 and B220. For BTK intracellular staining detection, cells were stained with antiBTK (clone D3H5, Cell Signaling Technology Cat. 8547BC) primary and AF647-anti-Rabbit secondary antibodies with mouse FC block (BD553142). Flow cytometry analysis was performed on an Attune NxT Acoustic Focusing Flow Cytometer and data were analyzed using FlowJo and GraphPad Prism. B cells (CD45+/B220+/TCR β-) and T cells (CD45+/B220-/TCR β+) BTK MFIs were measured and degradation was calculated using the following equation:

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In vivo Tumor Implantation [3]
7–8-week-old female CB.17 SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcrIcoCrl) were subcutaneously implanted into the mid-flank with 1 x 107 cells of TMD8 cells resuspended in Hank’s Balanced Salt Solution (HBSS) and MatrigelTM at a ratio of 1:1. When tumors reached 60-175 mm3 , daily oral treatment of either NX-2127, ibrutinib, or vehicle was initiated. Body weight and tumor volumes were measured twice weekly. Tumors were measured using the formula of length x width2 x 0.5. Tumor growth inhibition (% TGI) was calculated using the equation [1 − (T − T0/C − T0)] × 100, where T and C represent the mean size of tumors in the treated (T) and control (C) groups, and T0 refers to the tumor size at randomization.

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Flow Cytometry Analysis of Canine Samples [3]
Beagle dogs (Canis familiaris) were dosed once with NX-2127 through either intravenous administration at 1 mg/kg or oral administration at 10 mg/kg. Animals were bled pre-dose and at intervals following dosing, and blood samples were collected into Cyto-Chex BCT tubes.

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Flow Cytometry Analysis of Non-Human Primate Samples [3]
Cynomolgus monkeys were dosed orally once daily with vehicle control or NRX-0492 or NX-2127 (10, 30, or 100 mg/kg) at BASi or Charles River Laboratories (Ashland, OH), respectively. Animals were bled on day 1 and 14 and blood samples were collected into Cyto-Chex BCT tubes (Streck, 213386).

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Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to <10% of baseline levels at doses as low as 1 mg/kg.[1]

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NX-2127-001 is a first-in-human, dose escalation (Phase 1a) and cohort expansion (Phase 1b) study designed to evaluate the safety, tolerability, and preliminary efficacy of NX-2127 in adult patients with relapsed/refractory B-cell malignancies with once daily oral dosing. Dose escalation will proceed using a modified Fibonacci design with 1 patient per cohort, proceeding to a standard 3 + 3 design based on protocol specified criteria. There will be up to 5 expansion cohorts in Phase 1b enrolling patients with CLL/SLL, DLBCL, FL, MCL, MZL, and WM. Key eligibility criteria include >2 two prior lines of therapy (>1 prior for WM); measurable disease; and an Eastern Cooperative Oncology Group performance status of 0 or 1. Approximately 130 patients (30 in Phase 1a, 100 in Phase 1b) will be enrolled and treated until disease progression or unacceptable toxicity. The primary objectives are to evaluate safety and tolerability and to determine the maximum tolerated dose (Phase 1a), and to evaluate the early clinical activity of NX-2127 in expansion cohorts (Phase 1b). The Phase 1a part of this study is currently enrolling in the United States. Clinical trial information: NCT04830137.[2]

NX-2127 possesses potent in vivo target degradation activity across species. Upon iv administration, NX-2127 demonstrated low clearance (Tables 4 and 5). Oral dosing of NX-2127 in mice resulted in dose-dependent plasma exposure (Figure 3A), moderate oral bioavailability, and dose-dependent degradation of BTK in mouse blood (Figure 3B). Single, oral doses of 0.3, 3, 10, and 30 mg/kg of NX-2127 in mice resulted in reduction of BTK levels to 81%, 36%, 21%, and 12% of baseline in circulating B cells after 24 h. In addition to mice, we conducted in vivo PK–PD experiments in rat, dog, and cynomolgus monkey. NX-2127 had moderate clearance and low oral bioavailability in rat. Highlighting the power of the catalytic mechanism of targeted protein degradation (TPD), NX-2127 promoted potent BTK degradation in dog and cyno, despite relatively low bioavailability and high clearance (Table 5). Oral doses of 10 mg/kg in dog and cyno resulted in reduction of BTK levels to 17% and 9% of baseline BTK levels, respectively.[3]

[1]. Nx-2127, a degrader of BTK and IMiD neosubstrates, for the treatment of B-cell malignancies. Blood, 2020, 136: 34.

[2]. A first-in-human phase 1 trial of NX-2127, a first-in-class oral BTK degrader with IMiD-like activity, in patients with relapsed and refractory B-cell malignancies. 2022.

[3].Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies. J Med Chem. 2024 Feb 22;67(4):2321-2336.

Zelebrudomide is an orally bioavailable chimeric targeting molecule (CTM) and targeted degrader of Bruton's tyrosine kinase (BTK), with potential immunomodulatory drug (IMiD) and antineoplastic activities. Zelebrudomide is comprised of a cereblon (CRBN)-binding moiety conjugated, via a linker, to a BTK-binding moiety. Upon administration, zelebrudomide targets and binds to BTK with its BTK-targeting moiety. Upon binding, the CRBN-binding moiety recruits CRBN, a component of the CRL4-CRBN E3 ubiquitin ligase complex. This catalyzes ubiquitination and proteasome-mediated degradation 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. This leads to an inhibition of the growth of malignant B-cells that overexpress BTK. In addition, zelebrudomide catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1), two transcription factors regulating T-cell function. This modulates the activity of the immune system and increases the activation of T-lymphocytes, thereby increasing T-cell-mediated anti-tumor effects. 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. CRBN, the substrate recognition component of the CRL4-CRBN E3 ubiquitin ligase complex, plays a key role in the ubiquitination of certain proteins. Compared to BTK inhibitors, zelebrudomide may overcome tumor resistance associated with BTK inhibitor-induced resistance mutations.

C39H45N9O5

719.831907987595

719.354

C, 65.07; H, 6.30; N, 17.51; O, 11.11

2416131-46-7

146559796

Light yellow to yellow solid powder

3.7

3

11

9

53

1380

1

C1(C(N)=O)=NC=C(N2CCCCC2)N=C1NC1=CC=C(C2CCN(C[C@@H]3CCN(C4C=CC5=C(C=4)C(=O)N(C4CCC(=O)NC4=O)C5=O)C3)CC2)C=C1

XLWJWCMQMBVNSG-ACXKHFGCSA-N

InChI=1S/C39H45N9O5/c40-35(50)34-36(43-32(21-41-34)46-15-2-1-3-16-46)42-27-6-4-25(5-7-27)26-13-17-45(18-14-26)22-24-12-19-47(23-24)28-8-9-29-30(20-28)39(53)48(38(29)52)31-10-11-33(49)44-37(31)51/h4-9,20-21,24,26,31H,1-3,10-19,22-23H2,(H2,40,50)(H,42,43)(H,44,49,51)/t24-,31?/m0/s1

3-[4-[1-[[(3S)-1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]pyrrolidin-3-yl]methyl]piperidin-4-yl]anilino]-5-piperidin-1-ylpyrazine-2-carboxamide

NX-2127; 2416131-46-7; Zelebrudomide; 2-Pyrazinecarboxamide, 3-[[4-[1-[[(3S)-1-[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-5-yl]-3-pyrrolidinyl]methyl]-4-piperidinyl]phenyl]amino]-5-(1-piperidinyl)-; 3-[4-[1-[[(3S)-1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]pyrrolidin-3-yl]methyl]piperidin-4-yl]anilino]-5-piperidin-1-ylpyrazine-2-carboxamide; NX2127; LSC67HA8DE; SCHEMBL21947733;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 100 mg/mL (138.92 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.)