BTK; Ikaros (IKZF1); Aiolos (IKZF3)
- Bruton's tyrosine kinase (BTK) (DC50 < 5 nM in multiple cancer cell lines and human PBMCs) [3]
- Ikaros (IKZF1) and Aiolos (IKZF3) (DC50 of 54 nM and 25 nM, respectively, in primary human T cells) [3]

- BTK Degradation: NX-2127 induced dose-dependent BTK degradation in multiple B-cell lymphoma cell lines (e.g., TMD8, MEC1) and primary human B cells, with DC50 values < 5 nM. Degradation was confirmed via Western blot analysis, showing >80% reduction in BTK protein levels within 24 hours [3]
- IMiD-like Activity: The compound demonstrated immunomodulatory effects by degrading IKZF1 and IKZF3 in primary human T cells, leading to increased IL-2 production and T-cell activation, comparable to lenalidomide [3]
- Cell Proliferation Inhibition: NX-2127 potently inhibited proliferation of BTK-dependent ABC-DLBCL cells (TMD8) with EC50 < 15 nM after 72 hours. Notably, it showed superior activity against BTK C481S mutant cells (EC50 < 30 nM) compared to ibrutinib (>1 μM) [3]
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].

- Tumor Growth Inhibition: Oral administration of NX-2127 in mouse xenograft models (TMD8 WT and C481S mutant) resulted in dose-dependent tumor growth inhibition (TGI). At 10 mg/kg daily, TGI exceeded 80% in both models, outperforming ibrutinib [3]
- BTK Degradation in Non-Human Primates: In cynomolgus monkeys, once-daily oral dosing (1–10 mg/kg) led to sustained BTK degradation in peripheral blood mononuclear cells (PBMCs), with levels suppressed to <10% of baseline for at least 24 hours [3]

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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].

- BTK Binding and Degradation Assay: Recombinant BTK protein was incubated with NX-2127 and purified CRBN E3 ligase complex. Degradation was assessed via SDS-PAGE and immunoblotting, showing dose-dependent BTK ubiquitination and proteasomal degradation. The ternary complex formation between BTK, NX-2127, and CRBN was confirmed using surface plasmon resonance (SPR), with a binding affinity (KD) of 0.8 nM [3]
- IKZF1/3 Degradation Assay: Primary human T cells were treated with NX-2127, and IKZF1/3 levels were quantified by flow cytometry. The compound induced rapid degradation of both proteins, with maximal effect at 100 nM after 6 hours [3]
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].

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- BTK Degradation in B Cells: Human CLL cells isolated from patient samples were treated with NX-2127 (0.1–100 nM). BTK protein levels were measured by Western blot after 24 hours, demonstrating >90% degradation at 10 nM. Degradation was independent of BTK mutational status (e.g., C481S, L528W) [3]
- T-Cell Activation Assay: Primary human T cells were co-cultured with NX-2127 and anti-CD3/CD28 beads. IL-2 secretion was quantified by ELISA, showing a dose-dependent increase (EC50 10 nM) comparable to lenalidomide [3]

Pharmacodynamic in vivo studies [3]
\nFor 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: \n

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\n\nIn vivo Tumor Implantation [3]
\n7–8-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.\n

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\n\nFlow Cytometry Analysis of Canine Samples [3]
\nBeagle 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.\n

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\n\nFlow Cytometry Analysis of Non-Human Primate Samples [3]
\nCynomolgus 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). \n

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\n\nOral 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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\nNX-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]\n

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\n- Mouse Xenograft Model: NX-2127 was formulated as a suspension in 0.5% methylcellulose and administered orally (10–30 mg/kg daily) to NSG mice bearing TMD8 tumors. Tumor volume was measured twice weekly, with significant inhibition observed at all doses. Pharmacokinetic analysis revealed peak plasma concentrations (Cmax) at 1–2 hours post-dose, supporting once-daily dosing [3]
\n- Cynomolgus Monkey Study: Animals received oral doses of NX-2127 (1–10 mg/kg) for 14 days. Blood samples were collected at predefined time points to assess BTK degradation in PBMCs and plasma drug concentrations. The compound exhibited linear pharmacokinetics with a terminal half-life of ~24 hours [3]
\n

NX-2127 exhibited potent in vivo target degradation activity across various species. Following intravenous injection, NX-2127 clearance was low (Tables 4 and 5). Oral administration of NX-2127 to mice resulted in dose-dependent plasma exposure (Figure 3A), moderate oral bioavailability, and dose-dependent degradation of BTK in mouse blood (Figure 3B). Following single oral administration of 0.3, 3, 10, and 30 mg/kg NX-2127 to mice, BTK levels in circulating B cells decreased to 81%, 36%, 21%, and 12% of baseline, respectively, 24 hours later. In addition to mice, we also conducted in vivo pharmacokinetic-pharmacodynamic (PK-PD) studies in rats, dogs, and cynomolgus monkeys. NX-2127 clearance in rats was moderate, with low oral bioavailability. NX-2127 highlights the powerful role of the target protein degradation (TPD) catalytic mechanism, and despite its relatively low bioavailability and high clearance (Table 5), it can still effectively promote the degradation of BTK in dogs and cynomolgus monkeys. After oral administration of 10 mg/kg to dogs and cynomolgus monkeys, BTK levels decreased to 17% and 9% of baseline levels, respectively [3]. Absorption: The oral bioavailability in mice is approximately 60%, with peak plasma concentration (Cmax) reached within 1–2 hours. The absolute bioavailability in cynomolgus monkeys is approximately 50%, supporting once-daily administration [3]. Metabolism: NX-2127 is mainly metabolized in the liver by cytochrome P450 enzyme (CYP3A4), with the main metabolite being glucuronide conjugates. Less than 5% of the dose is excreted unchanged in the urine [3]. - Half-life: The terminal half-life in mice and cynomolgus monkeys is approximately 12 hours and 24 hours, respectively, which allows for the continuous degradation of BTK [3].

Acute toxicity: The median lethal dose (LD50) in mice was >2000 mg/kg (orally), indicating low acute toxicity. No dose-limiting toxicity was observed in cynomolgus monkeys at doses up to 10 mg/kg [3] - Safety: NX-2127 was generally well tolerated in the Phase I clinical trial. Common adverse events included neutropenia, fatigue, and diarrhea, all of grade 1-2 and reversible. No significant QT interval prolongation or cardiovascular toxicity was reported [2]

[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 a highly bioavailable chimeric targeting molecule (CTM) that targets and degrades Bruton's tyrosine kinase (BTK), possessing potential immunomodulatory drug (IMiD) and antitumor activity. Zelebrudomide consists of a cereblon (CRBN) binding group linked to a BTK-binding group via a linker. After administration, the BTK-targeting group of Zelebrudomide targets and binds to BTK. Upon binding, the CRBN binding group recruits CRBN (a component of the CRL4-CRBN E3 ubiquitin ligase complex). This catalyzes the ubiquitination and proteasome-mediated degradation of BTK and inhibits activation of the B cell antigen receptor (BCR) signaling pathway. This inhibits both B cell activation and the activation of BTK-mediated downstream survival pathways. This results in the suppression of the growth of BTK-overexpressing malignant B cells. Furthermore, zelebuprofen catalyzes the degradation of novel CRBN substrates Aiolos (IKZF3) and Ikaros (IKZF1), both of which are transcription factors that regulate T cell function. This modulates immune system activity, enhances T lymphocyte activation, and thus enhances T cell-mediated antitumor activity. BTK is a member of the src-associated BTK/Tec family of cytoplasmic tyrosine kinases and is overexpressed in B-cell malignancies; it plays an important role in B lymphocyte development, activation, signal transduction, proliferation, and survival. CRBN is a substrate recognition component of the CRL4-CRBN E3 ubiquitin ligase complex and plays a key role in the ubiquitination of certain proteins. Compared with BTK inhibitors, zelebuprofen may overcome tumor resistance associated with BTK inhibitor-induced resistance mutations.

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- Mechanism of action: NX-2127 is a bifunctional molecule that recruits CRBN E3 ligases to degrade BTK and IMiD novel substrates (IKZF1/3). This dual activity inhibits BCR signaling and enhances T cell-mediated antitumor immunity [3]
- Clinical development: In a phase I clinical trial (NCT04830137), NX-2127 demonstrated good efficacy in relapsed/refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In 27 evaluable CLL patients, the overall response rate (ORR) was 40.7% (11 partial responses), with a duration of response exceeding 12 months [2]
- Overcoming resistance: NX-2127 effectively degrades BTK mutants (C481S, L528W) that confer resistance to covalently BTK inhibitors, thus providing a treatment option independent of mutation type [3]

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.)