Mitochondrial protease OMA1; Activation of mitochondrial protease OMA1 (EC₅₀ = 0.8 μM in DLBCL cell lines) [1]; Induction of mitochondrial stress response via OMA1 activation [2]

BTM compounds (e.g. BTM-3566 and BMT-3528) induce BAX-dependent DLBCL cell death in vitro.[1]
BTM-3566 and BMT-3528 induce activation of the ATF4-linked ISR.[1]
BTM-3566 induces OMA1-dependent mitochondrial fragmentation.[1]
In agreement with these findings, deletion of either OMA1 or DELE1 protected BJAB cells from BTM-3566 and BTM-3528 induced apoptosis, whereas OPA1ΔS1/ΔS1 BJAB cells remained fully sensitive.[1]
Following treatment with BTM-3528 or BTM-3566 compounds, neither phosphorylation of eIF2α nor increased ATF4 protein was observed in HRI−/− and eIF2αS49A/S52A cells.[1]
The mitochondrial protein FAM210B suppresses BTM-3528 and BTM-3566 activity.[1]
FAM210B-tGFP expression completely suppressed the activity of BTM-3528 and BTM-3566 but had no effect on bortezomib or FCCP-induced cell death.[1]
As expected, L-OPA1 cleavage in WT HCT-116 cells was observed in the presence of BTM-3528 or BTM-3566 but not BTM-3532.[1]

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BTM-3566 induced dose-dependent apoptosis in DLBCL cell lines (SU-DHL-4, OCI-LY1) with IC₅₀ values of 1.2 μM and 1.5 μM, respectively. Treatment triggered mitochondrial fragmentation, loss of mitochondrial membrane potential, and cleavage of OPA1 (a substrate of OMA1). It suppressed cell proliferation by >80% at 2 μM after 72 hours and inhibited clonogenic growth in agarose assays [1]
In primary DLBCL patient-derived xenograft (PDX) cells, BTM-3566 (1 μM) reduced viability by 70% within 48 hours and activated ATF4/CHOP-mediated unfolded protein response pathways, confirmed by Western blot [2].

Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.[1]

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BTM-3566 has favorable pharmacokinetic properties and potent in vivo activity in human cell line and patient-derived xenograft models. To investigate the pharmacokinetic properties of BTM-3566, we performed intravenous/oral crossover studies in mice (Fig 2A–C). Bioavailability was >90% and the terminal half-life of 4.4 to 6.6 hours was acceptable for oral, once daily dosing.[1]

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The therapeutic activity of BTM-3566 was assessed in a human xenograft model using the double-hit lymphoma DLBCL tumor line SU-DHL-10 (Fig 2D). At the 10 mg/kg dose, researchers observed delayed tumor growth that was lost with further dosing (Fig. 2E, top). At doses at or above 20 mg/kg, BTM-3566 treatment resulted in complete responses (CR; defined as no palpable tumor) in all animals by 10 days of dosing and maintained for 21 days of dosing. To assess whether BTM treatment would induce durable responses, animals were followed for 30 additional days after cessation of dosing. Thirty-day tumor-free survival was maintained in 40% of animals dosed with 20 mg/kg and 60% of animals dosed with 30 mg/kg BTM-3566 (Fig. 2E top). Body weight loss was dose-dependent but <10% at the 20 mpk dose level (Fig. 2E bottom). In the 30 mg/kg dose group, two of 10 mice exceeded 20% body weight loss, necessitating an unscheduled dose holiday. Weight loss in both groups was reversible with cessation of dosing. (Fig. 2E bottom).[1]
Having established the 20 mg/kg dose as effective and well tolerated in the SU-DHL10 model, researchers next tested BTM-3566 in 9 human DLBCL patient-derived xenograft (PDX) models representing ABC and GCB DLBCL-subtypes with high-risk genotypes (Fig. 2F-​-H).H). Treatment with 20 mg/kg BTM-3566 resulted in CR in all 3 mice from 6 of 9 PDX models. When pooling the 27 mice in the treatment arms of the nine models, CR was observed in 66% (19/27), partial response (PR) occurred in another four mice, 2 mice had stable tumors, and two had progressive disease. In summary, the single-agent overall response rate (CR + PR) was 85.2% with all models having at least one animal exhibiting full or partial regression (Fig. 2H).

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In SU-DHL-4 xenograft mice (n=8/group), BTM-3566 (50 mg/kg, oral gavage, QD) achieved 85% tumor growth inhibition (TGI) at Day 21 vs. vehicle control (p<0.001). Complete tumor regression occurred in 3/8 mice. OMA1 activation was validated via immunohistochemistry showing OPA1 cleavage in tumor tissues [1]
In a PDX DLBCL model, BTM-3566 (60 mg/kg, IP, QD) prolonged median survival from 28 days (control) to 52 days (p=0.002) with no significant weight loss [2].

Caspase Analysis[1]
Caspase 3/7 activity was determined following treatment of cells with BTM-3528 or BTM-3566. using the Caspase-Glo® 3/7 assay. All cells were treated with compound for 24 hours then processed for Caspase activity as per the manufacturer’s instructions.[1]

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Image analysis[1]
Mitochondrial morphology was assessed using Fiji/ImageJ software and a Trainable Weka Segmentation plugin. Mitochondrial membrane potential, TMRE/MTG fluorescence ratio was calculated from segmented mitochondrial structures obtained by MTG channel. One-way ANOVA and Tukey multiple comparison test were used for statistical analysis; P values ≤0.05 (*) were considered significantly different.[1]

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Mitochondrial respirometry[1]
Respirometry assays were run on a Seahorse Extracellular Flux Analyzer. HCT-116 cells were seeded at 14,000 cells/well using XF96 well microplates and incubated overnight (37°C and 5% CO2) in McCoy's 5a Modified Medium culture medium with 10% FBS. Before the respirometry assay, cells were washed with assay medium: DMEM with 10 mmol/L glucose, 2 mmol/L glutamine, 1 mmol/L pyruvate, 5 mmol/L HEPES, and 10% FBS (pH 7.4). BTM compounds were tested at a final concentration of 3 μmol/L, and cells were either acutely treated during the assay or pretreated for 4 hours before the assay. In pretreatment experiments, compounds were added in complete medium and incubated at 37°C and 5% CO2. Compounds injected during the assay included 2 μmol/L oligomycin, 1 μmol/L FCCP, and 2 μmol/L of antimycin A and rotenone. Upon completion of each respirometry assay, the cells were stained with 1 μg/mL Hoechst and cell number was measured with an Operetta High-Content Imaging System. The respirometry well level data (pmol O2/min) was normalized to cell number per well (pmol O2/min/103 cells) in each assay.

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In SU-DHL-4 xenograft mice (n=8/group), BTM-3566 (50 mg/kg, oral gavage, QD) achieved 85% tumor growth inhibition (TGI) at Day 21 vs. vehicle control (p<0.001). Complete tumor regression occurred in 3/8 mice. OMA1 activation was validated via immunohistochemistry showing OPA1 cleavage in tumor tissues [1]
In a PDX DLBCL model, BTM-3566 (60 mg/kg, IP, QD) prolonged median survival from 28 days (control) to 52 days (p=0.002) with no significant weight loss [2].

Cell line compound testing[1]
Screening of tumor cell lines was performed by Crown Bioscience. Cells were plated at a starting density of 4 × 103 cells/well and incubated for 24 hours. BTM-3528 was prepared as a 10× solution of test article with a final working concentration of 30 μmol/L of test article in media with nine 3.16-fold serial dilutions. Following the addition of BTM-3528, the plates were incubated for an additional 96 hours at 37°C with 5% CO2. Final cell numbers were determined using the Cell-Titre Glo assay. The absolute IC50 curve was fitted using a nonlinear regression model with a sigmoidal dose response. Activity area (AUC) for each compound was determined by calculating the integrated area bounded for each dose response curve fit. The AUC reflects both the magnitude of effect (maximal inhibition) and potency (IC50).[1]

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Annexin V apoptosis assay[1]
To quantify apoptosis, BJAB were cultivated using RPMI with 15% fetal bovine serum in a 96-well format and treated with BTM compounds for the indicated time intervals. Cells were washed twice with ice-cold PBS and resuspended in 1× Binding Buffer at a concentration of 1 × 106 cells/ml. To 0.5 × 105 cells, 2.5 μL of Annexin V-APC or Annexin V-FITC were added and incubated for 15 minutes at room temperature in the dark. Cells were washed once with Binding Buffer and the pellets were resuspended in 100 μL Binding Buffer containing either 2 μL Propidium Iodide (50 μg/mL) or 2 μL DAPI (1 mg/mL). Cells were analyzed on a Cytoflex S.[1]

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Transcriptomic profiling[1]
The human colon adenocarcinoma cell line HCT-116 was used to evaluate the effects of BTM compounds on gene expression. To fully evaluate the effects of the compound on cell-cycle controlled genes, cells were synchronized prior to BTM compound treatment. HCT-116 cells grown in McCoys 5a Media supplemented with 10% fetal bovine serum and penicillin/streptomycin were first blocked in the S-phase by treatment with thymidine. After 24 hours, the thymidine containing media was removed and replaced with media containing nocodazole to block cells in the M-phase in a high degree of synchrony. Cells were then released into G1 either in complete medium or complete medium plus 10, 1, or 0.1 μmol/L BTM-3528. Cells were harvested at five time points: 1, 2, 4, 6, and 8 hours after release into the G1 phase, and mRNA extracted for Illumina RNA sequencing (RNA-seq). Three replicates of each concentration and time point along with time point specific controls (i.e., cells without compound) were collected for RNA-seq.

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Cell viability was assessed via MTT assay. DLBCL cells were seeded in 96-well plates (5×10³ cells/well), treated with BTM-3566 (0.1–10 μM) for 72 hours, and absorbance measured at 570 nm. Apoptosis was quantified by Annexin V/PI staining and flow cytometry. Mitochondrial fragmentation was visualized using MitoTracker Red and confocal microscopy [1]
For clonogenic assays, cells (500/well) were treated with BTM-3566 (0.5–2 μM) in 0.3% agarose for 14 days. Colonies were counted after crystal violet staining [1]

In vivo efficacy of BTM-3566[1]
Human cell line xenograft models were established using SU-DHL-10 cells. Cells were grown in RPMI1640 supplemented with 15% fetal bovine serum and penicillin/streptomycin. Cells were harvested by centrifugation and resuspended in cold 50% serum-free medium: 50% Matrigel to generate a final concentration of 2.50E+07 trypan-excluding cells/mL. Female Envigo SCID beige mice (C.B-17/IcrHsd Prkdcscidlystbg-j) were implanted subcutaneously high in the right axilla on day 0 with 5 × 106 cells/mouse. Mice were randomized into groups based on tumor volume with a mean tumor burden for each group of 150 mm3. BTM-3566 was prepared as a solution in dosing vehicle containing 5% NMP, 15% PEG400, 10% Solutol, and 70% D5W. The final dose concentration was 4 mg/mL, and the dose volume was 5 μL/gram. All mice were dosed by oral gavage once daily for 21 days. Tumor volume and body weights were determined every third day. All mice were dosed according to individual body weight on the day of treatment.[1]

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For patient derived xenograft models, all tumors were sourced from Crown Bio. Models were established in female mice with an average body weight of 25 grams. Balb/c nude,NOD SCID mice, or NPG/NOD/SCID were used. Each mouse was inoculated subcutaneously in the right flank region with fresh tumor derived from mice bearing established primary human cancer tissue. Mice were randomized into vehicle or treatment groups with a mean tumor burden of 200 mm3. All mice were dosed once daily by oral gavage for 21 days. Tumor volume and body weights were determined three times per week. [1]

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Pharmacokinetic Analysis of BTM-3566 in mouse blood[1]
Blood was collected from a tail vein snip into K2-EDTA tubes. Plasma was isolated and a 20 mL sample was protein precipitated with 200 L of acetonitrile containing 100 ng/mL diclofenac, tolbutamide and labetalol as internal standards. The mixture was vortex-mixed and centrifuged at 13000 rpm for 15 min, 4 ℃. An 80 mL aliquot of the supernatant was transferred to a sample plate and mixed with 80 μL water, then the plate was shaken at 800 rpm for 10 min. A 1 L aliquot was injected on to a Waters ACQUITY UPLC BEH C18 2.1*50mm, 1.7µm reverse phase column using a two-component mobile phase gradient. Mobile phase A was 0.1% trifluoracetic acid in water and mobile phase B 0.1% TFA in acetonitrile. Plasma BTM-3566 was detected using electrospray ionization and multiple reaction monitoring mass spectrometry (BTM-3528 [M+H]+ m/z 522.10>203.1. All data were analyzed using single compartment analysis in WinNonLin.[1]

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Xenograft efficacy: NOD/SCID mice bearing SU-DHL-4 tumors (200 mm³) were randomized. BTM-3566 was formulated in 10% DMSO + 40% PEG300 + 50% saline. Administered orally (50 mg/kg, QD) for 21 days. Tumor volume measured biweekly [1]
PDX model: NSG mice implanted with primary DLBCL tumors received BTM-3566 (60 mg/kg) via intraperitoneal injection (QDX14). Drug was dissolved in 5% ethanol + 95% corn oil [2]
PK study: CD-1 mice (n=3/group) received single oral (30 mg/kg) or IV (5 mg/kg) doses. Plasma collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dose [3].

To investigate the pharmacokinetic properties of BTM-3566, researchers conducted an intravenous/oral crossover study in mice. Bioavailability >90%, and a terminal half-life of 4.4 to 6.6 hours is acceptable for once-daily oral administration. [1]

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Oral bioavailability in mice: 92% (30 mg/kg orally vs 5 mg/kg intravenously). Peak plasma concentration (Cₘₐₓ) = 8.7 μg/mL, Tₘₐₓ = 1 hour. Plasma half-life (t₁/₂) = 4.2 hours. AUC₀–₂₄ = 42.3 μg•h/mL [3]
Tissue distribution: The highest concentrations were found in the liver (AUC₀–₂₄ = 89 μg•h/g) and spleen (AUC₀–₂₄ = 76 μg•h/g) [3].

Oral bioavailability: 92% in mice (30 mg/kg orally, 5 mg/kg intravenously). Peak plasma concentration (Cₘₐₓ) = 8.7 μg/mL, Tₘₐₓ = 1 hour. Plasma half-life (t₁/₂) = 4.2 hours. AUC₀–₂₄ = 42.3 μg•h/mL [3]
Tissue distribution: The highest concentrations were found in the liver (AUC₀–₂₄ = 89 μg•h/g) and spleen (AUC₀–₂₄ = 76 μg•h/g) [3].

[1]. Targeting aggressive B-cell lymphomas through pharmacological activation of the mitochondrial protease OMA1. Mol Cancer Ther. 2023 Aug 30.

[2]. BTM-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma. Blood. Volume 138, Supplement 1, 23 November 2021, Page 684.

[3]. Pharmacokinetics and pharmacodynamics of the novel OMA1 activator BTM 3566 in a mouse model of diffuse large B-cell lymphoma. Cancer Research, 2023, 83(7_Supplement): 2803-2803.

Diffuse large B-cell lymphoma (DLBCL) is a highly aggressive and rapidly proliferating tumor whose ability to adapt to uncontrolled growth-induced stress (e.g., hypoxia, amino acid deficiency, and accumulation of misfolded proteins) heavily depends on the ATF4-mediated integrated stress response (ISR). This paper demonstrates that ISR overactivation is a potential therapeutic target for DLBCL. We describe a novel class of compounds, represented by BTM-3528 and BTM-3566, that activate ISR via the mitochondrial protease OMA1. Treatment of tumor cells with these compounds leads to OMA1-dependent DELE1 and OPA1 cleavage, mitochondrial fragmentation, eIF2α kinase HRI activation, cell growth arrest, and apoptosis. The mechanism by which BTM-3528 and BTM-3566 activate OMA1 differs from that of mitochondrial electron transport inhibitors because these compounds induce OMA1 activity without causing acute changes in respiration. We further discovered that the mitochondrial protein FAM210B is a negative regulator of the activity of BTM-3528 and BTM-3566. Overexpression of FAM210B inhibits OMA1 activation and apoptosis. Notably, FAM210B expression is almost absent in healthy germinal center B lymphocytes and derived B-cell malignancies, revealing a fundamental molecular vulnerability that BTM compounds target. Both compounds induced rapid apoptosis in various diffuse large B-cell lymphoma (DLBCL) cell lines derived from activated B cells, germinal center B cells, and MYC rearrangement lymphomas. Once-daily oral administration of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 out of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-in-class approach to selectively activating mitochondrial integrated stress response (ISR) for the treatment of diffuse large B-cell lymphoma (DLBCL). [1] Treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r-DLBCL) is extremely challenging, especially for patients who have failed high-dose chemotherapy, stem cell transplantation, or CAR-T cell therapy. r/r-DLBCL exhibits high heterogeneity at both the clinical and molecular levels, thus necessitating the development of novel therapies to improve patient outcomes regardless of molecular subtype. This article introduces BTM-3566, a first-in-class compound active against a variety of B-cell malignancies, but with particularly significant efficacy against DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in diffuse large B-cell lymphoma (DLBCL) cell lines in vitro and achieves complete tumor regression in vivo in a DLBCL PDX mouse model carrying gene alterations associated with poor prognosis. [2] BTM-3566 is an oral small molecule drug based on a pyrazolothiazole backbone for the treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and completes cell killing in DLBCL cell lines, with an IC50 value of approximately 200-500 nM. DLBCL cell lines sensitive to this drug include ABC, GCB, and double-hit and triple-hit lymphoma cell lines. Pharmacokinetic studies in mice have shown that the drug is suitable for once-daily administration, with oral bioavailability > 50% and a serum half-life of approximately 6 hours. A 14-day dosing study in mice and dogs showed that the drug was well tolerated at therapeutic doses. BTM-3566 also showed stability in human hepatocytes (IC50 < 5 ml/minkg) and good in vitro safety. In a xenograft model using the double-hit DLBCL cell line SU-DHL-10, BTM-3566 treatment resulted in complete tumor regression in all tumor-bearing animals. More importantly, no tumor growth was observed within two weeks after drug withdrawal, indicating that BTM-3566 treatment achieved a durable complete remission in this double-hit DLBCL model. Extended studies in human DLBCL PDX models containing a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, showed that all cell lines responded, with complete tumor regression achieved in 6 of the 8 tested PDX models (Table 1). [2] Transcriptomic and proteomic analyses showed that BTM-3566 strongly activated the ATF4 integration stress response (ISR), manifested as phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. We used CRISPR-Cas 9 gene knockout technology to identify HRI as the most specific of the four eIF2α kinases in the human genome that BTM-3566 induces eIF2α phosphorylation, ATF4 ISR activation, and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased reactive oxygen species (ROS) production, or impaired mitochondrial ATP synthesis, which leads to enhanced mitochondrial protein homeostasis, including activation of the mitochondrial protease OMA1. We found that BTM-3566 can activate OMA1, but not in the manner of a classic mitochondrial toxin. BTM-3566 treatment only takes 30 minutes to induce OMA1-dependent OPA1 processing and mitochondrial fragmentation without causing a decrease in mitochondrial oxygen consumption or membrane depolarization. These data suggest that BTM-3566 represents a new class of compounds that can activate the mitochondrial protease OMA1. [2]

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A gene expression-based sensitivity analysis of BTM-3566 on more than 400 cancer cell lines showed that the mitochondrial membrane protein FAM210B was negatively correlated with the response to BTM-3566. Notably, overexpression of FAM210B completely inhibited OMA1 activation and led to complete resistance to BTM-3566-induced apoptosis in DLBCL cell lines BJAB and Burkitt lymphoma cell line Ramos. Therefore, FAM210B can serve as a strong predictor of BTM-3566 sensitivity and reveals a novel regulatory mechanism for OMA1 activation. [2] In summary, we describe here a novel and highly effective mitochondrial ISR activator that is well tolerated in mice and dogs, has good pharmacokinetic properties, and induces significant DLBCL regression in vivo. An IND application for B-cell malignancies is expected to be completed in the first quarter of 2022 and the first human clinical trial is expected to begin in the first half of 2022. [2] BTM-3566 is a first-in-class OMA1 activator that triggers mitochondrial proteolysis, thereby leading to apoptosis in DLBCL cells. It overcomes the resistance of MYC-driven lymphoma to standard therapies such as R-CHOP[1]
The mechanism involves OMA1-mediated OPA1 cleavage, leading to mitochondrial cristae remodeling and cytochrome c release[2]
A phase I trial (NCT pending) is planned for relapsed/refractory DLBCL[3].

C24H23F4N3O2S2

525.581937074661

525.116

2228857-70-1

134564305

Light yellow to yellow solid powder

7.3

1

10

6

35

799

0

S1C(N2C(C(=O)O)=C(C3C=CC=C(C=3)F)C(C)=N2)=NC(=C1SC(C)C)C1=CCC(C(F)(F)F)CC1

LDSVVHIOWFIJNE-UHFFFAOYSA-N

InChI=1S/C24H23F4N3O2S2/c1-12(2)34-22-19(14-7-9-16(10-8-14)24(26,27)28)29-23(35-22)31-20(21(32)33)18(13(3)30-31)15-5-4-6-17(25)11-15/h4-7,11-12,16H,8-10H2,1-3H3,(H,32,33)

4-(3-fluorophenyl)-5-methyl-2-[5-propan-2-ylsulfanyl-4-[4-(trifluoromethyl)cyclohexen-1-yl]-1,3-thiazol-2-yl]pyrazole-3-carboxylic acid

BTM-3566; 2228857-70-1; BTM3566; SCHEMBL20214005; LDSVVHIOWFIJNE-UHFFFAOYSA-N;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)