YAP-TEAD (IC50 = 9 nM)[1]

IAG933 and its analogs are potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. [2]
IAG933 is a direct YAP-TEAD protein-protein interaction disruptors (PPIDs) that targets interface 3 and has entered a clinical trial in 2021[3].

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IAG933 improves response to JDQ443 by inducing apoptosis[2]
Despite the impact of selective KRASG12C inhibitors on mutant cancers, their clinical effectiveness is generally less pronounced than RTK inhibitors. Overcoming resistance to KRASG12C inhibitors remains a challenge, prompting ongoing clinical trials that investigate combination therapies12. In line with the findings obtained from allosteric TEAD inhibitors, IAG933 and the Novartis KRASG12C inhibitor JDQ443 displayed strong combination benefit in a panel of KRASG12C-mutated NSCLC and CRC cell lines (Fig. ​(Fig.6a).6a). IAG933 compared favorably to other JDQ443 candidate partners, such as inhibitors of SHP2, MEK, ERK or PIKα, by causing a notable shift in maximal growth inhibition across cell lines (Extended Data Fig. ​Fig.9a).9a). In long-term proliferation assays, we observed robust and sustained inhibition of cell growth when combining subefficacious concentrations of JDQ443 and IAG933, which modestly delayed cell proliferation as single agents (Extended Data Fig. ​Fig.9b).9b). Consistently, in vivo, upfront addition of IAG933 deepened responses to JDQ443 in NCI-H2122 NSCLC xenografts (Fig. ​(Fig.6b),6b), with this combination outperforming JDQ443 plus the SHP2 inhibitor TNO155 (Extended Data Fig. ​Fig.9c).9c). This antitumor combination effect was also observed in a PDX model of NSCLC, with no tumor regrowth observed for 30 days after end of treatment (Fig. ​(Fig.6c6c).

IAG933 and YTP-75 achieve dose-dependent antitumor efficacy[2]
IAG933 was assessed in mouse MSTO-211H cell-derived xenograft (CDX) models at single doses between 30 and 240 mg per kg of body weight (mg kg−1) administered by oral gavage. Dose-related blood exposure was observed with a time at maximal concentration (Tmax) of ~1–2 h, correlating with a dose/exposure-dependent TEAD target gene inhibition commencing at ~2 h after dosing (Fig. 3a,b). The in vivo blood IC50 for target gene inhibition of 64 nM was slightly higher than the in vitro IC50 of 11–26 nM for MSTO-211H cells (Fig. ​(Fig.1c).1c). An in vivo reporter assay, using luciferase expression under TEAD-responsive elements in orthotopic pleural MSTO-211H tumors, showed rapid and profound loss of bioluminescence following a single dose of IAG933 (Fig. ​(Fig.3c),3c), followed within a few hours by a rebound to baseline due to the relatively short half-life of IAG933 in mice. Similar PK/PD findings were observed for the IAG933 analog YTP-75 (Extended Data Fig. 5a,b), demonstrating deep and quick TEAD in vivo transcriptional inhibition by both compounds.

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IAG933 erradicates tumors in a rat model at tolerated doses[2]
Extending our in vivo studies beyond mice, we also evaluated IAG933 in a subcutaneous MSTO-211H rat xenograft model. Target gene inhibition kinetics after single-dose IAG933 were similar to those observed in the mouse model (Extended Data Fig. 7a,b), whereas the averaged CCN2/ANKRD1/CCN1 IC50 of 20 nM was approximately threefold lower and similar to the in vitro IC50 values. After 2 weeks of daily dosing, tumor stasis was observed at 10 mg kg−1, and complete regression was seen at 30 mg kg−1 in four of five animals (Fig. ​(Fig.3g).3g). Exposures were dose proportional, and no compound accumulation was detected over 12 days of daily treatment (Extended Data Fig. ​Fig.7c).7c). No body weight loss was observed, and treatments were well tolerated. Comparing rat and mouse model response curves established a dosing equivalence between 30 mg kg−1 once a day in rats and 240 mg kg−1 once a day in mice (Extended Data Fig. ​Fig.7d7d).

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IAG933 activity in mesothelioma and Hippo-altered xenografts[2]
Mesothelioma pathogenesis frequently involves genetic alterations in tumor suppressor genes of the Hippo signaling cascade, including NF2 and LATS1/LATS2, in an estimated 32–50% of cases9,37–39. We explored the antitumor efficacy of YTPs in differing mesothelioma genetic backgrounds in a panel of nine human-derived xenograft (PDX) mouse models treated daily with YTP-75. Significant tumor responses were observed in seven of nine models, with deep tumor regressions in three NF2-altered models and durable tumor stasis in four other models without reported Hippo alterations (Fig. ​(Fig.4a4a and Extended Data Fig. ​Fig.7e).7e). Interestingly, the two tumor models that did not respond displayed the lowest basal expression of TEAD target genes (Fig. ​(Fig.4a).4a). NF2 mutations have also been detected at low prevalence (~1–2%) in other solid tumors9,38,40. To explore YTP activity in such cases, we assessed IAG933 in an NF2-altered PDX model of triple-negative breast cancer (5938-HX) and YTP-75 in a CDX model of NF2-altered lung carcinoma (NCI-H292). Both models showed an antitumor response to treatment, but while 5938-HX underwent tumor regression (Fig. ​(Fig.4b),4b), the NCI-H292 model showed a lesser inhibition of tumor growth (Fig. ​(Fig.4c4c).

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IAG933 combination treatment improves RTK inhibitor efficacy[2]
Co-inhibition of EGFR and TEAD by osimertinib and VT104, respectively, has previously been shown to enhance osimertinib tumor response in NSCLC models43. Elevated YAP activity has been described in HER2-positive cancers and relapsing cancers3,8, and YAP–TEAD activation has been linked to trastuzumab resistance8,44. Moreover, recent data suggest that TEAD activation maintains a minimal residual disease under RTK inhibitor treatment6,43. Therefore, co-inhibition of TEAD could be essential for eradicating RTK-mediated cancers and achieving tumor elimination.
Consistent with this concept, IAG933 plus osimertinib showed enhanced antitumor benefit, leading to rapid regression in the EGFR-mutated NCI-H1975 CDX model of NSCLC (Fig. ​(Fig.5a).5a). Furthermore, IAG933 plus the MET inhibitor capmatinib induced profound tumor shrinkage in the EBC-1 MET-amplified CDX model of lung cancer, while no activity was seen for IAG933 alone (Fig. ​(Fig.5b).5b). Despite modest single-agent effects by IAG933 in a panel of seven HER2-amplified cell lines from various cancer indications, dose-dependent combination activity was seen for IAG933 with the HER2 inhibitor lapatinib (Fig. ​(Fig.5c),5c), and prolonged combination activity after the end of treatment was observed in lengthier in vitro studies (Fig. ​(Fig.5d).5d). In vivo, the HER2-amplified NCI-N87 gastric carcinoma xenograft model underwent complete tumor regression with the combination of YTP-75 and trastuzumab (Fig. ​(Fig.5e).5e). Hence, a combination benefit is observed with YTPs in cancer models that are driven by different RTKs, indicating a shared underlying mechanism and presenting an opportunity for combining these therapeutic agents.

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IAG933 combination shows benefit in BRAFV600E-altered tumors[2]
Because activating mutations in BRAF also drive oncogenic reliance on the MAPK pathway, we explored the combination potential of YTPs in the setting of BRAFV600E-mutant disease by combining IAG933 with the BRAF inhibitor dabrafenib, the MEK1/MEK2 inhibitor trametinib and/or the ERK1/ERK2 inhibitor LTT462. The combinations of dabrafenib + IAG933, dabrafenib + LTT462 + IAG933 and dabrafenib + trametinib + IAG933 showed benefit in short-term cell viability assays (Fig. ​(Fig.7a).7a). Consistent with an adaptive role for TEAD activity on MAPK pathway inhibition, increased expression of TEAD-responsive genes was noted after dabrafenib + trametinib treatment without a YTP, which was prevented by concomitant TEAD inhibition with the IAG933 analog YTP-10 (Fig. ​(Fig.7b).7b). Stronger antitumor responses were seen in the BRAFV600E-mutated CRC CDX model HT-29 with the triple combination of dabrafenib + LTT462 + IAG933 than with single-agent treatments (Fig. ​(Fig.7c).7c). Similarly, the triple combination of dabrafenib + trametinib + YTP-75 showed stronger antitumor activity in the BRAFV600E-mutated CRC xenograft model 5238-HX than either dabrafenib + trametinib or dabrafenib + trametinib + cetuximab, resulting in a sustained tumor regression across the 21-day study period (Fig. ​(Fig.7d7d). TEAD and RAF/MAPK blockade benefit in non-KRASG12C PDAC Apart from the clinically targetable G12C variant, therapeutic suppression of KRAS-driven oncogenesis remains challenging52. To address non-KRASG12C-mutant tumors, effective inhibition of downstream RAF, MEK and/or ERK effectors may offer potential therapeutic options. In this context, IAG933 could represent a promising combination opportunity, considering the encouraging combination outcomes achieved with mutant-specific inhibitors (Figs. ​(Figs.66 and ​and77 and Extended Data Fig. ​Fig.10).10). We investigated this hypothesis in PDAC cells bearing various KRAS alleles. The addition of YTP-75 to trametinib plus the RAF inhibitor naporafenib significantly enhanced growth inhibition in a panel of 23 PDAC cell lines (Fig. ​(Fig.8a),8a), consistent with results obtained from a mouse clinical trial53, including 12 PDAC PDXs with different KRAS mutations (7 G12D, 2 G12V, 2 Q61H and 1 G12R), where 8 models (66%) showed tumor regression or near stasis with the triple combination (Fig. 8b,c). Strong induction of TEAD transcriptional activity by trametinib + naporafenib was observed and prevented by YTP-13 cotreatment in a luciferase-based reporter system in SUIT-2 PDAC cells (Fig. ​(Fig.8d),8d), and this triple combination was shown to inhibit both DUSP6 and TEAD-responsive ANKRD1 gene expression in a panel of three PDAC lines (Fig. ​(Fig.8e8e).

Surface plasmon resonance assay[2]
Surface plasmon resonance assay measurements were acquired with human TEAD1209–426, TEAD2221–447, TEAD3218–435 and TEAD4217–434 as previously described. The four N-biotinylated TEAD proteins were tagged with AviTag and immobilized on sensor chips, and the binding of different concentrations of YTP-3 and YTP-32 was measured at 298 K. The data were globally fitted with a 1:1 interaction model using Biacore T200 evaluation software to determine the dissociation constants (Kd) measured at equilibrium.

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TR-FRET assays[2]
Different compounds were tested in a TR-FRET assay as previously reported24. The lipid-binder compounds (K-975 and VT104) targeting the myristate/palmitoyl pocket are inactive in TR-FRET because the TEAD4 protein used in this assay is fully acylated.

Selectivity assessment in colony formation assays with clones derived from SF-268 cells[2]
The SF-268 cell line was engineered, and a clone bearing a double mutation in TEAD1 (V406A/E408A) was established as follows. The targeting sequence of TEAD1 (gtgcattcgctgtttcaaat) was cloned into the pNGx_006 vector (pUC/ori, U6 promoter for tracrRNA/chimera, CMV promoter for SPyCas9 and puromycin selection). SF-268 cells (2 × 105) were electroporated with 1.5 μg of pNGx_006_sgTEAD1 and 0.5 μg of single-stranded oligonucleotide for TEAD1V406A and TEAD1E408 (ttaacaggtggtaacaaacagggatacacaagaaactctactctgcatggcctgtgcattcgctgtttcaaatagtgaacacggagcacaacatcatatttacaggcttgtaaaggactg) using a Neon Transfection System with the following parameters: voltage 1,300 V, pulse 20 ms and pulse number 2. Single clones were seeded after puromycin selection and characterized by Sanger sequencing. For the colony formation assay, SF-268 clones 18 and 23 were seeded at low density (1,000 cells per well in six-well plates) 24 h before treatment. Test compound (IAG933) was distributed into the assay plates in a five-point threefold serial dilution starting at a top concentration of 10 μM. DMSO was used as a control, and DMSO content was normalized to the highest volume in all compound-treated wells. Medium containing compound was renewed twice a week. After an incubation period of 11 days under regular cell culture conditions (37 °C, 5% CO2), cells were fixed with 3.7% formaldehyde for 10 min, and colonies were stained with crystal violet.

Animal treatments[2]
Most compounds were administered at the indicated doses by oral gavage with the following formulations. IAG933 was formulated in 0.5% methylcellulose and 0.1% Tween-80 in 100 mM phosphate buffer (pH adjusted to 8). VT104 and K-975 were formulated in 100% Maisine CC. YTP-75 was formulated in 30% PEG300 and 50 mM acetate buffer (pH adjusted to 5.5). YTP-13 was formulated in 5% PEG300 and 50 mM acetate buffer (pH adjusted to 4.8). LTT462, dabrafenib and trametinib were formulated in 20% MEPC4 in water. JDQ443, TNO155, osimertinib and capmatinib were formulated in 0.5% methylcellulose and 0.1% Tween-80 in water. Other compounds were administered by intraperitoneal injection. Antibodies to trastuzumab and cetuximab and MRTX1133 compound were formulated in Dexolve.

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PD in vivo studies[2]
Animals were assigned into groups of n = 3–5 per time point and treatment. Blood, plasma and tumor samples for PK and PD analyses were collected. Blood samples were collected on ice and stored at –20 °C until further processing. Plasma and tumor samples were snap-frozen on dry ice and stored frozen at –80 °C until further processing. The in vivo TEAD reporter assay was performed with the MSTO-211H STB-Luc orthotopic pleural mesothelioma tumor model. For each measurement, mice were injected intraperitoneally with luciferin (150 mg kg−1). Exactly 20 min later, the mice were imaged with an IVIS Spectrum while conscious and restrained for less than 1 min.

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In vivo efficacy studies[2]
Treatment was initiated when the tumors engrafted in the flank were at least 100 mm3, and random enrollment was applied. Efficacy studies, tumor response and relapse were reported with the measures of tumor volumes at the start of treatment. For efficacy studies on ectopic models, animals were randomized into treatment groups based on tumor volume. Tumor size was measured using a caliper and calculated using the formula length × width2 × π/6. As a measure of efficacy, the percent T/C value was sometimes calculated at the end of the experiment or at best response using the formula (Δtumor volume treated/Δtumor volume control) × 100. In the case of tumor regression, the tumor response was quantified using the formula –(Δtumor volume treated/tumor volume treated at start) × 100. Statistical analyses were performed using GraphPad Prism. For efficacy studies on pleural orthotopic models, viable tumor burden was assessed by measurements of GLuc from 20 μl of blood collected in microvette EDTA-coated tubes, and samples were stored at –20 C. Coelentrazine (Nanolight) substrate solution was added (100 μl of a 100 mM solution) to each well of 96-well white plates, and 5 μl of blood was added in triplicate. Bioluminescence was measured with a CentroXS LB960 Luminometer for 2 s.

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Bioanalytical method for detection of compounds in blood, plasma and tumors[2]
Concentrations of IAG933 and YTP-75 in total blood, plasma and tissues were determined by a ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) assay. Frozen tissue samples were pulverized to powder using CryoPrep according to manufacturer’s instructions or homogenized in an equal volume of HPLC water using the Fast Prep-24 system. Samples (about 25 mg, exact weight collected) of blood, plasma or tissue (in the form of powder or homogenate) were mixed with 25 µl of internal standard (1 µg ml–1) and extracted by the addition of 200 µl of acetonitrile to precipitate proteins. After sonication for 5 min, samples were centrifuged, and supernatants (70 µl) were mixed with 60 µl of HPLC water before the analysis of 5-µl aliquots by UPLC–MS/MS. Samples were injected onto a reverse-phase column using formic acid in water and formic acid in acetonitrile as mobile phases. The column eluent was directly introduced into the ion source of the triple quadrupole mass spectrometer. Electrospray positive ionization multiple reaction monitoring was used for MS/MS detection of the analyte. PK parameters were calculated from the mean values with the linear trapezoidal rule by using a noncompartmental model for extravascular dosing.

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Combination assays in matrix format[2]
The effect of compound combinations on cell proliferation was assessed by ATP quantification using CellTiter-Glo reagent. Cells were seeded at 300–700 cells per well in white-walled, clear-bottomed 384-well plates and incubated overnight at 37 °C before the addition of serial compound dilutions or vehicle control in a matrix format using an HP300 digital dispenser, and treatments were applied in triplicate. After incubation for 5–7 days in the presence of compounds, cell viability was monitored using CellTiter-Glo following the supplier’s instructions. Data were analyzed using the in-house program Combination Analysis Module. To enable differentiation of cytotoxic from cytostatic compound effects, the number of viable cells on the day of compound addition (day 0) was also assessed in a separate cell plate and used to calculate the extent of cell viability suppression. Depending on whether the CellTiter-Glo signal for a given point in the concentration matrix was above or below day 0, the latter suggesting cell death due to compound treatment, a ‘growth inhibition’ (GI) value was calculated as follows: T < D0: GI = 100 × {1 – [(S – D0)/D0]}; T ≥ D0: GI = 100 × [1 – (S – D0)/(V – D0)], where D0 is day 0, V is vehicle control, and S is signal. This formula leads to a scale where 0 corresponds to no compound effect compared to vehicle, 100 corresponds to growth arrest (that is, signal on endpoint equal to signal on day 0), and 200 corresponds to complete cell killing. In Fig. ​Fig.6a,6a, threefold dilutions were used for IAG933 starting from 5.595 µM for NSCLC and 3 µM for CRC cell lines and fourfold dilutions for JDQ443 starting with 1.6 µM as the highest compound concentrations.

[1]. Preparation of biaryl derivatives as YAP/TAZ-TEAD protein-protein interaction inhibitors. World Intellectual Property Organization, WO2021186324 A1. 2021-09-23.

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.[2]

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The Hippo signaling pathway is a highly conserved pathway that plays important roles in the regulation of cell proliferation and apoptosis. Transcription factors TEAD1-4 and transcriptional coregulators YAP/TAZ are the downstream effectors of the Hippo pathway and can modulate Hippo biology. Dysregulation of this pathway is implicated in tumorigenesis and acquired resistance to therapies. The emerging importance of YAP/TAZ-TEAD interaction in cancer development makes it a potential therapeutic target. In the past decade, disrupting YAP/TAZ-TEAD interaction as an effective approach for cancer treatment has achieved great progress. This approach followed a trajectory wherein peptidomimetic YAP-TEAD protein-protein interaction disruptors (PPIDs) were first designed, followed by the discovery of allosteric small molecule PPIDs, and currently, the development of direct small molecule PPIDs. YAP and TEAD form three interaction interfaces. Interfaces 2 and 3 are amenable for direct PPID design. One direct YAP-TEAD PPID (IAG933) that targets interface 3 has entered a clinical trial in 2021. However, in general, strategically designing effective small molecules PPIDs targeting TEAD interfaces 2 and 3 has been challenging compared with allosteric inhibitor development. This review focuses on the development of direct surface disruptors and discusses the challenges and opportunities for developing potent YAP/TAZ-TEAD inhibitors for the treatment of cancer.[3]

C27H26CLF2N3O4

529.962852954865

529.157

2714434-21-4

156855755

White to off-white solid powder

3.7

3

8

7

37

793

2

CNC(=O)C1=CN=C(C(=C1C2=C3C[C@@](OC3=CC(=C2Cl)F)([C@@H]4CCCN4)C5=CC=CC=C5)F)OCCO

HUVOYQMXUNTUAI-DCFHFQCYSA-N

InChI=1S/C27H26ClF2N3O4/c1-31-25(35)17-14-33-26(36-11-10-34)24(30)22(17)21-16-13-27(20-8-5-9-32-20,15-6-3-2-4-7-15)37-19(16)12-18(29)23(21)28/h2-4,6-7,12,14,20,32,34H,5,8-11,13H2,1H3,(H,31,35)/t20-,27-/m0/s1

4-[(2S)-5-chloro-6-fluoro-2-phenyl-2-[(2S)-pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2-hydroxyethoxy)-N-methylpyridine-3-carboxamide

YAP-TEAD-IN-3; IAG933; 2714434-21-4; IAG-933; NVP-IAG933; SCHEMBL23834952; GTPL13367; IAG933?;

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: 125 mg/mL (235.87 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.)