IDH1R132H/isocitrate dehydrogen

Ivosidenib (AG-120) (0-13 μM; 48 hours) suppresses many IDH1-R132 mutants with comparable potency and IC50 values: IDH1-R132H (IC50=12 nM); IDH1-R132C (IC50=13 nM); IDH1-R132G (IC50=8 nM); IDH1-R132L (IC50=13 nM); and IDH1-R132S (IC50=12 nM), respectively [3]. Ivosidenib (AG-120) (0-13 μM; 48 hours) suppresses many IDH1-R132 mutants with comparable potency and IC50 values: IDH1-R132H (IC50=12 nM); IDH1-R132C (IC50=13 nM); IDH1-R132G (IC50=8 nM); IDH1-R132L (IC50=13 nM); and IDH1-R132S (IC50=12 nM), respectively [3].

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Biochemical and cell biology profiling revealed that IvosidenibAG-120 inhibited several IDH1-R132 mutants with potency similar to that seen for R132H (Table 3) and was highly selective for IDH1 isoforms, showing no inhibition of IDH2 (WT or mutant) isoforms at micromolar concentrations (Table S2). Ivosidenib/AG-120 at 100 μM did not inhibit multiple dehydrogenases tested (Table S3).[3]

In vitro, AG-120 exhibited rapid-equilibrium inhibition against the mIDH1-R132 homodimer. Kinetic studies of binding to demonstrate mode of action were inconclusive due to persistent prebound NADP(H) in all soluble mIDH1 enzyme preparations (Supporting Information, Figures S1 and S2). Surprisingly, AG-120 demonstrated slow-tight binding inhibition against the IDH1-WT homodimer (Figure S3 and S4).[3]

AG-120/Ivosidenib also showed good cellular potency across multiple mIDH1-R132 endogenous and overexpressing cell lines (Table 3), indicating its potential for use across all mIDH1-R132 cancers. [3]

IDH mutations have been shown to block normal cellular differentiation via epigenetic and metabolic rewiring.1,3−5 To determine the effect of mIDH1 inhibition in primary human AML blast cells, mIDH1-R132H, mIDH1-R132C, and IDH1-WT, bone marrow or peripheral blood samples from patients (Table S5) were treated with Ivosidenib/AG-120 in an ex vivo assay. Living blast cells were sorted and cultured in medium containing cytokines (at a density of 0.5 × 106 cells/mL) in the presence or absence of AG-120. In mIDH1 samples, AG-120 reduced the level of intracellular 2-HG by 96% at the lowest tested dose (0.5 μM) and by 98.6% and 99.7%, respectively, at 1 and 5 μM (Figure 2). 2-HG was not measurable in multiple IDH1-WT patient samples assessed. AG-120 induced differentiation of primary mIDH1-R132H and mIDH1-R132C (but not IDH1-WT) blast cells from patients with AML treated ex vivo, as shown by enhanced ability to form differentiated colonies in methylcellulose assays, increased levels of cell-surface markers of differentiation, and increases in the proportion of mature myeloid cells [3].

Twelve hours after therapy, AG-120 (gavage delivery; 50 mg/kg and 150 mg/kg) produced maximal inhibition (92.0% and 95.2% at the 50 mg/kg and 150 mg/kg dosages, respectively) and rapidly decreased tumor 2-HG concentrations [3].

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PK studies of Ivosidenib performed in Sprague–Dawley rats, beagle dogs, and cynomolgus monkeys showed rapid oral absorption, low total body plasma clearance (CLp) and moderate to long half-life (t1/2) (Table S4). Although moderate exposure reduction was observed in a repeat-dose study in rodents (data not shown), no exposure reduction occurred in cynomolgus monkeys, and in patients with cancer a long t1/2 and accumulation of Ivosidenib/AG-120 following multiple doses were observed.

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Following a single oral dose of 50 mg/kg to rats with an intact blood–brain barrier, Ivosidenib/AG-120 exhibited brain penetration of 4.1% (AUC0–8h [brain]/AUC0–8h [plasma]). However, brain penetration is likely to be higher in glioma patients who have a compromised blood–brain barrier. Given that AG-120 is very potent and well tolerated, it has the potential to achieve therapeutic concentration in the brain, and its therapeutic benefit in glioma is being evaluated in clinical trials.

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AG-120/Ivosidenib showed robust tumor 2-HG reduction in female nude BALB/c mice inoculated with HT1080 cells. Each mouse received a single oral dose of vehicle or AG-120 at 50 or 150 mg/kg by gavage. Tumor 2-HG concentration declined rapidly, with maximum inhibition (92.0% and 95.2% at the 50 mg/kg and 150 mg/kg doses, respectively) achieved at ∼12 h post dose. Tumor 2-HG concentrations approached baseline levels 48–72 h following a single dose of AG-120 (Figure 1), consistent with the reversible nature of AG-120 inhibition [3].

Determination of compound inhibition potency against the mIDH1-R132H enzyme reaction using a diaphorase/resazurin coupled system [3]
In the primary reaction, the reduction of α-KG acid to D-2-hydroxyglutarate (2-HG) is accompanied by a concomitant oxidation of NADPH to NADP. The amount of NADPH remaining at the end of the reaction time is measured in a secondary diaphorase/resazurin reaction in which the NADPH is consumed in a 1:1 molar ratio with the conversion of resazurin to the highly fluorescent resorufin. Uninhibited reactions exhibit a low fluorescence at the end of the assay, while reactions in which the consumption of NADPH by mIDH1- R132H has been inhibited by a small molecule show a high fluorescence. The primary reaction was performed in a volume of 50 L 1X Buffer (150 mM NaCl, 20 mM Tris 7.5, 10 mM MgCl2, 0.05% w/v bovine serum albumin [BSA]), contained 2 nM mIDH1-R132H, 1 mM α-KG, and 4 M NADPH, and was conducted for 60 minutes at 25°C. To perform the secondary reaction, 25 L of 1X buffer containing 36 g/mL diaphorase and 30 mM 10 resazurin was added to the primary reaction and incubated for a further 10 minutes at 25°C. Florescence was read on a Spectramax plate reader at Ex 544 Em 590. Recombinant protein was expressed and purified as previously described. 5 Compounds or compound dilutions were prepared in 100% dimethyl sulfoxide (DMSO) concentration and diluted 1:100 into the final reaction. mIDH1-R132C was assayed under similar conditions, with the exception that the 1X Buffer was 50 mM K2HPO4 pH 6.5, 40 mM NaHCO3, 5 mM MgCl2, 10% glycerol, 0.03% w/v BSA.

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Assay of the IDH1-WT enzyme reaction for determination of inhibitor potency [3]
IDH1-WT enzyme was assayed in a modified version of the assay used for mIDH1-R132H. Since this enzyme converts NADP to NADPH stoichiometrically with the conversion of isocitrate to α-KG, NADPH product can be continuously assayed by direct coupling to the diaphorase/resazurin system and reading resorufin production at Ex 544 Em 590. Assays were conducted in 50 L of 1X Buffer (150 mM NaCl, 20 mM Tris pH 7.5, 10 mM MgCl2, 0.05% (w/v) BSA, 2 mM beta-mercaptoethanol [B-ME]) containing 50 M NADP, 70 M DL-isocitrate, and 31.2 ng/mL IDH1-WT enzyme (reaction time 1 or 16 hours). The direct coupling system comprised 20 g/mL diaphorase and 40 M resazurin.

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Assay of the IDH2-WT enzyme reaction for determination of inhibitor potency[3]
Inhibitory potency of compounds against the IDH2-WT enzyme was determined in a coupled assay to diaphorase. In this assay, production of NADPH by IDH2-WT was linked to a concomitant reduction of resazurin to the highly fluorescent resorufin. Enzyme was diluted to 0.06 g/mL in 40 L 1X Assay Buffer (150 mM NaCl, 50 mM potassium phosphate pH 7, 10 mM MgCl2, 10% glycerol, 2 mM B-ME, 0.03% BSA), to which 1 L of compound was added in DMSO. The mixture was incubated for 16 hours at room temperature (RT). The reaction was started with the addition of 10 L of Substrate Mix (200 M isocitrate, 175 M NADP, 60 g/mL diaphorase, 200 M resazurin, in 1X Assay Buffer), and run for 30 minutes at RT. The reaction was halted with the addition of 25 L of 6% sodium dodecyl sulfate and read on a Spectramax Plate Reader at Ex544/Em590.

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Assay of the mIDH2-R140Q and mIDH2-R172K enzyme reaction for determination of inhibitor potency[3]
Inhibitory potency against the mIDH2-R140Q and mIDH2-R172K enzymes was determined in an endpoint assay in which the amount of NADPH remaining at the end of the reaction was measured by the addition of a large excess of diaphorase and resazurin. mIDH2-R140Q 11 was diluted to 0.25 g/mL in 40 L 1X Assay Buffer (150 mM NaCl, 50 mM potassium phosphate pH 7.5, 10 mM MgCl2, 10% glycerol, 2 mM B-ME, 0.03% BSA) and incubated for 16 hours at 25°C in the presence of 1 L of compound in DMSO. The reaction was started with the addition of 10 L of Substrate Mix (20 M NADPH, 8 M α-KG, in 1X Assay Buffer) and incubated for 1 hour at 25°C. Then, remaining NADPH was measured by the addition of 25 L of Detection Mix (36 g/mL diaphorase, 18 M resazurin in 1X Assay Buffer), incubated for 5 minutes at 25°C, and read as described above. mIDH2-R172K was assayed as for mIDH2-R140Q with the following modifications: 1.25 g/mL of protein was used, the Substrate Mix contained 50 M NADPH and 6.4 M α-KG, and the compound was incubated for 1 hour before starting the reaction.

Cells were seeded in their respective growth media at a density of 5000 (U87MG, HCCC9810, COR-L 105) or 2500 (HT1080) cells/well into 96-well microtiter plates and incubated overnight at 37°C and 5% CO2. The next day, AG-120 was prepared in 100% DMSO as a 10 mM stock and then diluted in media for a final concentration of 0.1% DMSO. Highest concentration dose was 3 µM. Medium was removed from the cell plates and 200 µL of the compound dilutions were added to each well. For neurospheres, compounds and cells (40,000/well) were plated together at the same time. After 48 hours of incubation with compound at 37°C, 100 µL of media was removed from each well and analyzed as described below. The cell plates were then allowed to incubate another 24 hours. At 72 hours post compound addition, a 10 mL/plate of Promega Cell Titer Glo reagent was thawed and mixed. The cell plate was removed from the incubator and allowed to equilibrate to RT. Then 100 µL of reagent was added to each well of media. The cell plate was placed on an orbital shaker for 10 minutes and then allowed to sit at RT for 20 minutes. The plate was then read for luminescence with an integration time of 500 ms to determine any compound effects on growth inhibition (half maximal inhibition of cell proliferation, GI50).[3]

Generation of HT1080 mIDH1-R132C xenografts[3]
All animal studies were approved by the Institutional Animal Care and Use Committee and conducted in compliance with all national and local guidelines and regulations. HT1080 mIDH1-R132C cells were grown and 3 × 106 cells were inoculated subcutaneously on the flank of female BALB/c mice. When tumors reached approximately 200 mm3 the mice were randomized into dosing groups according to tumor size and treated with AG-120. Mice were dosed orally by gavage with a single dose of AG-120 at 50 or 150 mg/kg (n = 21 per dose group). Blood and tumor tissue samples were collected at 1, 3, 6, 12, 24, 48, and 72 hours following the dose (n = 3 at each time point) and were analyzed for AG-120 and 2-HG via LC-MS/MS.

Absorption, Distribution and Excretion
Following oral administration, ivosidenib is rapidly absorbed. After a single oral dose, the Cmax is 4503 ng/mL in patients with relapsed or refractory AML, 4820 ng/mL in newly diagnosed AML patients receiving azacitidine, and 4060 ng/mL in patients with cholangiocarcinoma. Steady-state plasma concentrations are reached within 14 days. The steady-state Cmax is 6551 ng/mL in patients with relapsed or refractory AML, 6145 ng/mL in newly diagnosed AML patients receiving azacitidine, and 4799 ng/mL in patients with cholangiocarcinoma. The Tmax is 2 to 3 hours. High-fat meals increase ivosidenib exposure.
After oral administration of ivosidenib, approximately 77% of the dose is excreted in the feces, of which 67% is excreted as the unchanged parent drug. Approximately 17% of the dose is excreted in the urine, of which 10% is excreted as unmetabolized ivosidenib.
In patients with relapsed or refractory AML, the steady-state apparent volume of distribution is 403 L; in newly diagnosed AML patients receiving azacitidine concurrently, the steady-state apparent volume of distribution is 504 L; and in patients with cholangiocarcinoma, the steady-state apparent clearance is 706 L.
In patients with relapsed or refractory AML, the steady-state apparent clearance is 5.6 L/h; in newly diagnosed AML patients receiving azacitidine concurrently, the steady-state apparent clearance is 4.6 L/h; and in patients with cholangiocarcinoma, the steady-state apparent clearance is 6.1 L/h.
Metabolism/Metabolites
Ivosidenib is primarily metabolized via CYP3A4 oxidation. The exact chemical structures of the CYP3A4-mediated oxidative metabolites have not been fully elucidated. Ivosidenib can also be metabolized via secondary metabolic pathways such as N-dealkylation and hydrolysis.

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Biological Half-Life
In patients with relapsed or refractory AML, the steady-state terminal half-life is 58 hours; in newly diagnosed AML patients concurrently receiving azacitidine therapy, the steady-state terminal half-life is 98 hours; and in patients with cholangiocarcinoma, the steady-state terminal half-life is 129 hours.

Hepatotoxicity
Elevated serum transaminase levels are common during ivosidenib treatment, occurring in approximately 15% to 20% of patients, but only 1% to 2% of patients have transaminase levels exceeding five times the upper limit of normal. Clinical use of ivosidenib is limited, but no cases of acute liver injury with symptomatic or jaundice have been found. Due to limited clinical experience with IDH inhibitors, the potential for liver injury is unclear. In premarketing studies, ivosidenib treatment was associated with differentiation syndrome in 5% to 20% of patients, sometimes severe and life-threatening. Differentiation syndrome is characterized by rapid myeloid cell proliferation and respiratory distress symptoms, accompanied by hypoxemia, pulmonary infiltration, and pleural effusion. Other manifestations include renal impairment, fever, lymphadenopathy, bone pain, peripheral edema, and weight gain. Liver dysfunction may also occur, but is usually masked by more severe systemic manifestations. Differentiation syndrome typically develops within 2 to 8 weeks of starting treatment and can be quite severe. Treatment includes discontinuing ivosidenib and, in severe cases, using corticosteroids and hydroxyurea. Once differentiation syndrome resolves, the patient can restart ivosidenib.
Probability score: E (Unproven but suspected cause of clinically significant liver injury).
Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
There is currently no clinical information regarding the use of ivosidenib during lactation. Because ivosidenib binds to plasma proteins at a rate of 92% to 96%, its concentration in breast milk may be low. However, its half-life is approximately 93 hours, so it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during ivosidenib treatment and for one month after administration.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein binding
In vitro experiments showed that ivosidenib binds to plasma proteins at a rate of 92-96%.

[1]. N Engl J Med.2018 Sep 20;379(12):1186;
[2]. Drugs.2018 Sep;78(14):1509-1516;
[3]. ACS Med Chem Lett.2018 Jan 19;9(4):300-305.

Ivosidenib is a tertiary amide formed by the condensation of the carboxyl group of (2S)-1-(4-cyanopyridin-2-yl)-5-oxopyrrolidine-2-carboxylic acid with the secondary amino group of (2S)-2-(2-chlorophenyl)-N-(3,3-difluorocyclobutyl)-2-[(5-fluoropyridin-3-yl)amino]acetamide. It has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with acute myeloid leukemia (AML) harboring isocitrate dehydrogenase-1 (IDH1) mutations. It is an anti-tumor drug and also an EC 1.1.1.42 (isocitrate dehydrogenase) inhibitor. It belongs to the monochlorobenzene, cyanopyridine, pyrrolidine-2-one, organofluorine compounds, tertiary amides, and secondary amides. Ivosidenib is a first-in-class isocitrate dehydrogenase-1 (IDH1) inhibitor. IDH1 is an enzyme that is frequently mutated and overexpressed in certain cancers, leading to abnormal cell growth and proliferation. Ivosidenib inhibits mutated IDH1, blocking its enzymatic activity and thus suppressing further differentiation of cancer cells. In July 2018, ivosidenib received accelerated approval from the U.S. Food and Drug Administration (FDA) for the treatment of relapsed/refractory acute myeloid leukemia in adults. Currently, ivosidenib is approved for the treatment of newly diagnosed acute myeloid leukemia in the elderly (in combination with azacitidine or as monotherapy), as well as for the treatment of locally advanced or metastatic cholangiocarcinoma and relapsed or refractory myelodysplastic syndromes in adults. This drug is only effective in patients carrying susceptible IDH1 mutations. In February 2023, the European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP) gave a positive opinion on ivosidenib and recommended its approval for marketing in the treatment of acute myeloid leukemia and cholangiocarcinoma. In May 2023, the EMA officially approved the drug.
Ivosidenib is an isocitrate dehydrogenase 1 (IDH1) inhibitor. Its mechanism of action is as an isocitrate dehydrogenase 1 inhibitor, a cytochrome P450 3A4 inducer, and a cytochrome P450 2C9 inducer.
Ivosidenib is an oral, small-molecule isocitrate dehydrogenase-1 inhibitor used to treat adult acute myeloid leukemia (AML) and is an anti-tumor drug. Elevated serum transaminases during Ivosidenib treatment occur at a moderate rate and are suspected to be the cause of a small number of clinically significant cases of acute liver injury.
Ivosidenib is an oral isocitrate dehydrogenase type 1 (IDH1) inhibitor with potential anti-tumor activity. After administration of AG-120, it specifically inhibits a mutated form of IDH1 in the cytoplasm, thereby inhibiting the production of the carcinogenic metabolite 2-hydroxyglutaric acid (2HG). This may lead to the induction of differentiation and inhibition of proliferation in IDH1-expressing tumor cells. IDH1 is an enzyme in the citric acid cycle that is mutated in various cancers; it initiates and drives cancer growth by blocking cell differentiation and catalyzing the production of 2HG. Ivosidenib is a small molecule drug, currently in Phase IV clinical trials (covering all indications), and was first approved in 2018. It currently has 3 approved indications and 7 investigational indications. This drug carries a black box warning from the U.S. Food and Drug Administration (FDA). Pharmacodynamics Ivosidenib is an anti-tumor drug effective against cancers carrying susceptible IDH1 mutations, indicating elevated levels of the oncogenic metabolite D-2-hydroxyglutaric acid (D-2HG) in cancer cells. Ivosidenib reduces D-2HG levels in a dose-dependent manner by inhibiting the IDH1 enzyme. Ivosidenib inhibits both mutant and wild-type IDH1 but not IDH2. Somatic point mutations in a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer novel functions in cancer cells, leading to the production of the oncogenic metabolite D-2-hydroxyglutaric acid (2-HG). Elevated 2-HG levels are associated with epigenetic alterations and impaired cell differentiation. IDH1 mutations have been found in various hematologic malignancies and solid tumors. This article reports the discovery of AG-120 (ivosidenib), an IDH1 mutant enzyme inhibitor that exhibits significant 2-HG-reducing effects in tumor models and can induce differentiation in primary AML patient samples in vitro. Preliminary data from a Phase I clinical trial recruiting cancer patients with IDH1 mutations demonstrate that AG-120 has acceptable safety and clinical activity. These compelling preclinical data provide a theoretical basis for advancing AG-120 into clinical development. Previously, enasidenib (an active agent targeting mIDH2) and AG-120 (ivosidenib, an active agent targeting mIDH1), described in this article, have been identified, representing a novel cancer therapy based on cell differentiation. AG-120 is a potent mIDH1 inhibitor with a favorable non-clinical and clinical safety profile, demonstrating good clinical activity in both solid tumors and hematologic malignancies in a Phase 1 clinical trial. Interim results from an ongoing Phase 1 clinical trial in patients with relapsed/refractory mIDH1 AML showed an overall response rate of 42% and a complete response rate of 22% (median duration of complete response was 9.3 months). Long-term disease stability was observed in patients with previously treated non-enhancing mIDH1 gliomas and in patients with previously heavily treated mIDH1 cholangiocarcinoma, with a median progression-free survival of 3.8 months and a 6-month progression-free survival rate of 40%. ¹⁷ In these two single-arm phase 1 studies, AG-120 has demonstrated acceptable safety to date. ^15-18 AG-120 is currently in late-stage clinical development in adult mIDH1 AML (ClinicalTrials.gov NCT03173248) and previously treated advanced mIDH1 cholangiocarcinoma (NCT02989857). [3]

C28H22CLF3N6O3

582.97

582.139

C, 57.69; H, 3.80; Cl, 6.08; F, 9.78; N, 14.42; O, 8.23

1448346-63-1

Ivosidenib;1448347-49-6;(R,S)-Ivosidenib;2070009-31-1

89699486

White to off-white solid

1.5±0.1 g/cm3

854.3±65.0 °C at 760 mmHg

470.4±34.3 °C

0.0±3.2 mmHg at 25°C

1.651

0.38

1

9

7

41

1050

1

ClC1=C([H])C([H])=C([H])C([H])=C1C([H])(C(N([H])C1([H])C([H])([H])C(C1([H])[H])(F)F)=O)N(C1C([H])=NC([H])=C(C=1[H])F)C([C@]1([H])C([H])([H])C([H])([H])C(N1C1C([H])=C(C#N)C([H])=C([H])N=1)=O)=O

WIJZXSAJMHAVGX-XADRRFQNSA-N

InChI=1S/C28H22ClF3N6O3/c29-21-4-2-1-3-20(21)25(26(40)36-18-11-28(31,32)12-18)37(19-10-17(30)14-34-15-19)27(41)22-5-6-24(39)38(22)23-9-16(13-33)7-8-35-23/h1-4,7-10,14-15,18,22,25H,5-6,11-12H2,(H,36,40)/t22-,25?/m0/s1

(S)-N-((S)-1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide

trade name: Tibsovo® , (R,S)-Ivosidenib; (R,S)-AG-120; AG-120 (racemic); AG120; AG 120 (racemic); 1448346-63-1; AG-120 (Ivosidenib); IDH1 Inhibitor 8; AG-120 Racemate; (2S)-N-(1-(2-chlorophenyl)-2-((3,3-difluorocyclobutyl)amino)-2-oxoethyl)-1-(4-cyanopyridin-2-yl)-N-(5-fluoropyridin-3-yl)-5-oxopyrrolidine-2-carboxamide; AG-120 (racemic); AG 120; 2-(2-CHLOROPHENYL)-2-{1-[(2S)-1-(4-CYANOPYRIDIN-2-YL)-5-OXOPYRROLIDIN-2-YL]-N-(5-FLUOROPYRIDIN-3-YL)FORMAMIDO}-N-(3,3-DIFLUOROCYCLOBUTYL)ACETAMIDE; RG-120; RG 120; RG120 (racemic)

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)

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.)