DPP-IV (IC50 = 3.5 nM)

Vildagliptin inhibits apoptosis to increase β-cell survival. Additionally, vildagliptin stimulates cell division [2].

Vildagliptin raises plasma active GLP-1 levels in the islets of db/db mice when administered orally once daily at a dose of 35 mg/kg [2]. Vildagliptin (10 µmol/kg; oral) in obese male Zucker rats dramatically lowers glucose excursions and increases insulin secretion [1].

DPP-IV Inhibition Measurement ex Vivo.Rat, Human, Monkey Plasma Assays.[4]
Human, rat, or monkey plasma was used as the source of DPP-IV in the assay. The standard assay was modified from a previously published method. Five μL of plasma was added to 96-well flat-bottom microtiter plates, followed by the addition of 5 μL of 80 mM MgC12 in assay buffer (25 mM HEPES, 140 mM NaC1, 1% RIA-grade BSA, pH 7.8). After a 5-min preincubation at room temperature, the reaction was initiated by the addition of 10 μL of assay buffer containing 0.1 mM substrate (H-Gly-Pro-AMC; AMC is 7-amino-4-methylcoumarin). The plates were covered with aluminum foil (or kept in the dark) and incubated at room temperature for 20 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 2 μL additions, and the assay buffer volume was reduced to 13 μL. A standard curve of free AMC was generated using 0−50 μM solutions of AMC. The curve generated, which was linear, was used for interpolation of substrate consumption (catalytic activity in nmoles substrate cleaved /min).

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DPP-II Inhibition Measurement in Vitro. [4]
An extract of bovine kidney homogenate, partially purified by ion-exchange and adenosine deaminase chromatography, was used as the source of DPP-II in the assay. The standard assay was modified from a previously published method. 47 Twenty micrograms of DPP-II-containing fraction diluted to a final volume of 60 μL in assay buffer (0.2 M Borate, 0.05 M Citrate, pH 5.3) was added to 96-well flat-bottom microtiter plates, followed by the addition of 10 μL of 10 mM o-phenanthroline (to inhibit aminopeptidase activity) and 20 μL of 5 mM substrate (H-Lys-Ala-AMC; AMC is 7-amino-4-methylcoumarin). The plates were incubated at 37 °C for 30 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 20 μL additions, and assay buffer volume is reduced to 50 μL. A standard curve of AMC was generated using 0 to 100 μM of AMC. The curve generated, which was linear, was used for interpolation of catalytic activity (in nmoles substrate cleaved/min).

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Vildagliptin (LAF-237; NVP-LAF 237) has an IC50 of 2.3 nM, which inhibits DPP-4. Figure 2 represents vildagliptin, an N-substituted glycyl-2-cyanopyrrolidine. With an inhibitory concentration (IC50) of approximately 2–3 nmol/L, it is a strong, reversible, and competitive inhibitor of DPP-4 in both humans and rodents in vitro. Crucially, vildagliptin exhibits high specificity inhibition of DPP-4 in comparison to other analogous peptidases, wherein its IC50 surpasses 200 mol/L.

In Vitro Studies.DPP-IV Inhibition Measurement in Vitro:  Caco-2 Assay. [4]
An extract from human colonic carcinoma cells (Caco-2; American Type Culture Collection; ATCC HTB 37) was used as the source of DPP-IV in the assay. The cells were differentiated to induce DPP-IV expression as described by previously. Cell extract was prepared from cells solubilized in lysis buffer (10 mM Tris-HC1, 0.15 M NaC1, 0.04 T.I.U. (trypsin inhibitor unit) aprotinin, 0.5% nonidet-P40, pH 8.0) then centrifuged at 35 000g for 30 min at 4 °C to remove cell debris. The assay was conducted by adding 20 μg of solubilized Caco-2 protein, diluted to a final volume of 125 μL in assay buffer (25 mM Tris-HC1 pH 7.4, 140 mM NaC1, 10 mM KC1, 1% bovine serum albumin) to 96-well flat-bottom microtiter plates. The reaction was initiated by adding 25 μL of 1 mM substrate (H-Ala-Pro-pNA; pNA is p-nitroaniline). The reaction was run at room temperature for 10 min, and then 19 μL of 25% glacial acetic acid was added to stop the reaction. Fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 30 μL additions, and the assay buffer volume was reduced to 95 μL. A standard curve of free p-nitroaniline was generated using 0−100 μM pNA in assay buffer. The curve generated, which was linear, was used for interpolation of substrate consumption (catalytic activity in nmoles substrate cleaved /min).

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Post-Proline Cleaving Enzyme (PPCE) Inhibition Measurement in Vitro. [4]
A cytosolic extract of human erythrocytes, partially purified by ion-exchange chromatography, was used as the source of PPCE in the assay. The standard assay is modified from a previously published method. PPCE-containing fraction (350 ng protein) diluted to a final volume of 90 μL in assay buffer (20 mM NaPO4, 0.5 mM EDTA, 0.5 mM DTT, 1% BSA, pH 7.4) was added to 96-well flat-bottom microtiter plates, followed by the addition of 10 μL of 0.5 mM substrate (Z-Gly-Pro-AMC; AMC is 7-amino-4-methylcoumarin). The plates were incubated at room temperature for 30 min. After incubation, fluorescence was measured using a CytoFluor II fluorometer (excitation 380 nm/ emission 460 nm). Test compounds and solvent controls were added as 20 μL additions, and the assay buffer volume was reduced to 70 μL. A standard curve of free AMC was generated using 0 to 5 μM solutions of AMC. The curve generated, which was linear, was used for interpolation of catalytic activity (in nmoles substrate cleaved/min).

Animal/Disease Models: Male db/db mice (BKS) and wild-type mice [2]
Doses: 35 mg/kg
Route of Administration: po (oral gavage); one time/day; for 6 weeks
Experimental Results: Increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44).

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Animal/Disease Models: Obese male Zucker rat [1]
Doses: 10 µmol/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: Oral
Experimental Results:Dramatically diminished blood sugar fluctuations and stimulated insulin secretion.\n

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\n\nIn Vivo Obese Male (fa/fa) Zucker Rat Studies.[1]
\nEffect of Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (Vildagliptin (NVP LAF 237; DSP7238; LAF237) ) on DPP-IV Activity, Active GLP-1 Levels, and Glucose and Insulin Excursions. Studies were performed on obese male Zucker (fa/fa) rats (Charles River Labs, Cambridge, MA); controls (n = 9) and Vildagliptin (NVP LAF 237; DSP7238; LAF237) -treated (n = 9). These rats were purchased at 7 weeks of age, cannulated at 7.5 weeks, and studied beginning at around 11 weeks of age. In the morning of the oral glucose tolerance test (OGTT), the rats were “fasted” by removing food before the lights were turned on, after which they were transferred to the experiment room at 8:00 a.m.. Vildagliptin (NVP LAF 237; DSP7238; LAF237)  was dissolved in vehicle solution (0.5% carboxymethylcellulose (CMC) and 0.2% Tween 80). The cannulas were connected to sampling tubing (PE-100, 0.034 in. i.d. × 0.06 in. o.d.), which were filled with saline. After 30−40 min cage acclimation, a 0.5 mL baseline blood sample was taken at t = −15 min, and the rats were then orally dosed with CMC or Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (10 μmol/kg), after which additional baseline blood samples were taken at t = −5, −2.5, and 0 min. The animals were then administered an oral glucose solution (10% glucose, 1 g/kg) immediately after t = 0‘. The rest of the samples were taken at 1, 3, 5, 10, 15, 20, 30, 45, 60, 75, and 90 min. Throughout the OGTT, an equal volume of donor blood was used to replace the blood withdrawn during sampling. Donor blood was obtained from donor rats through cardiac puncture. The collected blood samples (0.5 mL) were immediately transferred into chilled Eppendorf tubes containing 50 μL of EDTA:  trasylol (25 mg/mL of 10 000 trasylol) and used for the measurement of glucose and insulin levels and DPP-IV activity. Larger blood samples (0.75 mL) were collected at t = −15, 0, 5, 10, 15, and 30 min for GLP-1 (7−36 amide) measurements. To these tubes, the DPP-IV inhibitor valine pyrrolidide was added to yield a final concentration in the blood of 1 μM. Technical difficulties with obtaining blood samples after minute 20 for one rat in both the CMC and Vildagliptin (NVP LAF 237; DSP7238; LAF237)  groups resulted in the inability to calculate glucose and insulin AUC data for those rats, leading to AUC data with an n = 8/group. Measurement of plasma glucose was made using a modification of a Sigma Diagnostics glucose oxidase kit. DPP-IV activity was measured in plasma samples obtained at −5, 0, 20, 45, and 90 min DPP-IV activity as previously described in the above ex vivo rat plasma experimental. Plasma levels of GLP-1 (7−36 amide) were measured using the GLP-1 (active) Elisa Kit.\n

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\n\nIn Vivo Cynomolgus Monkey PK/PD Studies Using 8c and Vildagliptin (NVP LAF 237; DSP7238; LAF237) . [1]
\nKetamine-anesthetized male healthy cynomolgus monkeys received either 8c (n = 2) or Vildagliptin (NVP LAF 237; DSP7238; LAF237)  (n = 3) (dissolved in CMC/Tween-80) by oral gavage (1.007 μmol/kg), and by intravenous administration (0.399 μmol/kg) (dissolved in saline). For iv study, compound was administered (0.4 mL/kg over 1 min) in 0.9% saline as vehicle. Different monkeys were used for each dosage regimen. Basal blood samples were collected at −10 min and immediately prior to administration of compound. Blood samples were collected at 0.03, 0.08, 0.17, 0.25, 0.33, 0.42, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 7, 12, and 25 h postdose for both routes of administration. Blood was obtained into heparin-coated syringes, transferred to microcentrifuge tubes, and centrifuged to separate the plasma. The plasma was stored at −80 °C in fresh microcentrifuge tubes until assay. DPP-IV activity was measured in a similar manner was as previously described in the above ex vivo rat and human plasma experimentals. Plasma DPP-IV activities were calculated and expressed as ‘percent of baseline' to reduce variability due to individual differences in plasma enzyme activity. Area-under-curve (AUC) values for DPP-IV activity were calculated from time (hours after dose) vs effect (percent inhibition) curves from individual animals using the trapezoidal method. The ratio of dose-normalized effect AUC for oral/intravenous administration routes was taken as an estimate of effect bioavailability. Parent drug concentrations were determined using an HPLC/MS/MS method with a limit of quantification of 1 ng/mL. Pharmacokinetic parameters were calculated using noncompartment modeling, and the AUC was calculated using the linear trapezoidal method. Absolute oral bioavailability was calculated by (AUC0-∞po × 399)/(AUC0-∞iv × 1007).\n

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\n\nVildagliptin was orally administered to db/db mice for 6 weeks, followed by evaluation of beta cell apoptosis by caspase3 activity and TUNEL staining method. Endoplasmic reticulum stress markers were determined with quantitative RT-PCR, immunohistochemistry and immunoblot analysis.\n
\nResults: After 6 weeks of treatment, vildagliptin treatment increased plasma active GLP-1 levels (22.63±1.19 vs. 11.69±0.44, P<0.001), inhibited beta cell apoptosis as demonstrated by lower amounts of TUNEL staining nuclei (0.37±0.03 vs. 0.55±0.03, P<0.01) as well as decreased caspase3 activity (1.48±0.11 vs. 2.67±0.13, P<0.01) in islets of diabetic mice compared with untreated diabetic group. Further, vildagliptin treatment down-regulated several genes related to endoplasmic reticulum stress including TRIB3 (tribbles homolog 3) (15.9±0.4 vs. 33.3±1.7, ×10⁻³, P<0.001), ATF-4(activating transcription factor 4) (0.83±0.06 vs. 1.42±0.02, P<0.001) and CHOP(C/EBP homologous protein) (0.07±0.01 vs. 0.16±0.01, P<0.001).\n
\nConclusions: Vildagliptin promoted beta cell survival in db/db mice in association with down-regulating markers of endoplasmic reticulum stress including TRIB3, ATF-4 as well as CHOP.[5]\n

Absorption
Vildagliptin is rapidly absorbed after oral administration on an empty stomach. Peak plasma concentration is reached 1.7 hours after administration. Plasma concentrations of vildagliptin increase approximately dose-proportional. Food delays the time to peak concentration (Tmax) to 2.5 hours and reduces plasma concentration (Cmax) by 19%, but has no effect on total drug exposure (AUC). The absolute bioavailability of vildagliptin is 85%.
Elimination Pathway
Vildagliptin is primarily eliminated through metabolism. After oral administration, approximately 85% of the radiolabeled vildagliptin dose is excreted in the urine, and approximately 15% is excreted in the feces. Of the dose excreted in the urine, approximately 23% is unmetabolized parent compound.
Volume of Distribution
The mean volume of distribution at steady state after intravenous administration of vildagliptin is 71 L, suggesting that it is primarily distributed extravascularly.
Clearance
In healthy subjects following intravenous administration of vildagliptin, the total plasma clearance and renal clearance were 41 L/h and 13 L/h, respectively.
Metabolism/Metabolites
Approximately 69% of orally administered vildagliptin is cleared via a non-cytochrome P450 enzyme-mediated metabolic pathway. Based on results from a rat study, DPP-4 is involved in the partial hydrolysis of vildagliptin. Vildagliptin is metabolized in the kidneys to pharmacologically inactive cyano (57%) and amide (4%) hydrolysates. LAY 151 (M20.7) is a major inactive metabolite, a carboxylic acid formed via cyano hydrolysis, accounting for 57% of the administered dose. Other reported circulating metabolites include N-glucuronide (M20.2), N-amide hydrolysate (M15.3), and two oxidation products, M21.6 and M20.9.

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Biological Half-Life
The mean elimination half-life after intravenous administration is approximately 2 hours. The elimination half-life after oral administration is approximately 3 hours.

Protein binding rate: Vildagliptin has a plasma protein binding rate of 9.3%. Vildagliptin is evenly distributed in plasma and erythrocytes.

[1]. Combination of the dipeptidyl peptidase IV inhibitor LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] with the angiotensin II type 1 receptor antagonist valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphen. J Pharmacol Exp Ther. 2008 Dec;327(3):683-91.

[2]. The synergistic effect of valsartan and LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] on vascular oxidative stress and inflammation in type 2 diabetic mice. Exp Diabetes Res. 2012;2012:146194.

[3]. Vildagliptin/Pioglitazone Combination Improved The Overall Glycemic Control In Type I Diabetic Rats. Can J Physiol Pharmacol. 2018 Aug;96(8):710-718.

[4].1-[[(3-hydroxy-1-adamantyl)amino]acetyl]-2-cyano-(S)-pyrrolidine: a potent, selective, and orally bioavailable dipeptidyl peptidase IV inhibitor with antihyperglycemic properties. J Med Chem. 2003 Jun 19;46(13):2774-89.

[5]. Dipeptidyl peptidase-4 inhibitor, vildagliptin, inhibits pancreatic beta cell apoptosis in associationwith its effects suppressing endoplasmic reticulum stress in db/db mice. Metabolism. 2015 Feb;64(2):226-35.

Vildagliptin is an amino acid amide. Vildagliptin (LAF237) is an oral hypoglycemic agent that selectively inhibits dipeptidyl peptidase-4 (DPP-4). It is used to treat type 2 diabetes, a condition in which glucagon-like peptide-1 (GLP-1) secretion and insulin secretion are impaired. Vildagliptin inhibits DPP-4, preventing the degradation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both of which are incretins that promote insulin secretion and regulate blood glucose levels. Increased levels of GLP-1 and GIP ultimately improve glycemic control. Clinical trials have shown that vildagliptin carries a relatively low risk of hypoglycemia. In 2008, the European Medicines Agency approved oral vildagliptin for the treatment of type 2 diabetes in adults, either as monotherapy or in combination with metformin, sulfonylureas, or thiazolidinediones, for patients with inadequate glycemic control after monotherapy. It is marketed under the brand name Galvus. Vildagliptin also has a fixed-dose combination formulation, Eucreas, used in combination with metformin for adult patients whose glycemic control is inadequate after monotherapy. Currently, vildagliptin is being investigated in the United States. Vildagliptin is a cyanopyrrolidine-based, orally bioavailable dipeptidyl peptidase-4 (DPP-4) inhibitor with glycemic activity. The cyano group of vildagliptin is hydrolyzed, and the resulting inactive metabolites are primarily excreted in the urine. Vildagliptin is a pyrrolidine nitrile derivative and a potent inhibitor of dipeptidyl peptidase-4, used to treat type 2 diabetes. Indications Vildagliptin is indicated for the treatment of type 2 diabetes in adults. As monotherapy, vildagliptin is indicated for adult patients whose glycemic control is inadequate with diet and exercise alone and who are unsuitable for metformin due to contraindications or intolerance. Vildagliptin can also be used in combination with metformin, sulfonylureas, or thiazolidinediones for adult patients whose glycemic control remains inadequate despite receiving the maximum tolerated dose of monotherapy. Vildagliptin is also marketed in combination with metformin for the treatment of adult patients with type 2 diabetes who have not responded adequately to vildagliptin or metformin monotherapy. This fixed-dose formulation can be used in combination with sulfonylureas or insulin (i.e., triple therapy) as adjunctive therapy to diet and exercise in adult patients who have not achieved adequate glycemic control with monotherapy or dual therapy. Vildagliptin is indicated as adjunctive therapy to diet and exercise to improve glycemic control in adult patients with type 2 diabetes; it can also be used in patients who are not suitable for metformin due to contraindications or intolerance. It can be used in combination with other diabetes medications, including insulin, when they do not provide adequate glycemic control (see Sections 4.4, 4.5, and 5.1 for available data on different combination therapy regimens).
Vildagliptin is indicated as adjunctive therapy to diet and exercise to improve glycemic control in adults with type 2 diabetes: it can be used as monotherapy in patients who are unsuitable for metformin due to contraindications or intolerance. It can be used in combination with other diabetes medications, including insulin, when they fail to provide adequate glycemic control.

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Pharmacodynamics
Vildagliptin improves glycemic control in type 2 diabetes by enhancing glucose sensitivity of pancreatic β-cells and promoting glucose-dependent insulin secretion. Elevated GLP-1 levels enhance α-cell sensitivity to glucose, thereby promoting glucagon secretion. Vildagliptin increases the insulin/glucagon ratio by increasing incretin levels, thereby reducing fasting and postprandial hepatic glucose production. Vildagliptin does not affect gastric emptying or insulin secretion or blood glucose levels in individuals with normal glycemic control. Clinical trials have shown that daily administration of 50-100 mg vildagliptin in patients with type 2 diabetes significantly improves β-cell markers, proinsulin/insulin ratio, and indicators of β-cell responsiveness in frequently sampled postprandial glucose tolerance tests. Vildagliptin also improves glycated hemoglobin (HbA1c) and fasting plasma glucose (FPG) levels. Mechanism of Action: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are incretins that regulate blood glucose levels and maintain glucose homeostasis. It is estimated that the activity of GLP-1 and GIP contributes more than 70% to the insulin response in oral glucose tolerance tests. They stimulate insulin secretion in a glucose-dependent manner through the G protein-coupled GIP and GLP-1 receptor signaling pathways. In addition to its effects on insulin secretion, GLP-1 is also involved in promoting islet anogenesis and differentiation and attenuating islet β-cell apoptosis. Incretins also have extrapancreatic effects, such as promoting adipogenesis and regulating myocardial function. In type 2 diabetes, GLP-1 secretion is impaired, and the insulin-stimulating effect of GIP is significantly weakened. Vildagliptin exerts its hypoglycemic effect by selectively inhibiting dipeptidyl peptidase-4 (DPP-4). DPP-4 is an enzyme that rapidly cleaves and inactivates GLP-1 and GIP released from intestinal cells. DPP-4 cleaves oligopeptides, with the cleavage site located after the second amino acid at the N-terminus. Inhibition of DPP-4 significantly prolongs the half-life of GLP-1 and GIP, thereby increasing the level of circulating active incretin hormones. The duration of vildagliptin's inhibitory effect on DPP-4 is dose-dependent. Vildagliptin reduces fasting blood glucose, postprandial blood glucose, and glycated hemoglobin (HbA1c) levels. It enhances glucose sensitivity in α and β cells and promotes glucose-dependent insulin secretion. Lowered fasting and postprandial blood glucose levels are accompanied by improved postprandial lipid and lipoprotein metabolism.

C17H27N3O3

321.41458439827

339.215

2133364-01-7

Vildagliptin;274901-16-5;(2R)-Vildagliptin;1036959-27-9

167996054

Typically exists as solid at room temperature

4

6

3

24

523

3

N(C12CC3CC(CC(C3)(O)C1)C2)CC(N1CCC[C@H]1C#N)=O.O

MVOBUCAQTXEOGS-XQOPLDTQSA-N

InChI=1S/C17H25N3O2.2H2O/c18-9-14-2-1-3-20(14)15(21)10-19-16-5-12-4-13(6-16)8-17(22,7-12)11-16;;/h12-14,19,22H,1-8,10-11H2;2*1H2/t12-,13+,14-,16?,17?;;/m0../s1

(2S)-1-[2-[[(5S,7R)-3-hydroxy-1-adamantyl]amino]acetyl]pyrrolidine-2-carbonitrile;dihydrate

LAF-237 dihydrate; Vildagliptin dihydrate; 2133364-01-7; (2S)-1-[2-[[(5S,7R)-3-hydroxy-1-adamantyl]amino]acetyl]pyrrolidine-2-carbonitrile;dihydrate NVPLAF 237 dihydrate; LAF 237 dihydrate NVP-LAF-237 dihydrate; LAF237 dihydrate; NVP-LAF 237 dihydrate

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.)