Thyroid hormone beta receptor/(TR)-β

MB07811 is the liver-targeted prodrug of the novel TR agonist 3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy methylphosphonic acid (MB07344) and has been shown to have oral cholesterol lowering activity in a variety of animal models (Erion et al., 2007). MB07344 exhibits a TRβ binding affinity Ki of 2.17 ± 0.03 nmol·L−1, and a Ki TRα/Ki TRβ ratio of 15.8, whereas MB07811 has low TR affinity (>12 µmol·L−1). We report that MB07811/MB07344 had adjunctive activity when given in combination with atorvastatin in all three species. These results are supportive of the potential for TR agonists, such as MB07811, to have clinical utility as a treatment to further lower cholesterol in those patients who do not successfully achieve cholesterol goals with statin treatment alone[1].

In this study, researchers evaluated the activity of a liver-targeted prodrug, MB07811 , of a novel TH receptor beta agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys. Key results: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811  (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy.[1]

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Activity of MB07811  in normal and hypercholesterolaemic rabbits[1]
These experiments were conducted to determine the oral efficacy of MB07811  in the normal and hypercholesterolaemic rabbit models. For the evaluation in normal rabbits, following baseline TPC measurements, animals were randomized into control (n = 6) and MB07811 groups (10 mg·kg−1·day−1, n = 6). Drug treatment (MB07811, 200 p.p.m. in diet) was instituted for 4 weeks with daily monitoring of food intake and body weight. TPC measurements were obtained weekly as previously described. In hypercholesterolaemic rabbits, a separate cohort of rabbits was placed on a diet containing 0.2% cholesterol. After 4 weeks, when TPC levels had increased to 13.0–15.5 mmol·L−1, animals were randomized to control (n = 7) and MB07811-treated (n = 8) experimental groups. MB07811 animals were treated with MB07811 (added to 0.2% cholesterol diet) at a dose of 5 mg·kg−1·day−1 for 2 weeks followed by 10 mg·kg−1·day−1 for an additional 5 weeks. TPC, body weight and food intake measurements were obtained weekly.

Adjunctive activity of MB07811  in combination with atorvastatin in normocholesterolaemic rabbits[1]
To evaluate the oral activity of MB07811  when administered as adjunctive treatment to atorvastatin, an experimental design similar to the MB07344 ± atorvastatin study described above was employed. The experimental groups (n = 6/group) were non-drug control, atorvastatin alone (3 mg·kg−1·day−1), MB07811 alone (10 mg·kg−1·day−1) and atorvastatin (3 mg·kg−1·day−1) + MB07811 (10 mg·kg−1·day−1). The duration of atorvastatin exposure prior to initiation of MB07811 treatment was increased to 3 weeks to ensure that TPC levels were stable prior to administration of MB07811. All other aspects of the protocol were identical to the MB07344 ± atorvastatin study. As with the first combination study, there were no significant effects on either body weight or food intake among the experimental groups.

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MB07811  and atorvastatin monotherapy/combination efficacy in dogs[1]
A study was conducted in six male and six female dogs utilizing a three-way crossover design consisting of two male and two female dogs/group, three treatment groups/cycle, and three treatment cycles. The experimental groups were MB07811 (30 mg·kg−1·day−1), atorvastatin (10 mg·kg−1·day−1) or a combination of MB07811  (30 mg·kg−1·day−1) and atorvastatin (10 mg·kg−1·day−1) administered by oral gavage daily for seven consecutive days. The dose of atorvastatin was chosen as 10 mg·kg−1·day−1 as this has been determined to be the no-adverse-effect level in chronic studies in dogs (C. Wilker, pers. comm.). A washout period of a minimum of 14 days was imposed between each cycle. With this experimental design, each dog received all three treatment regimens over the course of the study. Venous blood samples for blood cholesterol measurements were obtained in each animal at t = 0 h before the first dose (pre-dose) and then 24 h after the 7th dose for each treatment cycle. Both MB07811 and atorvastatin were formulated as uniform suspensions in 0.5% carboxymethyl cellulose, and 1% Lutrol in water and administered by oral gavage at 2 mL·kg−1.

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MB07811  and atorvastatin monotherapy efficacy in monkeys[1]
Six male and six female cynomolgus monkeys were instrumented with electrocardiogram leads, pressure transducers, and telemetry units and allowed to recover for at least 1 week prior to study initiation. MB07811  (0.03–30 mg·kg−1·day−1 in half-log increments), atorvastatin (0.3–30 mg·kg−1·day−1 in half-log increments) or vehicle (polyethylene glycol 400) was administered orally to groups of three male and three female monkeys on a daily basis for 7 days. Blood was drawn prior to dosing on day 1 (pre-dose) and approximately 24 h after the final dose on day 8 to assess serum cholesterol and clinical chemistry parameters. Treatment stopped on day 8 and the animals were subjected to a washout period of at least 7 days prior to the next treatment. Animals were cycled through 6–8 treatments over the course of the study. Vehicle was administered on two separate cycles, so all animals received vehicle.

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MB07811  and atorvastatin monotherapy/combination efficacy and pharmacokinetics in monkey[1]
A study was conducted to evaluate both the cholesterol lowering activity and pharmacokinetics of MB07811  and atorvastatin alone and in combination in cynomolgus monkeys. A total of six male and six female non-naïve cynomolgus monkeys were assigned to the study. Groups of four monkeys (two males and two females) were placed under one of three oral treatment regimens: (i) 3 mg·kg−1·day−1 of MB07811 for 8 days; (ii) 3 mg·kg−1·day−1 of atorvastatin for 8 days; and (iii) a combination of 3 mg·kg−1·day−1 of MB07811 and 3 mg·kg−1·day−1 of atorvastatin for 8 days. The vehicle for all treatments was polyethylene glycol-400. Prior to treatment and at 24 h following the 7th dose, blood samples were collected from the femoral vein or artery and serum prepared for analysis of total cholesterol. Blood was also collected on the final day (day 8) of compound administration at 0 (pre-dose), 0.5, 1, 2, 4, 8, 12 and 24 h post last dose (day 9) for each regimen for pharmacokinetic evaluation. For this latter purpose, plasma proteins from plasma samples (50 µL) were precipitated by addition of methanol (75 µL). After 20 min of centrifugation (Eppendorf microfuge) at 1500× g and room temperature, the resulting supernatant was collected and concentrations of MB07811, AT and their respective active metabolites MB07344 and hydroxy-atorvastatin were measured by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) as described below. Non-compartmental pharmacokinetic analysis was performed on the resulting plasma concentration-time profiles for each analyte using WinNonLin (ver. 1.1; Scientific Consultants, Cary, NC). The pharmacokinetic variables measured were defined as follows: AUClast, the area under the curve from the time of dosing (t = 0) to the last measurable concentration (24 h); Cmax, maximum observed concentration; MRT, mean residence time from the time of dosing to the time of the last measurable concentration and t1/2, terminal half-life estimated via linear regression of the time versus log concentration plot.

In Vitro Metabolism.[2]
MB07811  was efficiently converted to MB07344 and the glutathione conjugate 2 by liver microsomes prepared from male Sprague–Dawley (SD) rats. The Vmax, Km, and CLint (intrinsic clearance = Vmax/Km) values were 2.74 ± 0.12 nmol·min−1·mg−1, 18.8 ± 3.06 μM, and 145 ± 24.5 μl·min−1·mg−1, respectively. Conversion was inhibited by clotrimazole (100% at 1 μM, Ki = 24 nM), suggesting that CYP3A is the predominant CYP responsible for prodrug conversion. Neither MB07811  nor MB07344 inhibited CYP3A at 10 μM.

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LC-MS/MS analysis[1]
A 10 µL plasma extract aliquot was injected onto a Gemini C18 column (5 µm, 2 × 50 mm, Phenomenex) fitted with a Gemini C18 guard column (5 µm, 4.0 × 3.0 mm) and eluted with a gradient consisting of mobile phase A (20 mmol·L−1 N,N-dimethylhexylamine and 10 mmol·L−1 propionic acid in 20% methanol) and B (20 mmol·L−1 N,N-dimethylhexylamine and 10 mmol·L−1 propionic acid in 80% methanol) at a flow rate of 0.4 mL·min−1 (0 min, 60% B; 0–0.5 min, 60–100% B; 0.5–6 min, 100% B; 6–6.1 min, 100–60% B; 6.1–9 min, 60% B). The injector temperature was set at 10°C. Elution times for MB07344, MB07811 , atorvastatin and hydroxyatorvastatin were approximately 2.7, 4.9, 2.3 and 2.2 min respectively. MB07811, MB07734, AT and hydroxyatorvastatin were detected using the MS/MS mode (513/63.1 for MB07811, 363.3/63.1 for MB07344, 557.5/278.4 for AT and 573.5/278.4 for hydroxyatorvastatin) and quantified by comparison of peak areas to standard curves obtained by adding known concentrations of the analytes to blank monkey plasma. Calibration curves ranging from 1 to 3000 ng·mL−1 for MB07344 and MB07811 (LOQ of 1 ng·mL−1) and from 0.1 to 3000 ng·mL−1 for atorvastatin (LOQ of 0.1 ng·mL−1) were generated. Although there is more than one possible isomer of hydroxyatorvastatin, this assay could not distinguish between them. As hydroxyatorvastatin standards were not available, plasma concentrations of this analyte were estimated using the atorvastatin calibration curve.

High intracellular levels of MB07344 were detected in freshly isolated rat hepatocytes incubated with MB07811 (Fig. 2A), indicating that MB07811 distributes readily into hepatocytes and is converted to MB07344 (Cmax = 1.03 ± 0.00 nmol per 106 cells; tmax = 1.5 h). Hepatocytes incubated with MB07344 also exhibited high intracellular MB07344 levels (1.77 ± 0.05 nmol per 106 cells; tmax = 1 h) (Fig. 2A), suggesting that MB07344, like other negatively charged phosphonates, may enter hepatocytes via organic anion transporters[2].

Adjunctive activity of MB07811  in combination with atorvastatin in normocholesterolaemic rabbits[1] To evaluate the oral activity ofMB07811  when administered as adjunctive treatment to atorvastatin, an experimental design similar to the MB07344 ± atorvastatin study described above was employed. The experimental groups (n = 6/group) were non-drug control, atorvastatin alone (3 mg·kg−1·day−1), MB07811 alone (10 mg·kg−1·day−1) and atorvastatin (3 mg·kg−1·day−1) + MB07811 (10 mg·kg−1·day−1). The duration of atorvastatin exposure prior to initiation of MB07811 treatment was increased to 3 weeks to ensure that TPC levels were stable prior to administration of MB07811. All other aspects of the protocol were identical to the MB07344 ± atorvastatin study. As with the first combination study, there were no significant effects on either body weight or food intake among the experimental groups.

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Rat Pharmacokinetics.[2]
Pharmacokinetic parameters for MB07344 (i.v.) and MB07811  (i.v. and per os) in male SD rats were determined from the temporal profile of MB07811 and MB07344 in plasma, using the methods described in SI Appendix. First-pass hepatic extraction (EH) was determined by measuring MB07811 levels after oral administration of MB07811 (3 mg/kg) to catheterized male SD rats (n = 4–5 per group) and by using the equation EH = (AUCpv − AUCsys)/AUCpv, wherein AUCpv and AUCsys represent AUC values derived from the portal vein (pv) and carotid artery (sys) plasma concentration-time profiles. Biliary excretion and enterohepatic recirculation were assessed by measuring MB07344 levels in plasma and bile collected from naïve and bile duct-cannulated SD rats (n = 3 per group) administered MB07344 (10 mg/kg, i.v.). The tissue distribution of MB07811 was evaluated in male SD rats (n = 4 per group) administered [14C]-MB07811 (5 mg/kg, per os). Tissues harvested after killing the animals 3 and 24 h after dosing were processed and analyzed directly by liquid scintillation counting. Mass balance studies were conducted by administration of [3H]-MB07344 [2 mg/kg, 45.5 mCi (1 Ci = 37 GBq)/mmol] or [14C]-MB07811 (5 mg/kg, 20.6 mCi/mmol) to male SD rats (n = 6) and monitoring mean cumulative radioactivity excreted in urine, wash, and feces at 0, 12, 24, 48, 72, and 96 h. Detailed procedures and additional results for the above studies are reported in SI Appendix.

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mRNA Expression Analysis.[2]
Normal male SD rats (63–74 days old) were administered vehicle (water or CMC/Lutrol) or drug (T3, KB-141, or MB07811 ) at 1×, 3×, and 10× the CF rat ED50 (T3 in water at 0.012, 0.036, and 0.12 mg/kg; KB-141 in water at 0.05, 0.15, and 0.5 mg/kg; and MB07811  in CMC/Lutrol at 0.4, 1.2, and 4.0 mg/kg). Animals (n = 6 per group) were killed at 3, 8, and 24 h after oral dosing, and selected tissues were harvested under anesthetization with 2.5% isoflurane. Tissues were either removed and snap-frozen in liquid nitrogen (pituitary and thyroid gland) or freeze-clamped (liver, heart, soleus muscle, spleen, and kidney). mRNA expression was analyzed by using quantitative real-time PCR. Hepatic LDLR mRNA was measured in thyroidectomized male SD rats treated with T3 (0.5 mg/kg) or MB07811 (5 mg/kg). Detailed procedures are reported in SI Appendix.

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Safety Pharmacology Studies.[2]
In separate studies, normal male SD rats were treated daily with MB07811  to assess effects on cardiac function, glycemic control, and the THA. Procedures are briefly described below, with additional details found in SI Appendix.

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Cardiac function.[2]
MB07811  was administered daily in PEG400 to SD male rats (n = 6 per group) by oral gavage. Vehicle and KB-141 (1 mg/kg) groups were also included in the study. In separate experiments, T3 and KB-141 were administered by oral gavage (n = 6 per group). On day 7 after the start of dosing, animals were anesthetized with isoflurane, and the left ventricle was cannulated with a high fidelity catheter-tip transducer via the right carotid artery. Left ventricular pressure, its first derivative (LV dP/dt), and heart rate (via led I ECG) were digitally recorded. Systolic and diastolic aortic pressures were measured by retracting the catheter into the proximal aorta.

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THA.[2]
Male SD rats (20 groups, n = 5–6 per group, and one group for baseline measurements, n = 8) were treated with vehicle (0.5% CMC/1% Lutrol F68), MB07811  (3 or 30 mg/kg), or KB-141 (0.1 or 1 mg/kg) once daily (per os). After 1, 2, 4, and 7 weeks of treatment, one group from each treatment cohort was weighed and then killed by decapitation without anesthesia to minimize stress effects on the THA. Serum obtained from trunk blood collected at killing was used for measurement of total T3, total T4, free T3, free T4), and TSH, which was measured by using the rat TSH [125I]-Biotrak assay system with magnetic separation (Amersham Biosciences, Piscataway, NJ).

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Efficacy Studies.[2]
DIO mice were derived from ≈4-week-old normal male C57BL/6 mice fed a high-fat diet [60% fat by kcal (1 cal = 4.18 J)] for ≈170 days and then assigned to 10 groups (n = 8 per group) such that all groups had comparable cholesterol levels and BW. Mice were dosed orally once daily with vehicle (0.5% CMC/1% Lutrol F68), MB07811  (0.3, 1, 3, 10, and 30 mg/kg), or KB-141 (0.03, 0.1, 0.3, and 1 mg/kg). Blood glucose and plasma cholesterol and TGs were measured at baseline from blood obtained via tail nick. After 2 weeks of treatment, animals were killed by cervical dislocation, and blood was collected by cardiac puncture for measurement of cholesterol, TGs, glucose, and THs. In addition, hepatic TGs were measured after removal and weighing of the liver. Additional details are described in SI Appendix, as are studies evaluating T3, KB-141, and MB07811  in the 24-h CF-rat assay and cholesterol-fed C57BL/6 and LDLR−/− mice.

The average pharmacokinetic parameters derived from the individual concentration-time profiles of each analyte (MB07811, MB07344, atorvastatin and hydroxyatorvastatin) are shown in Tables 1 and 2. There were no significant differences in the pharmacokinetics of either MB07811 or atorvastatin and their respective metabolites when given in combination compared with each as monotherapy. In addition, the MB07344/MB07811 and hydroxyatorvastatin/atorvastatin ratios of AUClast values observed after monotherapy treatment (i.e. 29 and 1.7 respectively) remained relatively unchanged following combination therapy (i.e. 28 and 1.3 respectively) in male or female animals. These data indicate that the major routes of absorption, metabolism and elimination of MB07811 and atorvastatin were largely unaffected by MB07811 and atorvastatin co-administration.[1]

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Pharmacokinetic Studies.[2]
Upon i.v. administration, MB07344 exhibited moderate clearance in the male SD rat (0.28 ± 0.00 liter·kg−1·h−1 with a distribution volume of 0.39 ± 0.05 liter/kg and a plasma half-life of 1.27 ± 0.26 h (Table 1). In contrast, MB07811 (SD rat, i.v.) was cleared rapidly (11.6 ± 1.9 liters·kg−1·h−1) with a distribution volume of 14.8 ± 4.0 liters/kg and a half-life of 1.23 ± 0.15 h. Higher relative oral bioavailability was demonstrated for MB07811 (39%) compared with MB07344 (<1%, data not shown). Oral administration of MB07811 resulted in significant first-pass hepatic extraction [≈55%; Fig. 2B and supporting information (SI) Appendix, Fig. 6]. [2]

SD rats administered a dose of 5 mg/kg [14C]-MB07811 per os excreted 28% and 93% of radioactivity after 3 and 24 h, respectively. Mass balance studies showed that MB07344 (2 mg/kg, i.v. bolus) was eliminated by the biliary system with only 2% of the total radioactivity recovered after 96 h in the urine compared with 98% in the feces. Bile samples collected from bile duct-cannulated rats administered MB07344 i.v. showed that MB07344 was excreted in the bile and that 50% of the dose was excreted within 1 h (Fig. 2C). Last, bile-diverted rats exhibited plasma MB07344 levels (area under the curve (AUC0–24 h) = 48.1 ± 13.7 mg·h/liter) similar to those seen in normal rats (AUC0–24h = 35.1 ± 8.5 mg·h/liter), suggesting that MB07344 is not subject to enterohepatic recirculation (SI Appendix, Fig. 7). [2]

HPLC separation of the extractable radioactive metabolites indicated that 3 h after oral dosing with [14C]-MB07811, 63 ± 7% and 54 ± 5% of the radioactivity in the plasma and liver, respectively, coeluted with MB07344. Levels of MB07811 were below the limit of quantitation in both tissues. The highest tissue concentrations of radioactivity were associated with the stomach, small intestine, large intestine, mesenteric lymph nodes, and the liver, with lower concentrations found in spleen, adrenal, kidney, and heart tissues (Fig. 2D) and very low levels (<5% of the liver concentration) measured in bone† and 16 other tissues examined (SI Appendix, Table 3). After 24 h, the liver contained the highest concentrations of radioactivity followed by fat, pancreas, skin, and kidney, with all other nongastrointestinal tissues containing <3% of the liver concentration.[2]

[1]. Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys. Br J Pharmacol. 2009 Feb;156(3):454-65.
[2]. Targeting thyroid hormone receptor-beta agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index. Proc Natl Acad Sci U S A . 2007 Sep 25;104(39):15490-5.

Pharmacodynamics
MB07811's TR beta receptor selectivity and liver targeting could harness the efficacy of the approach, while avoiding extra-hepatic activation of TR alpha and TR beta receptors that may lead to therapy limiting side effects. These benefits may include: reduction of LDL and total cholesterol, Reduced liver and serum triglycerides, reduced Lp(a) and enhanced clearance of liver fat.

C28H32CLO5P

514.97744846344

514.168

C, 65.30; H, 6.26; Cl, 6.88; O, 15.53; P, 6.01

852948-13-1

916055-16-8 (racemate);852948-13-1 (2R4S);915233-90-8 (2S4R);

15942005

Solid powder

1.27±0.1 g/cm3(Predicted)

660.6±55.0 °C(Predicted)

77-82 °C

8.084

1

5

7

35

708

2

CC1=CC(=CC(=C1CC2=CC(=C(C=C2)O)C(C)C)C)OC[P@@]3(=O)OCC[C@H](O3)C4=CC(=CC=C4)Cl

LGGPZDRLTDGYSQ-JADSYQMUSA-N

InChI=1S/C28H32ClO5P/c1-18(2)25-14-21(8-9-27(25)30)15-26-19(3)12-24(13-20(26)4)32-17-35(31)33-11-10-28(34-35)22-6-5-7-23(29)16-22/h5-9,12-14,16,18,28,30H,10-11,15,17H2,1-4H3/t28-,35+/m0/s1

(2R,4S)-4-(3-chlorophenyl)-2-((4-(4-hydroxy-3-isopropylbenzyl)-3,5-dimethylphenoxy)methyl)-1,3,2-dioxaphosphinane 2-oxide

VK-2809; VK 2809; VK2809; MB 07811; MB07811; MB-07811; MB-07344; MB 07344; MB07344; ChEMBL457748;

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.)