VDR/vitamin D receptor

Chemically synthesized 20S-(OH)D3 is biologically active [1]
The biological activity of 20S-(OH)D3, measured in human normal epidermal keratinocytes, immortalized HaCaT keratinocytes and hamster AbC1 melanoma cells, showed similar effects to those reported for P450scc generated compound.

First, we tested the effect on keratinocytes proliferation and found that treatment of normal human epidermal keratinocytes with 220S-(OH)D3 and 1,25(OH)2D3 at concentrations of 10−8 and 10−6 M for 24 h led to the inhibition of cell proliferation when compared to control (vehicle treated) cells (Figure 5). Similarly, 20S-(OH)D3 inhibited proliferation (as measured by 3H-thymidine incorporation into DNA) of HaCaT immortalized epidermal keratinocytes in a dose-dependent manner (Figure 6). The inhibitory effect was similar to that exerted by 1,25(OH)2D3, being seen at a concentration as low as 10−11 M (Figure 6). Interestingly, the precursor compound, 20S-(OH)-7DHC, also inhibited DNA synthesis, albeit at higher concentrations (≥10−10 M).

Next we tested the effect of 20S-(OH)D3 on the vitamin D receptor (VDR), involucrin and CYP24 gene-expression and found that 20S-(OH)D3 stimulated expression of VDR at comparable levels to 1,25(OH)2D3 (Figure 7A). However, it was less potent in the stimulation of the expression of involucrin (2 vs 5 fold) and CYP24 (60 vs 10,000 fold) in comparison to 1,25(OH)2D3 (Figure 7B and 7C). The more than 100 times difference between 1,25(OH)2D3 and 20S-(OH)D3 on the induction of CYP 24 expression is similar to that reported previously for biochemically synthesized 20S-(OH)D3 [18, 19, 33], and suggests that 20S-(OH)D3 will have only a minor influence on the inactivation (24 hydroxylation) of active forms of vitamin D3.

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20S-(OH)D3 inhibits cancer colony formation in monolayer and soft agar [2]
The antitumor activity of 20(OH)D3 was compared to that of 1,25(OH)2D3 by measuring their abilities to inhibit cancer cell colony formation. Firstly, we tested the effect of these compounds on HepG2 hepatocellular carcinoma colony growth and found that 20(OH)D3 and 1,25(OH)2D3 inhibit HepG2 proliferation in a concentration-dependent manner. 20S-(OH)D3 and 1,25(OH)2D3 at a concentration of 100 nM effectively reduced the CFU of colonies larger than 0.2 mm (54±14% and 50±6%, respectively) and larger than 0.5 mm (70±20% and 92±6%, respectively) in comparison to control (vehicle treated) cells (Figure 1A and B). The treatment with vitamin D3 derivatives at lower concentrations, 0.1 and 10 nM, had no significant effect on the total number of colonies larger than 0.2 mm (Figure 1A) but greatly prevented the formation of colonies larger than 0.5 mm (Figure 1B). 1,25(OH)2D3 inhibited formation of large colonies (>0.5 mm in size) to a greater extent than 20(OH)D3 at all three concentrations tested.

Next we tested the effect of 20S-(OH)D3 on MDA-MB-453 human breast carcinoma cell growth in soft agar. 20(OH)D3 and 25(OH)D3 at a concentration of 100 nM effectively inhibited the formation of MDA-MB-453 colonies larger than 0.2 mm (54±4% and 43±7% of CFU, respectively) in comparison to control (vehicle-treated) cells (Figure 2A and B). Colonies larger than 1.5 mm were not detected in either of the two 100 nM treatment groups. At 10 nM, 20(OH)D3 inhibited the formation of MDA-MB-453 colonies larger than 1.5 mm (Figure 2B) by 85±11% while the effect of 25(OH)D3 was not statistically significant.

Finally we tested the effect of 20S-(OH)D3 on colony formation by MCF7 breast cancer cells on soft agar. Due to the slow colony formation for this cell line, we prolonged the test period to 26 days in order to investigate the antiproliferative effect. 20(OH)D3 at a concentration of 100 nM inhibited formation of colonies larger than 0.2 mm by 78±28% (Figure 3A). However, 1α,25(OH)2D3 demonstrated a relatively stronger effect in this model and inhibited colony formation by 62±6% at a concentration as low as of 0.1 nM (Figure 3B).

High doses of 20S-(OH)D3 have no calcemic effect on mice after 3 weeks of treatment [2]
It has been reported that 20S-(OH)D3 has no effect on systemic calcium levels at a dose of 3 μg/kg for 7 consecutive days in a rat model. In the current study, we assessed the potential toxicity of 20(OH)D3 by administering a range of doses to C57BL/6 mice with the highest dose being 30 μg/kg. As positive controls, we used 25(OH)D3 and 1,25(OH)2D3 because they are reported to generate hypercalcemia at a concentration ≥1 μg/kg in mice or rats. As indicated in Figure 4, 20(OH)D3 did not display hypercalcemic activity (calcium=9.7±0.69 vs. 9.4±0.25 mg/dl for vehicle control) at even the highest dose tested (30 μg/kg) for the 3-week i.p. treatment. In contrast, 1,25(OH)2D3 at a dose of only 2 μg/kg caused the expected dramatic rise in calcium up to 14.6±0.48 mg/dl. 25(OH)D3 at a dose of 2 μg/kg showed a mild hypercalcemic effect elevating the serum calcium level to 11.5±0.48 mg/dl, beyond the 10.5 mg/dl upper range for normal serum calcium.

Metabolism of 20S-(OH)D3 by cytochrome P450scc and CYP27B1 [1]
Incorporation of 20S-(OH)D3 into phospholipid vesicles and measurement of its metabolism by P450scc was carried out as described previously. The incubation mixture comprised 510 μM phospholipid vesicles containing 20S-(OH)D3 at a ratio to phospholipid of 0.1 mol/mol, 2 μM bovine cytochrome P450scc, 15 μM adrenodoxin, 0.4 μM adrenodoxin reductase, 2 mM glucose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase and 50 μM NADPH. Samples were pre-incubated for 8 min, reactions started by the addition of NADPH and incubations carried out at 37°C with shaking for 6 min. After extraction with dichloromethane samples were analyzed by HPLC as described before using a C18 column (Grace 15 cm × 4.6 mm, particle size 7 μm). Metabolism of 20S-(OH)D3 by CYP27B1 (25-hydroxyvitamin D3 1α-hydroxylase) was measured by a similar procedure as described before

Colony forming assay [2]
The assay followed standard methodology as described previously. Briefly, cells were plated in 24-well plates at a density of 20 cells/9.6 cm2 in medium containing 5% charcoal-treated FBS, 1% antibiotic solution and 20S-(OH)D3, 1,25(OH)2D3, or 25(OH)D3 at graded concentrations, or ethanol (vehicle control). Cells were cultured at 37°C for 7 days with media being changed every 3 days. At the end of incubation, the colonies were fixed with 4% paraformaldehyde in phosphate buffer solution (PBS) overnight at 4°C, washed, stained with 5% crystal violet in PBS for 30 minutes, rinsed, and air-dried. The number and size of the colonies were measured using an ARTEK counter 880. Colony-forming units (CFU) were calculated by dividing the number of colonies by the number of cells plated and then multiplying by 100.

MCF7 cells were grown in soft agar as previously described. The tumorogenicity of HepG2, MCF7 and MDA-MB-453 cells were determined by their ability to form colonies in soft agar. Cells were grown in monolayer, trypsinized, and re-suspended (1,000 cells/well) in 0.25 ml medium containing 0.4% agarose and 10% charcoal-stripped serum. Cell suspensions were added to 0.8% agar layer in 24-well plates. 20S-(OH)D3, 25(OH)D3, or 1α,25(OH)2D3 were added from ethanol stocks to final concentrations ranging from 0.1–100 nM. An ethanol control was included in the assay. Two weeks later, colonies formed in soft agar were stained with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (0.5 mg/ml) and scored under a microscope. The number of units was calculated from the number of colonies formed divided by the number of cells seeded ×100.

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Cell proliferation assays [1]
1. MTS test [1]
HEKn keratinocytes were plated in 96-well plates, 10,000 cells/ well. After overnight incubation of cells, 1,25(OH)2D3 or chemically synthesized 20S-(OH)D3, initially dissolved in ethanol and then diluted in KGM medium containing 0.5% BSA, was added to the medium to achieve final concentrations of 0.01 μM or 1 μM, while in control cultures, the final concentration of ethanol vehicle was 0.1%. After 20 h of incubation with these compounds, 20μl of MTS/ PMS solution was added to the cells. Four hours later, absorbance was recorded at 490 nm using an ELISA plate reader. The number of viable cells was measured in six replicates.

2. DNA synthesis [1]
Testing of DNA synthesis was carried out as described previously. Cells were inoculated into 24-well plates at 5,000 – 25,000 cells/well, depending on cell type. After overnight incubation at 37°C, the cultures were placed in serum free media to synchronize cells at the G0/G1 phase of the cell cycle. After 24 h 20S-(OH)D3 was added with fresh media containing growth supplements and incubated for additional 72 h. After a defined period of time, [3H]-thymidine (specific activity 88.0 Ci/mmol) was added to a final concentration of 0.5 μCi/mL in medium. After 4 h of incubation at 37°C, media were discarded, cells precipitated in 10% TCA in PBS (phosphate-buffered saline) for 30 min, washed twice with 1 mL PBS and then incubated with 1 N NaOH/ 1% SDS (250 μL/well) for 30 min at 37°C. The extracts were collected in scintillation vials and 5 mL of scintillation cocktail was added to each. 3H-radioactivity incorporated into DNA was measured with a beta counter.

3. Colony forming assay [1]
The assay followed our standard methodology, as described previously. Briefly, cells were plated in six-well plates at a density of 192 cells/well in medium containing 5% ctFBS (Charcoal-treated fetal bovine serum), 1% antibiotic solution and 20S-(OH)D3 at graded concentrations or ethanol (vehicle control). Cells were cultured at 37°C for 7 days with media being changed every 3 days. At the end of incubation, the colonies were fixed with 4% paraformaldehyde in PBS overnight at 4°C, washed, stained with 5% crystal violet in PBS for 30 min, rinsed, and air-dried. The number and size of the colonies were measured using an ARTEK counter 880. Colony forming units were calculated by dividing the number of colonies by the number of cells plated and then multiplying by 100.

4. ApoTox-Glo Triplex Assay [1]
Melanoma cells were plated in 96- well plates, 10,000 cells/ well. After overnight incubation of cells, 1,25(OH)2D3 or 20S-(OH)D3 was added to the medium as described above. After 24 h of incubation with these compounds, 20 μl of Viability/Cytotoxicity Reagent containing both GF-AFC Substrate and bis-AAF-R110 Substrate was added to the cells. After 30 min of incubation at 37°C, fluorescence was recorded at 400 nm excitation / 505 nm emission for viability and 485 nm excitation / 520 nm emission for cytotoxicity using a microplate plate reader for fluorescence and luminescence. One hundred μl of Caspase-Glo 3/7 Reagent was further added to the cells, and after 30 min of incubation at room temperature, luminescence was recorded. Numbers of viable, cytotoxic and apoptotic cells were measured in four replicates. Real-time RT PCR The RNA from HEKn keratinocytes treated with 20S-(OH)D3 or 1,25(OH)2D3, was isolated using the Absolutely RNA Miniprep Kit. Reverse transcription (1 μg RNA/reaction) was performed using the Transcriptor First Strand cDNA Synthesis Kit. Real-time PCR was performed using cDNA diluted 5-fold in sterile water and a TaqMan PCR Master Mix. Reactions (in triplicate) were performed at 95°C for 5 min and then 45 cycles of 95°C for 10 sec, 60°C for 30 sec and 72°C for 30 sec. The primers and probes were designed with the universal probe library. Data were collected on a Roche Light Cycler 480. The amount of amplified product for each gene was compared to that for Cyclophilin B using a comparative CT method. A list of the primers used for RTPCR DNA amplification is shown in Table 2.

In vivo toxicity studies [2]
The potential toxicity of 20S-(OH)D3 was evaluated using male C57/BL6 mice. Mice weighed approximately 25–26 g and were seven weeks of age. Two positive control compounds, 1,25(OH)2D3 and 25(OH)D3, were obtained from xx. 20(OH)D3 in autoclaved sesame oil was administered by intraperitoneal (i.p.) injection (50 μl/mouse) once daily for three consecutive weeks to groups of five mice at doses of 5, 10, 20 and 30 μg/kg body weight. In the positive control group, 1,25(OH)2D3 or 25(OH)D3, was given at the dose of 2 μg/kg body weight by i.p. injection following the same pattern. Another group of five mice was injected with autoclaved sesame oil 50 μl /mouse daily, which served as a vehicle control. Clinical signs of toxicity and body weight were assessed twice a week throughout the experimental period. Terminal blood samples (800~1000 μl/mouse) were collected by cardiac puncture at the end of the three-week treatment. The serum (~300 μl/mouse) was immediately separated using BD Microtainer® tubes and stored at −20°C. All animals were sacrificed by cervical dislocation immediately after the blood collection and the main organs (heart, lung, liver, spleen, kidney, adrenal and one piece of skin) of each mouse were collected and stored separately in 10% buffered formalin phosphate solution for subsequent pathological analysis.

[1]. Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity. Steroids. 2010 Dec;75(12):926-35.
[2]. 20-hydroxyvitamin D₃ inhibits proliferation of cancer cells with high efficacy while being non-toxic. Anticancer Res. 2012 Mar;32(3):739-46.
[3]. Design, Synthesis and Biological Activities of Novel Gemini 20S-Hydroxyvitamin D3 Analogs. Anticancer Res. 2016 Mar;36(3):877-86.
[4]. Design, synthesis, and biological action of 20R-hydroxyvitamin D3. J Med Chem. 2012 Apr 12;55(7):3573-7.

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use. [1]

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Aim
To define the potential utility of 20-hydroxyvitamin D3 (20(OH)D3) as a tumorostatic agent, we assessed its in vitro antiproliferative activity and its in vivo toxicity.
Materials and Methods
The antitumor activity of 20(OH)D3 was tested against breast and liver cancer cell lines using colony formation assays. To assess in vivo toxicity, mice were injected with 5–30 μg/kg 20(OH)D3 intraperitoneally each day for 3 weeks. Blood and organ samples were collected for clinical pathology analyses.
Results
20(OH)D3 displays similar tumorostatic activity towards MDA-MB-453 and MCF7 breast carcinomas, and HepG2 hepatocarcinoma, in a dose-dependent manner. This compound is not hypercalcemic, does not cause detectable toxicities in liver, kidney, or blood chemistry in mice at a dose as high as 30 μg/kg. In contrast, both 25(OH)D3 and 1,25(OH)2D3 caused severe hypercalcemia at a dose of 2 μg/kg.
Conclusion
20(OH)D3 possesses high efficacy for inhibiting cancer cell proliferation in vitro and is non-toxic in vivo, supporting its further development as a potential anticancer therapeutic agent.[2]

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Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.[3]

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The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates that they have different behaviors in antiproliferative and metabolic studies.[4]

C27H44O2

400.637068748474

400.334

651734-12-2

651734-12-2

162367340

Typically exists as white solid at room temperature

6.2

2

2

6

29

657

4

CC(C)CCC[C@@](C)([C@H]1CCC\2[C@@]1(CCC/C2=C\C=C/3\C[C@H](CCC3=C)O)C)O

IQEQEOBGZMEDBQ-LWVSKBGXSA-N

InChI=1S/C27H44O2/c1-19(2)8-6-17-27(5,29)25-15-14-24-21(9-7-16-26(24,25)4)11-12-22-18-23(28)13-10-20(22)3/h11-12,19,23-25,28-29H,3,6-10,13-18H2,1-2,4-5H3/b21-11+,22-12-/t23-,24?,25-,26-,27-/m0/s1

(1S,3Z)-3-[(2E)-2-[(1S,7aS)-1-[(2S)-2-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol

20(OH)D3; 20-Hydroxyvitamin D3; 20S(OH)D3; (S,Z)-3-((E)-2-((1S,3aS,7aS)-1-((S)-2-Hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol; 20-Hydroxyvitamin D3; 651734-12-2; (1S,3Z)-3-[(2E)-2-[(1S,7aS)-1-[(2S)-2-hydroxy-6-methylheptan-2-yl]-7a-methyl-2,3,3a,5,6,7-hexahydro-1H-inden-4-ylidene]ethylidene]-4-methylidenecyclohexan-1-ol

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.)