Toll-like receptor 7/TLR7

AZ12441970 has improved antedrug characteristics [1]
The antedrug concept used in these compounds relies on the metabolite (acid) having reduced activity, compared with its parent (ester), and it was clear from the reporter assay for human and rat TLR7 (Table 1) that the acid of SM-324405 was, at best, only 10-fold less active than its parent ester. Having established that this adenine series of compounds showed biological activity comparable with R848, we sought compounds where the activity of the metabolite was further reduced and this led to a series of compounds exemplified by AZ12441970 (Table 1). This compound had a 0.5 log reduction in human TLR7 potency compared with SM-324405, though the rat TLR7 potency, pEC50= 6.6, was maintained (Table 1). The acid metabolite, AZ12443988, was less active than the acid of SM-324405 and had an ester/acid potency ratio of >60-fold for both human and rat TLR7. The true extent of the potency ratio value could not be determined as the activity of AZ12443988 was so low that no pEC50 could be determined over the range of concentrations up to 10 µM. As AZ12441970 also has a shorter plasma t1/2 than SM-324405, overall, AZ12441970 exhibited improved antedrug properties in vitro.

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Inhibition of the Th2 cytokine, IL-5, by AZ12441970 [1]
TLR7 agonists have the potential to treat allergic diseases, which are characterized by a Th2 phenotype, by rebalancing the immune response. We used PHA to polyclonally stimulate human PBMC and assessed the ability of the compounds to inhibit IL-5 production as a marker of Th2 cytokine modulation (Figure 4A and B). R848 dose-dependently inhibited the production of IL-5 with a pIC50 of 7.7. AZ12441970 and SM-324405 were potent inhibitors of IL-5 with a pIC50 of 8.7 and 7.9 respectively. The acid metabolite of AZ12441970 (AZ12443988) was much less active (pIC50= 5.4) compared with SM-324406 (pIC50= 6.8) as an inhibitor of IL-5 production, giving an ester/acid ratio of 1900 for AZ12441970. This was an improvement on the ratio of 13 for SM-324405/SM-324406. PHA also induced IL-13 in this assay and in an assessment of 10 TLR7 agonists we observed equivalent inhibition of IL-5 and IL-13 (Supporting Information Figure S1). IL-4, however, was not induced. In a murine assay where IL-5 was induced by addition of the antigen (OVA), R848 and AZ12441970 potently inhibited IL-5 production with pIC50 of 8.7 and 7.5 respectively (Figure 4C). AZ12443988 inhibited IL-5 production with a pIC50 of 6.0. Therefore, this class of TLR7 agonist suppresses Th2 cytokine production in both human and mouse T-cells.

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Identifying potential biomarkers of TLR7 agonist activity [1]
As TLR7 stimulation of PBMC results in production of IFN-α, we investigated whether this might be a mediator in the inhibition of IL-5. In both human PBMC and mouse splenocyte assays, addition of IFN-α resulted in inhibition of IL-5 (Figure 5). Therefore, IFN-α is a potential biomarker for TLR7 stimulation of plasmacytoid dendritic cells as well as a marker of suppression of Th2 cytokines. IFN-α has a short t1/2in vivo making it difficult to measure as a potential biomarker. To overcome this, we investigated gene products that are secondary markers of IFN-α activity. Addition of AZ12441970 to human PBMC induced IL-1RA (Figure 6A), and this induction was blocked with a neutralizing anti-IFN-α/β receptor antibody (Figure 6A). We confirmed that IFN-α induced IL-1RA (Figure 6B), and demonstrated that inclusion of the neutralizing antibody blocked the production of IL-1RA by IFN-α. This confirmed IL-1RA as a downstream marker of IFN-α production.

AZ12441970 shows efficacy in vivo with minimal systemic activity [1]
AZ12441970 activity in vivo was determined by giving it intratracheally to OVA-sensitized C57BL/6 mice that were then challenged with intratracheal antigen (Table 3). Pretreatment with 1 mg·kg−1 AZ12441970 by intratracheal administration significantly reduced eosinophilia in the bronchoalveolar lavage fluid by 87% and, although not significant, there was also a trend towards a reduction in the production of IL-5. When given intratracheally to naïve C57BL/6 mice, 0.1 mg·kg−1 AZ12441970 did not induce any increase in systemic IFN-α (Table 4) and treatment with a higher dose (1 mg·kg−1) of AZ12441970 resulted in only a modest increase in plasma IFN-α levels, when compared with the IFN-α induced by 1 mg·kg−1 R848. Plasma IL-1RA, IL-6 and TNF-α were similarly not induced by intratracheal dosing of AZ12441970, whereas all three cytokines were clearly induced by R848. This demonstrated the concept that local administration of AZ12441970 can mediate beneficial effects in a mouse allergy model with minimal systemic activation.

TLR reporter assays [1]
HEK293 cells, stably transfected with human TLR7 (pUNO expression vector) and pNiFty2-SEAP reporter plasmid were maintained in Dulbecco's modified Eagle's medium, FCS 10% (v/v), 2 mM l-glutamine, non-essential amino acids, 10 µg·mL−1 blasticidin S and 10 µg·mL−1 zeocin. The sequence used was represented by the European Molecular Biology Laboratory Nucleotide Sequence Database sequence AF240467. Cells were seeded in tissue culture treated clear flat bottom polystyrene 96 well plates at 10 000 cells per well. Dose–response curves were generated by addition of test compounds and incubation for 20 h at 37°C in an atmosphere of 5% CO2. The secretory alkaline phosphate (SEAP) released was quantified using p-nitrophenyl phosphate as a substrate, and the absorbance at 405 nm was determined by a microplate reader.

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Plasma stability determinations [1]
The test compounds (initial concentration of 1 µM) were added to human or rat plasma (prepared by centrifuging blood collected in EDTA tubes at 1800× g) at 37°C in a total volume of 0.5 mL. Incubations were for 10 min at 37°C with samples taken at 0, 20 s, 40 s and 1, 2, 3, 5 and 10 min into acetonitrile. Supernatants were analysed by LC/MS/MS for the remaining parent compound, and t1/2 of the parent compound was determined.

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Splenocyte preparations [1]
Spleens were removed from CO2 asphyxiated male Brown Norway rats or from naïve female Balb/c mice (Harlan), following cervical dislocation, and placed in a Petri dish containing RPMI 1640. The spleen was gently pushed through a 70 µm BD Falcon Cell Strainer to obtain a single cell suspension. Cells were centrifuged at 400× g for 5 min to obtain a cell pellet, the supernatant removed and cells resuspended in fresh RPMI 1640. The cells were centrifuged again and the cells resuspended in complete medium (RPMI-1640, fetal calf serum (FCS) 5% (v/v), 2 mM l-glutamine, 10 U·mL−1 penicillin, 10 µg·mL−1 streptomycin and 50 µM 2-mercaptoethanol).

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Peripheral blood mononuclear cell (PBMC) preparations [1]
Blood from healthy, consenting volunteers was collected into heparin and layered onto Lymphocyte Separation Medium 1077 (PAA, Pasching, Austria) and centrifuged at 700× g for 25 min. The PBMC layer was removed, diluted to 50 mL with PBS and centrifuged at 400× g for 10 min. The supernatant was removed, the pellet resuspended in 50 mL PBS and centrifuged at 300× g for 5 min. Finally the cells were washed in 50 mL PBS and the cells recovered by centrifuging at 200× g for 5 min. PBMCs were finally resuspended in assay medium (RPMI 1640 with 25 mM HEPES, FCS 10% (v/v), 2 mM l-glutamine, 10 U·mL−1 penicillin and 10 µg·mL−1 streptomycin).
Dog PBMC were prepared from dog blood (Animal Facilities, AstraZeneca R&D) collected into heparin and the PBMC prepared using the same protocol as for human PBMC.

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Splenocyte incubations [1]
Twenty microlitres of test compound or complete RPMI 1640 with DMSO 1% (v/v), vehicle control, were added to each well followed by 180 µL of splenocyte cell suspension (2 × 105 cells) in complete RPMI prepared as described earlier. Splenocytes and compound were incubated at 37°C in an atmosphere of air/CO2 (95/5 v/v) for the defined period of time.
Splenocyte proliferation was determined by addition of 0.0185MBq [3H]-thymidine to cellular assays at 44 h. After a further 6 h incubation, the cells were harvested onto glass fibre filter mats using a Tomtec filtration apparatus. The mats were dried, Betaplate Scint added, and filter-bound radioactivity was quantified with a MicroBeta 1450 Trilux.

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Mouse splenocyte IL-5 and IFN-γ [1]
Naïve female Balb/c mice were immunized by injection of 10 µg OVA + 1 mg Al(OH)3 in 100 µL by intraperitoneal injection on day 0. Eight days after immunization, spleens from OVA/Al(OH)3 sensitized mice were collected into RPMI 1640 medium and splenocytes prepared and incubated as described earlier. OVA was added to give a final concentration of 1 mg OVA mL-1 and incubations were for 5 d. The supernatant was removed for determination of the amount of IL-5 and IFN-γ produced.

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PBMC incubations [1]
Twenty microlitres of test compound or assay medium with dimethyl sulfoxide (DMSO) 1% (v/v), vehicle control, were added to each well followed by 180 µL of PBMC cell suspension (prepared as mentioned earlier) in assay medium (200 000 cells). PBMC and compound were incubated at 37°C in an atmosphere of air/CO2 (95/5 v/v) for the defined period of time.
For induction of IL-5, human PBMC were prepared and plated out with compounds as described earlier. Phytohaemagglutinin (PHA) was added at a final concentration of 5 µg·mL−1 and incubated for 44 h when the supernatant was removed for determination of the amount of IL-5 produced.
In assays where butyrylcholinesterase (BChE) was added to shorten the exposure time to antedrug, PBMC were plated out with BChE at a concentration of 1 U·mL−1 and incubations were initiated by addition of compound. After 24 h, 150 µL supernatant was removed for cytokine determinations and replaced with 150 µL fresh medium. At 44 h, [3H]-thymidine was added and proliferation determined as described earlier.

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Gene chip analysis [1]
Balb/c mouse splenocytes, Brown Norway rat splenocytes or human PBMC were incubated with compound, and after 4 h stimulation RNA was extracted using TRIzol® Reagent. Microarray analysis was performed on human (HG-U133 plus 2), mouse (MOE430), and rat (RAE230) Affymetrix chip sets according to standard protocols. Raw microarray data was normalized using the MAS5 algorithm within GeneChip Operating Software.

Inhibition of Inflammatory Cells in BALF (Efficacy) [1]
\nMale 8−10 weeks old Brown Norway rats were sensitized by intraperitoneal injection of ovalbumin (1 mg) together with aluminum hydroxide adjuvant (100 mg) in saline (1 mL) on day 0 and 7. Control (unsensitized/unchallenged) animals received vehicle (saline) alone at the same time points. On any one-day between days 14 and 18, rats were challenged by exposure to ovalbumin aerosol for 15 min generated from a 10 mg/mL ovalbumin solution by a nebulizer. Control animals were similarly exposed to saline aerosol for 15 min. Two hours before antigen challenge, rats were dosed with test compounds (suspended in saline) or vehicle by i.t. administration (dosing volume was 0.5 mL/kg). Twenty-four hours after antigen challenge, rats were sacrificed and the trachea was cannulated. The airway lumen was washed with 2 mL of saline, and this procedure was repeated six times (total volume of 12 mL). Infiltrated cells in BALF were stained with Turk solution and the number of nucleated cells was counted in a counting chamber. A differential count was made on a smear prepared with a cytocentrifuge and stained with Diff-Quick solution (May−Grunwald stain). At least 300 cells were counted in each BALF sample (magnification × 400).\n

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\n\nInduction of Systemic IFN (Side Effect) [1]
\nMale 8−10 weeks old Brown Norway rats were dosed with test compounds (suspended in saline) by i.t. administration (dosing volume was 0.5 mL/kg). At 2, 4, 6, and 24 h after i.t. administration, rats were anaesthetized with ether, and heparinized blood samples (about 0.3 mL) were collected via the caudal vein. Then plasma samples were prepared by centrifugation (12000 rpm for 10 min), and stored at −20 °C until analyzed for IFN. IFN titers in the plasma samples were determined in a CPE reduction assay (bioassay) using L929 and vesicular stomatitis virus (VSV).\n\nDetermination of pharmacokinetics In vivo [2]
\nAZ12441970 was formulated in 0.1% Tween80/0.6% NaCl/50 mM phosphate buffer pH 6.0 at a concentration of 0.5 mg·mL−1. Six female BALB/c mice were briefly anaesthetized with isoflurane then dosed intranasally with 50 µL of the formulation, giving a dose of 1 mg·kg−1 per mouse. This volume is sufficient to be inhaled into the lung rather than remain in the nasal cavity. At each time point, two animals were killed by an overdose of pentobarbital and blood taken from the vena cava into sodium fluoride (0.2 M final concentration) to prevent hydrolysis by esterase enzymes before mixing with the anticoagulant EDTA. Samples were quenched in methanol and frozen at −20°C. Lungs were excised and placed in vials containing 1 mL sodium fluoride (1.2 M) and immediately frozen at −20°C. The lungs were homogenized with eight parts water, and aliquots of the homogenate quenched with methanol. Standard curves were prepared from a known weight of the test compound AZ12441970 and the acid metabolite AZ12443988, added to lung homogenate or blood containing sodium fluoride and treated as earlier samples.\n\nAll samples were centrifuged and the supernatant analysed by LC/MSMS and the concentrations of AZ12441970 and AZ12443988 quantified.\n

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\n\nMouse OVA-induced allergic airways model [2]
\nFemale C57BL/6 mice were sensitized by subcutaneous injection of 10 µg of OVA adsorbed with 4 mg aluminium hydroxide adjuvant in 100 µL on Day 0 and 14. Animals were challenged by intratracheal (20 µL) administration of OVA (0.5 mg·mL−1) on Day 22. AZ12441970 (40 µL, dissolved in 0.1% Tween80/ 0.6% NaCl/50 mM phosphate buffer pH 6.0) was administered via the intratracheal route 24 h and 2 h prior to OVA challenge. Animals were killed under anaesthesia 48 h after the OVA challenge, and the number of eosinophils in bronchoalveolar lavage fluid was measured by FACS analysis as described previously (van Rijt et al., 2004). Briefly, bronchoalveolar lavage fluid cells were pre-incubated with anti-mouse CD16/CD32 monoclonal antibody 2.4G2 (BD Bioscience, San Diego, CA, USA) at 4°C for 15 min, then incubated with anti-mouse FITC-CD4(L3T4), FITC-CD8, FITC-B220 and PE-CCR3 (BD Bioscience). Number of CD4− CD8− B220− CCR3+ eosinophils was determined using a Becton Dickinson FACScan (Becton Dickinson). IL-5 in bronchoalveolar lavage fluid was measured by elisa (BD Bioscience).\n

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\n\nSystemic cytokine induction in mice [2]
\nAZ12441970 and R848 (dissolved in 0.1% Tween80/0.6% NaCl/50 mM phosphate buffer pH 6.0) were administered to naïve female C57BL/6 mice via the intratracheal route in a volume of 20 µL per mouse. Blood was collected 90 min later into heparinized syringes and plasma was prepared by centrifugation. Plasma was stored frozen until analysis. IFN-α in plasma was measured by the reporter assay system using L929/OAS cells and cytokines were determined by elisa or Luminex technologies.\n\n\n

Short-term exposure to the compound is beneficial for the induction of IFN-α [1]
To study the pharmacokinetic characteristics of AZ12441970, we used intranasal administration to ensure that the drug could reach the lungs. AZ12441970 is unstable in mouse plasma with a half-life of 0.22 minutes. Ten minutes after administration, 90% of the compound was metabolized in the lungs, generating acidic metabolites (Table 2). The levels of ester and acid metabolites decreased further within 150 minutes. At the three time points studied, the concentration of AZ12441970 in plasma was about 400 times lower than that in the lungs. Although the concentration of AZ12443988 in plasma was higher than that of the parent compound, this acid metabolite was also cleared from the blood.
Pharmacokinetic data indicate that AZ12441970 is not only rapidly metabolized in plasma, but also hydrolyzed when deposited in the lungs. BChE has been identified as the plasma esterase responsible for cleaving this resistant ester, as the selective inhibitor ethephon, a BChE inhibitor, blocks the conversion of AZ12441970 to its acidic metabolite in plasma (see Supplementary Info Figure S2). The IC50 value was 24 µM, consistent with the 11 µM value determined by Haux et al. (2000). To determine whether short-term exposure to the TLR7 agonist produces biological effects, we incubated PBMCs with BChE to promote rapid ester metabolism, thereby shortening the exposure time. Incubation with an equal concentration of the metabolite AZ12443988 showed that any acid produced by ester cleavage was biologically active. Higher concentrations of AZ12441970 were required in the presence of BChE to produce IFN-α and its downstream marker IL-1RA (Figures 7A and B). The levels of these mediators produced in the presence of 1 µM AZ12441970 were comparable to those induced in the absence of BChE. All of these activities cannot be attributed to the acid, as it is inactive at concentrations up to 1 µM (Fig. 7B). When other endpoints of TLR7 stimulation, such as TNF-α production and PBMC proliferation, were measured, the addition of BChE almost completely inhibited any TLR7-mediated effects, even at the highest dose of AZ12441970 tested. These data suggest that short-term exposure is more likely to induce IFN-α production than TNF-α production and proliferation.

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TLR agonist activity characterization of SM-324405, AZ12441970 and their metabolites[1]
A synthetic chemistry research program yielded a TLR7 agonist prodrug that can be rapidly metabolized in plasma (Kurimoto et al., 2010). Subsequent research programs have identified a number of alternative compounds, such as AZ12441970. This article summarizes the biological activity and mechanism of action of this series of compounds, using the TLR7 agonists SM-324405 and AZ12441970 and their metabolites as examples (Table 1). These compounds are rapidly metabolized in human plasma with a half-life of 1–3 minutes, and rapidly metabolized in rat plasma with a half-life of less than 1 minute (Table 1). AZ12441970 and SM-324405 showed comparable or higher activity to the well-characterized TLR7/8 ligand R848 in human and rat TLR7 reporter gene assays (Table 1). R848 is active against human TLR8, while the other two compounds do not, as assessed by activating NF-κB in reporter cell lines. The plasma metabolite is an acid, and the acidic product of SM-324405 still showed significant activity in human and rat TLR7 reporter gene assays. However, the design of AZ12441970 resulted in a more than 60-fold reduction in the potency of its acidic metabolite AZ12443988 in human TLR7 reporter gene assays. Neither of these acidic metabolites showed any detectable TLR8 activity.

[1]. Biological characterization of a novel class of toll-like receptor 7 agonists designed to have reduced systemic activity. Br J Pharmacol. 2012 May;166(2):573-86.

[2]. Chemical Strategies to Enhance the Therapeutic Efficacy of Toll-like Receptor Agonist Based Cancer Immunotherapy. Acc Chem Res. 2020 Oct 20;53(10):2081-2093.

We have identified potent TLR7 agonist prodrugs and provided data suggesting that TLR7 agonist 9e with prodrug properties is a novel candidate for immunotherapy of allergic diseases. Background and Objectives: Toll-like receptor 7 (TLR7) agonists hold promise for the treatment of allergic diseases. However, the therapeutic application of small molecule TLR7 agonists is currently limited by their systemic activity, leading to adverse side effects. We have developed a series of selective TLR7 “prodrugs,” including SM-324405 and AZ12441970, which contain an ester group that is rapidly cleaved in plasma, thereby reducing systemic exposure. Experimental Methods: In vitro, we evaluated the agonistic activity of the parent ester and its acid metabolites towards TLR7 using reporter cells and primary cells from various species. Pharmacokinetics were evaluated after administration to the mouse lungs, and in vivo efficacy was assessed using a mouse allergic airway model. Main Results: These compounds are selective TLR7 agonists with no cross-reactivity to TLR8 and are metabolically unstable in plasma, with their acid metabolites exhibiting significantly reduced activity in multiple assays. These compounds inhibit IL-5 production and induce IFN-α, which in turn mediates IL-5 inhibition. When administered to the lungs, these compounds are rapidly metabolized, and short-term exposure to the “antidrug” activates the IFN pathway. AZ12441970 showed efficacy in a mouse model of allergic airway disease with minimal systemic IFN-α induction, consistent with its low plasma concentration. Conclusions and Implications: The biological and metabolic characterization of these TLR7 selective agonist “antidrug” compounds suggests a novel class of compounds that can be used for localized treatment of allergic diseases while reducing the risk of systemic side effects. Related Article: Kaufman and Jacoby commented on this article on pages 569-572 of this issue. To view the commentary, please visit http://dx.doi.org/10.1111/j.1476-5381.2011.01758.x. [1]

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Another strategy is to use antidrugs, which are locally active compounds designed to be rapidly metabolized into an inactive form after entering the bloodstream and to prevent systemic toxicity by losing their agonistic activity in the plasma environment. TLR agonist antidrugs have a cleavable linker at the key TLR binding site. This cleavable linker is sensitive to the plasma environment, so the TLR agonist is inactivated by the loss of the key binding site. SM-324405 and AZ12441970 are TLR 7 agonist antidrugs, and they contain ester bonds at their key TLR binding sites. These ester bonds are easily cleaved by plasma esterases, resulting in reduced agonistic activity. Loss of the TLR binding site leads to a decrease in peripheral blood mononuclear cell (PBMC)-induced interferon-α (IFN-α) secretion. [2]

C27H41N7O4

527.658945798874

527.322

C, 61.46; H, 7.83; N, 18.58; O, 12.13

929551-91-7

57733215

Typically exists as solid at room temperature

2.6

2

9

17

38

728

0

O=C1NC2C(N)=NC(=NC=2N1CCCN(CC1C=CC=C(CC(=O)OC)C=1)CCCN(C)C)OCCCC

OULXSRDSTASMNN-UHFFFAOYSA-N

InChI=1S/C27H41N7O4/c1-5-6-16-38-26-30-24(28)23-25(31-26)34(27(36)29-23)15-9-14-33(13-8-12-32(2)3)19-21-11-7-10-20(17-21)18-22(35)37-4/h7,10-11,17H,5-6,8-9,12-16,18-19H2,1-4H3,(H,29,36)(H2,28,30,31)

methyl 2-[3-[[3-(6-amino-2-butoxy-8-oxo-7H-purin-9-yl)propyl-[3-(dimethylamino)propyl]amino]methyl]phenyl]acetate

AZ12441970; 929551-91-7; SCHEMBL13798671;

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.)