Platelet-activating factor (PAF; Ki = 9.9 nM)

A pharmacological approach to neoplasia by differentiation therapy relies on the availability of cytodifferentiating agents whose antitumor efficacy is usually assayed first on malignant cells in vitro. Using murine erythroleukemia cells (MELCs) as the model, we found that WEB-2086/Apafant, a triazolobenzodiazepine-derived PAF antagonist originally developed as an anti-inflammatory drug, induces a dose-dependent inhibition of MELC growth and hemoglobin accumulation as a result of a true commitment to differentiation. MELCs treated for 5 days with 1 mM WEB-2086/Apafant show ≥ 85% benzidine-positive cells, increased expression of α- and β-globin genes, and down-regulation of c-Myb. This differentiation pattern, which does not involve histone H4 acetylation and is abrogated by the action of phorbol 12-myristate 13-acetate, recalls the pattern induced by hexamethylene bisacetamide (HMBA). In addition to MELCs, human erythroleukemia K562 and HEL and myeloid HL60 cells are massively committed to maturation by Apafant/WEB-2086 and, with some differences, by its analog, WEB-2170. This suggests that WEB-2086, structurally distant from other known inducers, might be a member of a new class of cytodifferentiation agents active on a broad range of transformed cells in vitro and useful, prospectively, for anticancer therapy due to their high tolerability in vivo [2].

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WEB-2086/Apafant reduces the adhesion of S. pneumoniae and NTHi to CSE-treated bronchial epithelial cells [4]
WEB-2086 decreased S. pneumoniae adhesion to CSE-treated BEAS-2B cells back to control levels at a concentration of 100 nM (P=0.0058) (Figure 4A–C). WEB-2086/Apafant at 10 μM, but not at lower concentrations, also reduced adhesion of the reference strain of NTHi to CSE-treated cells to control levels (P<0.0001) (Figure 4D). Adhesion of the clinical strain of NTHi to CSE-treated BEAS-2B cells was reduced to control levels by WEB-2086 at a concentration of 1 μM (Figure 4E). WEB-2086 was nontoxic to epithelial cells at the highest concentration tested, that is, IC50 >10 μM.

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Docking WEB-2086/Apafant to a molecular model of PAFr [4]
Initial models of PAFr produced by MODELLER and Iterative Threading ASSEmbly Refinement were very similar (r.m.s. deviation of 3 Å over 295 Cα atom pairs), and predicted that the same set of amino acids would contribute to the ligand binding site. A final consensus model of PAFr was generated in MODELLER using neurotensin receptor type 1 (NTR1) and C-C chemokine receptor 5 (CCR5) as template structures (Figure 5A), taking advantage of regions of local high sequence/structure similarity to each template. The natural PAF ligand and WEB-2086/Apafant were docked to the model using the AutoDock Vina package. In a search of the whole molecular surface, both PAF and WEB-2086 consistently docked into the same deep cavity on the predicted extracellular face of the PAFr model (Figure 5A). The cavity is substantially larger than the WEB-2086 compound (Figure 5B) and a fine-grained docking search of this pocket identified a number of potential docking poses.

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In this work, we tested a PAFr antagonist for its ability to inhibit the binding of NTHi and S. pneumoniae to CSE-pretreated airway epithelial cells. WEB-2086/Apafant was effective in significantly reducing the adhesion of both respiratory pathogens to CSE-exposed bronchial epithelial cells with demonstrated PAFr upregulation. Interestingly, the bacterial adhesion seen in the presence of WEB-2086 was comparable to control levels observed for the cultured epithelial cells for which PAFr expression had not been induced through CSE exposure. Furthermore, the level of bacterial adhesion was found to be proportionate to the level of PAFr expression on lung epithelial cells. In terms of drug tolerability, PAF antagonists, including WEB-2086, have been shown to be safe in both animals and humans, even at high doses over several months. Similarly, we did not find WEB-2086 to be toxic to BEAS-2B cells.

There are a number of limitations in this study. We used a commercial airway epithelial cell line, and further work is now needed on the anti-infective effects of Apafant/WEB-2086 using primary human cells, especially from smokers. Second, the ligand-docking studies are reliant on an in silico model of PAFr, which is derived from the tertiary structure of related GPCRs. X-ray crystallographic work is required to confirm the specific PAFr–WEB-2086 interactions.

In summary, we investigated the antiadhesion activity of WEB-2086/Apafant toward NTHi and S. pneumoniae, two major respiratory pathogens, on airway epithelium primed by CSE to upregulate PAFr expression. We demonstrated an association between PAFr upregulation and bacterial adhesion, the latter of which was reduced to control levels in the presence of the PAFr antagonist WEB-2086. Understanding the events leading to colonization of the lower respiratory tract of COPD patients with NTHi and pneumococcus, and to subsequent increase in local and systemic inflammation, may lead to translation into new management strategies for COPD. Given the apparent tolerance of WEB-2086 in human subjects and its ability to block the adhesion of S. pneumoniae and NTHi to respiratory epithelial cells, we are now in a position to undertake exploratory proof-of-concept studies of PAF antagonists in COPD clinical studies to ascertain if they positively affect day-to-day symptoms as well as AE. We have also begun to explore the tertiary structure of PAFr to design new chemical derivatives of WEB-2086 with enhanced binding affinity for PAFr. This opens up the possibility of a novel nonantibiotic, anti-infective drug for therapeutic interventions in COPD patients, who are prone to frequent bacterial exacerbations, and in smokers, more generally, who are vulnerable to episodes of pneumonia.

Platelet-activating factor (PAF) plays important roles in allergic reactions. In particular, there are many concerns about PAF, eosinophils, and the chronicity of allergic diseases. The purpose of the present studies is to elucidate the role of PAF in eosinophil activation at conjunctiva and to confirm the efficacy of Apafant (a potent PAF antagonist) ophthalmic solution in chronic experimental allergic conjunctivitis. Guinea pigs were actively immunized and allergic conjunctivitis was induced by repetitive instillation of 2.5% ovalbumin. PAF solution was topically applied and eosinophil activation was assessed by measuring the eosinophil peroxidase (EPO) activity in the tear fluid. Itch-scratching episodes and clinical symptoms scores were evaluated in the repetitive challenge conjunctivitis. From the instillation of PAF solution into guinea pig eyes, which were in a state of chronic allergic conjunctivitis, a significant increase in EPO activity was observed, and this increase was inhibited by pre-treatment with Apafant. In the repetitive challenge model, the animals treated with Apafant ophthalmic solution showed a significant reduction of clinical symptoms and the itch-scratch response in both the first and the second challenges. PAF has an activity, that induces mediator release from eosinophils in the conjunctival tissues and may be involved in the chronic phase of allergic conjunctivitis [1].

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We also found that a minimum dose of 30 mg/kg WEB-2086/Apafant, the most potent commercially available PAF-R antagonist, was needed to provide the same level of protection provided by 12.5 mg/kg acyl-PAF (Table 1, Fig. 2). These experiments suggest that low concentrations of the pharmacologically less active acyl-PAF and possibly other acyl-PAF-like lipids described in the literature (1, 3, 16) may dampen PAF-R signaling.[3]

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Reactivation of PAF-R for alkyl-PAF-induced lethality in Swiss albino mice pretreated with acyl-PAF or WEB-2086/Apafant [3]
Given our observation that acyl-PAF has a dampening effect on PAF-R activation, we next determined the duration of acyl-PAF-induced dampening action by assessing the reactivation of PAF-R in Swiss albino mice at various time points. Briefly, we first injected mice with acyl-PAF at a fixed dose of 12.5 mg/kg, which was chosen based on our previous experiments (Fig. 2). We then challenged the mice with a lethal dose of alkyl-PAF (250 µg/kg) at 3, 5, 7.5, or 10 h after the initial PAF-R dampening. When pretreated with acyl-PAF, none of the mice died after being challenged 3 h later with a lethal dose of alkyl-PAF. However, 50% of the mice died when challenged 5 or 7.5 h later, and 83% died when challenged 10 h later (Fig. 3). Thus, the complete PAF-R dampening effects lasted for only 3 h when acyl-PAF was used as the dampening agent. A different scenario was observed when WEB-2086/Apafant (30 mg/kg) was used as the PAF-R dampening agent. None of the mice died when they were pretreated with WEB-2086 and then challenged with a lethal dose of alkyl-PAF 5 h later. Only 17% died when challenged 10 h after the WEB-2086 pretreatment. Even in the groups that received the alkyl-PAF injection 30 or 45 h after the WEB-2086 pretreatment, 33% of the mice still survived (Fig. 3).

A stock solution (10 mM) of WEB-2086/Apafant and BN-52021 was prepared in DMSO, and a working solution (1 mM) was diluted with saline and used in aggregation studies. Platelet-rich plasma (PRP) was isolated from the blood of healthy volunteers using the method previously described by Zhou et al. Briefly, blood was drawn into citrated tubes (1:9; citrate-blood), and the tubes were centrifuged for 15 min at 45 g at 28°C to obtain PRP. The remaining cells were settled by centrifuging the tubes at 2,200 g for 15 min, and the platelet-poor plasma obtained was used for setting the blank and diluting the PRP. Platelet aggregation was performed with the use of 4 × 108 platelets/ml in a final volume of 250 μl with stirring at 1,200 rpm at 37°C for up to 6 min. An aliquot of alkyl-PAF taken from a 10 mM stock (in methanol) was evaporated under a stream of nitrogen and reconstituted in PBS containing 0.1% HSA, yielding a 1 mM working solution. Using this solution, aggregation was initiated by adding alkyl-PAF (8 µM final concentration) to the PRP. The aggregation effects of acyl-PAF were determined by using the same protocol, but a concentration of 800 µM acyl-PAF was needed to induce effects similar to those of alkyl-PAF. [3]

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To determine the effect of PAF-R antagonists on platelet aggregation mediated by acyl-PAF or alkyl-PAF, PRP was pretreated with WEB-2086/Apafant (10 μM) or BN-52021 (10 μM) for 5 min and then exposed to acyl-PAF (800 µM) or alkyl-PAF (8 µM). In all aggregation studies, the final concentrations of DMSO never exceeded 0.1%. The dampening effect of acyl-PAF on PAF-R was studied by pretreating or simultaneously exposing platelets with the stated amounts of acyl-PAF (5 nM to 800 μM) and then stimulating platelets with alkyl-PAF (8 µM). In some of the aggregation experiments, alkyl-PAF or acyl-PAF was first predigested with various concentrations of rPAF-AH (stock solution: 4 µg/ µl) for 30 min at 37°C and then monitored for its ability to induce aggregation. In some experiments, the effect of rPAF-AH on aggregation induced by acyl-PAF or alkyl-PAF was determined by treating PRP simultaneously with either alkyl-PAF (8 µM) or acyl-PAF (800 µM) and rPAF-AH (50–200 ng). All assays were performed by using a Chrono-log platelet aggregometer, and the data were recorded by using AGROLINK software [3].

Treatment with PAFr antagonists [4]
A 1 mM stock solution of Apafant/WEB-2086 was prepared in dimethyl sulfoxide (≤1% concentration) and then diluted in a medium to a final concentration of 10 μM, 1 μM, 100 nM, and 10 nM. After CSE treatment, the cells were exposed to PAFr antagonist for 1 hour, followed by challenge with FITC-tagged bacteria.

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Immunocytochemistry [4]
After exposure to FITC-tagged bacteria, the cells were washed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes at room temperature and rinsed before treatment with ice-cold acetone. Cells were blocked/permeabilized with 1% bovine serum albumin, 1% Triton-X-100 in PBS for 30 minutes, and incubated with a 1:100 dilution of anti-PAFr monoclonal antibody in blocking buffer overnight at 4°C in the dark. After thorough rinsing with PBS, an AlexaFluor 594-conjugated goat anti-mouse secondary antibody, diluted to 1:500 in blocking buffer, was added, which was followed by incubation at room temperature for 1 hour. The cells were rinsed and then stained with 4′,6-diamidino-2-phenylindole, diluted 1:5,000 in PBS, and then incubated in the dark at room temperature for 15 minutes. The cells were washed thrice with PBS before slides were mounted with fluorescent-mounting media.

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Microscopy and image analyses [4]
Micrographs were analyzed using an Olympus BX50 Fluorescence Microscope and CoolSnap Hq2 CCD camera. Five images were taken per well using each immunofluorescence channel to take pictures of 4′,6-diamidino-2-phenylindole (eukaryotic cell nuclei; 30 ms exposure, 405 nm), PAFr (PAFr expression; 1–3 second exposure, 594 nm), and FITC (bacteria; 1–3 second exposure, 488 nm) signals under ×400 magnification. NIH elements imaging software was used to perform eukaryotic and bacterial cell counts, while image merging was completed using Adobe Photoshop CS6 software. Cell tracing was achieved using Image-Pro Plus 7.0.

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BEAS-2B cell viability assay [4]
Cells were passaged in sterile clear-flat 96-well plates with PAFr antagonist diluted to concentrations used within bacterial adhesion assays and added as triplicates to incubate for 1 hour. Alamar Blue was then added to each well at a final concentration of 10% (w/v) before hourly absorbance readings (585 nm) were taken up to 8 hours and finally, at 24 hours using a Spectromax Spectrophotometer Microplate Reader. A positive control of 100% (w/v) Alamar Blue reduced by autoclaving was used in experiments. Cell viability data are representative of the three biological replicates and expressed as 50% inhibition (IC50) values as per the manufacturer’s guidelines.

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Quantitative real-time polymerase chain reaction [4]
Cells were seeded into sterile clear-flat bottom 24-well plates before stimulation with CSE (1%, 4 hours). RNA was prepared using the Reliaprep™ Mini RNA cell Miniprep system with total RNA quantified by Nanodrop 1000. Complementary DNA was then collected using the Promega complementary DNA synthesis kit. The level of PAFr transcript was determined by quantitative polymerase chain reaction using the Corbett Rotor-Gene 6000 system. Thermocycling controls were run as previously described.25 The relative change of PAFr mRNA expression was normalized to three reference genes (18S rRNA, β-actin, and β2-microglobulin) using the primers and recommended conditions provided by the manufacturer. Data were derived from two independent experiments performed in duplicate.

Eosinophil activation by the instillation of PAF [1]
PAF solution (0.1%) was topically applied into the conjunctival sac of naive guinea pigs or guinea pigs that had been subjected to the allergic conjunctivitis model, described above. Thirty minutes after the PAF instillation, ophthalmic lavage was collected and EPO activity in the lavage fluid was measured. To examine whether Apafant blocks the release of EPO into the lavage fluid, Apafant ophthalmic solution (0.1% or 1.0%) or a vehicle was bilaterally administered 15 and 5 min prior to the PAF instillation.

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Effect of Apafant/WEB-2086 on alkyl-PAF-induced death in Swiss albino mice [3]
A stock solution of Apafant/WEB-2086 (30 mg/ml) was prepared in DMSO, and aliquots were diluted with sterile PBS to 0.5 ml. Five groups of Swiss albino mice were injected with WEB-2086 (1, 5, 10, 20, or 30 mg/kg) and then challenged 3 h later with a lethal dose of alkyl-PAF (250 μg/kg). The control mice received only the vehicle used in the preparation of WEB-2086. The concentration of DMSO never exceeded 0.2% in these experiments.

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Reactivation of PAF-R for alkyl-PAF-induced lethality in Swiss albino mice pretreated with acyl-PAF or Apafant/WEB-2086 [3]
Swiss albino mice weighing 20–25 g were assigned to 4 groups comprising 6 mice each. The mice were injected intraperitoneally with acyl-PAF (12.5 mg/kg) and then challenged with a lethal dose of alkyl-PAF (250 μg/kg) at 3, 5, 7.5, or 10 h after the initial acyl-PAF injection. In a parallel experiment, 7 groups of mice were injected with ApafantWEB-2086 (30 mg/kg) for various time points such as 3, 5, 10, 15, 30, and 45 h, followed by a lethal dose of alkyl-PAF. The mice were monitored for survival for up to 6 days.

65889 mouse LD50 oral 3 gm/kg United States Patent Document., #4968794
65889 mouse LD50 intravenous 400 mg/kg United States Patent Document., #4968794

[1]. Apafant, a potent platelet-activating factor antagonist, blocks eosinophil activation and is effective in the chronic phase of experimental allergic conjunctivitis in guinea pigs. J Pharmacol Sci. 2004 Aug;95(4):435-42.

[2]. Specific PAF antagonist WEB-2086 induces terminal differentiation of murine and human leukemia cells. FASEB J. 2002 May;16(7):733-5.

[3]. Modulation of inflammatory platelet-activating factor (PAF) receptor by the acyl analogue of PAF. J Lipid Res. 2018 Nov;59(11):2063-2074.

[4]. An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke. Int J Chron Obstruct Pulmon Dis . 2016 Jul 25:11:1647-55.

The development and utilization of animal models are important to elucidate the pathophysiology of allergic conjunctivitis and to develop effective therapies. Several animal models of allergic conjunctivitis have been reported. In this study, we have used a guinea pig allergic conjunctivitis model in which allergic conjunctivitis was repetitively elicited by antigen challenge because this model has the following advantageous features: Firstly, the clinical symptoms of allergic patients, such as ocular itching, redness and edema, are well replicated in this animal model. Secondly, eosinophils as well as mast cells play roles in the symptoms caused by the repetitive antigen challenge, and eosinophil granule proteins such as EPO were detected in the tear fluid, indicating that the model is well correlated with the pathophysiology of patients with allergic conjunctivitis. Yasuda et al. have previously reported the benefits of repetitive antigen challenge models of allergic conjunctivitis in guinea pigs. In summary, we have demonstrated that PAF has an activity that induces mediator release from eosinophils in the conjunctival tissues and may be involved in the chronicity and exacerbation of allergic conjunctivitis. We have also demonstrated that Apafant ophthalmic solution inhibits eosinophil activation and is effective in the chronic phase of experimental allergic conjunctivitis. These results support the potential clinical utility of a topically active PAF antagonist for the treatment of allergic conjunctivitis, especially at the chronic phase. [1]

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Platelet-activating factor (PAF) is a potent inflammatory mediator that exerts its actions via the single PAF receptor (PAF-R). Cells that biosynthesize alkyl-PAF also make abundant amounts of the less potent PAF analogue acyl-PAF, which competes for PAF-R. Both PAF species are degraded by the plasma form of PAF acetylhydrolase (PAF-AH). We examined whether cogenerated acyl-PAF protects alkyl-PAF from systemic degradation by acting as a sacrificial substrate to enhance inflammatory stimulation or as an inhibitor to dampen PAF-R signaling. In ex vivo experiments both PAF species are prothrombotic in isolation, but acyl-PAF reduced the alkyl-PAF-induced stimulation of human platelets that express canonical PAF-R. In Swiss albino mice, alkyl-PAF causes sudden death, but this effect can also be suppressed by simultaneously administering boluses of acyl-PAF. When PAF-AH levels were incrementally elevated, the protective effect of acyl-PAF on alkyl-PAF-induced death was serially decreased. We conclude that, although acyl-PAF in isolation is mildly proinflammatory, in a pathophysiological setting abundant acyl-PAF suppresses the action of alkyl-PAF. These studies provide evidence for a previously unrecognized role for acyl-PAF as an inflammatory set-point modulator that regulates both PAF-R signaling and hydrolysis.[3]

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With this background in mind, we designed our study to examine the effect of the abundant PAF structural analogue acyl-PAF on PAF-R activation. We found that acyl-PAF by itself was not toxic to the mice under physiologic conditions unless it was given at a supraphysiologic dose (data not shown). Intriguingly, we found that acyl-PAF at a dose of 10–50 mg/kg protected mice from alkyl-PAF-induced mortality (Fig. 2). The inhibitory effects of acyl-PAF were then compared with those of the potent, synthetic PAF-R antagonist WEB-2086 (Fig. 3). We found that acyl-PAF displayed inhibitory effects when used at a dose of 12.5 mg/kg, whereas for WEB-2086, a minimum dose of 30 mg/kg was required to achieve the same effect. This suggests that acyl-PAF is a better antagonist of PAF-R-induced lethality than the well-studied antagonist WEB-2086 (Table 1, Fig. 2).
We next sought to define the duration of protection exerted by acyl-PAF and found that, 5 h after being pretreated with acyl-PAF, mice exhibited a loss of protection, indicating that the reactivation of PAF-R in response to alkyl-PAF had occurred (Fig. 3). Thus, the protection provided by acyl-PAF from alkyl-PAF-induced lethality was temporary and quite different from the protection provided by WEB-2086 (Fig. 3). One possible explanation for the short duration of protection provided by acyl-PAF is its susceptibility to degradation by endogenous PAF-AH, which results in the reactivation of PAF-R for subsequent injections of alkyl-PAF. On the other hand, WEB-2086 contains no ester bonds, is not a substrate for PAF-AH, and may follow a different P-glycoprotein-dependent slower catabolic route. We observed that repeated injection of acyl-PAF did not protect against alkyl-PAF-induced animal death under our experimental conditions, suggesting that the continuous presence of acyl-PAF may not serve as a natural antagonist for PAF-R (data not shown).[3]

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Background [4]
COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells.

Objective [4]
To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae.

Methods [4]
Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, Apafant/WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken.

Results [4]
PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. Apafant/WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure.

Conclusion [4]
WEB-2086Apafant/ represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD.

C22H22CLN5O2S

455.961

455.118

C, 57.95; H, 4.86; Cl, 7.77; N, 15.36; O, 7.02; S, 7.03

105219-56-5

65889

White to yellow solid powder

1.5±0.1 g/cm3

720.2±70.0 °C at 760 mmHg

186-188ºC

389.4±35.7 °C

0.0±2.3 mmHg at 25°C

1.734

1.32

0

6

4

31

691

0

CC1=NN=C2N1C3=C(C=C(S3)CCC(=O)N4CCOCC4)C(=NC2)C5=CC=CC=C5Cl

JGPJQFOROWSRRS-UHFFFAOYSA-N

InChI=1S/C22H22ClN5O2S/c1-14-25-26-19-13-24-21(16-4-2-3-5-18(16)23)17-12-15(31-22(17)28(14)19)6-7-20(29)27-8-10-30-11-9-27/h2-5,12H,6-11,13H2,1H3

3-[7-(2-chlorophenyl)-13-methyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-4-yl]-1-morpholin-4-ylpropan-1-one

DE-081; WEB2086; Apafant; 105219-56-5; triazolodiazepine; Apafanto; Apafantum; WEB2086; WEB-2086

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)

DMSO : ~40 mg/mL (~87.73 mM)

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.)