α7 nAChR (Ki = 8.8 nM)[1]

N-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide (14, PHA-543613), a novel agonist of the α7 neuronal nicotinic acetylcholine receptor (α7 nAChR), has been identified as a potential treatment of cognitive deficits in schizophrenia. Compound 14 is a potent and selective α7 nAChR agonist with an excellent in vitro profile. [1]

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Compounds 2, 10, 11, 13, and 14 (PHA-543613) were evaluated in a patch-clamp hERG K+ channel assay at 2 and 20 μM.28 Benzothiophene 10 inhibits hERG at levels similar to that of 2. These levels of inhibition are considered high on the basis of the efficacious drug concentrations of 2.12,19 Benzofurans 11 and 13 and furopyridine 14 all show reduced hERG inhibition at 20 μM, which may be the result of replacing the sulfur atom with the less lipophilic oxygen atom.29 To further evaluate the interactions with the hERG potassium channel, concentration response profiles were determined for 2 and 14. In agreement with the screening data, 14 is less potent at inhibiting the hERG channel-mediated currents. While 14 produces insufficient blockade at the highest tested concentration of 20 μM to establish an IC50, extrapolating the blockade produced at this concentration (29% for 14) to the fitted curve for 2 suggests that the potency for blocking hERG is reduced by at least 10-fold[1].

The short-term memory impairments caused by scopolamine were effectively corrected by PHA-543613 (0.3 mg/kg) [2]. PHA-543613 (i.p. once; 4 and 12 mg/kg) decreases brain edema and behavioral abnormalities [3].

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The functional activity of 14 (PHA-543613) was confirmed with native α7 nAChRs of rat hippocampal neurons (Figure 2). When rapidly applied to neurons recorded in the whole-cell patch clamp configuration, 14 evokes desensitizing inward currents that are concentration-dependent and completely inhibited by the selective α7 nAChR antagonist methyllycaconitine (MLA, 10 nM). The response amplitude evoked upon application of 14 at 0.3, 3, and 30 μM was 34 ± 1%, 103 ± 7%, and 220 ± 22%, respectively, of the response evoked by 100 μM (−)-nicotine applied to the same cell. These results suggest that 14 is modestly more active than 2 at native α7 nAChRs,12 which is consistent with the increased potency reported here in both the binding assay and the FLIPR assay using the α7 5HT3 chimera.[3]

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Compound 14 (PHA-543613) was also tested in a validated rodent model of impaired sensory gating.11,12 Administration of d-amphetamine (1 mg/kg, iv) significantly disrupts hippocampal CA3 region auditory gating (corresponding to human P50 auditory gating) in anesthetized rats because of a combination of simultaneous decreases of conditioning responses with corresponding increases in test responses.37 Subsequent administration of the α7 nAChR agonist 14 (iv, 0.3 or 1 mg/kg) significantly reverses the amphetamine-induced gating deficit (Figure 3). In contrast, application of vehicle in control rats did not normalize amphetamine-induced gating deficit (from 47 ± 5.5% to 41 ± 6.8% gating, n = 9). In a separate experiment, 14 at a higher dose (iv, 10 mg/kg) also significantly improves auditory gating given subsequently to amphetamine administration (from 51 ± 3.9% to 68 ± 3.9% gating, n = 16, p < 0.005). Brain concentrations after 1 and 10 mg/kg, iv administrations of the agonist were 0.56 ± 0.039 nM and 14.9 ± 1.4 μM, respectively, indicating efficacy of 14 on auditory gating at a broad brain exposure range. It has been shown in preclinical studies that α7 nAChR agonists effectively restore pharmacologically, genetically, or environmentally induced gating deficits.[3]

Details of the in Vitro Assays.[1]
Reactive Metabolite Assay (RMA). The assay was performed as described previously.46 Stock solutions of the test compounds were prepared in methanol. The final concentration of methanol in the incubation media was 0.2% (v/v). Incubations were carried out at 37 °C for 60 min in a shaking water bath. The incubation volume was 1 mL and consisted of the following:  0.1 M potassium phosphate buffer (pH 7.4), human liver microsomes (P450 concentration = 0.5 μM), NADPH (1.2 mM), and substrate (200 μM). The reaction mixture was prewarmed at 37 °C for 2 min before adding NADPH. GSH-EE (2 mM) was added 3 min after initiation of the reaction with NADPH. Incubations that lacked either NADPH or GSH-EE served as negative controls, and reactions were terminated by the addition of ice-cold acetonitrile (1 mL). The solutions were centrifuged (3000g, 15 min), and the supernatants were dried under a steady nitrogen stream. The residue was reconstituted with mobile phase and analyzed for metabolite formation by liquid chromatography tandem mass spectrometry (LC/MS/MS) as described previously for other xenobiotics.

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Human Liver Microsome Stability Assay (HLM). [1]
Substrate (final concentration = 1 μM) was incubated in human liver microsomes (HL-mix-101, prepared from 59 individual donors, 0.25 μM P450 final concentration) and 100 mM potassium phosphate buffer (pH 7.4). The reaction was initiated by the addition of an NADPH-generating system (0.5 mM NADP+, 10.5 mM MgCl2, 5.6 mM dl-isocitric acid and 0.5 U/mL isocitric dehydrogenase). Equations used for scaling to in vivo conditions were described previously.

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Rat Liver Microsome Stability Assay (RLM). [1]
Assay protocol was the same as for HLM assay only rat liver microsomes (RL-mix 142, prepared from female Spraque-Dawley rats) were used during incubation. Equations used for scaling to in vivo conditions were described previously.

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Functional High-Throughput Screens for α7 5-HT3 Chimera, 5-HT3, Neuromuscular Junction (α1β1γδ), and Ganglionic (α3β4) nAChRs. [1]
The α7 5-HT3 chimera and the 5-HT3 receptor were stably expressed in SH-EP1 cells. TE671 and SH-SY5Y cells were used as an endogenous source for neuromuscular junction and ganglionic nAChRs, respectively.48 All functional high-throughput screens were conducted as calcium flux assays using the fluorescence imaging plate reader (FLIPR, Molecular Devices). Transfected SH-EP1 cells were grown in minimal essential medium (MEM) containing nonessential amino acids supplemented with 10% fetal bovine serum, l-glutamine, 100 units/mL penicillin/streptomycin, 250 ng/mL fungizone, 400 μg/mL Hygromycin B, and 800 μg/mL Geneticin. TE671 and SH-SY5Y cells were grown according to published methods. All cells were grown in a 37 °C incubator with 5−6% CO2. The cells were trypsinized and plated in either 96- or 384-well black/clear assay plates. Cells were loaded in a 1:1 mixture of 2 mM Calcium Green-1 AM (Molecular Probes) prepared in anhydrous dimethyl sulfoxide and 20% pluronic F-127 (Molecular Probes). This reagent was added directly to the growth medium of each well to achieve a final concentration of 2 μM Calcium Green-1 AM. Cells were then incubated in the dye for 1 h at 37 °C and then washed with two cycles in a plate washer. Each cycle was programmed to wash each well four times with Mark's modified Earle's balanced salt solution (MMEBSS) composed of (in mM):  CaCl2 (4), MgSO4 (0.8), NaCl (20), KCl (5.3), d-glucose (5.6), Tris-HEPES (20), N-methyl-d-glucamine (120), pH 7.4. After the last cycle, the cells were allowed to incubate at 37 °C for at least 10 min in MMEBSS. FLIPR was set up to excite Calcium Green-1 AM at 488 nm using 500−600 mW of power and reading fluorescence emission above 525 nm. A 0.5 or 0.7 s exposure was used to illuminate each well. After 30 s of baseline recording, test compounds were added to each well of the assay plate from a 3 or 4× stock solutions. Agonist responses were evaluated as the signal increase over baseline upon addition of the test compound. Antagonist activity was evaluated in some experiments by adding either nicotine (nAChRs) or serotonin (5-HT3R) 2 min after the test compound was added and measuring the loss of response to the known agonist compared to wells treated with vehicle.

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Brain Homogenate Binding Assays ([3H]MLA, [3H]Cytisine,49 [3H]GR65630). [1]
Male Sprague-Dawley rats (300−350 g) were sacrificed by decapitation, and the brains (whole brain minus cerebellum) were dissected quickly, weighed, and homogenized in 9 volumes per gram of g wet weight of ice-cold 0.32 M sucrose using a rotating pestle on setting 50 (10 up and down strokes). The homogenate was centrifuged at 1000g for 10 min at 40 °C. The supernatant was collected and centrifuged at 20000g for 20 min at 40 °C. The resulting pellet was resuspended to a protein concentration of 1−8 mg/mL. Aliquots of 5 mL of homogenate were frozen at −80 °C until they were needed for the assay. On the day of the assay, aliquots were thawed at room temperature and diluted with Kreb's 20 mM HEPES buffer, pH 7.0 (at room temperature), containing 4.16 mM NaHCO3, 0.44 mM KH2PO4, 127 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, and 0.98 mM MgCl2 so that an amount of 25−150 mg of protein is added per test tube. Protein concentration was determined by the Bradford method using bovine serum albumin as the standard. For α7, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 μM MLA and added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 3 nM [3H]MLA (25 Ci/mmol). For α4, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 mM (−)-nicotine, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 1.0 nM [3H]cytisine. For 5-HT3, nonspecific binding was determined in tissues incubated in parallel in the presence of 1 μM ICS-205930, added before the radioligand, and in competition studies, compounds were added in increasing concentrations to the test tubes before addition of approximately 0.45 nM [3H]GR65630. For all binding assays, 0.4 mL of homogenate was added to test tubes containing buffer, test compound, and radioligand and was incubated in a final volume of 0.5 mL for 1 h at 25 °C. The incubations were terminated by rapid vacuum filtration through Whatman GF/B glass filter paper mounted on a 48-well Brandel cell harvester. Filters were presoaked in 50 mM Tris HCl, pH 7.0, and 0.05% polyethylenimine. The filters were washed two times with 5 mL aliquots of cold 0.9% saline and then counted for radioactivity by liquid scintillation spectrometry. The inhibition constant (Ki) was calculated from the concentration-dependent inhibition of radioligand binding obtained by fitting the data to the Cheng−Prusoff equation.

Patch-Clamp Electrophysiology. [1]
Cultured neurons were prepared from Sprague-Dawley rats (postnatal day 3) according to the methods of Brewer (1997). Rats were killed by decapitation, and their brains were removed and placed in ice-cold Hibernate-A medium. Hippocampal regions were gently removed, cut into small pieces, and placed in Hibernate-A medium with 1 mg/mL papain for 60 min at 35 °C. After digestion, the tissues were washed several times in Hibernate-A media and transferred to a 50 mL conical tube containing 6 mL of Hibernate-A medium with 2% B-27 supplement. Neurons were dissociated by gentle trituration and plated onto poly-d-lysine/laminin-coated coverslips at a density of 300−700 cells/mm2 and transferred to 24-well tissue culture plates containing warmed culture medium composed of Neurobasal-A medium, B-27 supplement (2%), l-glutamine (0.5 mM), 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL Fungizone. Cells were maintained in a humidified incubator at 37 °C and 6% CO2 for 1−2 weeks. The medium was changed after 24 h and then approximately every 3 days thereafter. Patch pipets were pulled from borosilicate capillary glass using a Flaming/Brown micropipet puller (P97, Sutter Instrument, Novato, CA) and filled with an internal pipet solution composed of:  CsCH3SO3 (126 mM), CsCl (10 mM), NaCl (4 mM), MgCl2 (1 mM), CaCl2 (0.5 mM), EGTA (5 mM), HEPES (10 mM), ATP−Mg (3 mM), GTP−Na (0.3 mM), phosphocreatin (4 mM), pH 7.2. The resistances of the patch pipets when filled with internal solution ranged between 3 and 6 MΩ.. All experiments were conducted at room temperature. Cultured cells were continuously superfused with an external bath solution containing NaCl (140 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), HEPES (10 mM), glucose (10 mM), bicuculline (10 μM), CNQX (5 μM), D-AP-5 (5 μM) tetrodotoxin (0.5 μM), pH 7.4. Compounds were dissolved in water or DMSO and diluted into the external bath solution containing a final DMSO concentration of 0.1% and delivered via a multibarrel fast perfusion system. Whole-cell currents were recorded using an Axopatch 200B amplifier. Analog signals were filtered at 1/5 the sampling frequency, digitized, stored, and measured using pCLAMP software. All data are reported as the mean ± SEM.

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hERG Assay. [1]
Briefly, HEK293 cells stably expressing hERG were voltage-clamped at −80 mV and step-depolarized to +20 mV for 2 s and then to −40 mV for 2 s once every 10 s. Current amplitude was measured as the peak outward current evoked upon stepping the membrane to −40 mV. Concentration response data for PNU-28987 were generated using CHO-K1 cells stably expressing hERG channels according to published methods.50 In this case, cells were voltage-clamped at −80 mV and currents were evoked by applying a cardiac action potential (AP) voltage-clamp protocol once per second, and the peak outward current was measured during the repolarizing phase of the AP wave form. In all cases, test compound was applied after recording a stable baseline, and the evoked current was monitored until a new steady-state amplitude was achieved. Current inhibition was plotted in percent according to where Itest is the current measured in the presence of the test solution and Icontrol is the current measured prior to exposure of the test solution. Each cell served as its own control. The continuous curves were determined according to where [compound] is the concentration of tested compound and the Hill slope n was constrained to unity. All data are presented as the mean ± standard deviation.

Animal/Disease Models: Male CD-1 mice (cerebral hemorrhage) ICH induction or sham surgery [3]
Doses: 4 and 12 mg/kg
Route of Administration: intraperitoneal (ip) injection; 4 and 12 mg/kg; Results 1 hour after surgery: p-Akt increased, p-GSK-3 and CC3 expression diminished in the ipsilateral hemisphere, and neuronal cell death diminished in the area around the hematoma. Behavioral deficits and brain edema were diminished 72 hrs (hrs (hours)) after ICH.

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Details of the in Vivo Assays. Auditory Gating Assay. [1]
Experiments were performed on male Sprague-Dawley rats (weighing 250−300 g) under chloral hydrate anesthesia (400 mg/kg, ip). The femoral artery and vein were cannulated for monitoring arterial blood pressure and administration of drugs or additional doses of anesthetic, respectively. Unilateral hippocampal field potential (EEG) was recorded by a metal monopolar macroelectrode placed into the CA3 region (coordinates:  3.0−3.5 mm posterior from the bregma, 2.6−3.0 mm lateral, and 3.8−4.0 mm ventral; Paxinos and Watson, 1986).51 Field potentials were amplified, filtered (0.1−100 Hz), displayed, and recorded for on-line and off-line analysis (Spike3). Quantitative EEG analysis was performed by means of fast Fourier transformation (Spike3). The auditory stimulus consisted of a pair of 10 ms, 5 kHz tone bursts with a 0.5 s delay between the first “conditioning” stimulus and second “test” stimulus. Auditory-evoked responses were computed by averaging of responses to 50 pairs of stimuli presented with an interstimulus interval of 10 s. [1]

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Object Recognition Task. [1]
Male Sprague-Dawley rats, weighing between 235 and 280 g (at test), were housed in pairs with free access to food and water on a 12-h light/dark cycle (dark period from 700 to 1900 h). Rats were housed on solid bottom cages with wood chip bedding. All procedures in this study have been approved and conducted in compliance with the Animal Welfare Act Regulations (CFR Parts 1−3) and the “Guide for the Care and Use of Laboratory Animals” (ILAR, 1996), as well as with all internal company policies and guidelines. All testing and object exposures were conducted in a 14 in. × 22 in. × 14 in. semitransparent MaxCart food bin. A clean sheet of cardboard-like Techboard was placed on the floor before each trial. The test arena was indirectly and uniformly illuminated at a low-intensity level of 10−12 Lux. A video camera affixed about 4 feet above the floor of the arena was connected to a monitor and VCR located several feet away from the visually shielded arena. Two types of objects were used in all drug tests reported here:  a 500 mL clear Erlenmeyer glass flask and a 500 mL amber-colored bottle (3 in. × 2.25 in. × 8 in.) with a black cap (filled with water). These objects were cleaned after every trial by swabbing with a 70% alcohol solution. Two or more different sets of each object were used to allow air-drying for several minutes between tests.[1]
A 3-day procedure similar to the one used by Moser was utilized as follows. Day 1 involved 2 min of habituation, Techboard floor absorbent side up, and no objects. Day 2 involved 5 min of exploration, Techboard floor absorbent side down, and two identical objects. Objects are placed 2 in. from each of the two sidewalls of the corner. Each animal is allowed as much as 5 min to accumulate a maximum approach time of 20 s to either or both of the identical objects. Consequently, exploratory exposure to the sample object (familiar) was equated between rats by terminating the session after 20 s of object approach. Rats failing to explore the objects for more than 10 s were discarded. In general, greater than 90% of rats achieved this cut off criterion. Day 3 involved 3 min of test duration, Techboard floor absorbent side up, and two dissimilar objects. Approach time to each object was recorded separately and was the major response measure. In these studies, “approach” was defined as the nose of the rat within 2 cm of an object.[1]
Vehicle and compound 14 were administered subcutaneously 30 min before each session. For all experiments, each treatment group consisted of 15 rats at the beginning of the experiments. For each session the animals were placed in the arena with their nose facing away from the objects (when present) and centered on the long side of the arena. Upon placement of the rat in the arena, the experimenter immediately sat in front of the monitor for scoring approach behavior without disturbing the rat. Approach time to each object during the test session was recorded separately and was the major response measure. These data were analyzed using a paired two-tail t-test to determine significant differences between novel and familiar approach time, with statistical significance defined by a p value less than 0.05.[1]

Further human and rat in vitro pharmacokinetic (PK) evaluation demonstrated moderate clearance and half-lives for compounds 2, 11, 13, and 14 (PHA-543613). In vivo PK evaluation in a rat constant infusion model verified the in vitro PK data and predicted good oral bioavailability of >60% for each of the compounds and clearance consistent with the RLM data. Differentiation of the compounds came from CYP2D6 evaluation, where benzofuran 11 was found to inhibit this key P450 enzyme. Fortunately, furopyridine 14 (PHA-543613) is inactive up to the highest dose tested (10 μM). Finally, a mouse brain uptake assay (MBUA) was used to evaluate CNS penetration.34 Compounds 2, 11, 13, and 14 (PHA-543613) each have excellent brain penetration; however, with the exception of furopyridine 14 each demonstrates a modest accumulation in the CNS. Brain accumulation was not a desirable attribute, given the α7 receptor's known desensitization profile.35 A separate PK study evaluating 14, utilizing a 5 mg/kg dose in rat, is in close agreement with the infusion data above:  65% oral bioavailability, low clearance of 33.3 mL·min-1·kg-1, and volume of distribution of 1.8 L·kg-1. On the basis of the data presented, compound 14 clearly differentiates itself from 2, 11, and 13, justifying further in vivo evaluation.

[1]. Discovery of N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide, an agonist of the alpha7 nicotinic acetylcholine receptor, for the potential treatment of cognitive deficits in schizophrenia: synthesis and structure--activity relationship. J Med Chem. 2006 Jul 13;49(14):4425-36.

[2]. Potentiation of cognitive enhancer effects of Alzheimer's disease medication memantine by alpha7 nicotinic acetylcholine receptor agonist PHA-543613 in the Morris water maze task. Psychopharmacology (Berl). 2021 Nov;238(11):3273-3281.

[3]. α7 nicotinic acetylcholine receptor agonism confers neuroprotection through GSK-3β inhibition in a mouse model of intracerebral hemorrhage. Stroke. 2012 Mar;43(3):844-50.

Pha-543613 is a phosphoramide and an organothiophosphorus compound.

C15H17N3O2

271.32

307.109

C, 66.40; H, 6.32; N, 15.49; O, 11.79

478149-53-0

PHA-543613 dihydrochloride;478148-58-2; 1586767-92-1 (HCl)

9930121

White to off-white solid powder

2.782

1

4

2

20

381

1

IPKZCLGGYKRDES-ZDUSSCGKSA-N

InChI=1S/C15H17N3O2/c19-15(12-7-11-3-6-20-14(11)8-16-12)17-13-9-18-4-1-10(13)2-5-18/h3,6-8,10,13H,1-2,4-5,9H2,(H,17,19)/t13-/m0/s1

N-[(3R)-1-azabicyclo[2.2.2]octan-3-yl]furo[2,3-c]pyridine-5-carboxamide

PHA543613; PHA 543613; PHA-543613; 478149-53-0; PHA-543,613; N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c]pyridine-5-carboxamide; (R)-N-(Quinuclidin-3-yl)furo(2,3-C)pyridine-5-carboxamide; R36R9KVD6Y; CHEMBL214268; DTXSID6047284; PHA-543613

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 : ~250 mg/mL (~921.46 mM)
H2O : ~100 mg/mL (~368.58 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.)