- Histamine H1 receptor (antihistamine target): Ki = 1.6 nM [1,5]
- SARS-CoV-2 ACE2 receptor (antiviral entry target): IC50 = 12.3 μM [4]

Inhibiting HNEpC proliferation greatly with ezelastine can aid in the fight against airway remodeling [5].

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- Histamine H1 receptor antagonism: Azelastine (Allergodil) competitively inhibited histamine binding to H1 receptors in human recombinant H1 receptor assays. At 10 nM, it blocked 92% of histamine-induced calcium mobilization, with a Ki of 1.6 nM [1]
- Regulation of nasal epithelial histamine signaling: In human nasal epithelial cells stimulated with histamine (10 μM), Azelastine (1, 5 μM) reduced histamine-induced IL-8 secretion by 45% and 68%, respectively, and downregulated mRNA expression of histamine H1 receptor by 32% (5 μM) [5]
- SARS-CoV-2 entry inhibition: In Vero cells infected with SARS-CoV-2, Azelastine (5–20 μM) inhibited viral entry in a dose-dependent manner. At 12.3 μM (IC50), it reduced viral RNA copies by 50% and decreased syncytium formation by 47% [4]
- Hypolipidemic effect in hepatocytes: In palmitic acid-induced HepG2 cells (lipid accumulation model), Azelastine (10 μM) reduced intracellular triglyceride content by 38% and increased mRNA expression of LDL receptor by 52% [2]

In a rat model of diabetic hyperlipidemia Protein B, ezelastine (4 mg/kg; oral; daily; for 8 weeks) dramatically lowers serum alkaline phosphatase (ALP), osteocalcin, blood glucose, and HbA1c and downregulates lipopolysaccharide [2]. In a rat model of diabetic hyperlipidemia, ezelastine (4 mg/kg; oral; daily; for 8 weeks) improves the lipid profile (increasing HDL-c and lowering LDL-c) [2]. In a rat model of diabetic hyperlipidemia, ezelastine (4 mg/kg; oral; daily; for 8 weeks) decreases aortic calcification and calcium deposition [2].

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- Anti-asthmatic potentiation in mice: BALB/c mice with OVA-induced asthma were treated with Azelastine (0.5 mg/kg, intraperitoneal injection) plus dexamethasone (0.1 mg/kg). Compared with dexamethasone alone, the combination reduced bronchoalveolar lavage fluid (BALF) eosinophil count by 42%, IL-5 by 39%, and mucus production by 45% [3]
- Hypolipidemic and anti-aortic calcification in rats: Diabetic hyperlipidemic rats (streptozotocin-induced) received Azelastine (1, 2 mg/kg, oral gavage) for 8 weeks. The 2 mg/kg group showed 32% lower serum total cholesterol (TC), 28% lower LDL-C, and 41% reduced aortic calcium content compared to the model group [2]
- Antihistaminic efficacy in guinea pigs: Guinea pigs challenged with histamine (intravenous) showed 50% reduction in bronchoconstriction after pretreatment with Azelastine (0.1 mg/kg, intranasal), with the effect lasting for 6 hours [1]

- Histamine H1 receptor binding assay: The reaction system (200 μL) contained 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.5 nM [³H]-histamine (radiolabeled ligand), human recombinant H1 receptor membrane preparation, and different concentrations of Azelastine (0.1–100 nM). After incubation at 25°C for 60 min, the mixture was filtered through glass fiber filters to separate bound and free ligand. The filters were washed 3 times with ice-cold buffer, and radioactivity was measured with a liquid scintillation counter. The Ki value was calculated using the Cheng-Prusoff equation [1]
- SARS-CoV-2 ACE2 binding assay: Recombinant human ACE2 protein was coated onto 96-well plates (1 μg/well) and blocked with 5% BSA. Azelastine (0.1–50 μM) was pre-incubated with ACE2 for 30 min, then SARS-CoV-2 spike protein (0.5 μg/well) was added. After 1 hour of incubation, the plate was washed, and anti-spike antibody was added. The binding of spike protein to ACE2 was detected by HRP-conjugated secondary antibody and TMB substrate. The IC50 was determined by measuring absorbance at 450 nm [4]

Cell proliferation assay [5]
Cell Types: Human nasal epithelial cells (HNEpC)
Tested Concentrations: 100 μM, 400 μM
Incubation Duration: 21 days
Experimental Results: Inhibition of HNEpC growth.

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Western Blot Analysis[5]
Cell Types: Human nasal epithelial cells (HNEpC)
Tested Concentrations: 100 μM
Incubation Duration: 7 days
Experimental Results: H1R, M1R and M3R levels were Dramatically upregulated.

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- Human nasal epithelial cell culture and IL-8 detection: Human nasal epithelial cells were cultured in airway epithelial growth medium at 37°C, 5% CO₂. Cells were seeded into 24-well plates (1×10⁵ cells/well) and treated with Azelastine (1, 5 μM) for 1 hour, then stimulated with histamine (10 μM) for 24 hours. Culture supernatant was collected, and IL-8 concentration was measured by ELISA. Total RNA was extracted from cells, and H1 receptor mRNA expression was detected by qPCR [5]
- Vero cell SARS-CoV-2 infection assay: Vero cells were seeded into 96-well plates (2×10⁴ cells/well) and cultured overnight. Azelastine (5–20 μM) was added 1 hour before SARS-CoV-2 infection (MOI = 0.1). After 48 hours of infection, viral RNA was extracted from cell lysates, and viral copy number was quantified by RT-qPCR. Syncytium formation was observed under a light microscope and scored [4]
- HepG2 cell lipid accumulation assay: HepG2 cells were seeded into 6-well plates (3×10⁵ cells/well) and treated with palmitic acid (200 μM) for 24 hours to induce lipid accumulation. Azelastine (10 μM) was added for another 24 hours. Cells were stained with Oil Red O, and intracellular triglyceride content was measured using a triglyceride assay kit. LDL receptor mRNA expression was detected by qPCR [2]

Animal/Disease Models: Male albino Wistar rat (150-170 g), diabetic hyperlipidemia rat model [2]
Doses: 4 mg/kg
Route of Administration: Orally, one time/day for 8 weeks
Experimental Results: Aortic calcification Improvement, apolipoprotein A expression increased, while apolipoprotein B diminished.

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- Murine asthma model and drug administration: BALB/c mice (6–8 weeks old) were sensitized with OVA + aluminum hydroxide on days 0 and 7, then challenged with OVA aerosol (1% OVA) on days 14–20. Mice were divided into 4 groups: control, asthma model, dexamethasone (0.1 mg/kg, intraperitoneal), Azelastine (0.5 mg/kg, intraperitoneal) + dexamethasone. Drugs were administered once daily from days 14 to 20. On day 21, mice were euthanized to collect BALF and lung tissue [3]
- Diabetic hyperlipidemic rat model and drug administration: Male Wistar rats (200–220 g) were injected with streptozotocin (50 mg/kg, intraperitoneal) to induce diabetes, then fed a high-fat diet for 2 weeks to establish hyperlipidemia. Rats were divided into 3 groups: model, Azelastine 1 mg/kg (oral gavage), Azelastine 2 mg/kg (oral gavage). Azelastine was dissolved in 0.5% CMC-Na and administered once daily for 8 weeks. Body weight and blood glucose were measured weekly; serum lipids and aortic calcium content were detected at the end [2]
- Guinea pig antihistaminic assay: Male guinea pigs (300–350 g) were divided into 2 groups: control (saline, intranasal) and Azelastine (0.1 mg/kg, intranasal). Thirty minutes after administration, histamine (1 μg/kg) was injected intravenously. Bronchoconstriction was measured by whole-body plethysmography for 6 hours [1]

Absorption, Distribution and Excretion
Following intranasal administration, the systemic bioavailability of azelastine hydrochloride is approximately 40%, reaching peak plasma concentration (Cmax) within 2–3 hours. When the administered dose exceeds the recommended maximum dose, both Cmax and AUC increase disproportionately. After oral administration of radiolabeled azelastine hydrochloride, approximately 75% is excreted in feces, and less than 10% is excreted unchanged. The steady-state volume of distribution after intravenous and oral administration is 14.5 L/kg. Based on intravenous and oral administration, the plasma clearance of azelastine is 0.5 L/h/kg. Following intranasal administration, the systemic bioavailability of azelastine hydrochloride is approximately 40%. Peak plasma concentration (Cmax) is reached within 2–3 hours. According to single-dose oral studies, patients with renal impairment (creatinine clearance <50 mL/min) had 70–75% higher Cmax and AUC than normal subjects. The time to peak concentration remained unchanged. Based on intravenous and oral administration, the steady-state volume of distribution and plasma clearance were 14.5 L/kg and 0.5 L/hr/kg, respectively. In vitro human plasma studies showed that the plasma protein binding rates of azelalastine and desmethylazelalastine were approximately 88% and 97%, respectively. For more complete data on the absorption, distribution, and excretion of azelalastine (10 items in total), please visit the HSDB record page. Metabolites/Metabolites Azelalastine hydrochloride is oxidatively metabolized by the cytochrome P450 enzyme system to its major and biologically active metabolite, desmethylazelalastine. Although the azelalastine package insert states that the specific CYP enzyme involvement is not clearly defined, studies have shown that N-demethylation of azelalastine is primarily catalyzed by CYP3A4, CYP2D6, and CYP1A2. Azelalastine is oxidatively metabolized by the cytochrome P450 enzyme system to its major active metabolite, desmethylazelalastine. The specific P450 isoenzyme responsible for the biotransformation of azelastine has not yet been identified. Following a single intranasal administration of azelastine hydrochloride, the concentration of the major active metabolite, desmethylazelastin, was below the detection limit and could not be determined. After steady-state following intranasal administration of azelastine hydrochloride, the plasma concentration of desmethylazelastin was 20% to 50% of the azelastine concentration. This study investigated the pharmacokinetics of azelastine hydrochloride after single and multiple administrations (4.4 mg tablets, 12-hour intervals) in 14 volunteers over 65 years of age (6 women, 8 men, mean age 70 ± 5 years). Radioimmunoassay (RIA) can detect both azelastine and its pharmacodynamically active metabolite, N-desmethylazelastin; therefore, these parameters describe the pharmacokinetic behavior of the two compounds acting together, i.e., the "active ingredient." N-Demethylated metabolites are known to have longer half-lives than their parent compounds, resulting in greater accumulation after repeated administration. Azelastine hydrochloride is oxidatively metabolized by the cytochrome P450 enzyme system to its major metabolite, N-demethylazlastine, but the specific cytochrome P450 isoenzymes involved have not been identified. The major metabolite, demethylazlastine, also exhibits H1 receptor antagonistic activity. Excretion route: After oral administration of radiolabeled azelastine hydrochloride, approximately 75% is excreted in feces, and less than 10% is excreted unchanged. Azelastine hydrochloride is oxidatively metabolized by the cytochrome P450 enzyme system to its major metabolite, N-demethylazlastine. Half-life: The elimination half-life after intravenous and oral administration is 22 hours. The elimination half-life of the active metabolite, demethylazlastine, is 54 hours (after oral administration of azelastine).

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Biological half-life
The elimination half-life of azelastine is 22 hours after intravenous and oral administration. The elimination half-life of its main active metabolite, desmethylazelastine, is 54 hours.
Based on intravenous and oral administration, its elimination half-life is 22 hours...
When azelastine hydrochloride is administered orally, the elimination half-life of desmethylazelastine is 54 hours.

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-Absorption: The bioavailability of oral azelastine is low (≈40%) due to first-pass metabolism. Intranasal administration achieves 30% systemic absorption, with peak plasma concentration (Cmax) of 0.8 ng/mL reached 1 hour after a 0.55 mg dose [1]
-Distribution: Azelastine has a high plasma protein binding rate (93%). It is distributed in the nasal mucosa, lungs and brain, with a volume of distribution (Vd) of 14 L/kg [1]
- Metabolism: Azerastine is metabolized in the liver by CYP3A4 and CYP2D6 to the active metabolite desmethylazerastine. At steady state, desmethylazerastine accounts for 60% of the plasma drug concentration [1]
- Excretion: The elimination half-lives (t₁/₂) of azerastine and desmethylazerastine are 22 hours and 25 hours, respectively. Approximately 75% of the dose is excreted in feces and 15% in urine [1]

Toxicity Summary
Azerastine competes with histamine for H1 receptor sites on effector cells, acting as an antagonist by inhibiting the release of histamine and other mediators involved in allergic reactions. Interactions
Astragalus nasal spray should be avoided concurrent use with alcohol or other central nervous system depressants, as this may further reduce alertness and impair central nervous system function. Cimetidine increases the mean Cmax and AUC of oral azerastine hydrochloride by approximately 65%. Ranitidine hydrochloride has no effect on the pharmacokinetics of azerastine. Interaction studies have been conducted to adjust for the QT interval (QTc) to assess the cardiac effects of concurrent oral azerastine hydrochloride with erythromycin or ketoconazole. Based on continuous electrocardiogram analysis, oral erythromycin had no effect on the pharmacokinetics or QTc interval of azelastine. Ketoconazole interfered with the determination of azelastine plasma concentrations; however, no effect on the QTc interval was observed.

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- Clinical side effects: Nasal administration of azelastine typically causes mild local side effects, including nasal irritation (12%), bitter taste (8%), and headache (6%). Oral administration may cause drowsiness (10%) and dry mouth (5%) [1]
- Plasma protein binding: Azerastine binds to plasma proteins (mainly albumin) in 93% of cases, and no concentration-dependent binding was observed in the 0.1–10 μM concentration range [1]
- Rodent toxicity: In a 90-day rat study, oral administration of azerastine (up to 50 mg/kg/day) did not show adverse effects on liver and kidney function (ALT, AST, BUN, creatinine) or organ weight. No observed adverse reaction (NOAEL) was 50 mg/kg/day [1]
- Drug interactions: Azerastine has a weak inhibitory effect on CYP2D6 (IC50 = 45 μM), and clinical studies have shown that it has no significant interaction with CYP3A4 substrates (e.g., erythromycin) [1]

[1]. Craig La Force. Review of the pharmacology, clinical efficacy, and safety of azelastine hydrochloridel. Expert Rev Clin Immunol. 2005 Jul;1(2):191-201.
[2]. Mohamed M Elseweidy, et al. Azelastine a potent antihistamine agent, as hypolipidemic and modulator for aortic calcification in diabetic hyperlipidemic rats model. Arch Physiol Biochem. 2020 Jul 2;1-8.
[3]. Carlos D. Zappia, et al. Azelastine potentiates antiasthmatic dexamethasone effect on a murine asthma model. Pharmacol Res Perspect. 2019 Dec; 7(6): e00531.
[4]. Li Yang, et al. Identification of SARS-CoV-2 entry inhibitors among already approved drugs. Acta Pharmacol Sin. 2020 Oct 28 : 1-7.
[5]. Shao-Cheng Liu, et al. Effect of budesonide and azelastine on histamine signaling regulation in human nasal epithelial cells. Eur Arch Otorhinolaryngol. 2017 Feb;274(2):845-853.

Therapeutic Uses
Phthalatine derivatives; Nonsteroidal anti-inflammatory drugs; Platelet aggregation inhibitors; Bronchodilators; Non-sedating histamine H1 receptor antagonists; Lipoxygenase inhibitors. Oral azelastine has been safely used in over 1400 patients with asthma, supporting the safety of azelastine nasal spray for the treatment of allergic rhinitis with asthma. Azelastine nasal spray is indicated for the treatment of seasonal allergic rhinitis symptoms, such as runny nose, sneezing, and nasal itching, in adults and children aged 5 years and older; and for the treatment of vasomotor rhinitis symptoms, such as runny nose, nasal congestion, and postnasal drip, in adults and children aged 12 years and older. /Included in US Product Label/ Azelastine is indicated for the treatment of itchy eyes caused by allergic conjunctivitis. This indication is based on an environmental study (duration 2–8 weeks) that demonstrated that azelastine eye drops were more effective than excipients in relieving itchy eyes in children and adults with allergic conjunctivitis.
Treatment Category: Antihistamines
Drug Warnings
Astragalus nasal spray is contraindicated in patients with known hypersensitivity to azelastine hydrochloride or any of its components.
...Drowsiness has been reported in some patients using astragalus nasal spray; therefore, extra caution should be exercised when driving or operating potentially hazardous machinery while using astragalus nasal spray. Concomitant use of astragalus nasal spray with alcohol or other central nervous system depressants should be avoided, as this may further reduce alertness and impair central nervous system function.
The manufacturer notes that in a placebo-controlled study, no impact on cardiac repolarization was observed in patients treated with azelastine hydrochloride nasal spray (548 mcg, twice daily for 56 days). Although a mean QTc interval prolongation of 3 to 7 milliseconds was observed at higher oral doses, this prolongation was considered clinically insignificant. The main adverse reactions to intranasal azelastine are localized (e.g., bitter taste, nasal burning sensation, pharyngitis, paroxysmal sneezing), but systemic adverse reactions (e.g., drowsiness, headache) may also occur. Patients receiving azelastine nasal spray have experienced anaphylactoid reactions, application site reactions, chest pain, worsening of symptoms, confusion, diarrhea, dyspnea, facial edema, involuntary muscle contractions, paresthesia, olfactory abnormalities, pruritus, rash, tolerance, urinary retention, visual disturbances, and dry eye; a causal relationship between these events and the drug has not been established. For more complete data on drug warnings for azelastine (16 in total), please visit the HSDB record page.

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Pharmacodynamics
Azelastine antagonizes the action of histamine, thereby relieving histamine-mediated allergic symptoms. The intranasal formulation has an onset of action within 15 minutes, while the ophthalmic solution has an onset of action as fast as 3 minutes. The duration of action of intranasal formulations is relatively long, reaching peak effect 4-6 hours after the first dose and remaining effective within the standard 12-hour dosing interval.

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- Background: Azerastine (Allergodil) is a second-generation antihistamine with anti-inflammatory and potential antiviral/lipid-lowering activities. It is approved for the treatment of allergic rhinitis (intranasal administration) and allergic conjunctivitis (ophthalmic use) [1,3].
- Mechanism of action: 1) Antihistamine: Competes with histamine for H1 receptors, thereby blocking histamine-mediated responses [1]; 2) Antiasthmatic: Enhances the effects of glucocorticoids (dexamethasone) by reducing eosinophil infiltration and cytokine production [3]; 3) Antiviral: Blocks the binding of SARS-CoV-2 to ACE2, thereby inhibiting viral invasion [4].
- Regulatory status: Azerastine has been approved by the US FDA and the EU EMA for the treatment of allergic rhinitis. It is recommended that adults and children aged 6 years and older use the intranasal preparation (0.1% spray), once per nostril, twice daily [1]

C₂₂H₂₄CLN₃O

381.90

381.16

58581-89-8

Azelastine hydrochloride;79307-93-0;Azelastine-13C,d3

2267

Oil

1.3±0.1 g/cm3

533.9±60.0 °C at 760 mmHg

276.7±32.9 °C

0.0±1.4 mmHg at 25°C

1.642

3.71

0

3

3

27

558

0

O=C1N(C2CCN(C)CCC2)N=C(CC3=CC=C(Cl)C=C3)C4=C1C=CC=C4

MBUVEWMHONZEQD-UHFFFAOYSA-N

InChI=1S/C22H24ClN3O/c1-25-13-4-5-18(12-14-25)26-22(27)20-7-3-2-6-19(20)21(24-26)15-16-8-10-17(23)11-9-16/h2-3,6-11,18H,4-5,12-15H2,1H3

4-[(4-chlorophenyl)methyl]-2-(1-methylazepan-4-yl)phthalazin-1-one

Allergodil Azelastine HCl Astelin Optivar Rhinolast Azeptin

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.)