α2a-adrenergic receptor ( EC50 = 0.45 nM )

Brimonidine (1 mg/kg) dramatically shields RGCs from elevated IOP-induced cell death in adult rats.[4] Brimonidine (0.0001%) BMD possesses no discernible neuroprotective effects and causes the loss of roughly 37% of the retinal ganglion cell (RGC) populati in adult Sprague-Dawley rats. Brimonidine (0.001% or 0.01%) results in the survival of 76 or 90%, respectively, of the retinal ganglion cell (RGC) population, and 0.1% brimonidine completely prevents RGC death in the first seven days following ischemi in adult Sprague-Dawley rats. [5]

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Brimonidine (1 mg/kg) dramatically shields RGCs from elevated IOP-induced cell death in adult rats. In adult Sprague-Dawley rats, brimonidine (0.0001%) BMD possesses no discernible neuroprotective effects and causes the loss of roughly 37% of the retinal ganglion cell (RGC) population. RGC mortality is completely avoided in the first seven days following ischemia in adult Sprague-Dawley rats when brimonidine (0.001% or 0.01%) is used. This results in the survival of 76 or 90%, respectively, of the RGC population.

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In the control eyes injected with saline, BDNF was present in a minority of the RGCs. Two days after Brimonidine injection, the number of BDNF-positive RGCs was increased from 55% to 166%, depending on brimonidine concentrations, when compared with those in the controls. In addition, the BDNF signal intensities in individual RGCs were elevated 50% in brimonidine-injected eyes compared with control eyes. Northern blot revealed a 28% increase of BDNF expression in the brimonidine group compared with the controls (P <.003). No significant difference was observed in BDNF receptor, trk B, expression between brimonidine, or BSS control groups. Conclusions: A single dose of a low concentration of intravitreal Brimonidine is sufficient to significantly increase endogenous BDNF expression in RGCs. These results suggest that brimonidine neuroprotection may be mediated through up-regulation of BDNF in the RGCs. The BDNF should be further investigated regarding its role in the neuroprotective effects reported with brimonidine. Clinical relevance: Brimonidine may be (potentially) used clinically as a neuroprotective agent in optic neuropathy, including glaucoma, and ischemic and traumatic optic neuropathy[2].

[3H]Brimonidine (UK 14304) fully agonistically interacts with alpha 2-adrenergic receptors. In the human brain, [3H]Brimonidine (UK 14304) labels a minimum of two distinct binding sites that share the features of an alpha 2-adrenergic binding site. Although to varying degrees at each site, GTP inhibits agonist binding at both of these locations.

Brimonidine intravitreal injection[2]
Animals were anesthetized with intraperitoneal injections of pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, Ill) (75 mg/kg). A 0.2% brimonidine (3.4mM) ophthalmic solution was serially diluted with balanced salt solution (BSS; Alcon Labs Inc, Forth Worth, Tex) from 100-fold to 4000-fold (100-, 500-, 1000-, 2000-, 4000-fold, respectively) to obtain final concentrations from 34µM to 0.85µM (34µM, 6.8µM, 3.4µM, 1.7µM, and 0.85µM, respectively). A single dose of 5 µL of diluted brimonidine solutions was injected into vitreous under a dissecting microscope, through a temporal postlimbus spot using Hamilton microinjector. A 30-gauge needle was first used to make a punch incision 0.5 mm posterior to the temporal limbus, and a Hamilton needle was then inserted through the incision approximately 1.5 mm deep and angled toward the optic nerve until the tip of needle was seen in the center of the vitreous. When the lens was occasionally involved, a hard resistance could be felt, and the eye was discarded and not used for the study. Since BSS was used to dilute brimonidine to obtain serial concentrations, 5 µL of BSS was used as a vehicle control and injected into the fellow eyes. Animals were humanely killed 48 hours following injection. At least 3 animals were used for each concentration of brimonidine. Two rats were given a brimonidine injection in only one eye, and the fellow eyes were not given any injection and were processed for in situ hybridization. Five rats with a 1.7µM brimonidine injection in one eye and a BSS injection in the fellow eye, were humanely killed 1 week after injection, and eyes were processed for Northern blot analysis.

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Tissue preparation[2]
Animals were humanely killed with overdose of pentobarbital. Eyes were enucleated, an incision was made in the cornea, and eyes were fixed immediately in 4% formaldehyde in 0.1M phosphate buffer (pH, 7.4). After 15 minutes in the fixative, lenses were removed, and eyes were cut along the corneal optic nerve axis into halves. Tissues were further fixed and cryoprotected overnight in 4% formaldehyde, 0.5% glutaraldehyde, and 20% sucrose in 0.1M phosphate buffer (pH, 7.4). Tissues were embedded in Tissue-Tek OCT compound and cryosectioned at a thickness of 10 µm at −21°C. The Brimonidine-injected and BSS-injected eye tissue sections were mounted on the same slide and processed identically so that sections could be directly compared, with as little processing variability as possible.

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Brain-derived neurotrophic factor receptor, trkB, and mRNA expression were also examined in Brimonidine- and BSS-injected eyes. Trk B cDNA clone was in pGEM-3Z with an insertion of 432 base pairs (bp), encoding a portion of the extracellular domain of mouse trk B receptor. This clone was used to generate pan probe to detect all forms of trk B receptor.28 Restriction enzymes Hind III and Bam HI were used to linearize the plasmid for the generation of antisense and sense probes, respectively.30 S-labeled antisense and sense trk B riboprobes were transcribed using the Riboprobe Gemini System. In situ hybridization was then performed as described previously.

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Image quantification[2]
To determine and compare the numbers of BDNF-positive ganglion cells in the retinas, cells were quantified using computer-enhanced video densitometry(Southern Micro Instruments, Atlanta, Ga). Brain-derived neurotrophic factor mRNA-positive cells were defined as those cells over which silver grains exceed 5 times the background value. Total cell number in the ganglion cell layer was also counted and used as a denominator. Thus, the percentage of BDNF-positive ganglion cells was determined. For each concentration of Brimonidine, at least 3 animals were used and 3 tissue sections were counted for each animal eye.

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Animal eyes injected with 1.7µM (2000-fold dilution) Brimonidine were used to determine and compare BDNF signal levels in individual ganglion cells between groups, with and without brimonidine injection. Twenty to 30 BDNF-positive cells were randomly selected from each tissue section, and 3 sections were used from each animal. Silver grain densities over individual BDNF-positive cells were determined using computerized densitometry as described previously. Three animals were included for the brimonidine or BSS group. A t test was used for statistical analysis between the 2 groups.

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Northern blot[2]
Two groups of rats were used for Northern blot analysis at 48 hours after intravitreal injections (17 rats), and at 1 week after injections (5 rats). Brimonidine (1.7µM) was injected intravitreally in one eye of each animal, and BSS in the fellow eyes. Animals were then humanely killed, and retinas were dissected out and pooled in each group. Total retinal RNAs were isolated as described previously.30 The antisense BDNF RNA probe was synthesized as described previously using [phosphorus-32{32 P} cytidine 5′-triphosphate. Northern blot analysis was performed using standard methods: the total RNA of 30 µg was separated on 1% agarose formaldehyde–denaturing gel. For the 1-week group, 10µg of RNA was used. The RNA was blotted to 0.2 µm of neutral nylon membranes and hybridized to a 32P-labeled BDNF probe (3 × 106cpm/mL). The membrane was then washed in graded SSC, dried, and exposed to a PhosphorImager plate. Relative abundance of mRNA was quantified by reading the plate. Both bands of BDNF mRNA expression were used to perform the densitometry. For accurate quantification, the same blot was stripped off and hybridized to 32P-labeled β-actin probe. The ratio of BDNF to actin densities was then used for comparison between the Brimonidine and BSS control groups. Northern analysis was repeated 5 times for the 48-hour group, and 3 times for the 1-week group.

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1 mg/kg

Rats

Absorption, Distribution and Excretion
Brimonidine readily penetrates the cornea following ocular administration to reach pharmacologically active concentrations in the aqueous humor and ciliary body, the putative sites of its IOP-lowering activity. Following ocular administration of 0.2% brimonidine solution, the peak plasma concentrations were achieved within 1 to 4 hours. In a clinical study of adult subjects with facial erythema of rosacea, brimonidine was cutaneously applied on facial skin in a repeated manner. While there was no drug accumulation in plasma, the highest peak plasma concentrations (Cmax) and AUC were 46 ± 62 pg/mL and 417 ± 264 pgxhr/mL, respectively.
Brimonidine and its metabolites are predominantly eliminated via urinary excretion, with 74% of the total dose being found in the urine.
The volume of distribution of brimonidine has not been established. In animal studies, brimonidine was shown to cross the placenta and enter into the fetal circulation to a limited extent. As its lipophilicity is relatively low, brimonidine is not reported to easily cross the blood-brain barrier.
The apparent clearance has not been studied. However, the systemic clearance of brimonidine is reported to be rapid. Approximately 87% of the total radioactive dose of brimonidine was shown to be eliminated within 120 hours following oral administration.

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Metabolism / Metabolites
Brimonidine is reported to be metabolized in the cornea. Brominidine that reaches the systemic circulation upon topical administration undergoes extensive hepatic metabolism mediated by hepatic aldehyde oxidases.
Metabolized primarily by the liver.
Route of Elimination: Urinary excretion is the major route of elimination of the drug and its metabolites.
Half Life: 2 hours [ophthalmic solution]

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Biological Half-Life
Following ocular administration of 0.2% brimonidine solution, the systemic half-life was approximately 3 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited information indicates that maternal use of brimonidine 0.2% ophthalmic drops do not adversely affect their nursing infants. However, some authors warn of possible CNS depression, apnea, lethargy, bradycardia with brimonidine and recommend against its use during breastfeeding. To substantially diminish the amount of drug that reaches the breastmilk after using eye drops, place pressure over the tear duct by the corner of the eye for 1 minute or more, then remove the excess solution with an absorbent tissue.
Topical brimonidine gel used to treat rosacea has not been studied during breastfeeding. It is unlikely that the topical gel would affect the breastfed infant, but the manufacturer states that it should not be used during nursing. Until more data are available, an alternative topical agent might be preferred
◉ Effects in Breastfed Infants
A woman used brimonidine 0.2%, timolol gel-forming solution 0.5%, dipivefrin 0.2%, and dorzolamide 0.5% drops for glaucoma while nursing a newborn. The frequency of medication use and extent of nursing were not stated. All medications were given immediately after nursing with punctal occlusion of the tear duct. The infant's vital signs were closely monitored with no signs of bradycardia or apnea.
A woman was using ophthalmic drops containing 0.5% timolol and 0.2% brimonidine twice daily in the right eye for 6 months. During this time, she breastfed her infant (extent not stated) apparently without harm to her infant.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
The protein binding of brimonidine has not been studied.

[1]. Invest Ophthalmol Vis Sci . 2001 Nov;42(12):2849-55.

[2]. Arch Ophthalmol . 2002 Jun;120(6):797-803.

[3]. J Glaucoma . 1997 Aug;6(4):250-8.

[4]. Brain Res . 2001 Sep 21;913(2):133-9.

[5]. Exp Eye Res . 2002 Feb;74(2):181-9.

Brimonidine Tartrate is the tartrate salt form of brimonidine, an imidazole derivative and a selective alpha-2 adrenergic receptor agonist. Upon ocular administration, brimonidine tartrate acts on the blood vessels causing them to constrict which leads to a decrease in the production of aqueous humor. Brimonidine tartrate also enhances the outflow of aqueous humor. This drug is used in the treatment of glaucoma to reduce intraocular pressure.
A quinoxaline derivative and ADRENERGIC ALHPA-2 RECEPTOR AGONIST that is used to manage INTRAOCULAR PRESSURE associated with OPEN-ANGLE GLAUCOMA and OCULAR HYPERTENSION.
See also: Brimonidine (has active moiety); Brimonidine Tartrate; Brinzolamide (component of); Brimonidine Tartrate; timolol maleate (component of) ... View More ...

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Drug Indication
Mirvaso is indicated for the symptomatic treatment of facial erythema of rosacea in adult patients.
Treatment of rosacea. Brimonidine is a quinoxaline derivative, a secondary amine and a member of imidazoles. It has a role as an adrenergic agonist, an antihypertensive agent and an alpha-adrenergic agonist.
Brimonidine is an alpha-adrenergic agonist and 2-imidazoline derivative that was first introduced in 1996. It is considered to be a third generation alpha-2 aadrenergic receptor agonist, since it displays preferential binding at alpha-2 adrenoceptors over alpha-1 receptors. Brimonidine displays a higher selectivity toward the alpha-2 adrenergic receptors than [clonidine] or [apraclonidine], which are also alpha-2 adrenergic agonists. Alpha-2 adrenergic agonists are members of the ocular hypotensive agent drug class that are used in the chronic treatment of glaucoma. Early treatment and management of glaucoma, which predominantly involves the lowering of intraocular pressure, is critical since glaucoma is considered to be a common cause of blindness worldwide. Ophthalmically, brimonidine is used to lower intraocular pressure by reducing aqueous humor production and increasing uveoscleral outflow. Because it is oxidately stable, brimonidine is associated with fewer reports of ocular allergic reactions compared to other alpha-2 adrenergic agonists. The ophthalmic solution of brimonidine was first approved by the FDA in 1996 as Alphagan and brimonidine is the only selective alpha-adrenergic receptor agonist approved for chronic treatment in glaucoma. Brimonidine is also found in ophthalmic solutions in combination with [brinzolamide] under the market name Simbrinza for the reduction in intraocular pressure. Unlike nonselective beta-blockers used in ocular hypertension, brimonidine is not associated with significantly adverse cardiopulmonary side effects. Thus brimonidine is an effective and safe alternative to beta-blockers, in patients with, or at high risk for, cardiopulmonary disease. The topical form of brimonidine was approved by the FDA in August 2013 for the symptomatic treatment of persistent facial erythema of rosacea in adults. It is marketed under the brand name Mirvaso. Brimonidine is the first topical treatment approved for facial erythema of rosacea.
Brimonidine is an alpha-Adrenergic Agonist. The mechanism of action of brimonidine is as an Adrenergic alpha-Agonist.
Brimonidine is an imidazole derivative and a selective alpha-2 adrenergic receptor agonist. Upon ocular administration, brimonidine acts on the blood vessels causing them to constrict which leads to a decrease in the production of aqueous humor. Brimonidine also enhances the uveoscleral outflow of aqueous humor. This reduces intraocular pressure.
Brimonidine is only found in individuals that have used or taken this drug. It is a drug used to treat glaucoma. It acts via decreasing aqueous humor synthesis. [Wikipedia] A topical gel formulation, marketed under the name Mirvaso, was FDA approved on August 2013 for the treatment of rosacea. Brimonidine is an alpha adrenergic receptor agonist (primarily alpha-2). It has a peak ocular hypotensive effect occurring at two hours post-dosing. Fluorophotometric studies in animals and humans suggest that Brimonidine has a dual mechanism of action by reducing aqueous humor production and increasing uveoscleral outflow.
A quinoxaline derivative and ADRENERGIC ALHPA-2 RECEPTOR AGONIST that is used to manage INTRAOCULAR PRESSURE associated with OPEN-ANGLE GLAUCOMA and OCULAR HYPERTENSION.
See also: Brimonidine Tartrate (has salt form).

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Drug Indication
**Opthalmic** Indicated for lowering intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension as monotherapy or combination product with [brinzolamide]. **Topical** Indicated for the treatment of persistent (non-transient) facial erythema of rosacea in adults 18 years of age or older.
FDA Label
Treatment of conjunctival hyperaemia

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Mechanism of Action
In the eye, alpha-1 adrenoceptors play a role in vasoconstriction, mydriasis, eyelid retraction, and elevation of intraocular pressure (IOP) whereas alpha-2 adrenoceptors are responsible for IOP reduction via a complex Gi-coupled signaling cascade pathway. Activation of alpha-2 receptors leads to inhibition of adenylyl cyclase and reduction of cyclic AMP levels. As a result, there is a decrease in norpinephrine (NE) release at the synaptic junction, NE-induced stimulation of beta-2 adrenoceptors, and production of aqueous humor by the ciliary epithelium. An elevated IOP is the most significant risk factor for developing glaucomatous optic neuropathy, which is associated with progressive visual field loss and functional disability if left untreated. Regardless of the etiology of the disease, the aim of current therapies for glaucoma is to reduce IOP, as reduction of IOP significantly reduces the risk of progression of vision loss even when IOP is already within the normal range. When administered ophthalmically, brimonidine is rapidly absorbed into the eye, acts as an agonist at ocular alpha-2 adrenoceptors and lowers IOP via a dual mechanism of action. It is proposed that initial dosing of the drug causes a reduction in aqueous humour production and chronic dosing leads to an increase in uveoscleral outflow. Brimonidine does not affect episcleral venous pressure. By reducing IOP, brimonidine aims to reduce the likelihood of glaucomatous visual field loss in ocular hypertension, and slow the progression of visual field defect in established open-angle glaucoma. When applied topically on skin, brimonidine reduces erythema through direct vasocontriction of small arteries and veins. As brimonidine mediates a potent peripheral vasoconstrictive activity by selectively working on the alpha-2 adrenoceptors, the use of brimonidine is thought to be efficacious for the treatment of facial erythema of rosacea, which is thought to arise from vasomotor instability and abnormal vasodilation of the superficial cutaneous vasculature of the face.

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Pharmacodynamics
Brimonidine is a highly selective alpha-2 adrenergic receptor agonist that is 1000-fold more selective for the alpha2-adrenergic receptor than the alpha1-adrenergic receptor. This characteristic gives the drug some therapeutic advantages, since it reduces the risk of systemic side effects, such as systemic hypotension, bradycardia, and sedation. In addition, there is a reduction in the risk for developing alpha-1 mediated ocular unwanted effects, such as conjunctival blanching, mydriasis, and eyelid retraction. However, despite high alpha-2 receptor specificity, brimonidine may still produce alpha-1 adrenoceptor-mediated ocular effects, such as conjunctival vasoconstriction. Brimonidine has a peak ocular hypotensive effect occurring at two hours post-dosing. In a randomized, double-blind clinical study, ocular administration of 0.2% brimonidine in healthy volunteers resulted in a 23% reduction of mean intraocular pressure from baseline at 3 hours following administration. In comparative studies consisting of patients with open-angle glaucoma or ocular hypertension, the ocular hypotensive effect of brimonidine was maintained during treatment periods of up to 1 year. Brimonidine mediates vasoconstrictive effects and it was shown to exhibit anti-inflammatory properties in _ex vivo_ human skin model and _in vivo_ inflammation models. In a clinial trials consisting of adults with moderate to severe facial erythema of rosacea, brimonidine was shown to improve the extent of redness at 3 hours after application, compared to placebo. It was shown to be a potent vasoconstrictor of human subcutaneous vessels with a diameter of less than 200 µm. In _in vivo_ mouse inflammation models, brimonidine displayed anti-inflammatory properties by inhibiting edema. In a randomized, double-blind study, brimonidine reduced erythema for the 12 hours of the study in a dose-dependent manner. When adminsitered systemically, brimonidine was shown to cause cardiovascular effects by decreasing blood pressure, decreasing heart and respiratory rate, and prolonging the PR interval in the electrocardiogram. This is due to the targeting of adrenoceptors by the drug. Although the clinical significance has not been established, there is evidence that brimonidine exhibits neuroprotective activity in experimental models of cerebral ischemia and optic nerve injury. _In vitro_ studies show that brimonidine mediated protective effects on neuronal cells from kainate acid insult and on cultured retinal ganglion cells from glutamate-induced cytotoxicity, which is a possible mediator of secondary neuronal degeneration in human glaucoma. Neuroprotective actions of brimonidine were also demonstrated in rat models of acute retinal ischemia and chronic IOP elevation. It has been proposed that brimonidine may exert neuroprotective effects on the retina and optic nerve by enhancing intrinsic retinal ganglion cell survival mechanisms and/or induction of neuronal survival factors, such as bFGF. However, further investigations are needed to conclude on these possible therapeutic benefits of the drug.

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The present study examined the influence of alpha-2 adrenoceptor ligands on circling behavior in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. The alpha-2 adrenoceptor agonists, clonidine and UK 14304, inhibited both the ipsilateral rotation induced by the indirect dopaminergic agonist, methylphenidate, and the contralateral circling induced by the direct dopaminergic agonist, apomorphine. In contrast, the alpha-2 adrenoceptor antagonists, idazoxan and (+/-)-efaroxan, enhanced the circling induced by either methylphenidate or apomorphine. The facilitating activity of efaroxan was stereoselective because the (+)-enantiomer mimicked the effect of (+/-)-efaroxan, whereas the (-)-enantiomer was essentially inactive, thus indicating a mediation by alpha-2 adrenoceptors. Upon administration alone, the above-mentioned compounds did not modify spontaneous circling behavior, except for UK 14304, which decreased, and (+)-efaroxan, which slightly increased, the ipsilateral rotation. We conclude that activation and antagonism of alpha-2 adrenoceptors inhibit and enhance, respectively, the circling behavior evoked by both direct and indirect dopaminergic agonists. Although a modulation of dopamine release may be involved in some of these drug effects, the effects on apomorphine-induced circling indicate an influence of alpha-2 adrenoceptor compounds on nigrostriatal neurotransmission at sites downstream from the dopaminergic neurons themselves. These findings support the notion of a potential benefit of alpha-2 adrenoceptor antagonists in the treatment of Parkinson's disease.[3]

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Objectives: Brimonidine tartrate ophth, an alpha(2)-adrenergic agonist, is widely used as an antiglaucoma agent for lowering intraocular pressure. Recent studies suggest that brimonidine may be neuroprotective for retinal ganglion cells (RGCs) following optic nerve crush injury. Brain-derived neurotrophic factor (BDNF), a potent neuroprotective factor present in the RGCs, promotes RGC survival in culture and following optic nerve injury. We tested the hypothesis that a possible mechanism of brimonidine neuroprotection is through up-regulation of endogenous BDNF expression in the RGCs. Methods: A single dosage of brimonidine tartrate ophth solution (0.85-34 microM) was injected intravitreally into Sprague-Dawley rat eyes. The fellow eyes of each animal were injected with balanced salt solution (BSS) and used as control eyes. To determine BDNF messenger RNA expression, animal eyes were enucleated and processed for in situ hybridization, or retinas were isolated and processed for Northern blot analysis using rat BDNF radiolabeled riboprobes.[4]

C15H16BRN5O6

442.22

441.028

C, 40.74; H, 3.65; Br, 18.07; N, 15.84; O, 21.71

70359-46-5

Brimonidine; 59803-98-4; Brimonidine-d4 D-tartrate; 1316758-27-6; Brimonidine-d4; 1184971-51-4

54405

White to off-white solid powder

432.6ºC at 760 mmHg

207-208ºC (dec.)

215.4ºC

6

9

5

27

442

2

BrC1C2C(C([H])=C([H])C=1N([H])C1=NC([H])([H])C([H])([H])N1[H])=NC([H])=C([H])N=2.O([H])[C@@]([H])(C(=O)O[H])[C@]([H])(C(=O)O[H])O[H]

QZHBYNSSDLTCRG-LREBCSMRSA-N

InChI=1S/C11H10BrN5.C4H6O6/c12-9-7(17-11-15-5-6-16-11)1-2-8-10(9)14-4-3-13-8;5-1(3(7)8)2(6)4(9)10/h1-4H,5-6H2,(H2,15,16,17);1-2,5-6H,(H,7,8)(H,9,10)/t;1-,2-/m.1/s1

5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine;(2R,3R)-2,3-dihydroxybutanedioic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: 100 mg/mL (226.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)