Histamine H₁ Receptor

Astemizole is an H1-histamine receptor antagonist with a long duration of action permitting once daily administration. Its efficacy in seasonal and perennial allergic rhinitis has been convincingly demonstrated, and several comparative studies suggest that astemizole is at least as effective as some other H1-histamine receptor antagonists. A few smaller studies have shown beneficial effects on the symptoms of allergic conjunctivitis and chronic urticaria (but not atopic dermatitis). While astemizole appears to share with other H1-histamine receptor antagonists a tendency to increase appetite and cause weight gain after prolonged use, it offers the important advantage of an absence of significant central nervous system depression or anticholinergic effects with usual doses. Thus, astemizole offers a worthwhile improvement in side effect profile over 'traditional' H1-histamine receptor antagonists, especially in patients bothered by the sedative effects of these drugs[2].

Astemizole (po, 10 and 30 mg/kg) and (iv, 1 and 3 mg/kg) had no effect on respiratory rate, heart rate, or blood glucose, even at the high doses of 30 mg/kg and 3 mg/kg. It also had no effect on breast cancer and exercise capacity in marmosets of average weight. However, astemizole at 30 mg/kg (po) and 1 mg/kg (iv) can prolong the QT interval and induce premature ventricular contractions [3]. Astemizole (po, 3) and 30 mg/kg) at a dose of 3 mg/kg, preperfume control values (C) for ventricular rate, QT interval, and QTcF were 31 beats/min, 319 ms, and 256, whereas these were 31 beats/min, 331 ms, and 270 respectively at a dose of 30 mg/kg in mice. In addition, astemizole at a dose of 30 mg/kg (po) may cause torsade de pointes ventricular tachycardia by inhibiting hERG K+ channels [4].

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In conscious, freely moving common marmosets, astemizole administered orally at 30 mg/kg or intravenously at 3 mg/kg caused severe prolongation of the QT interval (approximately 80% increase compared to pre-dose values) and induced ventricular arrhythmias. In one out of three animals at each of these doses, polymorphic ventricular tachycardia (torsades de pointes) was observed, occurring 4-5 hours post oral administration and 1-2.5 hours post intravenous administration [3].
At lower doses (10 mg/kg, po and 1 mg/kg, iv), astemizole prolonged the QT interval (approximately 50% increase at 1 mg/kg iv) but did not induce torsades de pointes in the tested animals (n=3 per dose) [3].
Astemizole, at the tested doses (up to 30 mg/kg po and 3 mg/kg iv), had no significant effects on respiratory rate, heart rate, systolic/diastolic blood pressure, body temperature, or locomotor activity in the marmosets [3].

Binding characteristics of astemizole were studied in vitro in various receptor binding models and in vivo by determining the occupancy of histamine H1 receptors in guinea pig lung and cerebellum. In vitro, astemizole was found to have a high affinity for histamine H1 receptors, but great difficulties were encountered in proving this because of its high affinity for nonspecific binding sites. Since the equilibrium conditions were not reached in vitro, the real affinity of astemizole remains unclear and its receptor profile must be interpreted with caution. Nevertheless, the drug is certainly much more potent on histamine H1 receptors than on serotonin S2 and adrenergic alpha 1-receptors. Moreover, it was found to be devoid of antimuscarinic and antidopaminergic properties. The most striking property of this drug is its extremely slow dissociation rate from H1 receptors when assayed in vitro using [3H]-pyrilamine. Ex vivo experiments were performed in guinea pigs; astemizole was given orally to the animals, and the occupancy of H1 receptors in the lung and the cerebellum was determined in vitro by the [3H]-pyrilamine binding assay. Astemizole was found to occupy H1 receptors in lung at very low doses. Here again the most striking receptor binding property was its very long duration. The occupancy of H1 receptors in lung began to decline only 4-6 days after administration of the drug. However, there was a marked difference between the occupancy of peripheral and central receptors; indeed, in contrast to pyrilamine, astemizole at pharmacological doses did not reach the H1 receptors in the cerebellum, presumably because the drug does not readily cross the blood-brain barrier[1].

The purpose of this study was to evaluate a telemetry system for examining the cardiovascular system in the conscious common marmoset. Parameters obtained were blood pressure, heart rate, respiratory rate, ECG, body temperature and locomotor activity, and these were continuously recorded on a data recorder via the telemetry system and then processed by a computerized system. Diurnal rhythms of blood pressure, heart rate, body temperature and locomotor activity were observed in this system. We studied the effects of astemizole (antihistamine) and nicardipine (Ca2+ channel blocker) on cardiovascular parameters. Astemizole at 30 mg/kg (p.o.) and at 1 to 3 mg/kg (i.v.), prolonged QT interval and induced ventricular extrasystole. Torsades de pointes occurred in one of three cases at 3 mg/kg (i.v.) and 30 mg/kg (p.o.), while it did not affect the blood pressure, respiratory rate and body temperature. Nicardipine at 30 mg/kg (p.o.) caused sustained hypotension and tachycardia. These results demonstrate the usefulness of the telemetry system using the common marmoset for evaluating the cardiovascular effects of drugs under physiological conditions.[3]

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Since astemizole in an oral dose of 50 mg/kg/day was recently reported to exert anti-cancer effect in mice, we evaluated its proarrhythmic potential using the atrioventricular block dogs in order to clarify its cardiac safety profile. An oral dose of 3 mg/kg prolonged the QT interval without affecting the QTc (n = 4), whereas that of 30 mg/kg increased the short-term variability of repolarization and induced premature ventricular contractions in each animal, resulting in the onset of torsade de pointes in 1 animal (n = 4). Thus, proarrhythmic dose of astemizole would be lower than anti-cancer one, limiting its re-profiling as an anti-cancer drug.[4]

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Animal Model: Male common marmosets (Callithrix jacchus), weighing 300-500g, aged 1-2 years, were used [3].
Surgical Preparation: Under ketamine anesthesia (10 mg/kg, i.m.), a telemetry transmitter was implanted into the peritoneal cavity. A catheter for blood pressure measurement was inserted into the abdominal aorta. Two unipolar ECG electrodes were implanted subcutaneously at specific chest locations to simulate a CM-V5 lead configuration. Animals were allowed 2-3 weeks to recover post-surgery [3].
Drug Formulation & Administration: For oral administration, astemizole was suspended in a 0.5% carboxymethylcellulose (CMC) solution to achieve a concentration of 6 mg/ml. For intravenous administration, astemizole was first dissolved in ethanol containing 5% 0.1N hydrochloric acid (10 mg/ml stock), then diluted with physiological saline to a final concentration of 1.5 mg/ml prior to injection [3].
Dosing & Groups: Animals received astemizole at oral doses of 10 and 30 mg/kg (n=3 per dose, crossover design) and intravenous doses of 1 and 3 mg/kg (n=3 per dose, crossover design) [3].
Parameter Monitoring: Using a telemetry system, systolic/diastolic blood pressure, heart rate, respiratory rate (derived from BP waveform), body temperature, locomotor activity (via signal strength change), and a continuous ECG signal were recorded. Data were collected for 30 seconds every 5 minutes, from 2 hours before to 24 hours after drug administration. ECG parameters (PR, QRS, QT, QTc) were analyzed offline [3].

Absorption, Distribution and Excretion
Rapidly absorbed from the gastrointestinal tract. Protein binding: 96% Time to peak concentration: 1 hour. It is unclear whether astemizole is distributed into human milk. Astemizole is distributed into canine milk. Oral bioavailability of astemizole is reduced by 60% when taken with food. Metabolism/Metabolites Almost entirely metabolized in the liver and primarily excreted in feces. Oral astemizole is well absorbed but undergoes extensive first-pass metabolism to form O-desmizole. Desmizole is generated in the microsomal system of the human small intestine and liver, suggesting that cytochrome P450 (P450) may be involved in the first-pass metabolism of astemizole. However, the P450 involved in the O-demethylation of astemizole has not been identified, and 12 known drug metabolites have been excluded from this process. During the P450 identification study, it was found that the activity of astemizole O-demethylation in the rabbit small intestine was higher than that in the liver (approximately 3-fold). These data suggest that CYP2J may be involved, as this subfamily of P450 is highly expressed in the rabbit small intestine. Three metabolites of astemizole, namely desmethylastemizole (DES-AST), 6-hydroxyastemizole (6OH-AST), and norestemizole (NOR-AST), were detected in the human liver microsomal system in a ratio of 7.4:2.8:1. Recombinant P450 enzyme and antibody experiments showed that CYP3A4 plays a minimal role in the major metabolic pathway of astemizole (i.e., the formation of DES-AST), although CYP3A4 may mediate the minor metabolic pathways of 6OH-AST and NOR-AST. Recombinant CYP2D6 catalyzes the formation of 6OH-AST and DES-AST. However, studies of human liver microsomes have shown that P450 alone plays a crucial role in the generation of DES-AST. Second-generation relatively non-sedating histamine H1 receptor antagonists (H1-RAs) are widely used globally to treat the symptoms of allergic rhinoconjunctivitis and chronic urticaria. Although pharmacokinetic and pharmacodynamic information on these drugs is incomplete, it is currently sufficient to optimize treatment regimens. This article summarizes the published pharmacokinetic and pharmacodynamic information on these H1-RAs and points out areas where more data is needed. Serum concentrations of most second-generation H1-RAs are relatively low and are typically determined using radioimmunoassay. Peak plasma concentrations occur within 2 to 3 hours after oral administration. Due to the lack of intravenous formulations, their bioavailability has not been adequately studied. Most H1 receptor antagonists (H1-RAs) are metabolized in the hepatic cytochrome P450 system: terfenadine, astemizole, loratadine, azelastine, and ebastine have one or more active metabolites, which are present in serum at higher concentrations than their parent compounds and can therefore be determined by high-performance liquid chromatography (HPLC). Cetirizine, the active metabolite of the first-generation H1 receptor antagonist hydroxyzine, is poorly metabolized in vivo and is primarily excreted via the kidneys. Levocabastine is also primarily eliminated through excretion. The serum elimination half-lives of different H1-RAs vary considerably; the serum elimination half-lives of terfenadine, astemizole, loratadine, cetirizine, azelastine, and ebastine, as well as the active metabolites of terfenadine, loratadine, and ebastine, are all 24 hours or less. The serum elimination half-life of the active metabolite of azelastine (desmethylazelastine) is approximately 2 days, while that of astemizole (desmethylazelastine) is 9.5 days. Wide tissue distribution is observed from the few published studies calculating the apparent volume of distribution of second-generation H1 receptor antagonists. The half-lives of H1 receptor antagonists in children are generally shorter than in adults; currently, there is no published information on the pharmacokinetics of astemizole, loratadine, azelastine, or ebastine in children. In some elderly individuals, the half-lives of terfenadine, loratadine, and cetirizine may be longer than in young, healthy adults. Pharmacokinetic data on second-generation H1 receptor antagonists in patients with hepatic impairment are scarce. Cetirizine has a prolonged half-life in patients with renal impairment. Currently, pharmacokinetic information on H1 receptor antagonists in neonates, pregnant women, or lactating women is lacking. This study investigated the anti-allergic effects of astemizole and its metabolites in rats and guinea pigs. All tested astemizole metabolites showed greater activity than the parent compound in inhibiting histamine-induced ileal and bronchoconstriction in guinea pigs. Desmethylastemizole's ability to inhibit mepiracetam binding in the cerebellum of guinea pigs was comparable to that of astemizole. In both heterologous passive cutaneous anaphylaxis (PCA) and homologous PCA models, the inhibitory effects of these metabolites were nearly identical to those of astemizole. No H2 receptor antagonistic activity was observed with either astemizole or desmethylastemizole. Known metabolites of astemizole include 6-desmethylastemizole, desmethylastemizole, 2-(4-hydroxyphenyl)acetaldehyde, and 6-hydroxyastemizole. It is metabolized by CYP3A4. [Wikipedia]. It is almost entirely metabolized in the liver and primarily excreted in feces.
Half-life: 1 day

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Biological half-life
1 day
Elimination: Astemizole (and its hydroxylated metabolites) – multiple administrations, biphasic, with an initial half-life of 7 to 9 days (during which plasma concentration decreases by 75%) and a terminal half-life of approximately 19 days.
Multiple administrations: 7 to 9 days (initial); 19 days (terminal).
The half-life of the active metabolite of astemizole (desmethylastemizole) is 9.5 days.

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The literature cites pharmacokinetic data from previous studies: In conscious dogs, oral administration of 3 mg/kg and 30 mg/kg of astemizole achieved peak plasma concentrations (Cmax) of 13 ng/mL (28.3 nmol/L) and 178 ng/mL (388 nmol/L), respectively. In healthy young adults, oral administration of 30 mg/person achieved a Cmax of 1.9 ng/mL (4.14 nmol/L). The protein binding rate of astemizole was 96.7%. Based on this, the estimated peak plasma free drug concentration (Cmax) of dogs in this study after receiving doses of 3 mg/kg and 30 mg/kg were 0.93 nmol/L and 12.8 nmol/L, respectively [4].

Toxicity Summary
Astemizole competitively binds to H1 receptors in the gastrointestinal tract, uterus, large blood vessels, and bronchial smooth muscle with histamine. This reversible binding of astemizole to H1 receptors inhibits histamine-induced skin edema, erythema, and pruritus. Because the drug does not readily cross the blood-brain barrier and preferentially binds to peripheral rather than intrabrain H1 receptors, its inhibitory effect on the central nervous system is minimal. Astemizole may also act on H3 receptors, producing adverse reactions.

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Protein Binding
96.7% Toxicity Data
LD50: 2052 mg/kg (mice) (A308) Interactions
This study evaluated the effect of a standard dose of the macrolide antibiotic erythromycin on the pharmacokinetics of a single dose of the H1 receptor antagonist astemizole. The study participants were 18 healthy young adults (9 men and 9 women). The study employed a two-way crossover design, with participants randomly assigned to either group to receive either erythromycin (two 250 mg tablets) or a placebo (two tablets) daily for 10 consecutive days. On the morning of the fourth dose of erythromycin or placebo, each participant orally administered a single 30 mg dose (three 10 mg tablets) of astemizole. Blood samples were collected for 14 consecutive days following astemizole administration to determine serum concentrations of astemizole and its major metabolite N-desmethylastemizole, thereby characterizing the in vivo distribution kinetics of astemizole and its major metabolite N-desmethylastemizole. Furthermore, electrocardiogram (ECG) rhythm recordings were performed 24 hours before astemizole administration, 12 hours after administration, and after the last dose (erythromycin or placebo) in both study phases, and the corrected QTc interval was estimated based on the recordings. During each treatment period, pharmacokinetic parameters of astemizole and N-desmethylastemizole, including Cmax, tmax, AUC0-∞, oral clearance (CLoral), half-life, and volume of distribution (V), were measured. Analysis of variance (ANOVA) comparing data from the erythromycin treatment period and the placebo treatment period revealed no significant differences in any of the parameters for N-desmethylastemizole. On the other hand, during erythromycin treatment, the oral clearance (CLoral) of astemizole decreased by 34%, the volume of distribution increased by 24%, and the half-life was prolonged by 84%. Overall, all QT intervals appeared to be unaffected by erythromycin treatment. Since the in vivo distribution kinetics of N-desmethylastemizole are not affected by erythromycin, the changes in astemizole pharmacokinetics cannot be attributed to its N-demethylation effect. Therefore, it is difficult to determine the clinical significance of the changes in astemizole pharmacokinetics. Since there was no significant difference in the mean QTc interval and erythromycin treatment had no effect on the pharmacokinetics of N-desmethylastemizole, standard doses of erythromycin are unlikely to increase the risk of torsades de pointes or related ventricular arrhythmias in patients taking astemizole. To assess drug interactions, we investigated the effects of chemical inhibitors on the metabolism of the antihistamine astemizole. Using rabbit small intestine and liver microsomes as animal models to simulate human first-pass metabolism, we screened for chemical inhibitors of astemizole O-demethylation. In the rabbit small intestine, ebastine, arachidonic acid, α-naphthylflavonoids, ketoconazole, tranylcypromine, troglitazone, and terfenadine significantly inhibited astemizole O-demethylation. In humans, these inhibitors also reduced astemizole O-demethylation in the small intestine and liver microsomes. However, almost all of these chemicals showed significantly higher inhibition rates in the small intestine than in the liver. This suggests that the role of cytochrome P450 may differ in different tissues. All chemicals inhibited O-demethylation of astemizole in recombinant CYP2J2 microsomes. The results indicate that CYP2J2 is involved in O-demethylation of astemizole in the human small intestine and liver; however, its effect in the liver is less pronounced than in the small intestine. CYP2J2 inhibitors may have a greater effect on the small intestine than on the liver during first-pass metabolism. Since the inhibitory profiles of astemizole O-demethylation differ between the liver and small intestine, it can be speculated that another p450 enzyme may be involved in astemizole O-demethylation in the human liver. In the rabbit microsomal system, the same metabolites as in humans were qualitatively detected, and the inhibitory profiles of the chemicals in the microsomes were similar to those in humans. Concomitant use of astemizole with clarithromycin, erythromycin, or tromethorphan is contraindicated. Concomitant use of astemizole with other macrolide antibiotics (e.g., azithromycin) is not recommended until further evaluation is conducted. This study aimed to evaluate the effect of intraperitoneal administration of astemizole (single or 7-day administration) on the anticonvulsant activity of antiepileptic drugs induced by maximal electroshock (ESW) in mice. The intraperitoneal antiepileptic drugs included magnesium valproate, carbamazepine, phenytoin sodium, and phenobarbital. Adverse effects were assessed using a pole climbing test (motor ability) and a passive avoidance task (long-term memory). Immunofluorescence was used to determine the concentrations of antiepileptic drugs in brain tissue and plasma. Astemizole (single or 7-day administration at doses of 2–6 mg/kg) lowered the ESW threshold, while lower doses had no effect on this parameter. Astemizole (1 mg/kg) had no significant effect on the protective effect against maximal ESW injury (both acute and 7-day administration). Similarly, acute astemizole (2 mg/kg) was ineffective. Following prolonged administration, astemizole (2 mg/kg) significantly reduced the protective efficacy of phenobarbital and phenytoin, as evidenced by increases in their ED50 values (the 50% effective dose required to protect 50% of the test animals from maximal electroshock injury) from 21.1 mg/kg and 10.4 mg/kg to 34.0 mg/kg and 19.2 mg/kg, respectively. Astemizole (2 mg/kg) had no effect on the protective effects of the other antiepileptic drugs. Furthermore, astemizole (2 mg/kg) had no effect on the plasma free concentrations or intracerebral concentrations of the studied antiepileptic drugs. Additionally, this H1 receptor antagonist did not impair long-term memory or motor coordination upon acute administration. However, continuous 7-day treatment with astemizole (2 mg/kg) significantly reduced the TD50 value of phenobarbital (the 50% toxic dose required to cause motor impairment in 50% of the animals), while carbamazepine, sodium valproate, and sodium phenytoin had no such effect. Similarly, phenobarbital and phenytoin sodium, used alone at doses equivalent to the ED50 for maximal electroshock, or in combination with astemizole, impaired long-term memory in mice. These results suggest that caution may be warranted when using astemizole in patients with epilepsy.
For more complete data on interactions with astemizole (out of 15), please visit the HSDB record page.

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Non-human toxicity values
Rats oral LD50 >2560 mg/kg
Rats subcutaneous LD50 355 mg/kg
Rats intravenous LD50 28 mg/kg
Mice oral LD50 2560 mg/kg
For more complete data on non-human toxicity values of astemizole (out of 6), please visit the HSDB record page. This study showed that astemizole at a dose of 30 mg/kg could induce cardiotoxicity in common marmosets, characterized by QT interval prolongation and polymorphic ventricular tachycardia (torsades de pointes). The oral dose was 3 mg/kg, and the intravenous dose was 3 mg/kg. The literature also indicates that overdose of astemizole in humans is associated with QT interval prolongation and torsades de pointes [3].

[1]. In vitro and in vivo binding characteristics of a new long-acting histamine H1 antagonist, astemizole. Mol Pharmacol. 1982 Mar;21(2):294-300.

[2]. Astemizole. A review of its pharmacodynamic properties and therapeutic efficacy. Drugs. 1984 Jul;28(1):38-61.

[3]. Development of telemetry system in the common marmoset--cardiovascular effects of astemizole and nicardipine. J Toxicol Sci. 2002 May;27(2):123-30.

[4]. Possibility as an anti-cancer drug of astemizole: Evaluation of arrhythmogenicity by the chronic atrioventricular block canine model. J Pharmacol Sci. 2016 Jun;131(2):150-3.

Therapeutic Uses
Antihistamine; Histamine H1 receptor antagonist. Antihistamines. In June 1999, the manufacturer withdrew products containing astemizole from the U.S. and Canadian markets. Antihistamines are indicated for the prevention and treatment of perennial and seasonal allergic rhinitis, vasomotor rhinitis, and allergic conjunctivitis caused by inhaled allergens and foods. /Antimazoles/ Note: Products containing astemizole were withdrawn from the U.S. and Canadian markets by the manufacturer in June 1999. For more complete data on the therapeutic uses of astemizole (7 types), please visit the HSDB record page. Drug Warnings Astemizole has been shown to have various adverse effects on cardiac electrophysiology, including changes in repolarization, T-wave inversion notching, significant TU waves, QT interval prolongation, first- and second-degree atrioventricular block, ventricular tachycardia or ventricular fibrillation, and torsades de pointes. In rare cases, astemizole has been shown to induce torsades de pointes syndrome, a condition characterized by QT interval prolongation and life-threatening ventricular tachycardia. Studies have found that the drug prolongs cardiac repolarization time when its metabolic clearance is impaired (e.g., in liver disease or when taking drugs that inhibit the cytochrome P450 3A family). In vitro studies have shown that this effect is due to blocking one or more cardiac potassium channels that determine the duration of the action potential. The safety of antihistamines during pregnancy has not been established; therefore, such drugs should not be used in pregnant women or women who may become pregnant unless the potential benefit outweighs the potential risk to the fetus. Some manufacturers warn that antihistamines should not be used in late pregnancy because of the potential for serious reactions (e.g., seizures) in newborns and preterm infants. Patients taking astemizole should be advised to take the medication only as needed and not to exceed the prescribed dose. The manufacturers of astemizole note that patients should be advised not to take astemizole on demand (“prn”) for rapid symptom relief. In addition, although loading dose regimens were previously recommended when seeking rapid onset of action, this regimen is no longer recommended due to the risk of cardiotoxicity. For more complete data on drug warnings for astemizole (8 in total), please visit the HSDB record page. Pharmacodynamics Astemizole is a second-generation H1 receptor antagonist. It does not readily cross the blood-brain barrier and therefore does not cause drowsiness or central nervous system depression at normal doses. Astemizole is a second-generation selective histamine H1 receptor antagonist used to relieve symptoms of allergic diseases such as allergic rhinitis. This study used a marmoset telemetry model to assess its arrhythmogenic potential (QT interval prolongation and torsades de pointes) as part of a cardiovascular safety pharmacology assessment [3].

C28H31FN4O

458.57034

458.248

C, 73.34; H, 6.81; F, 4.14; N, 12.22; O, 3.49

68844-77-9

2247

White to off-white solid powder

1.2 g/cm3

627.3ºC at 760 mmHg

172.9ºC

333.2ºC

1.623

5.362

1

5

8

34

599

0

COC1=CC=C(C=C1)CCN2CCC(CC2)N=C3NC4=CC=CC=C4N3CC5=CC=C(C=C5)F

GXDALQBWZGODGZ-UHFFFAOYSA-N

InChI=1S/C28H31FN4O/c1-34-25-12-8-21(9-13-25)14-17-32-18-15-24(16-19-32)30-28-31-26-4-2-3-5-27(26)33(28)20-22-6-10-23(29)11-7-22/h2-13,24H,14-20H2,1H3,(H,30,31)

1-[(4-fluorophenyl)methyl]-N-[1-[2-(4-methoxyphenyl)ethyl]piperidin-4-yl]benzimidazol-2-amine

R 43512; Hismanal; R43512; astemizole; 68844-77-9; Hismanal; Histaminos; Paralergin; Laridal; Retolen; Astemison; Paralergin; R-43512; NSC 329963; NSC329963; NSC-329963; Astemizole; Histaminos; Laridal; Retolen

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: ~125 mg/mL (~272.6 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (13.63 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 62.5 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: ≥ 6.25 mg/mL (13.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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