The formation of bone marrow megakaryocytes is efficiently inhibited by angrelide (IC50=26 nM) [1]. One special medication that prevents bone marrow megakaryocytopoiesis is anagrelide. Only megakaryocyte proliferation was suppressed by angrelide (0.05, 0.3, and 1 µM), but not that of non-megakaryocytic cells. The fact that anagrelide causes cell proliferation to relocate to non-megakaryocytic compartments suggests that it does not have cytotoxic properties [2]. Although anagrelide only exhibits mild action in the GIST48 cell line, it can cause cytotoxicity in the GIST882 cell line at submicromolar concentrations (IC50= 16 nM) [3]. In vitro, anagrelide can decrease the growth of gastrointestinal stromal tumor (GIST) cells and increase apoptosis [3].

Anagrelide is efficacious in xenograft mouse models of GIST with KIT exon 9 mutations, which may represent treatment problems because these GISTs require large daily doses of imatinib [3]. Anagrelide suppresses GIST development in patient-derived mice xenograft models. Anagrelide has therapeutic potential in the treatment of GIST. Anagrelide exhibits anticancer efficacy in gastrointestinal stromal tumor xenograft models [3]. Anagrelide (5 mg/kg/bid) suppresses or decreases tumor growth in GIST2B, GIST9, and GIST882 models [3].

Cell proliferation assay[2]
Cell Types: megakaryocytes and non-megakaryocytes
Tested Concentrations: 0.05, 0.3, 1 µM
Incubation Duration: 12 days
Experimental Results: Inhibition of megakaryocyte growth only at each concentration tested

Animal/Disease Models: Adult female athymic mouse GIST2B, GIST3, GIST9, GIST882 model [3]
Doses: 5 mg/kg/bid
Route of Administration: 5 mg/kg/bid or combined with anagrelide and imatinib ( given simultaneously) dose and schedule as a single agent); for 10 days
Experimental Results: Three of the four models (GIST2B, GIST9, GIST882) inhibited or diminished tumor growth. The most potent effect was observed in the GIST2B model, which contains the KIT exon 9 mutation leading to the p.A502_Y503 duplication. The tumor volume of this model diminished by 68% after 10 days of treatment.

Absorption, Distribution and Excretion
Following oral administration, the bioavailability of anagrelide is approximately 70%. When taken on an empty stomach, peak plasma concentration (Cmax) is reached within 1 hour after administration (Tmax). Co-administration with food slightly decreases Cmax and increases AUC, but this is not clinically significant. After metabolism, urinary excretion of metabolites appears to be the primary route of elimination for anagrelide. Less than 1% of the administered dose is excreted unchanged in the urine, while approximately 3% and 16-20% of the administered dose are excreted as 3-hydroxyanagrelide and RL603, respectively. Following oral administration of 14C-anagrelide in humans, over 70% of the radioactive material is excreted in the urine. Current patient steady-state plasma concentration-time data indicate that anagrelide does not accumulate in plasma after repeated dosing. The volume of distribution (VolD) is 12 L/kg body weight. Limited data suggest a linear relationship between possible doses of 500 mg (0.5 mg) and 2 mg. Studies have found that anagrelide bioavailability is reduced by an average of 13.8% when taken after a meal. When 0.5 mg of anagrelide is taken after a meal, peak plasma concentration is reduced by an average of 45%, and the time to peak concentration is delayed by 2 hours… For more complete data on the absorption, distribution, and excretion of anagrelide (6 items), please visit the HSDB record page.

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Metabolism/Metabolites
Anagrelide is extensively metabolized in the liver via cytochrome P450 1A2 (CYP1A2) to produce two major metabolites: 6,7-dichloro-3-hydroxy-1,5-dihydroimidozolo[2,1-b]quinazolin-2-one (3-hydroxyanagrelide) and 2-amino-5,6-dichloro-3,4-dihydroquinazolin (RL603). The 3-hydroxy metabolite is considered pharmacologically active, with similar platelet-lowering efficacy and therapeutic effect to the parent drug, but its PDE3 inhibition efficacy is 40 times that of the parent drug. Anagrelide is widely metabolized; less than 1% is recovered unchanged in urine. The half-lives (t_1/2) of anagrelide and its active metabolite 3-hydroxyanagrelide are approximately 1.5 hours and 2.5 hours, respectively. The plasma half-life is 1.3 hours when taken on an empty stomach with 0.5 mg… Elimination: The plasma half-life is 1.3 hours (0.5 mg taken on an empty stomach). Note: When anagrelide is taken after a meal, the plasma half-life is prolonged to 1.8 hours. Steady-state plasma concentration measurements indicate that anagrelide does not accumulate in plasma after repeated dosing.

Hepatotoxicity
In pre-registration studies, anagrelide was not associated with elevated serum enzymes or clinically significant liver injury. Since its approval, only one published abstract reports a case of progressive, ultimately fatal cholestasis following liver transplantation with anagrelide, but no other reports of anagrelide hepatotoxicity have been found in the literature. In large, long-term follow-up studies, transient elevations of serum enzymes have been occasionally observed without jaundice or other symptoms. The anagrelide product label lists enzyme abnormalities as adverse events but does not mention clinically significant liver injury, hepatitis, or jaundice. However, overall clinical experience with anagrelide is limited. Probability score: E (Unlikely, but suspected as a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of anagrelide during lactation. The manufacturer recommends against use in breastfeeding women and within one week of the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on breastfeeding and breast milk
No published information found as of the revision date.

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Drug Interactions
Bioavailability studies assessing potential interactions between anagrelide and other drugs have not been conducted. Common drugs that are co-administered with anagrelide include aspirin, acetaminophen, furosemide, iron supplements, ranitidine, hydroxyurea, and allopurinol. The most commonly co-administered cardiac drug is digoxin. Although drug interaction studies have not been conducted, there is currently no clinical evidence that anagrelide interacts with any of the above drugs.
Case reports suggest that sucralfate may interfere with the absorption of anagrelide.

[1]. Comparison of the biological activities of Anagrelide and its major metabolites in haematopoietic cell cultures. Br J Pharmacol. 2005 Oct;146(3):324-32.

[2]. Comparison between Anagrelide and hydroxycarbamide in their activities against haematopoietic progenitor cell growth and differentiation: selectivity of Anagrelide for the megakaryocytic lineage. Leukemia. 2006 Jun;20(6):1117-22.

[3]. Anagrelide for Gastrointestinal Stromal Tumor. Clin Cancer Res. 2019 Mar 1;25(5):1676-1687.

Therapeutic Uses

Antocytosis
Anagrelide is indicated for reducing platelet count and the risk of thrombosis in patients with myeloproliferative disorders and for improving related symptoms. /US product label contains/
Drug Warnings
While most adverse events reported during anagrelide treatment are mild and their frequency decreases with continued treatment, serious adverse events have been reported in patients with myeloproliferative disorders of various etiologies. These adverse reactions include: congestive heart failure, myocardial infarction, cardiomyopathy, cardiomegaly, complete atrioventricular block, atrial fibrillation, cerebrovascular accident, pericarditis, pulmonary infiltration, pulmonary fibrosis, pulmonary hypertension, pancreatitis, gastric/duodenal ulcers, and seizures. In clinical trials involving 942 patients with myeloproliferative disorders, the most common adverse reactions (incidence ≥5%) of anagrelide were: headache 43.5%; palpitations 26.1%; diarrhea 25.7%; fatigue 23.1%; other edema 20.6%; nausea 17.1%; abdominal pain 16.4%; dizziness 15.4%; other pain 15.0%; dyspnea 11.9%; flatulence 10.2%; vomiting 9.7%; fever 8.9%; peripheral edema 8.5%; rash (including urticaria) 8.3%; chest pain 7.8%; anorexia 7.7%; tachycardia 7.5%; pharyngitis 6.8%; malaise 6.4%; cough 6.3%; paresthesia 5.9%; back pain 5.9%; pruritus 5.5%; and indigestion 5.2%. Anagrelide is recommended for patients with renal insufficiency (creatinine ≥2 mg/dL) when the potential benefit outweighs the potential risk. Patients should be closely monitored for signs of nephrotoxicity during anagrelation treatment.
In patients with known or suspected heart disease, caution should be exercised only if the potential benefit outweighs the potential risk. Due to the positive inotropic effect and side effects of anagrelation, cardiovascular assessment is recommended before treatment and close monitoring is advised during treatment. Therapeutic doses of anagrelation may cause adverse cardiovascular reactions, including vasodilation, tachycardia, palpitations, and congestive heart failure.
For more complete data on anagrelation (11 in total), please visit the HSDB record page.

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Pharmacodynamics
Anagrelation reduces platelet count by inhibiting transcription factors required for the synthesis and maturation of thrombopoietin. The drug has a relatively short residence time in the body and therefore needs to be taken twice or four times daily. However, given that the pharmacological action of anagrelation depends on the gradual inhibition of thrombopoietin, it may take 7 to 14 days after administration to reflect a reduction in platelet count—therefore, the dose adjustment of anagrelation should not exceed 0.5 mg/day in any given week. Animal studies suggest that anagrelide may impair female fertility. Women of reproductive age should be informed of the potential adverse effects of this medication on fertility before starting treatment.

C10H7N3OCL2

256.08808

254.996

68475-42-3

Anagrelide hydrochloride;58579-51-4;Anagrelide hydrochloride monohydrate;823178-43-4

135409400

Typically exists as solid at room temperature

1.8±0.1 g/cm3

376.5±52.0 °C at 760 mmHg

280 °C

181.5±30.7 °C

0.0±0.9 mmHg at 25°C

1.791

1.96

1

2

0

16

360

0

O=C1N=C2NC3=C(C(Cl)=C(Cl)C=C3)CN2C1

OTBXOEAOVRKTNQ-UHFFFAOYSA-N

InChI=1S/C10H7Cl2N3O/c11-6-1-2-7-5(9(6)12)3-15-4-8(16)14-10(15)13-7/h1-2H,3-4H2,(H,13,14,16)

6,7-dichloro-3,5-dihydro-1H-imidazo[2,1-b]quinazolin-2-one

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.)