Doxofylline (5, 10 µM; 48 h) reduces PGE2, NO release, and mitochondrial ROS generation in 16HBE cells, demonstrating strong protection against LPS-induced epithelial inflammation[1]. LPS-induced expression of NADPH oxidase subunits and TXNIP 16HBE cells is suppressed by doxofylline (5, 10 µM; 48 h)[1]. Doxofylline (5, 10 µM; 48 h) attenuates LPS-mediated SIRT1 reduction and prevents LPS-induced NLRP3 inflammasome activation and IL-1b and IL-18 secretion[1]. In BM cells, doxofylline (0.1–10 µM; 15 min) dramatically inhibits leukocyte migration induced by fMLP (formyl–methionyl–leucyl–phenylalanine)[2].

In mice, doxofylline (0.3, 1 mg/kg; ip; single) reduces inflammation brought on by LPS in the lungs[2]. Doxofylline (0.3 mg/kg; ip; pre-treat; single) suppresses the production of LPS-induced ICAM-1 in vivo and dramatically decreases cell adherence to vascular tissue[2].

Cell Viability Assay[1]
Cell Types: 16HBE cells
Tested Concentrations: 5, 10 µM
Incubation Duration: 48 h
Experimental Results: Weakened LPS-induced NO and PGE2 in a dose-dependent manner. Exerted dose-dependent inhibition on LPS-induced mitochondrial ROS production and NADPH oxidase subunits expression. Suppressed LPS- induced TXNIP expression and NLRP3 inflammasome activation at the protein level in a dose-dependent manner. Inhibited LPS-induced secretion of IL-1b and IL-18.

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Cell Viability Assay[2]
Cell Types: BM cells (from naive mice)
Tested Concentrations: 0.1-10 µM
Incubation Duration: 15 min (pretreat)
Experimental Results: Notably suppressed positive migration of BM cells in response to fMLP.

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (6 to 8weeks old)[2].
Doses: 0.3, 1 mg/kg
Route of Administration: intraperitoneal (ip)injection; single.
Experimental Results: Dramatically inhibited the migration of neutrophils and the release of IL-6 and TNF-a into the lung lumen. Increased the bone marrow leukocyte numbers to levels similar to those seen in the saline-treated group. Notably decreased the number of circulating leukocytes in comparison to LPS-treated mice. Dramatically decreased accumulation of neutrophils in the peribronchial area.

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Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (6 to 8weeks old)[2].
Doses: 0.3 mg/kg
Route of Administration: intraperitoneal (ip)injection; pre-treat; single.
Experimental Results: Dramatically decreased the adhesion of cells to the vascular tissue, but not the rolling of cells along the vessel wall in mice. Dramatically decreased the expression of ICAM-1 induced by LPS.

Absorption, Distribution and Excretion
After repeated administrations doxofylline reaches the steady-state in about 4 days. Following oral administration of 400 mg doxofylline twice daily for 5 days in adults with chronic bronchitis, the peak plasma concentrations (Cmax) at steady state ranged from 5.78 to 20.76 mcg/mL. The time to reach maximum concentration (Tmax) was 1.19 ± 0.19 hours. The absolute bioavailability of doxofylline in healthy subjects was 63 ± 25%.
Less than 4% of an orally administered dose is excreted unchanged in the urine due to extensive hepatic metabolism.
Doxofylline demonstrates a short distribution phase following intravenous administration of 100 mg given in adults with chronic bronchitis. As methylxanthines are distributed to all body compartments, doxofylline may be detected in breast milk and placenta.
Following oral administration of 400 mg doxofylline twice daily for 5 days, the total clearance was 555.2 ± 180.6 mL/min.

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Metabolism / Metabolites
Doxofylline is thought to undergo hepatic metabolism which accounts for 90% of total drug clearance. β-hydroxymethyltheophylline was detected in the serum and urine after oral administration of 400 mg given in healthy subjects. The circulating metabolite was devoid of any significant pharmacological activity.

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Biological Half-Life
Following administration of a single intravenous dose of 100 mg over 10 minutes in adults with chronic bronchitis, the elimination half life of doxofylline was 1.83 ± 0.37 hours. Following oral administration of 400 mg twice daily for 5 days in adults with chronic bronchitis, the mean elimination half life was 7.01 ± 0.80 hours.

Protein Binding
At pH 7.4, the fraction of plasma protein binding is about 48%.

[1]. The protective effect of doxofylline against lipopolysaccharides (LPS)-induced activation of NLRP3 inflammasome is mediated by SIRT1 in human pulmonary bronchial epithelial cells. Artif Cells Nanomed Biotechnol. 2020 Dec;48(1):687-694.

[2]. Doxofylline, a novofylline inhibits lung inflammation induced by lipopolysacharide in the mouse. Pulm Pharmacol Ther. 2014 Apr;27(2):170-8.

[3]. Doxofylline: a promising methylxanthine derivative for the treatment of asthma and chronic obstructive pulmonary disease. Expert Opin Pharmacother. 2009 Oct;10(14):2343-56.

Doxofylline is an oxopurine that is a derivative of xanthine, methylated at N-1 and N-3 and carrying a 1,3-dioxolan-2-ylmethyl group at N-7, used in the treatment of asthma. It has a role as a bronchodilator agent, an antitussive and an anti-asthmatic drug. It is functionally related to a 7H-xanthine.
Doxofylline is a methylxanthine derivative with the presence of a dioxolane group in position 7. As a drug used in the treatment of asthma, doxofylline has shown similar efficacy to theophylline but with significantly fewer side effects in animal and human studies. In contrast with other xanthine derivatives, doxofylline does not significantly bind to adenosine alpha-1 or alpha-2 receptors and lacks stimulating effects. Decreased affinity for adenosine receptors may account for the better safety profile of doxofylline compared to theophylline. Unlike theophylline, doxofylline does not affect calcium influx and does not antagonize the actions of calcium channel blockers which could explain reduced cardiac adverse reactions associated with the drug. The anti-asthmatic effects of doxophylline are mediated by other mechanisms, primarily through inhibiting the activities of the phosphodiesterase (PDE) enzyme.

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Drug Indication
Indicated for the treatment of chronic obstructive pulmonary disease (COPD), bronchial asthma and pulmonary disease with spastic bronchial component.

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Mechanism of Action
The main mechanism of action of doxofylline is unclear. One of the mechanisms of action of is thought to arise from the inhibition of phosphodiesterase activity thus increasing the levels of cAMP and promoting smooth muscle relaxation. The interaction of doxofylline with beta-2 adrenoceptors was demonstrated by a study using nonlinear chromatography, frontal analysis and molecular docking. Serine 169 and serine 173 residues in the receptor are thought to be critical binding sites for doxofylline where hydrogen bonds are formed. Via mediating the actions of beta-2 adrenoceptors, doxofylline induces blood vessel relaxation and airway smooth muscle relaxation. There is also evidence that doxofylline may exert anti-inflammatory actions by reducing the pleurisy induced by the inflammatory mediator platelet activating factor (PAF) according to a rat study. It is suggested that doxofylline may play an important role in attenuating leukocyte diapedesis, supported by mouse preclinical studies where doxofylline administration was associated with inhibited leukocyte migration across vascular endothelial cells in vivo and in vitro.Unlike theophylline, doxofylline does not inhibit tumor necrosis factor-induced interleukin (IL)-8 secretion in ASM cells.

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Pharmacodynamics
Doxofylline is a methylxanthine bronchodilator with potent bronchodilator activity comparable to that of theophylline. In animal studies, doxofylline demonstrated to attenuate bronchoconstriction, inflammatory actions and the release of thromboxane A2 (TXA2) when challenged with platelet-activating factor. Doxofylline does not demonstrate direct inhibition of any histone deacetylase (HDAC) enzymes or known PDE enzyme isoforms and did not act as an antagonist at A2 or A2 receptors. The affinity for adenosine A1, A2A and A2B receptors are reported to be all higher than 100 µM. It only displays an inhibitory action against PDE2A1 and antagonism at adenosine A(2A) at high concentrations. A study demonstrated that doxofylline interacts with β2-adrenoceptors to induce blood vessel relaxation and airway smooth muscle relaxation. In dog studies, doxofylline decreased airway responsiveness at a dose that did not affect heart rate and respiratory rate.

C11H14N4O4

266.25

266.101

69975-86-6

Doxofylline-d6;1219805-99-8;Doxofylline-d4;1346599-13-0

50942

White to off-white solid powder

1.6±0.1 g/cm3

505.2±53.0 °C at 760 mmHg

144-146ºC

259.3±30.9 °C

0.0±1.3 mmHg at 25°C

1.700

-0.7

0

5

2

19

398

0

HWXIGFIVGWUZAO-UHFFFAOYSA-N

InChI=1S/C11H14N4O4/c1-13-9-8(10(16)14(2)11(13)17)15(6-12-9)5-7-18-3-4-19-7/h6-7H,3-5H2,1-2H3

7-(1,3-dioxolan-2-ylmethyl)-1,3-dimethylpurine-2,6-dione

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.39 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 (9.39 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: ≥ 2.5 mg/mL (9.39 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 65 mg/mL (244.13 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.)