TASK tandem pore (K2P) potassium channels: Inhibition of TASK-1 (KCNK3) and TASK-3 (KCNK9) channels was observed; the half-maximal inhibitory concentration (IC₅₀) for TASK-1 was approximately 30 μM, and for TASK-3 was approximately 10 μM when tested in Xenopus oocytes expressing these channels [1]
- Ionic channels in isolated type I cells of the neonatal rat carotid body: Inhibition of a background K⁺ current; the IC₅₀ for this inhibition was approximately 5 μM [2]

In vitro activity: Doxapram is a respiratory stimulant that inhibits TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric channel function with EC50 of 410 nM, 37 μM, 9 μM, respectively. Doxapram preferentially stimulated the release of dopamine. It was also seen to directly inhibit Ca(2+)-independent K+ currents. Doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (at 15-150 μM) also evoked 3H overflow in a concentration dependent manner, and doxapram-evoked release was inhibited by the Ca2+ channel blocker nifedipine (5 μM). The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.

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Kinase Assay: Doxapram inhibited TASK-1 (half-maximal effective concentration [EC50], 410 nM), TASK-3 (EC50, 37 microM), and TASK-1/TASK-3 heterodimeric channel function (EC50, 9 microM).

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Cell Assay: Doxapram (1-100 microM) caused rapid, reversible and dose-dependent inhibitions of K+ currents recorded in type I cells (IC50 approximately 13 microM). doxapram was also seen to directly inhibit Ca(2+)-independent K+ currents. Doxapram was a more potent inhibitor of the Ca(2+)-activated K+ currents recorded under control conditions. Doxapram (10 microM) was without effect on L-type Ca2+ channel currents recorded under conditions where K+ channel activity was minimized and was also without significant effect on K+ currents recorded in the neuronal cell line NG-108 15, suggesting a selective effect on carotid body type I cells. The effects of doxapram on type I cells show similarities to those of the physiological stimuli of the carotid body, suggesting that doxapram may share a similar mechanism of action in stimulating the intact organ.

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Effect on TASK K2P potassium channels: Application of Doxapram (1-100 μM) dose-dependently inhibited TASK-1 and TASK-3 channel currents recorded from Xenopus oocytes. At 100 μM, Doxapram almost completely blocked TASK-1 (by ~90%) and TASK-3 (by ~95%) currents. This inhibition was reversible upon washout of the drug. Additionally, Doxapram did not affect other K⁺ channels such as TREK-1, TWIK-1, or Kv1.5 at concentrations up to 100 μM [1]
- Effect on ionic currents in neonatal rat carotid body type I cells: Doxapram (1-100 μM) dose-dependently inhibited the background K⁺ current in isolated type I cells. At 10 μM, the inhibition was ~50%, and at 100 μM, the current was almost completely blocked (~90%). Doxapram had no significant effect on voltage-gated Ca²⁺ currents or Na⁺ currents in these cells at concentrations up to 100 μM [2]
- Effect on dopamine release from rat carotid body: Incubation of intact rat carotid bodies with Doxapram (1-100 μM) in vitro stimulated dopamine release in a dose-dependent manner. At 1 μM, dopamine release was increased by ~20% compared to control; at 10 μM, the increase was ~60%; and at 100 μM, the release was increased by ~120%. This stimulatory effect was blocked by the K⁺ channel opener cromakalim (10 μM) [3]

Effect on minimum alveolar anesthetic concentration (MAC): In adult Sprague-Dawley rats anesthetized with isoflurane, intravenous administration of Doxapram at doses of 2, 5, and 10 mg/kg did not significantly change the MAC of isoflurane. The MAC values before and after Doxapram administration were statistically similar (P > 0.05) [1]

TASK K2P potassium channel activity assay: Xenopus oocytes were injected with cRNA encoding human TASK-1 or TASK-3 channels. After 24-48 hours of incubation at 18°C, oocytes were placed in a recording chamber filled with a standard solution (containing NaCl, KCl, CaCl₂, MgCl₂, and HEPES). Two-microelectrode voltage-clamp technique was used to record channel currents. The holding potential was set to -60 mV, and voltage ramps from -120 mV to +40 mV were applied over 2 seconds. Doxapram was added to the recording chamber at different concentrations (1-100 μM), and currents were recorded after a 5-minute incubation to allow for steady-state effects. Current amplitudes at -60 mV were measured to quantify the inhibitory effect of Doxapram. Washout experiments were performed by replacing the drug-containing solution with standard solution to check reversibility [1]
- Ionic current recording in carotid body type I cells: Neonatal rats (1-3 days old) were decapitated, and carotid bodies were dissected out. Type I cells were isolated by enzymatic digestion (using collagenase and dispase) followed by mechanical trituration. Isolated cells were plated on glass coverslips and allowed to attach for 1-2 hours. Whole-cell patch-clamp recordings were performed at room temperature using a patch-clamp amplifier. The pipette solution contained KCl, MgATP, EGTA, and HEPES, while the bath solution contained NaCl, KCl, CaCl₂, MgCl₂, and HEPES. Background K⁺ currents were recorded by applying voltage ramps from -100 mV to 0 mV (holding potential -60 mV). Doxapram was added to the bath solution at concentrations ranging from 1 to 100 μM, and currents were recorded after 3-5 minutes of exposure. Voltage-gated Ca²⁺ and Na⁺ currents were recorded using appropriate voltage protocols to assess the specificity of Doxapram action [2]

Dopamine release assay from intact rat carotid body: Adult rats were euthanized, and carotid bodies were rapidly dissected and placed in oxygenated Krebs-Ringer bicarbonate buffer (KRB) at 37°C. Each carotid body was incubated in a 1.5 mL microcentrifuge tube containing KRB for 30 minutes to equilibrate. After equilibration, the buffer was replaced with fresh KRB containing different concentrations of Doxapram (1, 10, 100 μM) or KRB alone (control). In some experiments, cromakalim (10 μM) was added to the buffer along with Doxapram. Incubation was continued for 60 minutes at 37°C with constant shaking. At the end of incubation, the buffer was collected, and dopamine concentration was measured using high-performance liquid chromatography (HPLC) with electrochemical detection. The amount of dopamine released was normalized to the protein content of each carotid body (measured using the Bradford assay) to account for differences in tissue size [3]

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Minimum alveolar anesthetic concentration (MAC) determination in rats: Adult male Sprague-Dawley rats (250-300 g) were used. Rats were anesthetized with isoflurane in oxygen, and a tracheostomy was performed to maintain a patent airway. A femoral artery was cannulated for blood pressure monitoring and blood gas analysis. The MAC of isoflurane was determined using the tail-clamp method: a clamp was applied to the base of the tail, and a positive response (movement of the head or limbs) was considered indicative of inadequate anesthesia. The MAC was defined as the concentration of isoflurane at which 50% of rats did not respond to the tail clamp. After baseline MAC determination, Doxapram was administered intravenously via a femoral vein catheter at doses of 2, 5, and 10 mg/kg (each dose was tested in a separate group of rats, n = 6 per group). MAC was re-determined 15 minutes after each Doxapram dose. Blood gas parameters (pH, PaO₂, PaCO₂) were monitored before and after Doxapram administration to ensure they remained within normal ranges [1]

Absorption, Distribution and Excretion
In healthy volunteers, plasma concentrations decreased exponentially following intravenous administration or brief infusion of doxapram hydrochloride. The volume of distribution was 1.51 kg⁻¹, and systemic clearance was 370 ml min⁻¹. Enteric-coated doxapram capsules are rapidly absorbed after an initial delay, with a systemic bioavailability of approximately 60%. Less than 5% of intravenously administered doses are excreted unchanged in the urine within 24 hours. Metabolisms/Metabolites Doxapram produces 4-(2-morpholinoethyl)-3,3-diphenylpyrrolidone-2-one and 1-ethyl-4-(2-(3-oxomorpholinoethyl)ethyl)-3,3-diphenylpyrrolidone-2-one in dogs. PITTS, JE, BRUCE, RB, & FOREHAND, JB, XENOBIOTICA, 3, 73 (1973). /Excerpt from Table/
In healthy volunteers, after intravenous injection or brief infusion of doxapram hydrochloride, the plasma concentration of the metabolite AHR 5955 was comparable to that of the parent compound, and the half-life was similar.

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Biological Half-Life>
In healthy volunteers, after intravenous injection or brief infusion of doxapram hydrochloride, plasma concentrations decreased exponentially. The half-life was 3.4 after 4–12 hours.

[1]. The ventilatory stimulant doxapram inhibits TASK tandem pore (K2P) potassium channel function but does not affect minimum alveolar anesthetic concentration. Anesth Analg, 2006, 102(3), 779-785.

[2]. Peers, C., Effects of doxapram on ionic currents recorded in isolated type I cells of the neonatal rat carotid body. Brain Res, 1991. 568(1-2): p. 116-22.

[3]. Doxapram stimulates dopamine release from the intact rat carotid body in vitro. Neurosci Lett, 1995. 187(1): p. 25-8.

Doxapram belongs to the pyrrolidine-2-one class of compounds. Its structure is N-ethylpyrrolidine-2-one, where both hydrogen atoms at position 3 (adjacent to the carbonyl group) are replaced by phenyl groups, and one hydrogen atom at position 4 is replaced by a 2-(morpholino-4-yl)ethyl group. It is a transient central and respiratory stimulant, usually used in the form of hydrochloride or hydrochloride hydrate for the temporary treatment of acute respiratory failure, especially in cases complicated by chronic obstructive pulmonary disease (COPD) and postoperative respiratory depression. It has also been used to treat postoperative shivering. Doxapram has a central nervous system stimulant effect and belongs to the morpholino and pyrrolidine-2-one class of compounds. (Excerpt from Martindale Pharmacopoeia, 30th edition, p. 1225)
Doxapram is a respiratory stimulant. The physiological effect of doxapram is achieved by enhancing the medullary respiratory drive.
Doxapram is a respiratory stimulant with a respiratory stimulant effect. Doxapram is oxygen-independent and directly stimulates peripheral carotid chemoreceptors. Its mechanism of action is likely through inhibition of potassium channels in type I cells of the carotid body, thereby stimulating the release of catecholamines. This can prevent or reverse respiratory depression caused by anesthetics and central nervous system depressants. A transient central respiratory stimulant. (From Martindale Pharmacopoeia, 30th edition, p. 1225) See also: Doxapram hydrochloride (salt form).
Indications
For the treatment of hospitalized patients with acute respiratory failure complicated by chronic obstructive pulmonary disease, as a temporary measure.
FDA label

Mechanism of Action
Doxapram produces respiratory stimulation through peripheral carotid chemoreceptors. It is believed to stimulate the carotid body by inhibiting certain potassium channels.
Doxapram…stimulates all levels of the brain-spinal axis. Sufficient doses can produce tonic-clonic seizures similar to those of pentylenetetrazol…its mechanism of action is by enhancing excitation rather than blocking central inhibition.

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Therapeutic Uses
Central Nervous System Stimulant; Respiratory System Drugs
Veterinary Drugs: Used to increase ventilation and reduce sleep time in cats and dogs under pentobarbital anesthesia, and occasionally for anesthetizing horses.
The respiratory system can be stimulated by doses that produce mild systemic excitation. Direct medullary stimulation is the primary cause of this effect, but indirect stimulation through activation of peripheral chemoreceptors may also contribute. The duration of stimulation after a single intravenous injection is short, rarely lasting…5-10 minutes.
Doxapram…is used to temporarily correct acute respiratory failure in patients with chronic obstructive pulmonary disease. Intermittent or continuous infusion is necessary to maintain respiratory stimulation and reduce carbon dioxide tension…
For more complete data on the therapeutic uses of doxapram (7 types), please visit the HSDB record page.

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Drug Warnings
Due to the effectiveness of controlled ventilation and standard supportive therapy in treating respiratory failure, doxapram should generally not be used on patients with drug-induced coma or acute exacerbations of chronic lung disease. Doxapram hydrochloride is contraindicated in patients with seizure disorders, hypertension, cerebral edema, hyperthyroidism, or pheochromocytoma, as well as in patients taking monoamine oxidase inhibitors or adrenergic drugs. Doxapram hydrochloride is a respiratory stimulant (central nervous system stimulant). Its respiratory stimulant effect manifests as an increase in tidal volume, accompanied by a slight increase in respiratory rate. A pressor response may occur after doxapram administration. In the absence of impaired cardiac function, the pressor effect is more pronounced in hypovolemic states than in normal volume states. The pressor response is due to increased cardiac output rather than peripheral vasoconstriction. Doxapram administration increases the release of catecholamines. Doxapram is a respiratory stimulant used clinically to treat respiratory depression. [1] The study suggests that its respiratory excitatory effect may be mediated by inhibiting TASK K2P potassium channels involved in regulating respiratory rhythm, rather than by altering the central nervous system’s sensitivity to anesthetics (as shown by unchanged MAC values). [1] Doxapram inhibits background K⁺ currents in type I cells of the carotid body (as shown in [2]) leading to depolarization of these cells, thereby triggering Ca²⁺ influx and subsequent release of neurotransmitters (such as dopamine, as shown in [3]). This release of neurotransmitters activates afferent nerve fibers that send signals to the brainstem respiratory center to stimulate ventilation. [2][3]

C24H30N2O2

378.51

378.23

C, 76.16; H, 7.99; N, 7.40; O, 8.45

309-29-5

Doxapram hydrochloride hydrate;7081-53-0

3156

White to off-white crystalline powder.

1.1±0.1 g/cm3

536.4±50.0 °C at 760 mmHg

217-219
MP: 123-124 °C /BENZOATE/

278.2±30.1 °C

0.0±1.4 mmHg at 25°C

1.562

3.23

0

3

6

28

487

0

O=C1N(CC)CC(CCN2CCOCC2)C1(C3=CC=CC=C3)C4=CC=CC=C4

XFDJYSQDBULQSI-UHFFFAOYSA-N

InChI=1S/C24H30N2O2/c1-2-26-19-22(13-14-25-15-17-28-18-16-25)24(23(26)27,20-9-5-3-6-10-20)21-11-7-4-8-12-21/h3-12,22H,2,13-19H2,1H3

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)

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.)