The effects of doxapram (blocker of K2p channels) on resting membrane potential and synaptic transmission at the Drosophila neuromuscular junction.

The resting membrane potential of most cells is maintained by potassium K2p channels. The pharmacological profile and distribution of various K2p channel subtypes in organisms are still being investigated. The Drosophila genome contains 11 subtypes; however, their function and expression profiles have not yet been determined. Doxapram is clinically used to enhance respiration in humans and blocks the acid-sensitive K2p TASK subtype in mammals. The resting membrane potential of larval Drosophila muscle and synaptic transmission at the neuromuscular junction are pH sensitive. The present study investigated the effects of doxapram on membrane potential and synaptic transmission using intracellular recordings of larval Drosophila muscles. Doxapram (1 mM and 10 mM) depolarizes the muscle and appears to depolarize motor neurons, causing an increase in the frequency of spontaneous quantal events and evoked excitatory junction potentials. Verapamil (1 and 10 mM) paralleled the action of doxapram. These changes were matched by an extracellular increase in KCl (50 mM) and blocked by Cd2+. It is assumed that the motor nerve depolarizes to open voltage-gated Ca2+ channels in presynaptic nerve terminals because of exposure to doxapram. These findings are significant for building models to better understand the function of pharmacological agents that affect K2p channels and how K2p channels contribute to the physiology of tissues. Drosophila offers a genetically amenable model that can alter the tissue-specific expression of K2p channel subtypes to simulate known human diseases related to this family of channels.

Big Data Analyses for Continuous Evaluation of Pharmacotherapy: A Proof of Principle with Doxapram in Preterm Infants.

BACKGROUND: Drug effect evaluation is often based on subjective interpretation of a selection of patient data. Continuous analyses of high frequency patient monitor data are a valuable source to measuring drug effects. However, these have not yet been fully explored in clinical care. We aim to evaluate the usefulness and applicability of high frequency physiological data for analyses of pharmacotherapy. METHODS: As a proof of principle, the effects of doxapram, a respiratory stimulant, on the oxygenation in preterm infants were studied. Second-to-second physiological data were collected from 12 hours before until 36 hours after start of doxapram loading dose plus continuous maintenance dose in seven preterm infants. Besides physiological data, plasma concentrations of doxapram and keto-doxapram were measured. RESULTS: Arterial oxygen saturation (SpO2) increased after the start of doxapram treatment alongside an increase in heart rate. The respiratory rate remained unaffected. The number of saturation dips and the time below a saturation of 80%, as well as the area under the 80%-saturation-time curve (AUC), were significantly lowered after the start of doxapram. The AUC under 90% saturation also significantly improved after start of doxapram. Plasma concentrations of doxapram and keto-doxapram were measured. CONCLUSION: Using high-frequency monitoring data, we showed the detailed effects over time of pharmacotherapy. We could objectively determine the respiratory condition and the effects of doxapram treatment in preterm infants. This type of analysis might help to develop individualized drug treatments with tailored dose adjustments based on a closed-loop algorithm.

The ventilatory stimulant doxapram inhibits TASK tandem pore (K2P) potassium channel function but does not affect minimum alveolar anesthetic concentration.

TWIK-related acid-sensitive K(+)-1 (TASK-1 [KCNK3]) and TASK-3 (KCNK9) are tandem pore (K(2P)) potassium (K) channel subunits expressed in carotid bodies and the brainstem. Acidic pH values and hypoxia inhibit TASK-1 and TASK-3 channel function, and halothane enhances this function. These channels have putative roles in ventilatory regulation and volatile anesthetic mechanisms. Doxapram stimulates ventilation through an effect on carotid bodies, and we hypothesized that stimulation might result from inhibition of TASK-1 or TASK-3 K channel function. To address this, we expressed TASK-1, TASK-3, TASK-1/TASK-3 heterodimeric, and TASK-1/TASK-3 chimeric K channels in Xenopus oocytes and studied the effects of doxapram on their function. 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). Chimera studies suggested that the carboxy terminus of TASK-1 is important for doxapram inhibition. Other K2P channels required significantly larger concentrations for inhibition. To test the role of TASK-1 and TASK-3 in halothane-induced immobility, the minimum alveolar anesthetic concentration for halothane was determined and found unchanged in rats receiving doxapram by IV infusion. Our data indicate that TASK-1 and TASK-3 do not play a role in mediating the immobility produced by halothane, although they are plausible molecular targets for the ventilatory effects of doxapram.

Severe side effects and drug plasma concentrations in preterm infants treated with doxapram.

A high-performance liquid chromatography method has been developed for simultaneous determination of doxapram and its metabolites including ketodoxapram, the main and only active metabolite. The aim of the study was to evaluate this microtechnique and to report the cases involving severe adverse effects to determine toxic plasma levels in neonates. The method was found to be selective, and showed a good baseline separation of doxapram and metabolites. Recovery, linearity, intraday/interday precision, and limit of detection determined in aqueous solutions and in spiked plasma were satisfactory. The assay is simple, rapid, and plasma-sparing, which represents a true advantage in managing neonates. Case analysis was performed in two consecutive periods: 124 preterm infants in the first period and 173 in the second period. Severe toxic effects were observed in 4 cases in the first period, with doxapram plus keto-doxapram levels 9 mg/L. In the second period, only one case was observed. High-range plasma concentrations were significantly less frequent in the second period than in the first one. The authors conclude that measuring doxapram plus keto-doxapram in plasma may be of interest to avoid severe toxic effects in preterm neonates treated with doxapram.

Pharmacokinetics and metabolism of intravenous doxapram in horses.

The pharmacokinetics and metabolism of doxapram in horses administered intravenous (iv) doses of 0.275, 0.55 and 1.1 mg doxapram/kg bodyweight (bwt) were investigated. Plasma doxapram concentrations decreased rapidly after drug administration and the disappearance of doxapram from plasma was best described by a polyexponential equation. Median values of total body clearance were 10.9, 10.6 and 10.9 ml/min/kg bwt for the three doses and were independent of dose. The steady-state volume of distribution was approximately 1,200 ml/kg bwt and the median biological half-life ranged from 121 to 178 mins. Plasma protein binding of doxapram ranged from 76.0 to 85.4 per cent. The blood:plasma doxapram concentration ratio was approximately 0.8 and the affinity of the red blood cells for doxapram ranged from 2.0 to 2.8 indicating sequestration of doxapram in erythrocytes. Renal clearance of doxapram was a minor route of elimination. Metabolic clearance of doxapram appeared to be a major route of elimination. Four metabolites of doxapram were isolated from urine and were identified. The metabolites were: a) 1-ethyl-4-[(2-hydroxyethyl) amino]ethyl-3,3-diphenyl-2-pyr-rolidinone, b) a glucuronic acid or sulphuric acid conjugate of 1-ethyl-3-(hydroxyphenyl)-4-(2-morpholinoethyl)-3-phenyl-pyrrolidinone, c) 3,3-diphenyl-4-(2-morpholinoethyl)-2-pyrrolidinone and d) 1-(2-hydroxyethyl)-3,3-diphenyl-4-(2-morpholinoethyl)-2-pyr-rolidinon e. The rapid disappearance of doxapram from plasma immediately after iv administration was attributed to redistribution of the drug from plasma to other tissues. The short duration of clinical effect from doxapram may be attributed to redistribution of the drug from plasma and other well-perfused tissues, such as the brain, to less well-perfused tissues such as the skeletal muscles and adipose tissue. Continuous or repeated administration of doxapram could prolong the duration of clinical effect because re-distribution is less important as steady-state conditions are approached.

Interaction of doxapram and pentobarbital in mice.

We found a statistically significant increase in duration of pentobarbital-induced narcosis in doxapram-treated mice. The influence of doxapram (a respiratory stimulant) pretreatment on pentobarbital metabolism in mice was assessed by measurements of sleeping times, hypothermia, LD50 values, hepatic microsomal metabolism and relative plasma and brain levels of pentobarbital. When doxapram was given intraperitoneally 60 min. prior to administration of pentobarbital, doxapram potentiated pentobarbital-induced narcosis in a dose- and time-dependent manner, but had no effect on onset time. Doxapram potentiated hypothermia, increased acute toxicity, and prolonged the pentobarbital half-life in brain and plasma, but measurement of the concentration of pentobarbital in the brain and plasma immediately upon recovery from narcosis showed that there were no differences in any of the groups examined. Also, brain-to-plasma ratios of pentobarbital did not differ between the control and doxapram-treated groups. Doxapram competitively inhibited the hepatic metabolism of pentobarbital in 9000 x g supernatant incubation mixtures. The results obtained from these experiments indicate that inhibition of drug-metabolizing enzymes by doxapram may account for its enhancement of the duration of pentobarbital-induced narcosis.

Urinary metabolites of doxapram in premature neonates.

1. Urine samples from 20 premature neonates who received doxapram by i.v. infusion were analysed for drug metabolites by g.l.c-mass spectrometry. 2. In addition to doxapram, all urines contained at least one metabolite, but the known metabolite, 3-ketodoxapram, was detected in only 50% of the samples, and in some instances only in trace amounts. 3. Significant inter-individual differences in the metabolic pathways of doxapram were observed. 4. A total of six metabolites of doxapram were isolated three of which have not been observed previously in human or in dog. 5. Appropriate structures for the new metabolites have been deduced from their mass spectral fragmentation pathways, and are 1-ethyl-4-[2-(N-formyl-N-(2-hydroxy-ethyl)amino)ethyl]-3,3-diphenyl-2- pyrrolidinone (VII), 1-ethyl-4-[2-(4-morpholin-2-onyl)ethyl]-3,3-diphenyl-2-pyrro lidinone (IX) and 4-ethenyl-1-ethyl-3,3-diphenyl-2-pyrrolidinone (X).

Doxapram metabolism in human fetal hepatic organ culture.

The biotransformation of doxapram, a respiratory stimulant was studied with use of explants from human fetal livers (n = 15 fetuses) obtained from therapeutic abortions (gestational age, 10 to 20 weeks). Explants were cultured in Leibowitz medium and the media from cultured samples were collected before and at 3, 6, 12, and 24 hours after incubation with 2.5, 5.0, and 10 micrograms/ml doxapram. The concentrations of doxapram and its metabolites (AHR 0914, an analog of doxapram, AHR 5955 or ketodoxapram, and AHR 5904) were measured by high pressure liquid chromatography. Explant histopathology and alkaline phosphatase activity showed no direct toxic effects of the drug on liver tissue. The fastest rate of doxapram metabolism occurred during the first 3 hours of incubation (198 +/- 73.3, 438 +/- 63.3, and 538 +/- 62 ng/mg/hr liver protein at doxapram concentrations of 2.5, 5.0, and 10.0 micrograms/ml, respectively). At 3 hours of incubation, the amount of doxapram metabolized (nanogram per milligram of liver protein) was significantly higher (p less than 0.01) at doxapram concentrations of 10.0 (1616 +/- 186) and 5.0 microgram/ml (1315 +/- 190) than at 2.5 micrograms/ml (594 +/- 220). The oxidative pathway producing keto-doxapram, or AHR 5955 and AHR 5904, is more active than the de-ethylation producing the analog of doxapram AHR 0914. Data indicate substantial metabolism of doxapram by the human fetal lives.

Doxapram inhibits the in vitro oxidation of hexobarbital.

The effect of doxapram on the mouse liver microsomal hexobarbital metabolism in vitro was studied. Doxapram inhibited hexobarbital oxidase in a competitive manner, with inhibition constants between 2 and 10 mM. Doxapram induced a reverse type I spectral change with a spectral dissociation constant of about 0.1 microM. These results indicate that doxaprame is an inhibitor of microsomal drug metabolism in the mouse.

A pharmacokinetic study of doxapram in patients and volunteers.

1. Following intravenous bolus injections or brief infusions in healthy volunteers, plasma concentrations of doxapram declined in a multi-exponential fashion. The mean half-life from 4-12 h was 3.4 h (range 2.4-4.1h), the mean apparent volume of distribution was 1.5 1 kg-1 and the whole body clearance was 370 ml min-1. 2. Enteric-coated capsules of doxapram base were absorbed rapidly after an initial delay, and the systemic availability was about 60%. 3. Doxapram is extensively metabolized and less than 5% of an i.v. dose was excreted unchanged in the urine in 24 h. A metabolite (AHR 5955) was present in plasma in amounts comparable to the parent compound and had a similar half-life. 4. The disposition of doxapram appears to be similar in healthy volunteers and patients with respiratory failure. 5. The previously held belief that plasma concentrations fall rapidly when an infusion is stopped is only true following short duration infusions. The pharmacokinetic properties of doxapram are such that steady-state plasma concentrations will not be achieved for many hours with the recommended constant rate infusion régime.

The disposition of intravenous doxapram in man.

The pharmacokinetics of intravenous doxapram in healthy individuals is consistent with a three-compartment open model. Doxapram was administered by bolus injection (1.5 mg . kg-1) and by intravenous infusion (6.5 mg . kg-1 for 2 h) to 5 subjects on separate occasions. There was no significant difference in mean terminal plasma half-lives (355 and 448 min) or in mean total body clearances 5.9 and 5.6 ml . min-1 . kg-1) following i.v. bolus injection or infusion respectively. In 3 subjects plasma doxapram concentrations during and after i. v. infusion agreed with those predicted from pharmacokinetic values obtained from the bolus injection study. Since mean steady-state concentrations (9.9 microgram . ml-1) would be reached only after an extended interval (mean 15.2 h), a variable-rate infusion regimen was calculated to produce and maintain a concentration of 2 microgram . ml-1 from 15--25 min onwards. A regimen in which the infusion rate is reduced step-wise is recommended to achieve early near-constant plasma doxapram concentrations.

[Doxapram. Pharmocokinetic and clinical study]. / Le doxapram. Etude pharmacocinétique et clinique.

Two series of work are reported. The first concerns the pharmacocinetic study of the product after it was administered through different ways and the corresponding evolution of PaCO2. The second study concerns the effect on pulmonary arterial pressure, blood gases and some parameters of ventilatory mechanics, of a perfusion of 200 mg of this product over a two hours period.