Combined Cardiopulmonary Assessments Using Impedance and Digital Implants in Conscious Freely Moving Cynomolgus Monkeys, Beagle Dogs, and Göttingen Minipigs: Pharmacological Characterization and Social Housing Effects.

Respiratory monitoring, using impedance with implanted telemetry in socially housed animals, was not possible until the recent development of digital signal transmission. The objective of this study was to evaluate digital telemetry monitoring of cardiopulmonary parameters (respiratory rate, tidal volume, minute volume, electrocardiography (DII), systemic arterial blood pressure, physical activity, and body temperature) in conscious, single-housed, non-rodent species commonly used in toxicology studies following administration of positive/negative controls (saline, dexmedetomidine, morphine, amphetamine, and doxapram), and also, the effects of various social housing arrangements in untreated female and/or male cynomolgus monkeys, Beagle dogs, and Göttingen minipigs (n = 4 per species). Aggressions were observed in socially housed male minipigs, however, which prevented pair-housed assessments in this species. All tested pharmacological agents significantly altered more than one organ system, highlighting important inter-organ dependencies when analyzing functional endpoints. Stress-related physiological changes were observed with single-housing or pair-housing with a new cage mate in cynomolgus monkeys and Beagle dogs, suggesting that stable social structures are preferable to limit variability, especially around dosing. Concomitant monitoring of cardiovascular and respiratory parameters from the same animals may help reduce the number of animals (3 Rs) needed to fulfill the S7A guidelines and allows for identification of organ system functional correlations. Globally, the data support the use of social housing in non-rodents for safety pharmacology multi-organ system (heart and lungs) monitoring investigations.

Combination acetaminophen and doxapram potentiated hepatotoxicity in mouse primary cultured hepatocytes.

Doxapram-induced potentiation of acetaminophen-induced reductions in cell viability and apoptosis was examined in mouse primary cultured hepatocytes. Loss of viability following exposure of acetaminophen and/or doxapram in cultured hepatocytes was assessed by monitoring [3H]-thymidine incorporation and mitochondrial activity, and apoptosis of hepatocytes was determined by nuclear microscopic observation and from detection of a ladder-like DNA fragmentation pattern in agarose gel electrophoresis. The combination of acetaminophen (5 mM) and doxapram (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased lipid peroxide levels of hepatocytes. Hepatocytes exposed for 24 h to acetaminophen (5 mM) plus doxapram (100 microM) showed atrophy of nuclei including chromatin condensation and a ladder-like DNA fragmentation pattern characteristic of apoptosis. Antioxidant (N-acetylcysteine), iron-chelator (deferoxamine), intracellular calcium ion chelator (quin 2-AM), endonuclease inhibitor (aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitor (3-aminobenzamide) all improved the viability of cells and eliminated the ladder-like DNA fragmentation in cells exposed to acetaminophen plus doxapram. In conclusion, the combination acetaminophen and doxapram potentiated the reduction in cell viability and apoptosis in mouse primary cultured hepatocytes. We suggest that careful observation for hepatotoxicity is recommended when acetaminophen and doxapram are prescribed simultaneously.

Potentiation of acetaminophen hepatotoxicity and mortality by doxapram in mice.

Whether a single dose of doxapram (DOP) can modulate the acute toxicity and the hepatotoxicity induced by acetaminophen (AA) was examined. Pretreatment with DOP (40 mg/kg, i.p.) 30 min prior to the administration of AA dose-dependently potentiated the lethality of AA in both native mice and mice fasted for 18 h, and the potentiating activity was greater in fasted mice than in native mice. The hepatotoxicity of AA was assessed by plasma transaminases activity (glutamyl oxaloacetic transaminase, GOT; glutamyl pyruvic transaminase, GPT) and the amount of plasma lipid peroxides at 6, 12, 18, 24, 36 and 48h after the administration of AA and histopathological examination of liver sections at 24 h after the administration of AA. DOP (40 mg/kg, i.p.) did not increase the plasma transaminase activity or the lipid peroxides level significantly, whereas AA administration to DOP-treated animals produced earlier maximal elevation of transaminase and lipid peroxide values compared to AA alone. These findings indicate that mortality and hepatotoxicity of AA is potentiated by DOP in mice.

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.

Biphasic effect of doxapram on hypnotic activity of pentobarbital in mice.

The effect of doxapram, a respiratory stimulant, on the pentobarbital sleeping time was investigated in mice. The sleeping time induced by the intraperitoneal injection of pentobarbital was prolonged 0--120 min after the administration of doxapram (25-100 mg/kg, i.p.). The pretreatment with doxapram 60 min before had no effect on the anesthetic time induced by ether and on the sleeping time induced by the intracerebroventricular injection of pentobarbital, while increased the lethality of pentobarbital only slightly and the levels of pentobarbital in the plasma and brain significantly. The activities of pentobarbital oxidase and aminopyrine N-demethylase in the 9000 X g supernatant fraction of the liver were inhibited by the pretreatment with doxapram 60 min before the test. On the other hand, 12-24 hr after the injection of doxapram the pentobarbital sleeping time was markedly shortened. Thus, the biphasic effect of doxapram, prolongation at first and shortening later, on the pentobarbital sleeping time was observed. It is possible that doxapram inhibits the hepatic microsomal drug-metabolizing enzymes without an increase in the sensitivity of the central nervous system at first and stimulates these enzymes during the second phase.

A study of naloxone and doxapram as agents for the reversal of neuroleptanalgesic respiratory depression in the conscious rabbit.

The effectiveness of naloxone and doxapram in reversing the respiratory depressant actions of fentanyl and droperidol in the rabbit has been examined. Both drugs did not reverse fully the depression of respiratory frequency produced by the neuroleptanalgesic agents. Doxapram also failed to reverse fully the depression of minute volume produced by fentanyl and droperidol, although naloxone was adequate in this respect. However, analysis of arterialized venous blood showed that both naloxone and doxapram not only prevented the increase in PCO2 caused by fentanyl and droperidol, but caused also a significant decrease. A reduction in PCO2 was seen also when either naloxone or doxapram was given to untreated rabbits. With doxapram this appeared to be a result of pure respiratory stimulation. Naloxone also produced a reduction in standard bicarbonate.

A study of the enhanced toxicity of doxapram in rodents treated with narcotic analgesics.

It has been shown in both mice and rats that the LD50 value for doxapram is reduced in rodents treated with narcotic analgesics. In both species the site of the toxic interaction appears to be the cardiovascular system. Doxapram alone, in sub-lethal doses, appears to cause conduction defects in the heart and this action of doxapram is increased in rodents treated with morphine. The enhancement of the toxicity of doxapram by morphine appears to involve an action in the central nervous system probably not related to respiratory depression.

Interactions between morphine and doxapram in the rabbit and mouse.

Certain actions of doxapram, administered alone and in combination with morphine, have been examined in the rabbit and the mouse. Single doses of doxapram were capable of stimulating respiration in both species. There was an increase in tidal volume in the rabbit and an increase in respiratory rate in the mouse. In both species the duration of action of single doses of doxapram was less than 15 min. In morphine-treated rabbits and mice single doses of doxapram affected neither the time course nor the intensity of the respiratory depression. In the rabbit repeated doses of doxapram did not produce tachyphylaxis with respect to the effect on tidal and minute volumes, and effectively reversed the respiratory depressant actions of morphine. The usefulness of this action must be balanced against the enhanced toxicity of doxapram observed in morphine-treated mice.