Alternative method of oral administration by peanut butter pellet formulation results in target engagement of BACE1 and attenuation of gavage-induced stress responses in mice.

Development of novel therapeutic agents aimed at treating neurodegenerative disorders such as Alzheimer's and Parkinson's diseases require chronic and preferentially oral dosing in appropriate preclinical rodent models. Since many of these disease models involve transgenic mice that are frequently aged and fragile, the commonly used oro-gastric gavage method of drug administration often confounds measured outcomes due to repeated stress and high attrition rates caused by esophageal complications. We employed a novel drug formulation in a peanut butter (PB) pellet readily consumed by mice and compared the stress response as measured by plasma corticosterone levels relative to oral administration via traditional gavage. Acute gavage produced significant elevations in plasma corticosterone comparable to those observed in mice subjected to stress-induced hyperthermia. In contrast, corticosterone levels following consumption of PB pellets were similar to levels in naive mice and significantly lower than in mice subjected to traditional gavage. Following sub-chronic administration, corticosterone levels remained significantly higher in mice subjected to gavage, relative to mice administered PB pellets or naive controls. Furthermore, chronic 30day dosing of a BACE inhibitor administered via PB pellets to PSAPP mice resulted in expected plasma drug exposure and Aß40 lowering consistent with drug treatment demonstrating target engagement. Taken together, this alternative method of oral administration by drug formulated in PB pellets results in the expected pharmacokinetics and pharmacodynamics with attenuated stress levels, and is devoid of the detrimental effects of repetitive oral gavage.

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase.

BACKGROUND AND PURPOSES: Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH: Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS: In male rats with tMCAO, HYDAMTIQ (0.1-10 mg·kg(-1)) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS: Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.

Design, synthesis, SAR, and biological evaluation of highly potent benzimidazole-spaced phosphono-alpha-amino acid competitive NMDA antagonists of the AP-6 type.

A series of 2-amino-(phosphonoalkyl)-1H-benzimidazole-2-alkanoic acids was synthesized and evaluated for NMDA receptor affinity using a [3H]CPP binding assay. Functional antagonism of the NMDA receptor complex was evaluated in vitro using a stimulated [3H]TCP binding assay and in vivo by employing an NMDA-induced seizure model. Several compounds of the AP-6 type demonstrated potent and selective NMDA antagonistic activity both in vitro and in vivo. In particular, [R(-)]-2-amino-3-(5-chloro-1-phosphonomethyl-1H-benzoimidazol-2-yl)-propionic acid (1) displayed an IC(50) value of 7.1 nM in the [3H]CPP binding assay and an ED(50) value of 0.13 mg/kg (ip) in the NMDA lethality model. Compound 1, when administered intravenously as a single bolus dose of 3 mg/kg following permanent occlusion of the middle cerebral artery in the rat, reduced the volume of infarcted brain tissue by 45%. These results support a promising therapeutic potential for compound 1 as a neuroprotective agent.

Characterization of transient focal ischemia-induced increases in extracellular glutamate and aspartate in spontaneously hypertensive rats.

Using middle cerebral artery occlusion (MCAO) and in vivo microdialysis, we have evaluated the changes in extracellular concentrations of the excitatory amino acids (EAA) glutamate and aspartate during varying periods of MCAO (0, 30, 60 min) in the striatum of spontaneously hypertensive rats (SHR). A positive correlation between occlusion time-dependent elevations in EAAs and the resulting ischemic injury was observed. This is the first demonstration of the temporal profile of EAA efflux during transient focal ischemia in SHRs. Possible sources and mechanisms of ischemia-induced EAA efflux were examined during 60 min of MCAO. Removal of Ca(2+) from the microdialysis infusion media significantly attenuated ischemia-induced increases in both glutamate (from ischemic peak of 4892 +/- 1298 to 1144 +/- 666% of preischemic values) and aspartate (from 2703 +/- 682 to 2090 +/- 599% of preischemic values). Similarly, infusion of the voltage dependent Na(+) channel blocker tetrodotoxin (TTX; 10 microM) significantly attenuated MCAO-induced increases in glutamate (to 1313 +/- 648%) and aspartate (to 359 +/- 114%). Infusion of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK; 1 mM) also significantly attenuated the ischemia-induced increases in both EAAs (1285 +/- 508 and 1366 +/- 741% of the preischemic levels, respectively). These results indicate that during transient focal ischemia the increase in extracellular EAAs originates from both the neuronal pool, via conventional exocytotic release, and glial sources via the reversal of the GLT-1 transporter.

Design and synthesis of [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)-ethyl]phosphonic acid (EAA-090), a potent N-methyl-D-aspartate antagonist, via the use of 3-cyclobutene-1,2-dione as an achiral alpha-amino acid bioisostere.

The diazabicyclic amino acid phosphonate 15, [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]phosphonic acid, was identified as a potent NMDA antagonist. It contains the alpha-amino acid bioisostere 3,4-diamino-3-cyclobutene-1,2-dione and an additional ring for conformational rigidity. Compound 15 was as potent as CGS-19755 (5) in the [3H]CPP binding assay, the stimulated [3H]TCP binding assay, and the NMDA-induced lethality model in mice. A single bolus dose of compound 15, administered intravenously following permanent occlusion of middle cerebral artery (MCA) in the rat, reduced the size of infarcted tissue by 57%. Structure-activity relationship (SAR) studies have indicated that the six- and eight-membered ring derivatives had diminished activity and that the two-carbon side chain length was optimum for NMDA receptor affinity. Substitution on the ring was found to be counterproductive in the case of sterically demanding dimethyl groups and of no consequence in the case of an H-bonding hydroxyl group. Replacement of the phosphonic acid group by either a carboxylic acid or a tetrazole group was unproductive. The potent bicyclic NMDA antagonists were synthesized efficiently by virture of their achiral nature and the ease of vinylgous amide formation from squaric acid esters. Compound 15, being a unique NMDA antagonist structural type with a favorable preclinical profile, may offer advantages over existing NMDA antagonists for the treatment of neurological disorders such as stroke and head trauma. Compound 15 is currently under clinical evaluation as a neuroprotective agent for stroke.

Effect of hydroperoxy fatty acids on acylation and deacylation of arachidonoyl groups in synaptic phospholipids.

The effect of hydroperoxy fatty acids on reactions involved in the acylation-deacylation cycle of synaptic phospholipids was studied in vitro, using nerve ending fraction isolated from rat forebrain. 15-Hydroperoxyeicosatetraenoic acid (15-HPETE), 13-hydroperoxylinoleic acid (13-HP 18: 2), and hydroperoxydocosahexaenoic acid (22:6 Hpx), at 25 microM final concentration, all inhibited the incorporation of [1-14C]arachidonate into synaptosomal phosphatidylinositol (PI), phosphatidylcholine (PC), and triacylglycerides by 50-80%. The lowest effective concentration of 15-HPETE and 13-HP 18:2 resulting in significant inhibition of the reacylation of PI was 5 microM, whereas the inhibition of [1-14C]arachidonate incorporation into PC required 10 and 5 microM hydroperoxy fatty acids, respectively. Cumene hydroperoxide and tert-butyl hydroperoxide at concentrations of 100 microM did not inhibit reacylation of PI and PC. Synthesis of labeled arachidonoyl-CoA from [1-14C]arachidonate was decreased by about 50% by 25 microM hydroperoxy fatty acids both in synaptosomes and in the microsomal fraction. Use of [1-14C]arachidonoyl-CoA as a substrate, to bypass the fatty acid activation reaction, revealed that activity of acyltransferase was not affected significantly by 25 microM 15-HPETE and 13-HP 18:2. At the same time, however, the hydrolysis of labeled arachidonoyl-CoA was substantially enhanced. Exposure of synaptosomes to 25 microM fatty acid hydroperoxides did not affect significantly the endogenous concentrations of five major free fatty acids. It is concluded that (1) among synaptic phospholipids, reacylation of PI and PC is the most susceptible to the inhibitory action of fatty acid hydroperoxides, and (2) the enzymes affected by these compounds in nerve endings are arachidonoyl-CoA synthetase and hydrolase.

Transport of asparagine by rat brain synaptosomes: an approach to evaluate glutamine accumulation.

Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 microM and a Vmax value of 3.7 nmol/mg of protein/min at 28 degrees C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas alpha-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparagine was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20-30, and this value was reduced to 5-6 by withdrawal of sodium or addition of high [KCI]. A plot of log [Na+]o/[Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to I, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [Gln]i maintained by the brain in vivo.

Neuronal glutamine utilization: glutamine/glutamate homeostasis in synaptosomes.

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.

Neuronal glutamine utilization: pathways of nitrogen transfer studied with [15N]glutamine.

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.

Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes.

Crude striatum synaptosomes (P2 fraction) from Fischer 344 female rats were incubated in the presence of ADP-chelated Fe3 (0.5-50 microM) and ascorbate (250 microM). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by 14CO2 evolution from L-[1-14C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe3./ascorbate was found with 50% inhibition occurring at 2.5 microM Fe3 concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe3 in the absence of exogenous ascorbate was effective only above 25 microM. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P2 actions using sucrose gradients. Membrane microviscosity of the purified synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagnetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe3./ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulfhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulfhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulfhydryl content. It is concluded that iron-induced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.

Lipid hydroperoxides inhibit reacylation of phospholipids in neuronal membranes.

The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1-14C]arachidonic acid into synaptosomal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration-dependent manner, with 50% inhibition occurring at less than 25 microM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non-competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.

Glucose and synaptosomal glutamate metabolism: studies with [15N]glutamate.

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.

Adriamycin-induced lipid peroxidation in mitochondria and microsomes.

The effect of the anti-neoplastic agent adriamycin on the peroxidation of lipids from rat liver and heart mitochondria and rat liver microsomes was investigated. The extent of total lipid peroxidation was determined by assaying for malondialdehyde (MDA), while the degradation of unsaturated fatty acids was monitored using gas chromatography. For liver mitochondria and microsomes, the formation of MDA was dependent on the concentrations of adriamycin, Fe3+, and protein, as well as time. In the presence of 50 microM adriamycin and saturating amounts of NADH, 1.5 +/- 0.2 nmol MDA/mg protein/60 min was produced with liver mitochondria. Upon addition of 25 microM Fe3+, the amount of MDA generated was increased to 6.5 +/- 0.1 nmol/mg protein/60 min. Liver microsomes produced amounts which were approximately 2-fold higher under all conditions. No MDA formation could be detected in rat heart mitochondria. The addition of 50 microM chlorpromazine completely inhibited peroxidation, whereas 0.5 to 1.0 mM p-bromophenacyl bromide blocked MDA formation by 50%. Analysis of fatty acids by gas chromatography showed that there was about a 50% decrease in arachidonic and docosahexaenoic acids in liver mitochondria and microsomes, but no change in the fatty acid content of heart mitochondria when incubated with both 50 microM adriamycin and 25 microM Fe3+ for 1 hr. These results suggest that (1) therapeutic concentrations of adriamycin enhance the peroxidation of lipids in liver mitochondria and microsomes through an enzymatic mechanism, especially in the presence of Fe3+; and (2) toxicity of this drug may be related to the degradation of membrane lipids.

Involvement of sialic acid in high-affinity uptake of dopamine by synaptosomes from rat brain.

The high-affinity, sodium-dependent uptake of dopamine (DA) was inhibited by the pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. The inhibition was of a non-competitive type, resulting in a 40% decrease of Vmax. Neither basal nor depolarization-stimulated release of DA was affected. Treatment of synaptosomes with neuraminidase caused a 48% loss of sialic acid from the lipid-bound pool and a 80% decrease in the protein-bound fraction. The inhibition of DA uptake was found to be related linearly to the loss of sialic acid from the protein pool. It is postulated that a sialic acid moiety is involved in DA transport across the synaptosomal membrane.

Role of sialic acid in synaptosomal transport of amino acid transmitters.

Active, high-affinity, sodium-dependent uptake of gamma-aminobutyric acid and of the acidic amino acid D-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the gamma-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake.

Regional lipid peroxidation in rat brain in vitro: possible role of endogenous iron.

Lipoperoxidative capacity of various brain areas of aging rats was examined in vitro using the thiobarbituric acid test. Significant regional differences in the generation of lipid peroxides were found in freshly prepared homogenates from different areas of brain incubated under air. Incubation under oxygen resulted in marked stimulation of lipid peroxidation, with highest increases in hypothalamus (144%). Addition of exogenous Fe2+ and ascorbic acid resulted in stimulation of lipid peroxidation ranging from 10-fold in cortex to 20-fold in hypothalamus homogenates during incubation in air. A linear relationship was found between endogenous iron content in brain regions and their ability to produce lipid peroxides in vitro under oxygen for all areas except striatum. Several iron chelating agents effectively inhibited lipid peroxidation under hyperbaric oxygen whereas oxygen-free radical scavengers, as well as catalase and superoxide dismutase were not effective. It is concluded that regional differences in lipoperoxidative capacity of brain areas in vitro are in part governed by local endogenous iron content and may indicate regional susceptibility to oxidative damage.

Pharmacokinetics of paraldehyde disposition in the neonate.

The pharmacokinetic parameters controlling paraldehyde elimination were determined in nine infants infused with paraldehyde at the rate of 150 mg/kg/hr in a 5% solution in 5% dextrose for the treatment of status epilepticus. The mean +/- SEM values for the observed parameters were as follows: rate constant for the disposition of paraldehyde 0.0680 +/- 0.0071 hr,-1 half-life 10.2 +/- 1.0 hr; volume of distribution 1.73 +/- 0.20 L/kg; clearance 0.121 +/- 0.023 L/hr/kg. Phenobarbital administration prior to or within 24 hours of the cessation of paraldehyde infusion decreased both paraldehyde clearance and volume of distribution in a manner linearly related to the logarithm of the phenobarbital dose. The rate constant for paraldehyde elimination was decreased as a linear function of the logarithm of the combined dose of administered phenobarbital and phenytoin. No acetaldehyde was detected in any blood samples. Paraldehyde administration was not correlated with any adverse reactions or toxicities.

Metabolism of paraldehyde to acetaldehyde in liver microsomes. Evidence for the involvement of cytochrome P-450.

A concentration-dependent acetaldehyde (AcH) generation was observed when paraldehyde was incubated with the mouse liver microsomal fraction. The process, which exhibited a requirement for oxygen and NADPH and was inhibited by carbon monoxide, was found to have a Km of 17.9 mM with respect to paraldehyde and a Vmax of 40.1 nmoles/mg protein/min with respect to AcH formation. NADH was much less effective as an electron donor than NADPH, though a more than additive increase in AcH generation was observed when both of these nucleotides were added to the incubation. The rate of microsomal AcH generation from paraldehyde was increased 2.5-fold by pretreatment of the mice with phenobarbital but only 0.6-fold by pretreatment with 3-methylcholanthrene. Pretreatment with 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) resulted in 54% inhibition of the reaction rate. Addition of metopirone to the incubation inhibited AcH generation in a concentration-related fashion, the inhibition being greatest, proportionately, in microsomes from phenobarbital-pretreated animals. The above results conclusively indicate the involvement of cytochrome P-540 mixed function oxidase in the formation of AcH from paraldehyde by mouse liver microsomes. It is also postulated that this process may be accomplished in the reaction analogous to O-dealkylation.

Metabolism of [14C]paraldehyde in mice in vivo, generation and trapping of acetaldehyde.

The metabolic fate and the kinetics of paraldehyde metabolism after the i.p. administration of a 400 mg/kg dose of this agent were investigated in mice. Paraldehyde was found to have a biologic half-life in this species of 41.5 min, its disappearance from blood being governed by a single component exponential process with a rate constant of 0.0167 min-1. By using [14C]paraldehyde, it was found that the major process responsible for paraldehyde disappearance was its metabolic degradation to carbon dioxide, a two-step process; the first step of which was inhibited by pretreatment with SKF-525A. The rate constants for the two steps being 0.0121 and 0.0212 min-1, respectively; on the basis of these rate constants it was calculated this pathway would account for 72.3% of the administered dose at infinite time. A second major pathway for the disposition of paraldehyde was its excretion in expired air, which, at infinite time, would account for 9.6% of the dose. No acetaldehyde (AcH) could be detected in either the breath or the blood of mice after paraldehyde administration. Pretreatment with the aldehyde dehydrogenase inhibitors, pargyline or cyanamide, did not result in the accumulation or excretion of detectable amounts of AcH. Pretreatment of mice administered [14C]paraldehyde with both cyanamide and D-penicillamine,, an AcH sequestering agent, resulted, however, in urinary excretion of the 14C-labeled condensate of D-penicillamine and AcH showing AcH to be formed from paraldehyde. The above results indicate that paraldehyde is rapidly metabolized in vivo to carbon dioxide and that AcH is an intermediary product in this process.