We previously found that the atypical antipsychotic drugs (APDs) clozapine, olanzapine, quetiapine, and risperidone reduce PC12 cell death induced by hydrogen peroxide, N‐methyl‐4‐phenylpyridinium ion, or β‐amyloid peptide (Aβ25–35). Such neurotoxic substances have in common the capability of causing oxidative stress. Atypical APDs have been used in treating schizophrenia and in treating psychotic symptoms of patients with Alzheimer's disease (AD), in which Aβ is involved by causing oxidative stress. Therefore, we hypothesized that atypical APDs might alleviate oxidative stress in PC12 cells, thus protecting them from apoptosis. PC12 cells were seeded in plates or chambers for 24 hr and cultured for another 24 hr with olanzapine or quetiapine in the medium, and then the cells were cultured in the new medium containing Aβ25–35 and/or olanzapine, quetiapine, but not serum, for various periods. It was shown that cultures treated with olanzapine + Aβ25–35, or quetiapine + Aβ25–35, had significantly higher cell viabilities and lower rates of apoptosis compared with the cultures exposed only to Aβ25–35. In addition, the drugs blocked the activation of caspase‐3 caused by Aβ25–35. Furthermore, olanzapine and quetiapine prevented Aβ25–35‐induced overproduction of intracellular reactive oxygen species, Aβ25–35‐induced decrease in mitochondrial membrane potential, and Aβ25–35‐induced changes in activities of the key antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. In consideration of the wealth of evidence linking oxidative stress to the pathophysiology of schizophrenia and AD, these findings give us a new insight into the therapeutic actions of atypical antipsychotics in patients with the disorders. © 2005 Wiley‐Liss, Inc.

We have demonstrated recently that atypical antipsychotics possess neuroprotective actions in H2O2-mediated and serum-withdrawal models of cell death. In the present study, we compared the ability of atypical and typical antipsychotics to protect against an insult mediated by Abeta(25-35), an apoptogenic fragment of the Alzheimer's disease-related beta-amyloid (Abeta) peptide. Treatment of PC12 cell cultures with Abeta(25-35) did not significantly alter total cellular expression levels of Bax, a proapoptotic Bcl-2 family member, or levels of Bcl-XL, an antiapoptotic analogue. Treatment with Abeta(25-35), however, did result in mitochondrial translocation of Bax, which effectively increased the mitochondrial ratio of Bax to Bcl-X(L). This relative increase in proapoptotic molecules was reduced by pretreatment with atypical (quetiapine and olanzapine) and typical (haloperidol) antipsychotics. We also observed a selective increase in proapoptotic Bcl-XS immunodetection in haloperidol-treated cells, which was evident particularly in the mitochondrial compartment. This increase in proapoptotic molecules may account for the lower neuroprotective potential of haloperidol, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction assay. The disparate neuroprotective effects of atypical and typical antipsychotics/neuroleptics may be due to their respective abilities to regulate pro- and anti-apoptotic protein translocation and expression.

(R)-N-(2-Heptyl)-N-methyl-propargylamine (R-2HMP) and (R)-N-(2-heptyl)-propargylamine (R-2HPA) are analogs of R-deprenyl. R-Deprenyl, a selective monoamine oxidase B inhibitor, is a mechanism-based inactivator of purified CYP2B1. The aim of the present study was to determine whether R-2HMP and R-2HPA behaved like deprenyl with respect to inhibiting cytochrome P450 (CYP450) enzyme activity. The activities of CYP1A2 and CYP1A1 were assessed by measuring the deethylation of 7-ethoxyresorufin by liver microsomes obtained from control and beta-naphthoflavone-treated female Wistar rats, respectively. CYP2B1 activity was assessed by measuring depentylation of 7-pentoxyresorufin by liver microsomes obtained from phenobarbital-treated rats. The activity of CYP1A1 was unaffected by 100 microM concentrations of R-deprenyl, R-2HMP, or R-2HPA. In contrast, the activities of CYP1A2 and CYP2B1 were significantly decreased. In general, the percentage of CYP1A2 activity remaining in the presence of 100 microM of one of these propargylamines ranged from 45 to 56%, whereas 10% or less of CYP2B1 activity remained. No marked differences between the various propargylamines were observed. The IC(50) values for the inhibition of CYP2B1 activity by R-deprenyl, R-2HMP, and R-2HPA were found to be 2.6, 8.5, and 3.6 microM, respectively. The S-enantiomers of deprenyl, 2HMP, and 2HPA also inhibited the activity of microsomal CYP2B1. R-2HMP, R-2HPA, and S-2HPA were found to be mechanism-based inactivators of CYP2B1 activity. The inactivation constants k(inact) and K(I) were found to be as follows: R-deprenyl, 1.3 microM and 0.32 min(-1); R-2HMP, 0.8 microM and 0.08 min(-1); R-2HPA, 0.5 microM and 0.36 min(-1); and S-2HPA, 0.24 microM and 0.18 min(-1).

(R)-N-(2-Heptyl)-N-methylpropargylamine (R-2HMP) is a monoamine oxidase inhibitor and putative antiapoptotic agent analogous to (R)-deprenyl. In the rat, the major amine metabolites of R-2HMP have been identified as (R)-N-2-heptylmethylamine (R-2HMA), (R)-N-2-heptylpropargylamine (R-2HPA), and (R)-2-heptylamine (R-2HA). After R-2HMP was administered s.c. to male Wistar rats, it was observed that the greatest concentration was of the original drug followed in decreasing order by R-2HMA, R-2HPA, and R-2HA in brain, liver, and plasma at all times after administration. The greatest concentrations of the three metabolites were found in brain followed by liver and plasma, and the peak concentrations occurred between 15 and 30 min after administration. After oral administration, the liver contained the greatest concentrations of drug and metabolites, and, again, the peak concentrations occurred at about 15 min. In all cases, depropargylation appears to occur at a faster rate than demethylation. After s.c. administration, R-2HMP and its metabolites exhibited biexponential redistribution and elimination losses. Half-lives of the compounds in brain for the redistribution phase were: R-2HMP, 10 min; R-2HMA, 11 min; R-2HPA, 16 min; and R-2HA, 15 min.

Two series of drugs, the aliphatic-N-methyl propargylamines and the aliphatic propargylamines, have been synthesised and shown to be specific, irreversible, and potent monoamine oxidase B inhibitors and neural rescue agents. In the latter case, an absolute stereochemical requirement for the R isomer exists. Both series of compounds have been shown, in numerous in vitro and in vivo experimental paradigms, to be effective neuronal rescue agents. Candidates from both series exhibit excellent bioavailability and pharmacokinetics and offer opportunities for treating neurodegenerative disorders and stroke and cognitive decline in companion animals. (C) 1997 Wiley-Liss, Inc.

The types of aldehyde dehydrogenases (ALDH) present in human hair roots and in saliva were investigated. ALDH was detected by activity staining following separation of crude extracts by isoelectric focusing. Hair roots were found to express ALDH1, ALDH2, ALDH3, and ALDH4, whereas saliva expressed ALDH3. Two different patterns of ALDH3 were detected in hair roots collected from 42 donors, 40 expressed one pattern (variant I) and two another pattern (variant II) of activity staining. The variant I pattern of hair root ALDH3 changed with repetitive freezing and thawing of the sample, whereas the variant II pattern was stable. In contrast to hair root ALDH3, all patterns of ALDH3 activity in saliva were stable. The patterns of ALDH3 activity present in human hair roots that had been frozen and thawed twice matched those present in saliva collected from the same individual. Three polymorphisms of ALDH3 (variants I, II, and III) were detected in the 33 saliva samples analyzed. Variants I and II were inherited in each of three generations of a 10-member family.

The effects of the administration of selective and non-selective inhibitors of monoamine oxidase (MAO) on the concentrations of three trace acid metabolites [phenylacetic acid (PAA); m-hydroxyphenylacetic acid (mHPAA); and p-hydroxyphenylacetic acid (pHPAA)] and of an acid metabolite of dopamine [3,4-dihydroxyphenylacetic acid (DOPAC)] in the rat striatum were determined. Administration of brofaromine (1−100 mg/kg, s.c.) a type AMAO inhibitor, dose-dependently decreased DOPAC and mHPAA levels. pHPAA levels were decreased by 100 mg/kg brofaromine, but PAA levels were unaffected. Doses of deprenyl of less than 100 mg/kg, i.p., had no effect on any of the acids, while 100 mg/kg decreased DOPAC, mHPAA and pHPAA but not PAA levels. Clorgyline, pargyline and tranylcypromine treatment decreased the levels of DOPAC, mHPAA and pHPAA but not PAA. Administration of α-monofluoromethyldopa, an inhibitor of aromatic amino acid decarboxylase, decreased the levels of all four acids. It was concluded that deamination of the respective parent amine by type A MAO is primarily responsible for the synthesis of DOPAC and mHPAA, but that another pathway contributes to pHPAA synthesis. It appears that either PAA arises predominantly independently from the actions of MAO or that its removal via transport or further metabolism regulates its concentration.

Three methods were employed to assess whether human volunteers (Caucasian, Asian or Cree Indian) possessed the typical or atypical mitochondrial aldehyde dehydrogenase (ALDH2) isozyme. These methods were: (1) questioning individuals about facial flushing responses following alcohol consumption; (2) application of the ethanol skin patch test, and (3) direct analysis using isoelectric focusing and activity staining of ALDH activity in hair root samples. The results from the three methods were in good agreement and revealed that only the typical ALDH2 isozyme was expressed in Saskatchewan Cree Indians. In agreement with previous reports, the typical ALDH2 was expressed in the Caucasian group of subjects, while both the typical and atypical forms were expressed in the Asian subjects.

The effects of brofaromine, clorgyline (reversible and irreversible type A MAO inhibitors, respectively) and tranylcypromine (non-selective MAO inhibitor) on rat striatal levels of phenylethylamine, tryptamine, mtyramine and p-tyramine were determined. Brofaromine and clorgyline increased m- and p-tyramine levels, but not phenylethylamine levels. Brofaromine given at a dose of 100 mg/kg did increase tryptamine levels. Tranylcypromine increased the levels of all four amines greatly. The effects of chronic treatment with brofaromine on amine levels were not different from those following acute treatment. By contrast, chronic treatment with clorgyline caused greater increases in striatal m- and p-tyramine levels than did acute clorgyline. These data show that changes in the rat striatal levels of m-tyramine and p-tyramine may be used as in vivo indicators of the selectivity and reversiblity of inhibition of type A MAO, while tryptamine levels reflect non-selective inhibition of both types of MAO.

The types of isozymes of aldehyde dehydrogenase (ALDH) present in human lymphocytes has been investigated using isoelectric focusing of polyacrylamide gels followed by substrate-specific staining. Lymphocytes obtained from most individuals were found to contain both types I and II ALDH. This group of 'typical' individuals reported that they did not develop marked facial flushing or rapid heart rate after drinking alcohol nor did they develop an erythema to cutaneously applied ethanol. Lymphocytes obtained from 'atypical' individuals who do suffer from alcohol-induced flushing and rapid heart rate and who developed erythema to cutaneous ethanol displayed type II, but not type I, ALDH. Lymphocytes thus appear to be an easily accessible and suitable tissue for determining type I ALDH phenotype.

Phosphoinositide hydrolysis was studied in slices of rat striatum and frontal cortex which had been incubated with [(3)H]inositol to prelabel the inositol phospholipids. Dopamine (100 ?M to 10 mM) increased phosphoinositide hydrolysis to a maximum of about 200% compared to control in both areas. Noradrenaline (1 ?M to 1 mM) stimulated [(3)H]inositol phosphate formation to about 400% of control. Dopamine-stimulated phosphoinositide hydrolysis was completely blocked by prazosin; while spiperone and SCH 23390 were partial inhibitors. The ability of noradrenaline (5 to 100 ?M) to stimulate phosphoinositide hydrolysis was antagonized by co-incubation with dopamine (1-10 mM). Low concentrations of dopamine (10 nM and 1 ?M) did not affect total [(3)H]inositol phosphate formation, and ion exchange chromatography of the [(3)H]inositol phosphates failed to show any inhibitory effects on the individual fractions (mono-, bis- and tris-phosphates). Ten mM dopamine, on the other hand, increased the production of [(3)H]inositol mono- and bis-phosphates compared to control. It was concluded that dopamine acts as partial ?(1)-agonist in both the rat striatum and frontal cortex. As such, it increased phosphatidylinositol hydrolysis. Dopamine partially inhibited noradrenaline-stimulated phosphatidylinositol hydrolysis, but it did not inhibit basal rates of phosphatidylinositol hydrolysis.

The basal and 50 mM K+-stimulated release of m-tyramine (mTA), p-tyramine (pTA), tryptamine (TR) and phenylethylamine (PE) from striatal slices obtained from rats pretreated with a monoamine oxidase inhibitor (MAOI) was investigated. A K+-stimulated release of mTA and pTA was observed, but K+ did not stimulate either TR or PE release. The latter two amines, therefore, are unlikely to be conventional neurotransmitters in the rat striatum. The release of endogenous striatal pTA from control rats was also investigated. Veratridine stimulated endogenous pTA release, but 50 mM K+ did not. It is possible, therefore, that endogenous pTA can be release iin a transmitter-like fashion.

In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of α1-adrenergic receptors, for example, increases polyphos-phoinositide turnover, but stimulation of α2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inositol and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an α2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of α2, 5HT2, H2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation.

A number of inhibitors of L-aromatic amino acid decarboxylase (AAD) and monoamine oxidase (MAO) were tested to determine whether they also inhibited tyrosine aminotransferase (TAT). The AAD inhibitors carbidopa, NSD-1015, NSD-1034 and Ro4-5127 inhibited liver TAT. Carbidopa inhibited brain AAD and liver TAT equally well. In contrast, other AAD inhibitors (Ro4-4602 and alpha-monofluoromethyldopa) did not inhibit TAT. Phenelzine, an MAO inhibitor, inhibited liver TAT, but other MAO inhibitors (tranylcypromine and isocarboxazid) did not. Systemic administration of those drugs that were found to be inhibitors of TAT in vitro caused significant increases in rat brain p-tyrosine levels.

To determine whether α,α-dideutero substitution in the side chain of the tryptamine molecule can exert primary isotope effects and enhance its bioavailability, equimolar mixtures of tryptamine (T) and either α,α-dideutero-tryptamine (α,α-[2H2]T) or β,β-dideutero-tryptamine (β,β-[2H2]T) were injected i.p. into rats. The amounts of these amines in the brain, liver and plasma were then measured at various times following the injection, and ratios between the deuterated T and T were computed. The ratio remained close to unity in plasma, but exceeded unity in the liver and brain when α,α-[2H2]T and T were injected; however, when β,β-[2H2]T and T were injected, the ratios were unity in all cases at all times. In the presence of a monoamine oxidase inhibitor, the relative enrichment of α-,α-[2H2]T compared to T was reduced. It is concluded that α,α-dideutero substitution exerts a primary isotope effect during oxidative deamination so that much more of this amine penetrates into, and persists in, the brain.

The concentration of p-tyramine in the rat striatum was increased significantly by intraperitoneal injection of phenelzine (5 or 100 mg/kg). Unlike other monoamine oxidase (MAO) inhibitors, phenelzine had no effect on p-tyramine levels in the first 1-2 h following injection. The high dose of phenelzine increased the p-tyramine levels much more than the low dose. In addition, the high dose of phenelzine increased striatal p-tyrosine levels significantly 12 h after injection. Further studies showed that phenelzine inhibited the tyrosine aminotransferase activity of rat liver homogenates; the IC50 was 50 microM. Phenelzine also inhibited the aromatic L-amino acid decarboxylase activity of rat brain homogenate with an IC50 of 25 microM. Following intraperitoneal injection of 100 mg/kg phenelzine, the initial concentration of phenelzine in the striatum appears to be high enough to inhibit aromatic L-amino acid decarboxylase. It is suggested that the multiple enzyme inhibition caused by administration of high doses of phenelzine accounts for its unusual effects on striatal p-tyramine levels compared with other MAO inhibitors, i.e., its initial lack of effect on p-tyramine levels followed later by very large increases in p-tyramine levels.

Abstract: β‐Phenylethylamine (PE) hydrochloride injected intraperitoneally into rats was distributed evenly throughout the various regions of rat brain. Similarly, when a mixture of PE and α, α, β, β‐deuterated PE ([2H4]PE) was injected, no regional differences were observed in the ratios of the amounts of [2H4]PE and PE present; however, significantly more [2H4]PE than PE was present, although a 1:1 mixture had been administered. Further experiments in which the amounts of [2H4]PE and PE in whole rat brain, liver, and plasma were quantified confirmed this finding. The maximum [2H4]PE‐to‐PE ratios observed were 67 in whole brain 1 h after injection and 8 in liver and in plasma 45 min after injection. The whole brain [2H4]PE‐to‐PE ratios were decreased by pargyline pretreatment. Subsequent experiments showed that more α, α‐[2H4]PE than PE was present in whole brain, liver, and plasma of rats injected with an equimolar mixture of α, α‐[2H4]PE and PE. In contrast, β, β‐[2H4]PE was not enriched in comparison to PE under the same experimental conditions. We concluded that the basis for the enrichment of [2H4]PE and α, α‐[2H4]PE compared to PE was due to protection of the deuterated analogs from the actions of monoamine oxidase and perhaps aldehyde dehydrogenase; this protection led to pronounced deuterium substitution effects in vivo especially in the brain.

To determine whether the monoamine oxidase inhibitor phenelzine was metabolized in vivo to produce β-phenylethylamine (PE) and p-hydroxy-β-phenylethylamine [p-tyramine (pTA)], a deuterated analogue, α,α,,β,β-2H-phenelzine (d4-phenelzine) was synthesized and injected i.p. into rats. In the first experiment, rat striata from d4-phenelzine-treated rats were analyzed for the presence of d4-PE and d4-pTA at a time at which phenelzine was known to cause particularly large increases in striatal pTA. While d4-PE was found to be present in these rat striata at a concentration equivalent to the endogenous PE, no d4-pTA was present. The amounts of d4-PE produced at various times after the i.p. injection of 50 mg/kg d4-phenelzine were measured; at 1 hr post-injection, 371 ± 60, 1295 ± 682 and 1242 ± 394 ng/g (mean ± S.E.M.) d4-PE were present in whole brain, liver and kidney. Rat urine collected for a 24-hr period after this treatment contained (mean ± S.E.M.) 88.5 ± 14.0 μg d4-PE. These results clearly indicate that the antidepressant phenelzine was metabolized in vivo to produce the trace amine PE.