Manipulation of Neurotransmitter Levels Has Differential Effects on Formalin-Evoked Nociceptive Behavior in Male and Female Mice.

UNLABELLED: Changes in serotonin (5-hydroxytryptamine; 5-HT), noradrenaline (NA), and γ-aminobutyric acid (GABA) levels in the spinal cord are known to occur in response to nociceptive stimuli, yet little research has examined possible underlying sex differences in these changes and how they might affect nociception. We have used pharmacological approaches in a well established model of tonic nociception, the formalin test, to explore the effects of altering neurotransmitter levels on nociceptive responses in male and female C57BL/6 mice. The monoamine oxidase (MAO) inhibitor phenelzine (PLZ), its metabolite phenylethylidenehydrazine (PEH), and a derivative compound of PLZ, N(2)-acetylphenelzine (N(2)-AcPLZ), were used to increase endogenous levels of: GABA, 5-HT, and NA (PLZ); GABA alone (PEH); or 5-HT and NA only (N(2)-AcPLZ). Although both sexes had a reduction in second phase nociceptive behaviors with PEH pretreatment, the analgesic effect of PLZ was only observed in male mice. High performance liquid chromatography analysis revealed male mice had greater spinal cord increases in 5-HT and NA levels compared with female mice. Female mice, in contrast, had greater increases in GABA levels with pretreatments. With N(2)-AcPLZ pretreatment, only male mice had a reduction in second phase nociceptive behaviors despite similar increases in 5-HT and NA levels in both sexes. These findings suggest that male mice may utilize serotonergic and noradrenergic pathways more efficiently for the attenuation of nociceptive behavior and female mice are more dependent on alternate mechanisms. To our knowledge, these findings are the first on the antinociceptive properties of altering 5-HT, NA, and GABA levels with the MAO inhibitor PLZ and its derivatives in a model of tonic pain processing. They also reveal significant underlying sex differences associated with these treatments. PERSPECTIVE: The present study found that nociception in male and female mice may be regulated by different neurotransmitter systems. These results indicate that different pharmacological approaches may be needed to treat pain in both sexes.

A selective phenelzine analogue inhibitor of histone demethylase LSD1.

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.

Synthesis of N-propargylphenelzine and analogues as neuroprotective agents.

A series of N(1)- and N(2)-propargylphenelzine derivatives and analogues (1-7) was synthesized. In addition to their activity as monoamine oxidase inhibitors, two of the compounds, N(1)- and N(2)-propargylphenelzines (3 and 6), were found to be potent at preventing DSP-4-induced noradrenaline (NA) depletion in mouse hippocampus, suggesting that they have neuroprotective properties.

The antidepressant drug phenelzine produces antianxiety effects in the plus-maze and increases in rat brain GABA.

Research on the effects of antidepressant/ antipanic drugs in animal models of anxiety has yielded equivocal results, even after chronic drug regimens. In contrast, we found that the antidepressant/antipanic drug phenelzine, given acutely, produced a clear anxiolytic effect in the elevated plus-maze, a widely-used animal model of "anxiety" that is primarily sensitive to benzodiazepine-type anxiolytics (e.g., diazepam). Furthermore, the effective dose of phenelzine (15 mg/kg) administered to rats was associated with more than a 2- fold increase in whole brain levels of gamma-aminobutyric acid (GABA), whereas an ineffective dose of phenelzine (5.1 mg/kg) did not significantly change GABA levels. The N-acetylated metabolite of phenelzine, N2-acetylphenelzine, produced neither an anxiolytic effect in the elevated plus-maze nor a significant change in whole-brain levels of GABA. However, both phenelzine and N2-acetylphenelzine potently inhibited monoamine oxidase, a mechanism commonly thought to be involved in the therapeutic effects of monoamine oxidase inhibitors such as phenelzine in the treatment of depression in humans. These results suggest that the mechanism whereby phenelzine produces anxiolytic effects in the plus-maze model is unique to a facilitatory action on brain levels of GABA, in contrast to classical benzodiazepines, which produce anxiolytic effects by enhancing the affinity of the GABAA-receptor for GABA.

Chronic administration of the antidepressant phenelzine and its N-acetyl analogue: effects on GABAergic function.

The MAO inhibitor phenelzine (2-phenylethylhydrazine; PLZ) is used widely in psychiatry for the treatment of depression and panic disorder. Its N-acetyl metabolite, N2-acetylphenelzine (N2AcPLZ) is a reasonably potent nonselective inhibitor of monoamine oxidase (MAO) that causes elevation in brain levels of the biogenic amines. In the studies reported here, PLZ (0.05 mmol/kg/day), N2AcPLZ (0.10 mmol/kg/day) or vehicle were administered to male rats for 28 days s.c. with Alzet minipumps, and their effects on GABAergic function were examined. Whole brain concentrations of gamma-aminobutyric acid (GABA) were significantly elevated in the PLZ but not in the N2AcPLZ-treated group. PLZ was found to inhibit the anabolic enzyme glutamic acid decarboxylase (GAD) and, to a greater extent, the catabolic enzyme GABA transaminase (GABA-T). The results of these investigations suggest that the free hydrazine moiety in PLZ is crucial to producing the elevated levels of GABA, probably through inhibition of GABA-T. Despite the considerable increase in whole brain GABA levels in the PLZ-treated rats, there were no significant differences in GABAA or benzodiazepine receptor binding parameters (KD or Bmax) between the groups as measured using 3H-muscimol and 3H-flunitrazepam in radioligand binding assays.

Chronic administration of the antidepressant-antipanic drug phenelzine and its N-acetylated analogue: effects on monoamine oxidase, biogenic amines, and alpha 2-adrenoreceptor function.

Phenelzine (PLZ), a nonselective monoamine oxidase inhibitor, is widely used in psychiatry for the treatment of panic disorder and depression. The effects of chronic (28-day) administration of PLZ (0.05 mmol/kg/day) and its N-acetylated analogue, 1-acetyl-2-(2-phenylethyl) hydrazine (N2-acetylphenelzine; N2AcPLZ; 0.10 mmol/kg/day), on alpha 2-adrenoreceptor function were investigated by use of a behavioral test on days 21 and 22. Rats treated with PLZ or N2AcPLZ displayed a significant attenuation of the suppressant effects of clonidine on spontaneous locomotor activity, compared with controls; these results suggest a reduced sensitivity of alpha 2-adrenoreceptors. By day 28, both PLZ and N2AcPLZ had produced greater than 85% inhibition of monoamine oxidases A and B in the brain, heart, and liver. Both drugs induced significant elevations of whole-brain levels of noradrenaline, 5-hydroxytryptamine, and dopamine, compared with those in controls. The levels of acid metabolites of dopamine and 5-hydroxytryptamine (homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid) were significantly reduced in both groups of drug-treated animals.

Tissue levels and some pharmacological properties of an acetylated metabolite of phenelzine in the rat.

The metabolic generation of N2-acetylphenelzine by rats treated with phenelzine, and the activity of this metabolite as an inhibitor of monoamine oxidase enzymes in vivo were confirmed. The isomeric amide N1-acetylphenelzine was not a metabolic product of phenelzine and also did not inhibit monoamine oxidase enzymes. Levels of N2-acetylphenelzine in rat blood, after treatment with a dose (0.1 mmol.kg-1) of N2-acetylphenelzine sufficient to inhibit monoamine oxidase enzymes but not to increase brain levels of dopamine or noradrenaline, were higher than those generated metabolically from a higher dose (0.38 mmol.kg-1) of phenelzine which did increase brain levels of these biogenic amines. Metabolically derived N2-acetylphenelzine, therefore, probably does not contribute in any significant way to monoamine oxidase inhibition by phenelzine.

N2-acetylphenelzine: effects on rat brain GABA, alanine and biogenic amines.

The neurochemical properties of N2-acetylphenelzine were compared with those of phenelzine in a rat model. N2-Acetylphenelzine is a relatively potent inhibitor of monoamine oxidase-A and -B and causes increases in whole-brain levels of noradrenaline and 5-hydroxytryptamine, and decreases in homovanillic acid, 5-hydroxyindole-3-acetic acid, and 3,4-dihydroxyphenylacetic acetic after acute i.p. administration of the drug. Phenelzine is a more potent monoamine oxidase inhibitor than is N2-acetylphenelzine. The most marked difference in the profile was that N2-acetylphenelzine had no effect on whole brain levels of the amino acid neurotransmitters alanine and gamma-aminobutyric acid, whereas phenelzine caused dramatic increases. Acetylation of phenelzine at the N2 position presumably interferes with the inhibition of the transaminase enzymes for gamma-aminobutyric acid and alanine.

Studies on the properties of some peripheral MAO inhibitors.

1. Studies were carried out on three monoamine oxidase (MAO) inhibitors, two of which, debrisoquine and para- hydroxyphenelzine, are purported to be peripheral inhibitors and one, phenelzine, is a peripherally acting inhibitor, which has been included for comparitive purposes. 2. All three showed varying degrees of specificity towards MAO type A. 3. The action of debrisoquine was very rapid as was that of para- hydroxyphenelzine. 4. The inhibition caused by debrisoquine was competitive and reversible, while that caused by both phenelzine and para- hydroxyphenelzine was irreversible. 5. The inhibition caused by debrisoquine appeared to be unaffected by the pH of the medium.

Synthesis and pharmacological evaluation of acyl derivatives of phenelzine.

Acyl derivatives of phenelzine were required for pharmacological evaluation. Eight mono- and di-acyl derivatives were synthesized and characterized by gas chromatography, mass spectrometry, nuclear magnetic resonance and infrared spectrophotometry. Selective acylation was observed with both acetic anhydride and ethyl chloroformate. In aqueous medium, monoacylation yielded N1-acetyl- and N1-(ethoxy-carbonyl)-phenelzine exclusively, whereas in non-aqueous medium only N2-acetyl and N2-(ethoxycarbonyl) products were obtained. NMR temperature studies were conducted to ascertain the presence of rotational isomers and their ratios. At room temperature, one ethoxy-carbonyl and four phenelzine acetate derivatives were present as mixtures of rotamers. Preliminary evaluations of the MAO-inhibiting properties of acylated phenelzines indicate that a hydrogen atom on the N1-position of phenelzine and its derivatives is essential for activity.

Recent studies on the MAO inhibitor phenelzine and its possible metabolites.

Although N2-acetylphenelzine (N2AcPLZ) appears to be only a minor metabolite of phenelzine (PLZ), other investigations have demonstrated that it may be worthy of study as an antidepressant in its own right. In the present report, the possibility of ring hydroxylation as a metabolic route for PLZ was investigated in the rat. Indirect evidence for such a route was obtained using iprindole, a drug known to block ring hydroxylation. Treatment of rats with iprindole followed by PLZ was demonstrated to result in increased brain levels of PLZ and beta-phenylethylamine (control rats were treated with vehicle and then PLZ). The possibility that hydroxylation in the para-position might be a metabolic route for PLZ has led to interest in the possible use of analogues in which this position is blocked with a substituent. In preliminary acute studies at a dose of 0.1 mmol/kg p-chloro-PLZ was found to have a similar effect to PLZ on the inhibition of MAO and to lead to an elevation of catecholamines and 5-hydroxytryptamine (5-HT) in rat whole brain.

Metabolic acetylation of phenelzine in rats.

1-Acetyl-2-(2-phenylethyl)hydrazine (N2-acetylphenelzine) is identified as an acetylated metabolite of phenelzine in the rat. One hour after intraperitoneal administration of a high dose of phenelzine sulfate to rats, the blood and brain of the animals were extracted and analyzed by combined gas chromatography/electron impact mass spectrometry in the total ion and selected ion modes. This procedure provided unequivocal proof of the presence of N2-acetylphenelzine in these tissues. The other possible monoacetylated metabolite of phenelzine, 1-acetyl-1-(2-phenylethyl)hydrazine (N1-acetylphenelzine), and the diacetylated derivative, 1,2-diacetyl-2-(2-phenylethyl)hydrazine, were sought, but were not detected.

Potential prodrugs of phenelzine: N2-acetylphenelzine and N2-ethoxycarbonylphenelzine.

This report describes experiments conducted to determine whether simple monoacylated analogues of phenelzine might act as prodrug sources of phenelzine in vivo. The data suggest that hydrolysis to phenelzine may vary between analogues and that not all analogues retain the pharmacological properties of phenelzine.

Involvement of brain trace amines in the behavioural effects of phenelzine.

The MAO inhibitor phenelzine (PLZ) at a dose of 25 mg/kg does not affect the behavior of rats. In contrast, the equivalent dose of a deuterated analog (alpha, alpha, beta, beta-tetradeutero-PLZ, d4PLZ) elicits a biphasic behavioral syndrome in rats. In an attempt to correlate changes in cerebral monoamines with behavior, the concentration of various amines were measured at various times after the administration of either d4PLZ or PLZ (25 mg/kg). In general, PLZ and d4PLZ caused elevations in brain amine levels, particularly in the time period 2-12 hours after drug administration. Furthermore, d4PLZ increased the concentrations of serotonin (5-HT), phenylethylamine (PE), tryptamine (T), meta-tyramine (mTA), and 3-methoxytyramine (3-MT) to a greater extent than PLZ. Since the time course of behavioral excitation closely parallels the elevations in T and PE levels in the brain and since the percentage increases in PE and T levels following d4PLZ compared to PLZ treatment were substantially greater than those of the other amines, it was postulated that PE and T are involved in d4PLZ-induced behaviors.

Microbial metabolism of phenelzine and pheniprazine.

Phenelzine and pheniprazine were used as substrates for metabolic studies with Cunninghamella echinulata and Mycobacterium smegmatis. Metabolites were identified by means of gas-liquid chromatography and mass spectrometry. 1-Acetyl-2-(2-phenylethyl)-hydrazine and 1-acetyl-2-(1-methyl-2-phenylethyl)hydrazine were the major products of C. echinulata metabolism of phenelzine and pheniprazine, respectively. In addition, M. smegmatis produced a second metabolite from each substrate; these metabolites were unequivocally identified as N-acetylphenylethylamine and N-acetylamphetamine from phenelzine and pheniprazine, respectively.

Metabolism and pharmacokinetics of phenelzine: lack of evidence for acetylation pathway in humans.

Earlier studies have suggested that patients with slow acetylation phenotype were more likely to respond to phenelzine treatment and more likely to experience side effects. The metabolism of phenelzine has not been extensively investigated in humans because of limitations in analytical methodology. A labeled form of phenelzine is required for metabolite identification because these same compounds can arise from endogenous substances e.g., phenylacetic acid. The use of a stable isotope-labeled compound has the advantage that in vivo studies can be performed without radiation exposure. Site-specific, stable isotope-labeled phenelzine analogs were synthesized and used in metabolic and pharmacokinetic studies in humans. The authors were unable to detect N-acetylphenelzine in any urine or plasma samples. The major metabolites phenylacetic acid and parahydroxyphenylacetic acid constitute up to 79% of the administered dose excreted via the urine in the first 96 hours. These studies indicate that N-acetylation of phenelzine is not a significant metabolic pathway in humans, which helps explain recent clinical studies that failed to find an association between acetylation phenotype and clinical effects of phenelzine.

Hydrazonopropionic acid, a new class of hypoglycemic substances. 2. Influence of 2-(phenylethylhydrazono)- and 2-(2-cyclohexyl-ethylhydrazono)-propionic acid on redox systems, acid base status and monoamino oxidase activity.

Phenylethylhydrazono-propionic acid (PEHP) and cyclohexyl-ethylhydrazono-propionic acid (CHEHP) has been shown to lower blood glucose concentrations in various laboratory animals after intraperitoneal or oral application. The present study has been conducted to investigate, if these hydrazones may induce lactic acidosis like the biguanides or may cause unwanted side reactions due to an inhibition of monoamino oxidase like the corresponding hydrazines. It was found that intramitochondrial and cytoplasmatic redox ratios were not influenced at a hypoglycemic effective dosage (145 mumol/kg body weight) of these substances in fasted guinea pigs. An effect on the acid base status was not observed. Both substances lowered the concentration of ketone bodies in fasted guinea pigs. In vitro the activity of monoamino oxidase purified from guinea pig liver was much less inhibited by the hydrazones than by one of the corresponding hydrazines (phenelzine). Furthermore CHEHP (145 mumol/kg body weight) did not influence the serum activity of various liver enzymes and the concentration of triglycerides, cholesterol and uric acid in plasma of fasted guinea pigs. It is concluded that CHEHP meets basic requirements for a potential oral antidiabetic agent.

Hydrazonopropionic acids, a class of hypoglycemic substances. 1. Hypoglycemic effect of 2-(phenylethylhydrazono)- and 2-(2-cyclohexyl-ethylhydrazono)-propionic acid.

The two hydrazone-compounds 2-(phenylethylhydrazono)-propionic acid (PEHP) and 2-(2-cyclohexyl-ethylhydrazono)-propionic acid (CHEHP) significantly lowered the blood glucose level in several laboratory animals fasted 48 hours (guinea pigs, mice, hamsters and rats). In the guinea pig, PEHP produced a three times stronger hypoglycemic effect than phenelzine, its corresponding hydrazine. Conversely both hydrazono compounds decreased the monoamine oxidase activity much less, than phenelzine. CHEHP (145 mumol/kg) inhibited this enzyme by less than 14%. After oral administration both hydrazones (200 mumol/kg) also produced a distinct hypoglycemic effect. The blood glucose lowering properties of the two hydrazones were most manifest in fasted guinea pigs, diabetic mice and rats with streptozotozin diabetes.