Ildikó Miklya

Enhanced catecholaminergic and serotoninergic activity in rat brain from weaning to sexual maturity: Rationale for prophylactic (-)deprenyl (selegiline) medication

Food deprived rats in the late developmental phase of life (2 months of age) are significantly more active than those in the early postdevelopmental phase (4 months of age), pointing to enhanced catecholaminergic activity during the developmental phase. We therefore measured the resting release of dopamine from the striatum, substantia nigra and tuberculum olfactorium, and of noradrenaline from the locus coeruleus, as an indicator of the basic activity of catecholaminergic neurons in the brain, in 2,4,8,16 and 32 weeks old male and female rats. We also measured the release of serotonin from the raphe. Both in male and female rats, the resting release of transmitters from brain catecholaminergic and serotoninergic neurons between weaning and the end of the 2nd month of age, i.e. during the crucial developmental phase of their life, was significantly higher than either before or after that period, signalling a transition from a developmental to a postdevelopmental (aging) phase of life and indicating that safe and effective measures are needed to maintain the catecholaminergic system at a higher activity level during the postdevelopmental phase. Daily administration of low doses (0.01-0.25 mg/kg) of (-)deprenyl for 21 days significantly enhances the resting release of catecholamines and diminishes that of serotonin, providing a rationale for prophylactic medication with this drug during the postdevelopmental lifespan. We also show that (-)methamphetamine, the parent compound of (-)deprenyl and (-)1-phenyl-2-propylaminopentane (PPAP), a deprenyl analogue free of MAO-B inhibitory potency but otherwise possessing the same pharmacological profile as (-)deprenyl, act similarly, furnishing direct evidence that enhancement of catecholaminergic activity in the brain by multiple, small dose administration of (-)deprenyl is unrelated to MAO-B inhibition.

Phenylethylamine and tyramine are mixed-acting sympathomimetic amines in the brain.

On the helical strip of a capacitance vessel, the pulmonary artery of the rabbit, phenylethylamine (PEA) and tyramine act solely via displacement of noradrenaline from their storage sites and this effect is inhibited by desmethylimipramine (DMI). In contrast, on a resistance vessel, the perfused central ear artery of the rabbit, PEA enhances stimulation induced contractions in 0.2-0.8 microgram/ml concentration [catecholaminergic activity enhancer (CAE) effect], and increases smooth muscle tone (noradrenaline displacing effect) in 4-6 micrograms/ml concentration. This latter effect only is blocked by DMI. Tyramine acts similarly and is more potent than PEA. On the isolated brain stem PEA, tyramine and (-)methamphetamine are, in the presence of cocaine and DMI, highly potent enhancers of stimulation induced release of 3H-noradrenaline, 3H-dopamine and 3H-serotonin. Compounds with specific CAE effect in the brain, (-)deprenyl and 1-phenyl-2-propylaminopentane [(-)PPAP], antagonize tetrabenazine-induced depression of performance of rats in the shuttle box. PEA and tyramine, which are rapidly metabolized in vivo, are ineffective in this test up to 40 mg/kg, whereas (-)methamphetamine, the stable PEA derivative, is highly effective. Compounds with CAE effect enhance at low concentrations the slow inward Ca2+ current in the sino-auricular fibers of the frog heart and inhibit it in high concentration. PEA and tyramine enhance Ca2+ influx from 0.05 to 4 micrograms/ml and inhibit it in 8 micrograms/ml. In conclusion, PEA and tyramine stimulate primarily coupling of action potential to transmitter release in the catecholaminergic neurons in the brain and displace catecholamines in higher concentration only.

(−)Deprenyl and (−)1-phenyl-2-propylaminopentane, [(−) PPAP] act primarily as potent stimulants of action potential — transmitter release coupling in the catecholaminergic neurons

The activity of the catecholaminergic neurons in the rat brain is enhanced significantly 30 min after the subcutaneous injection of very small doses of (-)deprenyl (threshold doses: 0.01 mg/kg for noradrenergic neurons and 0.025 mg/kg for dopaminergic neurons). As a catecholaminergic activity enhancer (CAE) substance (-)deprenyl is about ten times more potent than its parent compound, (-)methamphetamine. While the (+)methamphetamine is 3-5 times more potent than (-)methamphetammine in releasing catecholamines, the (-)methamphetamine is the more potent CAE substance. The mechanism of the CAE effect of (-)deprenyl and (-)PPAP, a deprenyl-derived substance devoid of MAO inhibitory potency, was studied in rats by measuring: a) the release of catecholamines from striatum, substantia nigra, tuberculum olfactorium and locus coeruleus; b) the stimulation induced release of 3H-noradrenaline from the isolated brain stem; and c) the antagonistic effect against tetrabenazine-induced depression of learning in the shuttle box. The CAE effect was found to be unrelated: a) to the inhibition of MAO activity; b) to the inhibition of presynaptic catecholamine receptors; c) to the inhibition of the uptake of catecholamines; and d) to the release of catecholamines. It was concluded that (-)deprenyl and (-)PPAP act primarily as potent stimulants of action potential-transmitter release coupling in the catecholaminergic neurons of the brain. We show that both (-)deprenyl and (-)PPAP enhance the inward Ca2+ current in sino-auricular fibers of the frog heart. (-)PPAP was much more potent than either (+)PPAP or (-)deprenyl in this test.

Structure - Activity studies leading to ( - )1-(Benzofuran-2-yl)-2-propylaminopentane, (( - )BPAP), a highly potent, selective enhancer of the impulse propagation mediated release of catecholamines and serotonin in the brain

The catecholaminergic and serotoninergic neurons in the brain change their performance according to the physiological need via a catecholaminergic/serotoninergic activity enhancer (CAE/SAE) mechanism. Phenylethylamine (PEA), tyramine and tryptamine are the presently known endogenous CAE/SAE substances which enhance the impulse propagation mediated release of catecholamines and serotonin in the brain. A PEA derivative, (-)deprenyl (selegiline), known as a selective inhibitor of MAO-B, is for the time being the only CAE/SAE substance in clinical use. Aiming to develop a selective CAE/SAE substance much more potent than (-)deprenyl, a series of new 1-aryl-2-alkylaminoalkanes, structurally unrelated to PEA and the amphetamines, was designed and prepared. Among them, (-)1-(benzofuran-2-yl)-2-propylaminopentane ((-)BPAP) was selected as a promising candidate substance for further studies. (-)BPAP significantly enhanced in rats the impulse propagation mediated release of catecholamines and serotonin in the brain 30min after acute injection of 0.36nmol/kg sc. In the shuttle box, (-)BPAP was in rats about 130 times more potent than (-)deprenyl in antagonizing tetrabenazine induced inhibition of performance. (+/-)BPAP protected cultured hippocampal neurons from the neurotoxic effect of beta-amyloid in 10(-14)-10(-15)M concentration.

Pharmacological studies with endogenous enhancer substances: β-phenylethylamine, tryptamine, and their synthetic derivatives

The discovery of enhancer regulation in the mesencephalon and the concept that it plays a key role in the operation of innate and acquired drives [Neurochem. Res. 28 (2003) 1187] sets the trace amines (TAs) in their true physiological perspective. The regulation is defined as the existence of enhancer-sensitive neurons in the brain capable of working in a split-second on a high activity level due to endogenous enhancer substances. For the time being, only beta-phenylethylamine (PEA) and tryptamine are the experimentally analyzed examples. (-)-Deprenyl (selegiline), widely used in Parkinsons disease and Alzheimers disease today, and known as the first selective monoamine oxidase (MAO) type-B inhibitor for decades, was identified as a PEA-derived synthetic mesencephalic enhancer substance. An important and convincing confirmation of the enhancer concept was the recent development of a highly specific and potent tryptamine-derived synthetic mesencephalic enhancer substance, (-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP]. This substance, which is specific and hundreds of times more potent than selegiline, is now the best experimental tool to study the enhancer regulation in the mesencephalon and a promising candidate to significantly surpass the therapeutic efficiency of selegiline in depression, Parkinsons disease, and Alzheimers disease.

A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain

Aims: First proof to show that (−)‐deprenyl/selegiline (DEP), the first selective inhibitor of MAO‐B, later identified as the first &bgr;‐phenylethylamine (PEA)‐derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)‐1‐(1‐benzofuran‐2‐yl)‐N‐propylpentane‐2‐amine (BPAP), the tryptamine‐derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer‐sensitive brain regulations. Main methods: Longevity study disclosing the operation of tumor‐manifestation‐suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. Key findings: Whereas 20/40 saline‐treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001 mg/kg DEP: 15/40 rats; with 0.1 mg/kg DEP: 11/40 rats (P < 0.01); with 0.0001 mg/kg BPAP: 8/40 rats (P < 0.001); with 0.05 mg/kg BPAP: 7/40 rats (P < 0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB‐186 (Daoy); UW‐228‐2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine‐transporter‐2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi‐modal, bell‐shaped concentration‐effect curves of DEP and BPAP. Significance: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer‐sensitive TMS‐regulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor.

The selective enhancer substance (-)-BPAP counteracts the histological and functional consequences of an experimental stroke in rats

The effect of the newly developed, up to the present most potent and selective enhancer substance, R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane [(-)-BPAP], was studied in rats exposed to an experimental stroke. Permanent focal ischemia was induced by cortical photothrombosis in Wistar rats with Rose Bengal (20 mg/kg, i.v.), irradiated by a cold light source for 10 min. Rats were treated daily with (-)-BPAP (0.2 mg/kg, i.p.) for 3 days. The infarction was visualized with 2% TTC. The lesion size was evaluated by morphometry using image analysis software (AxioVison/Zeiss). The effect of (-)-BPAP on the learning ability of rats was measured in the shuttle box. The acquisition of a two-way conditioned avoidance reflex (CAR) was analyzed during 5 consecutive days. The rats were trained with 100 trials per day. The light-irradiation for 10 min after injection of Rose Bengal caused well-demarcated tissue damage in the cerebral cortex and decreased the performance of the lesioned rats in the shuttle box. (-)-BPAP treatment caused a statistically significant reduction of the infarct size (from 1.190 ± 0.096 mm2 to 0.256 ± 0.057 mm2; n = 18) and improved the performance of the rats (45.75 ± 15 to 75.67 ± 7.2; n = 12). The data are in excellent harmony with the series of papers which described the protective effect of (-)-BPAP in various types of enhancer-sensitive cultured cells. Acknowledgements This work was supported by the Health Scientific Council (ETT 140/2003, ETT 606/2006). from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

The basic difference in the pharmacological spectrum between methamphetamine and its derivative, the enhancer substance (-)-deprenyl

Beta-phenylethylamine (PEA), the parent compound of both methamphetamine and (-)-deprenyl, has two effects: it is a releaser of catecholamines and a natural enhancer substance. Methamphetamine, the closest mother compound of (-)-deprenyl, acts like PEA. (-)-Deprenyl was the first PEA derivative which lost the releasing property but preserved the enhancer effect. We elaborated a method on isolated rat brain stem, which allowed to measure the enhancer effect of (-)-deprenyl and to detect how the releasing effect of methamphetamine hinders the recognition of its enhancer effect. We fill up the intraneuronal transmitter stores of the noradrenergic neurons in an isolated rat brain stem with [3H]noradrenaline and thereafter continuously measure the amount of the labeled transmitter released from the organ. Electrical stimulation increases the amount of [3H]noradrenaline released into the bathing fluid. Due to its enhancer effect, 2 μg/ml (-)deprenyl significantly increases the amount of labeled transmitter to the same stimulation. The addition of 2 μg/ ml amphetamine or methamphetamine to the bathing fluid in the resting state rapidly leads to the continuous release of a high amount of [3H]noradrenaline into the organ bath and electrical stimulation is unable to further increase the amount of the labeled transmitter, thus hindering the detectability of the enhancer effect.