Joseph Knoll: The discovery of the enhancer regulation in the mammalian brain and the development of synthetic enhancer substances


Joseph Knoll’s further reply to Hector Warnes


            I fully agree with Dr. Hector Warnes’ remarks regarding the therapeutic efficiency of a 10 mg/day dose of selegiline. However, I wish to take this opportunity to point out the mistake that even deprenyl became well known in 1972 as the first selective inhibitor of B-type MAO and I discovered only 20 years later that the catecholaminergic activity enhancer (CAE) effect, exerted in low doses, is the primary important pharmacological effect of deprenyl; this is still not yet a matter of general knowledge.

            Due to its specific pharmacological spectrum, deprenyl, the only synthetic
β-phenylethylamine (PEA)-derivative devoid of the catecholamine-releasing property,
enlightened me to the idea that the catecholaminergic neurons belong to the enhancer-sensitive brain regulations and led me to study this hitherto unknown life important mechanism in the mammalian brain.

                Deprenyl is still registered in the text-books and used in research and therapy as the reference compound to block selectively MAO-B. Since the mid-1980s, however, deeper analysis of the characteristic enhancement of the catecholaminergic brain machinery in deprenyl-treated rats clarified that this effect is unrelated to the selective inhibition of MAO-B (Knoll, 1992). Thus, I started a structure-activity-relationship study to develop a deprenyl analog devoid of MAO inhibitory property and (-)-1-phenyl-2-propylaminopentane (PPAP), equally active on the catecholaminergic neurons as deprenyl, was selected for further studies (Knoll et al., 1992).

                The first study, which demonstrated that multiple, low dose administration of deprenyl enhances catecholaminergic activity in the brain and this effect is unrelated to MAO-B inhibition, allowed for the discovery of the enhancer sensitive brain regulations (Knoll and Miklya, 1994).


            PEA and its best known synthetic derivatives (amphetamine and methamphetamine) are strong releasers of catecholamines from their plasmatic pools. Even hordenine a PEA-class natural biogenic amine occurring in a number of plants, which like PEA, is binding to trace amine-associated receptors (TAAR1), is a releaser of catecholamines (Berry, 2007). Since the catecholamine releasing effect conceals the detectability of the enhancer-sensitive nature of the catecholaminergic neurons (Knoll, 2016). PEA’s primary physiological function as a natural enhancer substance, as well as the fact that amphetamine and methamphetamine are, like deprenyl, PEA-derived synthetic enhancer substances, remained unknown. The later realization that tryptamine is, like PEA, a natural enhancer substance signaled the elaboration of BPAP, the most selective and potent synthetic enhancer substance currently known (Knoll et al.,1999).

            Deprenyl is primarily a catecholaminergic enhancer (CAE) substance and is a weak enhancer of serotonergic neurons. BPAP, as a tryptamine-derivative, is a highly potent enhancer of serotonergic neurons, but it is, even as a CAE substance, much more potent than deprenyl. The catecholaminergic and serotonergic neurons were studied as the first models of the enhancer-sensitive brain regulations (Knoll, 2005).

            With regard to the molecular mechanism of action of the enhancer-substances, we found later that BPAP, injected in a dose of 0.0001 mg/kg, reversed the decrease in the electrical stimulation-induced [3H]dopamine release, evoked by 1 mg/kg of tetrabenazine in superfused rat striatal slices. Tetrabenazine is a VMAT2 inhibitor proposed to interact with the extravesicularly located dihydrotetrabenazine binding site that is distinct from the dopamine uptake site on VMAT2. Moreover, tetrabenazine also binds to intra-vesicular dopamine release sites of VMAT2, exhibiting high and low sensitivity in binding affinity. Both the extra- and intra-vesicular VMAT2 dopamine uptake and release sites may be involved in BPAP’s effect. Furthermore, BPAP, acting as a substrate inhibitor of VMAT2, may compete with dopamine for uptake into the vesicle, and may exhibit a low affinity binding to the dopamine uptake site on VMAT2, as suggested by its poor activity on resting [3H]dopamine release in superfused striatal slices of the rat. The biphasic concentration-response curve for BPAP to release [3H]dopamine following electrical stimulation that fits a two-site model of interaction supports the interaction of two different intra-vesicular sites: a high affinity (picomolar) site and a low affinity (μmolar) dopamine release site. A binding to the high affinity dopamine release site represents the specific catecholaminergic activity enhancer activity of BPAP (Knoll et al., 1999), whereas the low affinity site is responsible for BPAP’s non-specific enhancer effect of on [3H]dopamine release. Taking into account the dopamine uptake and release sites on VMAT2, to which tetrabenazine and BPAP bind, we concluded that the observed interaction of these two drugs in [3H]dopamine release may be related to a binding of BPAP to the high affinity intra-vesicular dopamine release site, which is, on the other hand, also sensitive to the tetrabenazine, the vesicular inhibitor. All in all, the molecular mechanism of the enhancer effect clarifies BPAP’s highly characteristic bi-modal, bell-shaped concentration effect curves (Knoll et al., 2002) and testifies to the idea that the enhancer-sensitive brain regulations represent a promising new brain research domain.

            The discovery of the enhancer regulation in the mammalian brain, the study of the catecholaminergic and serotonergic neurons as enhancer sensitive brain regulations,  the identification of PEA and tryptamine as natural enhancer substances, the proof  that selegiline/(-)-deprenyl (DEP) is the first PEA-derived synthetic enhancer substance devoid  of the catecholamine releasing property, and  the development of (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP) allowed for promising new  brain-research (Knoll 2001, 2003, 2012; Miklya, 2016).

            Prior to the discovery of the catecholaminergic activity enhancer effect of DEP and the discovery of the enhancer regulation in the mammalian brain, the hypothesis was proposed in 1981 that a progressively developing catecholaminergic and trace-aminergic deficiency is responsible for the biochemical lesion in the aging brain which leads to the age-related decline in sexual and learning performance and ultimately natural death soon proved that this effect of DEP is unrelated to the inhibition of  MAO-B (Knoll, 1982; Knoll and Miklya, 1995).

            Enhancer substances keep the catecholaminergic neurons on a higher activity level. For example: 6.8±0.18 nmol/g wet weight dopamine was released within 20 minutes from the substantia nigra isolated from saline treated rats and 14.8±0.36 nmol/g wet weight dopamine was released within 20 minutes from the substantia nigra isolated from rats treated with a single dose of 0.0001 mg/kg BPAP. Similarly, a single dose treatment with 0.0005 mg/kg BPAP increased the release of norepinephrine from the isolated locus coeruleus within 20 minutes from 4.7±0.10 (saline) to 15.4±0.55 nmol/g wet weight; and a three-week treatment once daily with 0.0001 mg/kg BPAP acted similarly (the brain areas were isolated 24 hours after the last injection). These ex vivo results from studies using isolated discrete rat brain regions are in complete harmony with the results of the in vivo shuttle box experiments and furnish unequivocal evidence that the treatment of rats with 0.0001 mg/kg BPAP transformed the silent catecholaminergic neurons into spontaneous firing entities.

            It is worth remembering the origin of the enhancer regulation concept.  “An eagle pounces upon the chosen victim with lightning speed. Reacting accordingly is a life-and-death matter. Both the attacker and the victim have a split second to respond. This promptness of activation in assault/escape behavior inspired the working hypothesis in the mid-1980s that an unknown, life important, enhancer regulation, capable to momentarily increase neuronal excitability, might operate in the mammalian brain. Since the cerebral catecholaminergic machinery is responsible for the general activation of the cortex, it was reasonable to expect that the catecholaminergic brain engine must be endowed with this capacity” (Knoll, 2016).

            It is well known from studies with rodents and primates that dopaminergic neurons are silent or spontaneously active (Marinelli, 2006). Our finding that the treatment of rats with 0.0001 mg/kg BPAP transformed the silent catecholaminergic neurons in the enhancer-sensitive dopaminergic neurons into spontaneous firing entities explains the promptness of activation in assault/escape behavior. It is hard to overestimate the therapeutic consequences of the fact that influenced by 0.0001 mg/kg BPAP, this highly potent synthetic enhancer substance was capable of dramatically transforming dopaminergic neurons’s operation. 

            One of the most crucially important conclusions regarding the unknown life important physiological effects of the catecholaminergic and serotonergic neurons is that these enhancer-sensitive regulations work in the uphill period of life, from weaning until sexual maturity, on a significantly higher activity level. Sexual hormones (estrone, testosterone) return the enhancer regulation to the pre-weaning level, putting into action the downhill period of life and the aging-related slow decay of the enhancer regulation continues until death (Knoll and Miklya 1995; Knoll et al., 2000). Thus, maintenance during the downhill period of life on a proper low dose of a synthetic enhancer substance slows the aging related decay of the enhancer sensitive brain regulations, improves the quality of life in the latter decades, prolongs life and delays/prevents the manifestation of enhancer-regulation-dependent illnesses, signaling that the enhancer regulation, due to aging-related decay, already surpassed the critical threshold (Knoll, 1994).         For example, we lose 13% of our dopamine in the decade after age 45. In the healthy population, the calculated loss of striatal dopamine is about 40% at the age of 75 which is about the average lifetime. As symptoms become visible only after the unnoticed loss of about 70% of striatal dopamine, in diagnosing Parkinson’s disease the neurologist selects subjects with the most rapidly aging striatal dopaminergic system (about 0.1% of the population.)

            At present DEP, the PEA-derived CAE substance is the only safe synthetic enhancer drug in world-wide clinical use. In research and therapy, DEP celebrates a 50-year history. Masses of patients are still treated with a daily dose (10 mg) of DEP, and masses of people take 1 mg DEP daily to slow the aging-related decay of their catecholaminergic brain engine (Miklya, 2016).

            Decades ago, based on the concept that the long term administration of DEP may improve the quality of life in the declining years (Knoll, 1982) and this effect of DEP is unrelated to the inhibition of MAO-B (Knoll and Miklya, 1995), a retrospective analysis in parkinsonian patients was performed. The long term (nine years) effect of treatment with Madopar alone (N=177) or in combination with DEP (N= 564) revealed a significant increase in  life expectancy in Madopar+DEP group regardless of the significant demographic differences between the two groups (Birkmayer et al., 1985).

            Lifelong preventive medication obviously requires unique drug-safeness. Due to their peculiar mechanism of action and safety margin, only the synthetic enhancer substances adhere to this requirement. BPAP exerts its specific enhancer effect in a subcutaneous dose as low as 0.0001 mg/kg, and 20 mg/kg, a 20 times higher dose, is tolerated without any sign of toxic effect. This is truly an exceptional safety margin (Knoll and Miklya, 1994).

            As shown in a recent longevity study performed with low doses of deprenyl (Knoll and Miklya, 2016), the regularly used 10 mg/day human dose has two effects: it inhibits MAO-B and exerts its non-specific enhancer effect. It remains for the future to clarify, in retrospect, the participation of MAO-inhibition versus enhancer effect in the benefits observed in patients treated with deprenyl.

            To illustrate with a final example the consequences of the discovery of the enhancer-sensitive brain regulations and the development of the first synthetic enhancer-substances I am citing a recently published example demonstrating the anti-aging effect of BPAP. We treated rats daily with saline versus 0.0001 mg/kg BPAP and measured in the shuttle box their ability to fix a conditioned avoidance reflex (CAR). Due to aging of the dopaminergic neurons, learning ability is subject to an age-related decline. We found in our recent study that the three month old group of saline-treated rats worked with full capacity and built on the fifth day of training, on average, nearly 90% of the CARs. Due to aging of the dopaminergic neurons, the group of 18-month-old saline-treated rats reached on the fifth day of training, on average, only less than 30% of the CARs. However, the group of 18-month-old rats treated daily with 0.0001 mg/kg BPAP produced on the fifth day of training, on average, more than 90% of the CARs (Knoll and Miklya, 2016). As discussed above, 0.0001 mg/kg BPAP specifically stimulates the enhancer-sensitive dopaminergic neurons and we surprisingly found that by treating rats daily with a proper synthetic enhancer substance, 18-month-old rats remained as efficient learners in the shuttle box as the most active three-month-old rats. This is an unprecedented novelty.

            As published step by step since the 1950s and summarized in monographs (Knoll 1969, 2005, 2012, 2016), the essence of our findings leading finally to the discovery of the enhancer-sensitive brain regulations and the development of deprenyl and BPAP, open a hitherto unknown possibility to improve the quality and the duration of human life via slowing brain aging.




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Knoll J, Miklya I, Knoll B, Dallo J. Sexual hormones terminate in the rat the significantly enhanced catecholaminergic/serotoninergic tone in the brain characteristic to the post-weaning period. Life Sci 2000; 67:765-73. DOI:10.1016/S0024-3205(00)00671-8.


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Joseph Knoll

February 16, 2017