Enhancer regulation

The thorough analysis of the dose-dependent effect of deprenyl on the release of

catecholamines and serotonin in physiological quantities, by the aid of HPLC from
isolated discrete rat brain regions, (dopamine from the striatum, substantia nigra and
tuberculum olfactorium, noradrenaline from the locus coeruleus and serotonin from
the raphe), revealed the existence of the enhancer regulation in the mesencephalic
neurons.
We treated rats with 0.01, 0.025, 0.05, 0.1 and 0.25 mg/kg deprenyl, respectively,
once daily for 21-days, isolated the discrete rat brain regions 24-hours after the last
injection and measured the biogenic amines released during 20 minutes from the
freshly isolated tissue samples. The amount of dopamine released from the substantia
nigra and tuberculum olfactorium clarified that the dopaminergic neurons worked on
a significantly enhanced activity level, even in the brain of rats treated with the lowest,
0.01 mg/kg dose of (-)-deprenyl. As this small dose of (-)-deprenyl leaves the B-type
monoamine oxidase (MAO-B) activity and the uptake of amines practically
unchanged, this study was the first unequivocal demonstration for the operation of a
hitherto unknown enhancer mechanism in the dopaminergic neurons that is stimulated
by deprenyl in very low doses, (Knoll and Miklya, 1994).
Further studies clarified the operation of the mesencephalic enhancer regulation and
allowed us to realize that _-phenylethylamine (PEA), (the parent compound of
deprenyl) is primarily an endogenous enhancer substance, (Knoll and Miklya, 1995;
Knoll et al., 1996 a,b,c).
PEA induces in higher concentrations, a continuous strong release of the
catecholamines from their intraneuronal stores, and this well known releasing effect
covered up completely the enhancer effect of this trace-amine in the brain, that was
classified as the prototype of the indirectly acting sympathomimetics (Knoll et al.,
1996c). Amphetamine and methamphetamine, PEA derivatives with a long lasting
effect, share with their parent compound the releasing property. Deprenyl was the first
PEA/methamphetamine derivative that maintained the enhancer effect of its parent
compounds but lost completely the releasing property. This peculiar change in the
pharmacological spectrum disclosed the operation of the enhancer regulation (Knoll et
al., 1996a).
We define this mechanism as follows. Endogenous mesencephalic enhancer
substances capable to enhance the excitability of the mesencephalic neurons between
broad limits, work in the brain, of which, for the time being, only PEA and tryptamine
are the experimentally analyzed examples. Through the enhancer regulation the
sensitive neurons can drastically change within a split second their activity.
This is a life important mechanism. When an eagle for example pounces upon a rabbit
with lightning speed, both the attacker and the potential victim have only a split
second to become properly activated. The chance for the eagle to obtain its food and
for the rabbit to save its life lies in the mechanism that specific endogenous enhancer
substances dynamically increase the performance of the appropriate enhancersensitive neurons, according to the need and the partner with the more efficiently
activated brain will reach its goal, (see Knoll, 2001, 2003 for review).

The catecholaminergic and serotonergic neurons in the mesencephalon are the best
models to study the enhancer regulation, as their physiologic function is to supply
continuously the brain with the required amounts of catecholamines and serotonin that
properly influence - activate or inhibit - billions of neurons. The significant
enhancement of the nerve-stimulation induced release of [3H]-noradrenaline, [3H]dopamine, and [3H]-serotonin from the isolated brain stem of the rat in the presence of
PEA (Fig. 1) or tryptamine (Fig. 2) is shown to illustrate the mesencephalic enhancer
regulation in function.
From a freshly isolated brain stem of a satisfactorily pretreated rat a stable amount of
the labeled transmitter is released for a couple of hours, (see Knoll and Miklya, 1995
for methodology). Electrical stimulation of the brain stem significantly increases the
outflow of transmitters. The calculated average amount of each of the labeled
transmitters released from the stimulated brain stem is the product of a surviving
population of specific neurons with large individual variation in their performance.
Neurons respond to stimulation in an "all or none" manner. Hence, prior to the
administration of PEA or tryptamine, only the high performing members of the
population responded with transmitter release to electrical stimulation. As PEA or
tryptamine enhance specifically the performance of the enhancer-sensitive neurons,
the stimulation-evoked release of the labeled transmitter changed accordingly.
The data in Fig. 1 and 2 show a remarkable quantitative difference between PEA and
tryptamine in their effectiveness on serotonergic neurons. A lower concentration of
tryptamine, (1.3 mol/l) proved to be much more potent than a much higher
concentration of PEA (16 mol/l) in enhancing the stimulation-evoked release of
serotonin. This indicates that the enhancer regulation in the catecholaminergic and
serotonergic neurons are not identical on the molecular level.
Deprenyl, the PEA-derived representative synthetic mesencephalic enhancer
substance
As deprenyl was world-wide known as the first selective inhibitor of MAO-B, it was
not by chance that hundreds of clinical studies with the drug were designed thereafter
in the firm belief that selective blockade of MAO-B was responsible for all the effects
that followed
deprenyl medication. Realizing that PEA, known to be a releaser of catecholamines, is
an endogenous enhancer substance and deprenyl is a PEA-derived synthetic enhancer
substance devoid of the catecholamine releasing property of its parent compound,
clarified that it is the enhancer effect of deprenyl that is responsible for the majority of
the beneficial effects of the drug described in various experimental and clinical
studies, (see Knoll, 1998, 2001 for review).
Being rapidly metabolized by MAO, PEA is short acting, and its enhancer effect can
be detected in in-vitro experiments only, (see Fig. 1 and 2). As deprenyl is not
metabolized, its effect is long lasting and it can reliably be measured in-vivo in a dosedependent manner. The most convenient method for in-vivo testing of the enhancer
effect of a compound is to measure the release of catecholamines and serotonin from
discrete brain areas by the aid of HPLC with electrochemical detection, (see Knoll
and Miklya, 1995 for details of methodology).

The subcutaneous administration of deprenyl enhanced the activity of the

catecholaminergic neurons in a dose-dependent manner, (see Knoll, 2003 for review).
As an example, Fig. 3 shows the enhancer effect of a single subcutaneous 0.05 mg/kg
dose of deprenyl on the noradrenergic, dopaminergic and serotonergic neurons.
Deprenyl is a PEA-derived enhancer substance and its in-vivo ineffectiveness on
serotonergic neurons is in harmony with the finding that in the in-vitro experiments,
too PEA was much less potent than tryptamine in enhancing the activity of the
serotonergic neurons, (compare Fig. 1 to Fig. 2).
The tryptamine-derived representative synthetic mesencephalic enhancer
substance
The discovery that tryptamine is also an endogenous enhancer substance, (Knoll,
1994) opened the way for the synthesis of a new family of enhancer compounds
unrelated to PEA and the amphetamines, and R-(-)-1-(benzofuran-2-yl)-2propylaminopentane _(-)-BPAP_ was selected as the reference compound for further
studies, (Knoll et al., 1999).
According to preclinical studies, (-)-BPAP is the presently known most potent
synthetic mesencephalic enhancer substance. The compound is now in the initial
phase of clinical trials.
Fig. 3 shows the enhancer effect of a single subcutaneous dose of 0.0005 mg/kg of ()-BPAP on noradrenergic, dopaminergic and serotonergic neurons. A comparison of
the effect of 0.0005 mg/kg (-)-BPAP with 0.05 mg/kg deprenyl shows; (i) the
substantially higher potency of (-)-BPAP than deprenyl in enhancing the activity of
the catecholaminergic neurons; and (ii) the high effectiveness of (-)-BPAP on
serotonergic neurons, whereas deprenyl was ineffective.
In a recent study the effect of (-)-BPAP was compared to that of the known stimulants
of catecholaminergic and/or serotonergic neurons (desmethylimipramine, fluoxetine,
clorgyline, lazabemide, pergolide, bromocriptine) on electrical stimulation induced
release of the labeled transmitters from the isolated brain stem of rats following the
incorporation of [3H]-noradrenaline or [3H]-dopamine or [3H]-serotonin by
preincubation into the transmitter stores. The study confirmed the selectivity of the
enhancer effect of (-)-BPAP (Miklya et al., 2003).
Fig. 4 shows the chemical structure of the two identified endogenous mesencephalic
enhancer substances, PEA and tryptamine, and their synthetic derivatives, deprenyl
and (-)-BPAP, used as reference compounds to study the mesencephalic enhancer
regulation.
The peculiar concentration dependency of the enhancer effect
Enhancer substances stimulate the enhancer-sensitive neurons in the mesencephalon
in a peculiar manner. Fig. 5 shows, as an example, the characteristics of the enhancer
effect of (-)-BPAP added to isolated locus coerulei of rats. We see two bell-shaped
concentration/effect curves. The one in the low nanomolar range, with a peak effect at
10-13 M concentration, is clearly demonstrating the existence of a highly sophisticated,
specific form of enhancer regulation in noradrenergic neurons. The second, with a
peak effect at 10-6 M concentration, shows the operation of a hundred million times

less sensitive, obviously nonspecific form of the enhancer regulation in these neurons,
(see Knoll et al., 2002 for details).
The great individual variation in sexual activity and learning performance in any
random population of mammals of the same strain is common experience.
Considering arguments speaking in favour for a key role of the enhancer regulation in
the modification of behaviour through experience training or practice, (see Knoll,
2003 for review), the discovery of the bell-shaped concentration/effect curve of the
enhancer substance in the low nanomolar concentration range offers a reasonable
explanation for this phenomenon. To illustrate it we refer to one of our earlier studies,
(Knoll et al., 1994).
In a longitudinal study performed from 1990 until 1994, we selected sexually highand low-performing male rats from a population of 1600 rats of the same strain and
tested continuously their sexual activity and their learning performance, in the shuttle
box until they died. We found at the start of the experiment 99 high-performing (HP)
rats that produced ejaculation in each of the four consecutive weekly mating tests
used for selection, and 94 low-performing (LP) rats, unable to produce a single
intromission in the four mating tests. The HP rats were significantly better performers
also in the shuttle box. The striking difference in performance remained until death.
In the light of the peculiar dose-dependency of the enhancer effect shown in Fig. 5 we
assume that out of the 1600 rats the 99 HP rats produced the enhancer substance
inciting the sexual drive at the peak of the bell-shaped concentration/effect curve,
while the 94 LP rats produced it at an inactive part of the curve; and the production of
the overwhelming majority of the population (1407 rats) fall between the two
extremes. The observed peculiar effect of the lifelong administration of deprenyl, the
synthetic mesencephalic enhancer substance, strongly supports this conclusion.
We treated the selected HP and LP rats from the 8th month of their life three times a
week, subcutaneously, with either 0.9 % NaCl or 0.25 mg/kg (-)-deprenyl until they
died. Their copulatory activity was tested once a week. Their learning performance
was measured in the shuttle box once in three months.
The salt-treated LP rats (N=44) never displayed ejaculation during their lifetime and
were extremely dull in the shuttle box. They displayed 16.362.47 conditioned
avoidance responses (CAR) during the first 36-week testing period. They lived
134.582.29 weeks. In contrast, their (-)-deprenyl-treated peers (N=48) became
sexually fully active, their mating performance was substantially increased. They
produced 46.853.91 CARs during the first 36-week testing period, and this level of
performance was maintained during the 108 weeks of testing. They lived 151.241.36
weeks, significantly (P<0.001) longer than their salt-treated peers.
The salt-treated HP rats (N=49) displayed 14.040.56 ejaculations during the first 36week testing period and due to aging they produced 2.470.23 ejaculation between
the 73-108th weeks of testing. They displayed 78.453.01 CARs during the first 36week testing period and 50.672.99 CARs between the 73-108th weeks of testing.
They lived 152.541.36 weeks. In contrast, the (-)-deprenyl-treated HP rats (N=50)
were significantly more active sexually than their salt-treated peers. They displayed
30.040.85 ejaculations during the first 36-week testing period and 7.400.32

ejaculation between the 73-108th weeks of testing. They were also significantly better
performers in the shuttle box, produced 113.983.23 CARs during the first 36-week
testing period and 81.682.14 CARs between the 73-108th weeks of testing. They
lived 185.301.96 weeks, significantly (P<0.001) longer than their salt-treated peers.
The age-related decline of the mesenecephalic enhancer regulation and its
consequences
From point of view of the anti-aging effect of the synthetic enhancer substances the
peculiar age-related changes in the mesencephalic enhancer regulation deserves
serious attention.
We observed already in the mid 1950s that food deprived rats in the late
developmental phase of life (2 months of age) were significantly more active than
those in the early post-developmental phase (4 months of age) (see Knoll, 1969 for
review). In the light of our present knowledge this means that the catecholaminergic
system in the mesencephalon operates at a higher activity level during the
developmental phase of life. In order to verify this conclusion we revisited this
problem in the mid 1990s when the HPLC technique allowed already to measure
exactly the low amounts of catecholamines released from the discrete brain areas.
We measured the activity of the catecholaminergic and serotonergic neurons in the
mesencephalon in groups of 2, 4, 8, 16 and 32 weeks old rats. We compared in this
way the state of the enhancer regulation in rats one week before weaning (2-week old
rats), one week after weaning (4-week old rats), at the age when sexual maturity was
reached (8-week old rats), and in two groups in their early phase of postdevelopmental longevity (16- and 32-week old rats). Both in male and female rats the
catecholaminergic and serotonergic neurons in the mesencephalon worked on a
significantly higher level from weaning until sexual maturity, (in the 4- and 8-week
old rats) than before, (2-week old rats) or after that period (16- and 32-week old rats).
Rats treated any time during their post-developmental phase of life with (-)-deprenyl,
showed a significantly higher catecholaminergic activity than their saline-treated
peers (Knoll and Miklya, 1995).
This explains why in series of studies, rats treated during their post-developmental
phase of life with deprenyl, performed better in behavioral tests, had slower agerelated decline in sexual and learning performances, and lived longer than their salinetreated peers, (see Knoll, 1998, 2001 for review).
As sexual hormonal regulation starts working in the rat at the and of the 2nd month of
age and we observed that the elevated level of enhancer regulation was terminated
exactly at this age, it was reasonable to assume that the enhanced activity of the
catecholaminergic engine of brain characteristic to the developmental period of life,
was dampened by sexual hormones. To check the validity of this assumption we
analyzed in detail the effect of estrone and testosterone in male and female rats. This
study proved that sexual hormones terminate the hyperactive phase of adolescence by
dampening the enhancer regulation, (Knoll et al., 2000). Thus, sexual maturity ends
the uphill period of life and this is the beginning of post-developmental longevity with
its slow continuous age-related decline until death.

Conclusion: The human catecholaminergic engine

A convincing example for the age-related decline of the catecholaminergic engine in
the human brain, is the rapid decline of the dopamine content of the striatum beyond
the age of 45 years. According to our present knowledge the nigrostriatal
dopaminergic neurons are the most rapidly aging neurons in the human brain. The
dopamine content of the human caudate nucleus decreases steeply, at a rate of 13%
per decade over age 45. (Ed.- See Fig. 6) We know that symptoms of Parkinson's
disease appear if the dopamine content of the caudate sinks below 30% of the normal
level. About 0.1% of the population over 40 years of age develop Parkinson's disease,
and prevalence increases sharply with age. Thus, the normal aging of the system is
slow enough to avoid the appearance of Parkinsonian symptoms within the average
lifespan for 99.9% of the human population.
Because of the lack of constancy of physiological age, we know that the variance
within a particular age group for any measurable parameter is large. In cross-sectional
studies, no single age emerges as the point of sharp decline in function. Any
individual may show different levels of performance, and the careful observer finds
many dissociations between "chronological" and "physiological" age. (Ed.- Antiaging
advocates may refer to Professor Knoll's "physiological" age as "biological" age).
Nevertheless, the age-related decline of sexual and learning performances hits
everybody on his/her own level.
Collating the results of experimental studies with clinical experiences there can be
little doubt that the slow, continuous decline of the mesencephalic enhancer regulation
during the post-developmental phase of life is in causal relationship with the decline
of sexual and learning performances with the passing of time, and contributes to the
manifestation of age-related neurological diseases. It therefore seems reasonable to
enhance the activity of the catecholaminergic engine of the brain from sexual maturity
until death via the administration of a safe, small daily dose of a synthetic
mesencephalic enhancer substance. At present deprenyl is the only available drug for
this purpose, and 1 mg is the proposed daily dose. This will work for decades. It will
improve the quality of life in the latter period of life, hopefully shifting the time of
natural death, with high probability decreasing the precipitation of age-related
depression, eliminating or at least significantly shifting the precipitation of the
symptoms of Parkinson's disease, and possibly reducing or delaying the onset of
Alzheimer's disease, (see recent reviews, Knoll, 2001 and 2003, for details).
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