Joseph Knoll’s response 2 to Hector Warnes’ response 2
Joseph Knoll: The Discovery of the Enhancer Regulation in the Mammalian Brain and the Development of the Synthetic Enhancer Joseph Substances
Clinicians are still convinced that the selective inhibition of MAO-B is primarily responsible for the beneficial therapeutic effects of selegiline/(-)-deprenyl (DEP). This misunderstanding is due to the negligence of the gradual recognition of the complicated pharmacological spectrum of DEP.
The first phase in selegiline (DEP) history was the recognition that DEP is free of the ‘cheese effect’ (Knoll et al. 1968).
This was finally proved in 1988. We compared all the known MAO inhibitors’ response to tyramine on rabbit arterial strips. Not only the best known and regularly used MAO inhibitors, but also all the newly published, and at that time, less known compounds were studied. Only DEP inhibited the response to tyramine (Abdorubo et al. 1988).
The second phase was the recognition that DEP is the first selective inhibitor of MAO-B (Knoll and Magyar 1972).
Prior to the discovery of the catecholaminergic activity enhancer (CAE) effect of DEP (Knoll 1998), it was my firm belief that the selective inhibition of MAO-B is responsible for the drug’s beneficial therapeutic effects. However, further studies, which for the first time revealed the true aphrodisiac effect of 0.25 mg/kg DEP, led to an unexpected observation which raised doubts regarding the supposed leading role of the selective inhibition of MAO-B in DEP’s therapeutic effects (Knoll 1982).
at that time when we discovered DEP’s true aphrodisiac effect, we developed U-1424, a new indane-derived potent selective inhibitor of MAO-B (Knoll et al. 1978). We performed with U-1424 exactly the same experiment as with DEP on sluggish, aged rats and found that the new compound did not possess an aphrodisiac effect (Knoll 1982). I concluded in my lecture that DEP exerts its aphrodisiac effect by more than one mechanism (Knoll 1982). For me, this finding was a serious warning that we needed to clarify DEP’s unknown mechanism, obviously unrelated to MAO-B inhibition.
We developed (-)-1-phenyl-2-propylaminopentane (PPAP), the DEP-analog containing a propyl-group attached to the nitrogen instead of the propargyl-group. The propyl-group is unable to covalently bind with the flavin in MAO-B rather than the propargyl-group in DEP. Thus, PPAP leaves MAO-B activity unchanged, however, as a central stimulant of the catecholaminergic neurons, PPAP proved to be as potent as DEP (Knoll et al. 1992).
The third phase was the recognition that DEP is a PEA-derived synthetic enhancer substance (Knoll et al. 1996).
Discovery of the enhancer-sensitive regulations in the mammalian brain opened a new domain in brain research (Knoll 2003, Knoll 2005). An enhancer-sensitive neuron is defined as one capable to change excitability and work immediately on a significantly higher activity level in the presence of a natural or synthetic enhancer substance (Knoll 2005).
The catecholaminergic and serotonergic neurons and their natural enhancers, β-phenylethylamine (PEA) for the catecholaminergic neurons and tryptamine for the serotonergic neurons, were identified as enhancer-sensitive brain regulators and selected as the first models to study the characteristics of the enhancer-sensitive brain regulations. Two synthetic enhancers were developed for the pharmacological analysis: Selegiline/(-)-deprenyl (DEP), the PEA-derived synthetic catecholaminergic activity enhancer (CAE) substance (Knoll 1998) and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP) the tryptamine-derived synthetic enhancer substance (Knoll et al. 1999).
BPAP, the selective and much more potent synthetic enhancer than DEP, is preferentially used as a specific marker to detect unknown enhancer-sensitive brain regulations. A bi-modal, bell-shaped concentration effect curve is characteristic to the enhancer substances. This peculiar behavior brought the distinction of the “specific” and “non-specific” enhancer effect to perfection. The bi-modal, bell-shaped nature of the enhancer effect, confirmed on the cultured rat hippocampal neurons (Knoll et al. 1999), was first precisely analyzed on the isolated locus coeruleus of rats (Knoll et al. 2002). In this test, BPAP enhanced the activity of the noradrenergic neurons in the femto/picomolar concentration range with a peak at 10-13 M (“specific” enhancer effect), and also in a 10 million times higher concentration range with a peak at 10-6 M (“non-specific” enhancer effect).
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 (Knoll et al. 2017).
The main effect of DEP is the enhancer effect
It is unquestionable that since the early 1960s DEP’s story would be full of surprises. DEP-research catalyzed us to the discovery of the enhancer regulation in the mammalian brain, to the realization that the catecholaminergic and serotonergic neurons are enhancer-sensitive units, and to the development of BPAP.
A recent study presents evidence that the enhancer effect of DEP and BPAP are responsible for the prolongation of mammalian life (Knoll and Miklya 2016). Rats treated three times a week with 0.0001 mg/kg BPAP, which is the peak dose exerting its “specific” enhancer effect, significantly prolonged the rat life. This study also showed that the 0.25 mg/kg dose of DEP, used from the beginning in the longevity studies, has two effects: it is the peak dose which completely blocks MAO-B in the brain, and it is also the peak dose which exerts the non-specific enhancer effect of DEP (Knoll and Miklya 2016).
Since the presently used 10 mg daily dose of DEP in therapy was originally selected as the one equivalent with the dose used in animals, it remains for the future to clarify the role of the non-specific enhancer effect of DEP in the therapeutic benefits observed in the last decades.
That the main effect of DEP is the enhancer effect is unquestionable.
As already noted, 0.25 mg/kg DEP is the peak concentration that elicits in rats both MAO-B inhibition and the “non-specific” enhancer effect. We established at the very beginning of the planned clinical trials with DEP that the 0.25 mg/kg dose of DEP, which selectively and completely blocks MAO-B activity in the rat brain, is equivalent with 10 mg/day DEP in humans, and this remains the standard daily therapeutic dose of DEP. Considering DEP’s already exactly verified pharmacological profile, it is obvious that DEP exerts the same two effects in humans as in rats.
As a matter of fact, it was the Deprenyl And Tocopherol Antioxidant Therapy Of Parkinsonism/Parkinson Study Group (PSG) which published the DATAtOP study’s results that DEP has a beneficial influence on the natural history of PD, which clearly proved, in light of the discovery of the CAE effect, that only the enhancer effect of DEP can be responsible for this unexpected, unknown and unique benefit. Tetrud and Langston (1989) published first in Science that DEP-treatment delayed the need for levodopa therapy. They found that the average time until levodopa was needed was 312.1 days in the placebo group and 548.9 days for patients in the DEP group. This finding was immediately confirmed (Parkinson Study Group 1989).
The original 1989 Science paper that reported the finding that DEP treatment is changing the natural history of PD was soon further confirmed by important multicenter studies, such as the French Selegiline Multicenter Trial (FSMT) (Allain et al. 1991), the Finnish Study (Myttyla et al. 1992), the Swedish PSG (Palhagen et al. 1998), and the Norwegian-Danish Study Group (Larsen et al. 1999).
When the DATATOP study was planned, DEP’s enhancer effect was unknown, so the organizers hypothesis was that the activity of MAO and the formation of free radicals predispose patients to nigral degeneration and contribute to the emergence and progression of PD. In accordance with their working hypothesis, they expected that DEP, the MAO inhibitor, α-tocopherol, the antioxidant, and the combination of the two compounds would slow the disease’s clinical progression.
They selected patients with early, untreated PD and measured the delay in the onset of disability necessitating levodopa therapy. In the first phase of the trial, 401 subjects were assigned to a-tocopherol or placebo and 399 subjects were assigned to DEP, alone or with a-tocopherol. Only 97 subjects who received DEP reached the “end” of the trial (i.e., the onset of disability necessitating levodopa therapy) during an average 12 months of follow-up compared with 176 subjects who did not receive DEP. The risk of reaching the end of the trial was reduced by 57% for patients who received DEP, and these patients also had a significant reduction in their risk of having to give up full-time employment (Parkinson Study Group 1989). Following the course of changes, the authors concluded in their next paper that DEP, but not a-tocopherol, delayed the onset of disability associated with early, otherwise untreated PD (Parkinson Study Group 1993). But over time, the DATATOP study also revealed that DEP did not reduce the occurrence of subsequent levodopa-associated adverse effects in patients. This fact still needs serious consideration (Parkinson Study Group 1996).
Idiosyncratic prescribing of DEP in combination with levodopa already led to false conclusions (Knoll 2010). Due to the inhibition of MAO-B, DEP-treatment allows for a 20-50% decrease in levodopa dose needed in PD. In patients who need levodopa, however, there is always a risk that the administration of DEP will enhance the side effects of levodopa which can only be avoided by properly decreasing the levodopa dose according to individual sensitivity.
An example of a multicenter clinical trial with improper combination of levodopa which led to confusion and misinterpretation, was the one performed by the PD Research Group in the United Kingdom (PDRG-UK) (Lees 1995). Quite unexpectedly, this group published an alarming paper claiming that parkinsonian patients treated with levodopa combined with DEP show an increased mortality in comparison with the patients treated with levodopa alone. This finding was in striking contradiction to all other studies published in a variety of countries. Comments uniformly pointed to substantial overdosing of levodopa (Dobbs et al. 1996, Knoll 1996, Olanow et al. 1996).
The outcome of the DATATOP study, the finding that DEP delayed the need for levodopa therapy, but a-tocopherol fell short of expectation, clearly proved that DEP exerts an unknown pharmacological effect of basic importance and a-tocopherol is devoid of this effect. Now we know that DEP, as a CAE substance, is an enhancer of the impulse propagation mediated release of catecholamines. A comparative pharmacological analysis of DEP and a-tocopherol proved that a-tocopherol is devoid of the enhancer effect (Miklya et al 2003). Since 0.25 mg/kg DEP selectively blocks MAO in the brain and also exerts in the same dose the non-specific enhancer effect (Knoll and Miklya 2016), it is obvious that DEP’s CAE effect was responsible for the delayed levodopa need (Knoll 2010).
This conclusion was also supported by the clinical trial with rasagiline, performed by the PSG. The trial revealed that unlike the early selegiline trials, rasagiline failed to demonstrate a decreased need for levodopa (Parkinson Study Group 2002). Even the results of additional studies (Olanow and Rascol 2010, Ahlskog and Uitti 2010), led to the conclusion that “based on current evidence, rasagiline cannot be said to definitely have a disease-modifying effect” (Robottom 2011). Similar to a-tocopherol, neither lazabemide nor rasagiline, the two selective MAO-B inhibitors used in PD, are also devoid of the CAE effect of DEP (Miklya 2014).
Since the mid-1980s, further analysis of the characteristic enhancement of the catecholaminergic brain machinery in DEP-treated rats rendered probable that this effect is unrelated to the selective inhibition of MAO-B. The development of PPAP, the DEP-analog devoid of a MAO inhibitory property, and an equally active stimulant of the catecholaminergic neurons as DEP, verified this suggestion (Knoll 1992). The first study which demonstrated that multiple, low dose administration of DEP 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 (AM and MAM) are strong releasers of catecholamines from their plasmatic pools. Since the catecholamine releasing effect conceals the detectability of the enhancer-sensitive nature of the catecholaminergic neurons (Knoll 2016), DEP’s primary physiological function as a natural enhancer substance, as well as the fact that AM and MAM are, like DEP, PEA-derived synthetic enhancer substances, remained unknown.
The later realization that tryptamine is like PEA, a natural enhancer (Knoll 1994), signaled the elaboration of BPAP as the most selective and potent synthetic enhancer substance currently known (Knoll et al. 1999).
The discovery of the enhancer-sensitive brain regulations and the development of synthetic enhancer substances clarified that both the developmental and post-developmental phases of mammalian life are under strict control of the enhancer-sensitive brain regulations. During the developmental period of life, from weaning until sexual maturity, the enhancer-sensitive neurons work on significantly higher activity level (Knoll and Miklya 1995). Sexual hormones immediately restore the pre-weaning low level of the enhancer-sensitive brain regulations and activate the post-developmental (aging) phase; due to the slow unbroken loss of the natural enhancers, the regressive effects of brain aging continue until death (Knoll et al. 2000).
The preliminary observations that enhancer-sensitive neurons do not age suggested that synthetic enhancer substances might prevent the regressive effects of brain aging. A carefully performed longevity study verified the suggestion (Knoll and Miklya 2016).
Since the enhancer-sensitive dopaminergic neurons are primarily responsible for the rat’s learning ability and we also know that, similar to the human brain, the dopaminergic neurons belong to a very rapidly aging brain-system (Knoll 2010), we selected the learning test to prove that the dopaminergic neurons do not age. We treated subcutaneously, three times weekly, groups of rats from sexual maturity until death with saline versus 0.0001 mg/kg BPAP, the peak dose of the synthetic enhancer with the specific enhancer effect. We measured in the shuttle box rats’ ability to fix during a 5-day daily training a conditioned avoidance reflex (CAR). We found in this study that 3-month-old saline-treated rats worked with full capacity in the shuttle box and built on the 5th day of training an average of nearly 90% of the possible 100% of CARs. Due to aging of the dopaminergic neurons, the 18-month-old saline-treated rats reached on the 5th day of training an average of less than 30% of the possible 100% of CARs. However, the group of 18-month-old rats treated from sexual maturity until death three-times weekly with 0.0001 mg/kg BPAP reached on the 5th day of training an average of over 90% of the possible 100% of CARs (Knoll and Miklya 2016). The proof that BPAP-treatment fully prevented the aging-related decay of the dopaminergic neurons shows promise that we may safely counteract in the future the regressive effects of brain aging and, thus, improve the quality and prolong the duration of human life.
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January 25, 2018