One plus one equals more than two …

Galantamine and Melatonin
Are Brain Synergistic

Improving Both Memory and Sleep

By Will Block

I n a new not-yet published study, Spanish scientists—from Autonomous University of Madrid and an affiliated hospital—provide experimental evidence on the synergistic neuroprotective effect of the combination of melatonin and galantamine in an Alzheimer’s disease (AD)-related pathology model.1

Melatonin Is Reduced or Even Absent in Alzheimer’s

Melatonin is a hormone released primarily by the brain’s pineal gland that helps anticipate the daily arrival of darkness and induces sleep. It is involved in the synchronization of circadian rhythms, harmonizing physiological functions such as sleep timing, blood pressure regulation, seasonal reproduction and many other activities. Significantly, melatonin’s levels have been found to be much reduced or even absent in AD, resulting in higher levels of insomnia.2


Significantly, melatonin’s levels have
been found to be much reduced or
even absent in AD, resulting in higher
levels of insomnia.


Galantamine provides a dual-mode action for boosting cholinergic function: 1) it inhibits the enzyme acetylcholinesterase, thereby boosting brain levels of acetylcholine, and 2) it modulates the brain’s nicotinic receptors so as to maintain their function. Galantamine also helps sleep. The cholinergic activity of galantamine may repair the sleep pattern by restoring regular functions of the hypothalamopituitary axis (HPA), impairment of which is associated with cognitive disorders and insomnia (see “Galantamine Enhances Sleep” in the April 2015 issue). The HPA is a complex set of direct influences and feedback interactions among three endocrine glands: the hypothalamus, the anterior pituitary (the expression of one subunit is regulated by the secretion of melatonin in response to light information transmitted to the pineal gland), and the adrenal cortex (see Fig. 1).


It is widely known that sleep
has an important role in
memory consolidation.


Poor Sleep Quality and AD

Figure 1. Basic HPA axis summary (corticotropin-releasing hormone=CRH, adrenocorticotropic hormone=ACTH, cortisol=CORT.
LEM1508Fig1_274.gif
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Is there a link between poor sleep quality and AD? To answer this question, a randomized, double-blind, parallel-group study was published last year in Clinical Interventions in Aging.2 The study utilized 80 subjects diagnosed with mild to moderate AD who were receiving standard therapy [acetylcholinesterase inhibitors (AChEI), such as galantamine, with or without memantine]. The subjects were equally divided between men and women, with an average age 75.3 years (range, 52–85 years). They were treated for 2 weeks with placebo and then randomized to receive 2 mg of melatonin or placebo nightly for 24 weeks, followed by 2 weeks placebo.

All subjects were tested using several well-known memory assessments: the AD Assessment Scale-Cognition (ADAS-Cog), the Mini-Mental State Examination (MMSE), and the Instrumental Activities of Daily Living (IADL)


From the Clinical Interventions in
Aging
study, it may be concluded that
supplemental melatonin has positive
effects on cognitive functioning and
sleep maintenance in AD patients.


As well, sleep qualities were tested, as assessed by the Pittsburgh Sleep Quality Index (PSQI), and a daily sleep diary was produced. Safety parameters were also measured, including adverse events, vital signs (heart rate and blood pressure), physical examinations, and laboratory tests.


When melatonin and galantamine
co-incubated with the toxic stimuli,
concentration-dependent
neuroprotection
resulted.


Melatonin Boosts Cognition and Sleep Efficiency


LEM1508_assessment_274.gif
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Subjects treated with melatonin for 24 weeks had significantly better cognitive performance than those treated with placebo, as measured by the IADL and MMSE. Sleep efficiency, as measured by the PSQI was also better with melatonin.

In the comorbid insomnia (PSQI ≥6) subgroup, melatonin treatment resulted in significant and clinically meaningful effects versus the placebo, in mean IADL, MMSE score (+1.5 versus −3 points), and sleep efficiency.

Median ADAS-Cog values (−3.5 versus +3 points) were significantly better with melatonin. Differences were more significant at longer treatment duration. Melatonin was well tolerated, with as few adverse events as placebo.

Evidence Links Poor Sleep to Memory Loss

Neurodegeneration, such as dementia and AD, frequently produces neurobehavioral symptoms, including sleep disturbances typically characterized by nighttime awakenings. It is widely known that sleep has an important role in memory consolidation. Furthermore, developing evidence links poor sleep to increased AD risk and memory loss.

From the Clinical Interventions in Aging study, it may be concluded that supplemental melatonin has positive effects on cognitive functioning and sleep maintenance in AD patients, particularly in those with insomnia comorbidity. This suggests a link between poor sleep and cognitive decline.


Combined therapy has opened a
broad line of research for scientists in
different fields.


Can Melatonin Together with Galantamine Provide Added Benefits?


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Noting that combination therapy is extensively used to treat different pathological diseases such as acquired immune deficiency syndrome (AIDS) and cancer, the Madrid researchers designed their new study to evaluate whether melatonin in combination with galantamine could provide added beneficial properties in a novel in vitro model of AD.1

In their experiment, they exposed hippocampal cultures—the hippocampus region of the brain is thought to be the center of emotion, memory, and the autonomic nervous system—to subtoxic concentrations of β-amyloid plus okadaic acid (βA/OA) for 4 days. β-amyloid plaque is known to be a benchmark for AD. Okadaic acid is a toxin produced by several species of microscopic algae, and is known to accumulate in shellfish, the consumption of which can make people sick.

β-amyloid Plus Okadaic Acid Increases Cell Death

Subjecting the hippocampal cultures to βA/OA increased cell death by 95%, which was mainly apoptotic (cell suicide) induced. The combination of βA/OA increased the measures of AD severity, including amyloid plaques, hyperphosphorylation of Tau, oxidative stress, and neuro­inflammation.

Melatonin and Galantamine Enhance Neuroprotection

Under these experimental conditions, melatonin and galantamine co-incubated with the toxic stimuli, provided a concentration-dependent neuroprotection. Maximal neuroprotective effect was achieved at 1 μM of melatonin and galantamine (about 0.25 mg melatonin and the same amount of galantamine; the molecular mass of melatonin is 232.278 g/mol and that of galantamine is 287.354 g/mol).

However, a combination of sub-effective concentrations of melatonin (1 nM) and galantamine (10 nM) provided a synergic anti-apoptotic effect and reduction of most of the AD-related pathological hallmarks observed in the βA/OA model. It was also the most effective. Consequently, the Madrid researchers suggest that supplementation of melatonin in combination with lower doses of AChEI could be an important strategy for AD patients.


The development of approaches to
reduce oxidative stress and
restore melatonin levels in AD
patients seem likely to provide
new therapeutic opportunities.


Major Hallmarks of AD: Plaques and Tangles

AD is an age-related neurodegenerative disease characterized by impaired memory, progressive loss of cognitive function, judgment and decision making, among other impediments. The brains of those with AD show extracellular βA plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated Tau protein, which are considered major hallmarks for this disease and, are related to neurodegeneration. The presence of plaques and tangles were shown in the hippocampal cultures (see above section, “βA/OA Increases Cell Death”).

However, glutamate excitotoxicity, free radical-mediated damage, and mitochondrial dysfunction are also found in AD, as they are in other neurodegenerative diseases. Nevertheless, AD is not fully explicated and is thereby considered to be a multifactorial disease.

Several One-Target Specific Drugs or Nutrients

In the treatment of complex diseases such as heart disease or cancer, the combination of several one-target specific drugs or nutrients has provided better results than a single one-target drug. This can be explained by the fact that diseases with a complex pathogenesis need to be approached with different drugs acting on the different pathways that lead to pathology.

Therefore, combined therapy has opened a broad line of research for scientists in different fields. In fact, several studies have already been carried out showing the usefulness of combination therapies for AD. As an example, consider the use of AChEI and memantine in patients with AD or CoQ10 and omega-3 fish oil for heart disease.

Melatonin Benefits for AD

As mentioned earlier, melatonin is a hormone synthesized mainly in the pineal gland. However, it is also produced in cells of the immune system and other tissues. These include those of the brain, airway epithelium, bone marrow, gut, ovary, testes, and skin. Melatonin and its metabolites possess potent antioxidant activity, and its therapeutic applications or preventive uses are based in part on this property.

In AD, the beneficial effects of melatonin involve protection of nuclear DNA, membrane lipids and cytosolic proteins. Because many lines of evidence have shown strong implications of oxidative stress in the origins and effects of AD—combined with the fact that melatonin levels are greatly reduced or even absent—the development of approaches to reduce oxidative stress and restore melatonin levels in AD patients seem likely to provide new therapeutic opportunities.


Sub-effective concentrations of
melatonin combined with
galantamine offer a synergic
neuroprotective effect in an in vitro
model
of oxidative stress.



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AChEI are used in the treatment of AD. But their clinical usefulness in some diseases is limited largely because of their accompanying adverse effects (confusion, hallucinations, extreme or sudden changes in behavior, nausea, or stomach pain) arising from excessive activation of the cholinergic system in some users. The AChEI galantamine is an alkaloid isolated from snowdrop and snowflake flowers that has been used for AD. It is a central acting, selective, reversible, and competitive AChEI, as well as an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. These properties are of value for improving cholinergic transmission, and galantamine has been shown to be protective in different models related to AD, ischemia, Huntington’s disease, and amyotrophic lateral sclerosis. And it is easier to take than other AChEI.

Returning to melatonin, there is evidence that it may have an effective positive influence on several of the AD hypotheses that explain the cause of the disease. As well, melatonin has negligible toxicity and is able to reduce the side effects and increase the efficacy of other drugs or nutrients. Moreover, previous data from other researchers have demonstrated that sub-effective concentrations of melatonin combined with galantamine offer a synergic neuroprotective effect in an in vitro model of oxidative stress.3

Galantamine Treatment for Arthritis

And there’s more … In a recent study of research done at several Egyptian Universities, galantamine significantly reduced all biomarkers of inflammation in rats, with the highest dose showing the best beneficial anti-inflammatory effect.1 These results were superior in magnitude to the reference drug leflunomide in most of the studied parameters. In conclusion, these results suggest that galantamine may represent a novel, inexpensive and effective therapeutic strategy in the treatment of rheumatoid arthritis.

  1. Gowayed MA, Refaat R, Ahmed WM, El-Abhar HS. Effect of galantamine on adjuvant-induced arthritis in Rats. Eur J Pharmacol. 2015 Jul 16. pii:S0014-2999(15)30164-3. doi: 10.1016/j.ejphar.2015.07.038. [Epub ahead of print]

The Goals of Combination Therapy

The Madrid study had two objectives: 1) to develop a new in vitro model that reproduces AD-related pathological hallmarks by combining the toxic stimuli βA/OA which leads to Tau protein hyperphosphorylation in hippocampal cultures and 2) to determine if subthreshold concentrations of melatonin and galantamine reduce AD-like pathology and apoptosis in the toxicity model of βA/OA. The results of this study could provide new insights on the validity of this combination therapy in AD and also to develop new multitarget melatonin-cholinergic compounds.

Cell Death Caused by β-Amyloid and Okadaic Acid Alone or in Combination

The researchers first performed concentration-response curves with βA and OA in order to evaluate their toxicity in hippocampal cultures. Then after 4 days, hippocampal cultures were exposed for another 4 days to increase concentrations of the toxic stimuli. βA increased the cell death of the hippocampal cultures in a concentration-dependent manner. At the concentration of 5 μM, it almost doubled basal cell death.


The association of subeffective
concentrations of melatonin and
galantamine caused a marked
reduction of AD benchmarks.


In the case of OA, significant cell death was achieved at the concentration of 3 nM and at 10 nM it almost tripled basal cell death. In order to develop a new neurotoxicity model that combined both alterations, i.e., βA and hyperphosphorylation of Tau, hippocampal cultures were treated with subtoxic concentrations of βA and OA as had previously been described in the human neuro­blastoma cell line SH-SY5Y.

The combination of 0.5 μM of βA with 1 nM of OA almost doubled (195±16 %) basal cell death measured as PI uptake; this cell death was significantly higher than that afforded by the toxic stimuli alone (108±12 % for βA and 147±14 % for OA). Most of the cells in CA1 treated with the combination of 0.5 μM of βA with 1 nM of OA were TUNEL-positive as compared to control; these results indicate that the βA/OA toxic stimuli principally caused apoptotic cell death in the hippocampal culture. TUNEL is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.

Effect of Sub-effective Concentrations of Melatonin and Galantamine on βA Aggregates and Tau Hyperphosphorylation

Once the researchers observed that sub-effective concentrations of melatonin plus galantamine could afford a significant protective effect, they were interested in knowing if our toxicity model would reproduce two of the major pathological hallmarks associated with AD, and if the combination of Mel/Gal could reduce these alterations. When they analyzed hyperphosphorylation of Tau they observed that βA/OA increased Tau phosphorylation by 2.2-fold while Mel/Gal treatment reduced it by 73 %. Therefore, their toxic stimuli reproduced both βA aggregates and hyperphosphorylation of Tau and, more interestingly, the association of subeffective concentrations of melatonin and galantamine caused a marked reduction of AD benchmarks.


Maximum protection was achieved
with 300 nM galantamine (56%
protection) and 10 nM melatonin
(50% protection), the highest level
for each.


Neuroprotective Mechanism of the Combination of Melatonin with Galantamine

Besides inflammation, the Madrid researchers also looked at radical oxygen species (ROS) production. The toxic stimuli βA/OA almost doubled ROS production, and such effect was almost reduced to basal levels when Mel/Gal was present in the hippocampal culture. The reduction of ROS production caused by the combination of Mel/Gal was blocked by a melatonin receptor antagonist drug and by a drug that inhibits the binding of acetylcholine to nicotinic acetylcholine receptors.

Finally, to confirm that the protective effect of the neuroprotectant combination Mel/Gal was mediated by melatonin and a7 nAChRs, the researchers measured cell viability by an indicator of cell death; both the antagonist and the inhibitor drug prevented the protective effect of the combination of Mel/Gal against bA/OA-induced toxicity. This confirmed that melatonin and a7 nAChRs receptors are plasma membrane targets for the protective actions of the combination Mel/Gal.

Not the First Study to Show Mel/Gal Synergy

This was not the first study suggesting that melatonin may help a cholinergic, such as galantamine, work more effectively. Both melatonin and galantamine are antioxidants and neuroprotective nutrients. In 2010, another research team published an investigation with the goal of evaluating a possible synergistic neuroprotective effect of sub-effective concentrations of combined galantamine and melatonin. 3

In this study, human neuroblastoma cells were subjected to mitochondrial oxidative stress by blockade of mitochondrial complexes with metabolic poisons. This lasted 24-hours and caused 40% of the cells to die. When the cells were incubated with increasing concentrations of galantamine (10-300 nM) or melatonin (0.3-10 nM) for 24 hours, followed by the same length period with the two poisons, the result was a concentration-dependent protection. Maximum protection was achieved with 300 nM galantamine (56% protection) and 10 nM melatonin (50% protection), the highest level for each.

The combination of sub-effective concentrations of galantamine (30 nM) and melatonin (0.3 nM) caused a synergistic and significant protection that was similar to the maximum protection afforded by effective concentrations of melatonin or galantamine alone. This protective effect was completely reversed when nicotinic and melatonin receptors were blocked by two drugs specific to these effects.


Compared to the neuroblastoma
model, the hippocampal culture
model is superior for the study of
neurodegeneration.


Madrid Provides More Experimental Evidence

In the present study, the Madrid researchers provide more experimental evidence on the synergic neuroprotective effect of the combination of sub-effective concentrations of melatonin and galantamine in an AD-related pathology model. Nanomolar concentrations of melatonin and galantamine improves AD markers including:

  1. β-amyloid aggregation

  2. Tau hyperphosphorylation

  3. Inflammation

  4. Oxidative stress in hippocampal cultures sub-chronically exposed to βA and OA.

Compared to the neuroblastoma model, the hippocampal culture model is superior for the study of neuro­degeneration. This model allowed the researchers to incubate the slices for subchronic periods (4–7 days). Thus, they could reduce the concentrations of the toxic stimuli to more pathophysiological ones.

In the work, they incubated the slices with βA or OA at concentrations 50-fold lower than concentrations used in the literature, and used a range of βAs which has shown to be as toxic as other βA fragments. Thus, they developed a new model that combines βA and Tau pathologies using subtoxic concentrations of the toxic stimuli, βA/OAs, which by themselves did not provide toxicity.

Under these experimental conditions, subtoxic concentration of βA/OA produced an increment of cell death, which was confirmed as apoptotic. Postmortem analysis of AD brains shows that there is DNA fragmentation in neurons and glia of hippocampus and cortex as detected by a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.


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The Madrid researchers’ results are also consistent with those obtained in the triple transgenic animal model of AD, 4 where the functional alteration and pathological hallmarks appear earlier compared to double transgenic animals. The most important finding was that the combination of low concentrations of melatonin (1 nM) and galantamine (10 nM), which did not afford protection by themselves, produced a potent neuroprotective, antioxidant and anti-inflammatory effect.

Melatonin with Other Drugs Enhances the Protective Effect of Melatonin

Recently, several studies have reported that simultaneous administration of melatonin with other drugs enhances the protective effect of melatonin, as well as the effect of the second drug. In a previous work, the Madrid researchers demonstrated that combination of sub-effective concentrations of melatonin and galantamine protects against oxidative stress in human neuroblastoma cells.

In this work, the effective concentrations of melatonin and galantamine were higher, probably because the toxic stimulus was more potent and it was applied during a shorter period. Here, they corroborated these data and expanded them to AD. By the use of the βA/OA model, some of the major pathological markers of AD are resembled, such as amyloid aggregates, Tau hyperphosphorylation, neuroinflammation, and oxidative stress. The researchers proved that combined therapy of melatonin and galantamine is beneficial as it restored all these pathological alterations. The potential effect of melatonin in the different AD hypothesis has been recently reviewed, and the conclusion is similar to the Madrid scientists’ results: that melatonin could prevent amyloid overproduction and reduce hyperphosphorylation of Tau; it is an antioxidant and free radical scavenger and modulates pro-inflammatory processes and, curiously, it could work as an anticholinesterase agent.


If melatonin supplementation is
provided, the dose of the AChEI could
be potentially reduced to as little as
one tenth its effective dose.


On the other hand, galantamine, which is one of the current treatments, has proven to be useful in multiple in vitro and in vivo AD models and in different clinical trials. However, AChEI revealed only a modest trend favoring active treatment over placebo. Glutamate excitotoxicity, mitochondrial dysfunction, and free radical-mediated damage are identified as pathophysiological mechanisms leading to neuronal death in neurodegenerative diseases. Neuroprotective properties of melatonin against these three processes, added to its regulatory effects on circadian disturbances, point to melatonin as a therapeutic substance in the symptomatic treatment of neurodegenerative diseases. The multiple neuroprotective effects of melatonin, which include its anti-apoptotic, anti-inflammatory, and antioxidant actions, are mediated by both receptor and nonreceptor pathways.


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However, it has been demonstrated that the neuroprotective effect of galantamine, a well-known AChEI used to treat AD, is associated with its ability to act on nicotinic receptors and to its antioxidant effect. Hence, both drugs could be good candidates to combat AD. In the current study, the researchers observed that the neuroprotective effect of subthreshold concentrations of melatonin and galantamine is specifically mediated by both melatonin and nicotinic receptors, as the blockade of those receptors with both a melatonin receptor antagonist drug and by a drug that inhibits the binding of acetylcholine to nicotinic acetylcholine receptors totally reversed their neuroprotective, antioxidant, and anti-inflammatory actions. Melatonin exerts neuroprotective effects in different models of AD.

It must be noted that melatonin production declines with aging due to dysfunction of the sympathetic regulation of pineal melatonin by the suprachiasmic nucleus. The loss of melatonin during aging contributes to oxidative stress accumulation because its antioxidant effect is lost, and thereby it contributes to the progression of diseases with a free radical component like neurodegenerative diseases or cardiovascular disease. Supplementation of melatonin in aging animals reduced AD deviant changes due, in part, to the antioxidant effect of melatonin at the mitochondrial level.

Results from this study indicate that there are several advantages for melatonin supplementation along with an AChEI supplement such as galantamine. From a clinical point of view, if melatonin supplementation is provided, the dose of the AChEI could be potentially reduced to as little as one tenth its effective dose. This would imply a significant reduction of cholinergic side effects, which while unlikely with galantamine is more apt to be so with other AChEI. This could contribute to increased therapeutic compliance and, indirectly, improved global efficacy of this combination of nutrients.


Therefore, melatonin could
improve galantamine therapy for
the treatment of AD.


Alternatively, since melatonin levels are significantly reduced or absent in AD patients, the addition of melatonin to standard AD treatments could help restore internal melatonin levels and its associated homeostatic actions. Remember that a recent report by Wade et al2 showed that the addition of melatonin to an acetylcholinesterase inhibitor, with or without memantine treatment, had positive effects on cognitive function and sleep maintenance in mild to moderate AD patients compared to placebo. There are also several studies that support the potential role of melatonin as an effective adjuvant in AD management.5,6

When considered as a whole, these results show that subjecting the hippocampal culture with the combination of low concentrations of βA and OA for 4 days causes βA, aggregates, hyperphosphorylation of Tau protein, oxidative stress, inflammation and glial alterations to form. Each of these are found in the brains of AD patients.

However, when the combined therapy of melatonin and galantamine were added at even sub-threshold concentrations, the nutrients synergized and were thus proven to be a powerful force for preventing cell death, βA aggregates, hyperphosphorylation of Tau protein, oxidative stress, and glial alterations. Therefore, melatonin could improve galantamine therapy for the treatment of AD.

References

  1. Buendia I, Parada E, Navarro E, León R, Negredo P, Egea J, López MG. Subthreshold Concentrations of Melatonin and Galantamine Improves Pathological AD-Hallmarks in Hippocampal Organotypic Cultures. Mol Neurobiol. 2015 Jun 17. [Epub ahead of print] PubMed PMID: 26081146.
  2. Wade AG, Farmer M, Harari G, et al. Add-on prolonged-release melatonin for cognitive function and sleep in mild to moderate Alzheimer’s disease: a 6-month, randomized, placebo-controlled, multicenter trial. Clin Interv Aging. 2014 Jun 18;9:947-61.
  3. Romero A, Egea J, García AG, López MG. Synergistic neuroprotective effect of combined low concentrations of galantamine and melatonin against oxidative stress in SH-SY5Y neuroblastoma cells. J Pineal Res. 2010 Sep;49(2):141-8.
  4. Rhein V, Song X, Wiesner A, et al. Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer’s disease mice. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):20057-62.
  5. Pappolla MA, Sos M, Omar RA, et al. Melatonin prevents death of neuroblastoma cells exposed to the Alzheimer amyloid peptide. J Neurosci. 1997 Mar 1;17(5):1683-90.
  6. Yang X, Yang Y, Fu Z, et al. Melatonin ameliorates Alzheimer-like pathological changes and spatial memory retention impairment induced by calyculin A. J Psychopharmacol. 2011 Aug;25(8):1118-25.


Will Block is the publisher and editorial director of Life Enhancement magazine.

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