Patent Publication Number: US-2022233546-A1

Title: Neurologist Formulated Nocturnal Nootropic Founded on a Novel Theory of Brain Aging

Description:
INTRODUCTION 
     Rational scientific explanations, or hypotheses, for maladaptive brain aging are well studied 1 . However, a trusted scientific explanation, or theory, on the mechanisms of brain aging is not well-established and currently under robust investigation 1 . Brain Theory is a proposed theory of brain aging developed by a physician-scientist neurologist and founded on high-quality level evidence, including but not limited to randomized clinical trials and critical appraisal of the scientific literature. A dietary supplement duly purposed to modulate maladaptive brain aging based on an evidence-based theory of brain aging as well as provide nootropic benefits, herein referred to as BrainTheory™ N o  12, does not currently exist. 
     Modulation of Biological Brain Aging 
     Brain Theory is founded on the principle that maladaptive brain aging is a manifestation of biological age (healthspan) as opposed to chronological age (lifespan) 2 . Early in life, molecular pathways controlled by genetic and epigenetic factors determine biological aging 2 . The heterogeneous accumulation of mutations and coding errors during a lifespan determines if the aging process is adaptive or maladaptive 2 . Bioactive, neuro-available substances in BrainTheory™ N o  12 are proposed to modulate molecular aging pathways that affect biological brain aging. 
     Nootropic Effects 
     The proposed nootropic effects of BrainTheory™ N o  12 are associated with the cognitive enhancement properties of each substance. Review of high-quality level evidence including but not limited to randomized controlled trials and critical appraisal of the scientific literature validates proposed claims of cognitive enhancement, bioactivity, neuro-availability, dose-dependence, nocturnal administration, safety, and tolerability. 
    
    
     PROPOSAL 
     Nocturnal Biological Brain Aging Processes 
     Biological brain aging processes are constant and dynamic. However, there are sleep-dependent functions vital to preservation of brain function including but not limited to clearance of pathological brain aging biomarkers 3  and learning and memory consolidation 4 . Moreover, neurodegenerative processes and dementia syndromes are preceded by clinical and/or subclinical disorganized sleeping patterns as well as impaired degradation and clearance of pathological proteins and toxic waste products 5 . Therefore, nocturnal administration of BrainTheory™ N o  12 (1-3 hours prior to onset of sleep) is recommended for peak performance. 
     R-Alpha-Lipoic Acid and the Neurological Reserve Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Neurological Reserve Hypothesis is also known as the Network Control Hypothesis 6, 7 . Brain size (brain reserve) and the brain&#39;s ability to grow, evolve, and adapt (cognitive reserve) decreases during the normal brain aging processe 6, 7, 8 . However, there is significant heterogeneity in the rate of decline of neurological reservese. 6, 7, 8 . This is due to marked between-persons differences in the molecular resilience of the brain&#39;s neural network 6, 7, 8 . Neurons with effective intercommunication have superior connectivity such that the ‘connectome’ is optimally activated and preserved 6, 7, 8 . 
     The substance in BrainTheory™ N o  12 that preserves brain and cognitive reserve is (R)-Alpha Lipoic Acid. The R-enantiomer is the bioactive and neuro-available form of Alpha Lipoic Acid, which is endogenous and produced in neuronal mitochondria 9 . (R)-Alpha Lipoic Acid is clinically shown to increase brain and cognitive reserve 9 . A randomized, controlled, double-blind human clinical trial showed that bioactive (R)-Alpha Lipoic Acid reduces yearly rate of brain atrophy in young persons with multiple sclerosis by nearly 70% with a good safety and tolerability profile 9 . Multiple sclerosis is a potential model of neurological reserve given that brain atrophy is part of the natural history and pathophysiology of this pathological disease process 9 . 
     Nootropic and Systemic Effects 
     Human studies demonstrate that bioactive (R)-Alpha Lipoic Acid is effective in alleviating symptoms of peripheral neuropathy 10  and improves glucose and fat metabolism for weight control 11 . Bioactive (R)-Alpha Lipoic Acid is superior to glutathione and independently enhances the systemic functions of glutathione, vitamins C, and vitamin E 12 . 
     Crocetin and the Reductive-Oxidative Stress Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Reductive-Oxidative Stress Hypothesis posits that neurons shift from a stable reduced state to an unstable oxidized state in normal brain aging 13  whereby reduction and oxidation are inverses, the latter of which is characterized by the loss of electrons, loss of hydrogen, gain of oxygen, and increase in oxidation number. 
     In maladaptive brain aging, a majority of neurons shift from a stable, reduced state to an unstable, oxidized state that propagates the accumulation of regulatory errors and inhibits the mitigation of salvage pathways 13 . The sequelae of this maladaptive process is decreased capacity to maintain optimal cognitive function with age. This is also the rationale behind the use of antioxidants and free radical scavengers in nutritional and dietary supplementation 13 . 
     The substance in BrainTheory™ N o  12 that mitigates redox stress is Crocetin. Crocetin is a bioactive, neuro-available component of saffron that has superior stability and blood-brain barrier penetrance compared to other saffron derivatives, including saffranal and crotin 14 . A randomized, controlled, double-blind human clinical trial demonstrated that bioactive saffron has similar effects to donepezil, a prescription medication for dementia, in mild to moderate Alzheimer&#39;s disease in under 6 months. The Alzheimer&#39;s Disease Assessment Scale-Cognitive Subscale that assesses word and procedural recall, naming, commands, construction and ideational praxis, orientation, word recognition, language, and comprehension was used for evaluation 15 . 
     Nootropic and Systemic Effects 
     Bioactive saffron is also superior to vitamin E for protection of the cell wall with less risk of toxicity. Cell wall stability determines the pathway of rate-limiting steps in brain aging 16 . Bioactive saffron regulates subclinical neurodegenerative processes that predate clinical manifestations of mild cognitive impairment 17, 18 . Human studies demonstrate that bioactive saffron has a similar effect to methylphenidate in attention deficit disorder 19 , contains antidepressant and analgesic properties comparable to fluoxetine 20 , and reduces opioid-related memory loss and opioid withdrawal symptoms 20 . Laboratory models demonstrate that bioactive saffron has anti-epileptic properties and clinical efficacy in mitigating neuronal damage in stroke, traumatic brain injury, and spinal cord injury 20 . 
     Curcumin and the Caloric Restriction Anti-Inflammation Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     Akin to localized macroscopic inflammation from physical injury, molecular inflammation occurs systemically on a microscopic level 21 . In a subset of individuals, inflammation occurs at an accelerated rate with a fulminant course akin to a cytokine storm 21 . In brain aging, activation of specific neuron subtypes, namely astrocytes and microglia, precipitate neuroinflammation 21 . 
     The sequelae of this maladaptive process is decreased capacity to maintain cognitive function with age and accelerated neurodegenerative processes 21 . The scientific literature suggests that neuroinflammation can be mitigated in a multitude of ways 21 . However, caloric restriction has the strongest evidence that demonstrates attenuation of pro-inflammatory astrocytic and microglial processes 21 . Given the practical limitations of caloric restriction, mimickers of caloric restriction are well studied and have similar mechanisms of action in regulating inflammatory pathways 21 . 
     The substance in BrainTheory™ N o  12 that regulates neuroinflammation is Curcumin. Curcumin is a bioactive and neuro-available component in turmeric that mimics caloric restriction in the brain and systemically 22 . A randomized, controlled, double-blind human clinical trial demonstrated that Curcumin improves working memory and attention in adults without dementia 22 . Tests for verbal memory (Buschke Selective Reminding Test), visual memory (Brief Visual Memory Test-Revised), and attention (Trail Making A) domains were used for evaluation 22 . Persons who underwent positron emission tomography to detect post-interventional changes in pathological proteins, including amyloid and tau, demonstrated a significant decrease in pathological proteins in brain regions including the amygdala 22 . 
     Nootropic and System Effects 
     Human studies demonstrate that Curcumin improves mood, anxiety, and fatigue in healthy adults 23 . Curcumin plays a preventative role in Alzheimer&#39;s and other neurodegenerative diseases 23, 24 . In athletes and non-athletes, Curcumin decreases biomarkers of stress and muscle damage 25 . Curcumin has anti-obesity properties that limit fat cell growth in metabolic syndrome as well as improves hyperglycemia, insulin resistance, hypertension, hyperlipidemia, and arthritis 26, 27 . 
     Methylcobalamin and Choline in the Homocysteine Metabolism Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     Homocysteine is an amino acid that requires post-production modification mainly via the transfer of a methyl group 28 . High levels of homocysteine lead to dysfunctional transfer of methyl groups 28 . The main sequelae of this maladaptive process is hypermethylation of DNA and proteins that accelerate brain aging 28 . An international consensus statement based on evidence from meta-analyses of human studies concluded that high levels of homocysteine are associated with age-associated neurodegenerative diseases including Parkinson&#39;s and Alzheimer&#39;s diseases, accelerated brain atrophy, chronic brain hypoxia, and ischemic stroke 29-34 . 
     The substance in BrainTheory™ N o  12 that regulates homocysteine methylation is Methylcobalamin 35 . Methylcobalamin is the bioactive and neuro-available form of vitamin B12 that has superior bioavailability compared to cyanocobalamin 35 . A meta-analysis of 15 human clinical studies demonstrated there is an association between hyperhomocysteinemia and cognitive impairment in persons with Parkinson&#39;s disease 38 . Mini-mental status examination of orientation, registration, attention, recall, naming, repetition, syntax, reading, commands, and copying was used for evaluation 38 . 
     The indirect methyl donor, Choline, and direct methyl donor, Betaine, also mitigate hypermethylation of DNA and proteins in accelerated brain aging 37 . Choline and Betaine are essential nutrients that additionally fortify the neural membrane, enhance neuro-metabolism, and regulate neurotransmitter synthesis 37 . Human clinical trials using Betaine or Choline supplementation demonstrate a significant reduction in plasma homocysteine concentrations 37 . 
     Nootropic and Systemic Effects 
     Human studies demonstrate that Methylcobalamin has clinical benefit in the duration, cardinal symptoms, and severity of Parkinson&#39;s disease as well as mild cognitive impairment, adaptive behavior in autism spectrum disorder, late-life depression, and peripheral neuropathy 38, 39, 40 . Choline and Betaine are essential in impaired folate-dependent methylation, especially in cases of folate deficiency, vitamin B12 deficiency, or alcoholism 37 . 
     Methylcobalamin is effective in non-neurological conditions including congenital birth defects, dysfunction calcium metabolism, and diseases of the cardiovascular, renal, and gastrointestinal systems 41 . Meta-analysis of epidemiological studies demonstrate an inverse relationship between serum Choline and Betaine concentrations and the incidence of colorectal, breast, nasopharyngeal, hepatocellular, and pulmonary neoplasms 42 . 
     5-Methyltetrahydrofolate and  Eucommia ulmoides  Oliver in the Neuroplasticity Neurotransmitter Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Neuroplasticity Neurotransmitter Hypothesis posits that the capacity for early neurodevelopment, post-injury neural recovery, and neural enhancement in adulthood, known as neuroplasticity, is modulated largely by neurotransmitters, the control of which is weakened in maladaptive brain aging 43, 44, 45 . This is validated in clinical, pathological, and imaging studies in human and animal models of normal aging and neurodegenerative disease 43, 44, 45 . 
     These neurotransmitters include acetylcholine, dopamine, serotonin, adrenaline, and norepinephrine that are critical for attention, learning, cognition, memory, impulse control, reward-motivation behavior, mood, and somatic manifestations of stress 43, 44, 45 . Significant inter-person heterogeneity in baseline neurotransmitter reserves determine the capacity for neuronal recruitment, activation, and communication in hippocampal, frontal-temporal-parietal, subcortical, limbic, auditory, and visual neural networks as well as cerebral blood flow patterns and rate of grey matter atrophy 43, 44, 45 . 
     The substance in BrainTheory™ N o  12 that facilitates neuroplasticity is 5-Methyltetrahydrofolate. 5-Methyltetrahydrofolate is the bioactive and neuro-available form of folate that is critical in the production of neuromodulatory neurotransmitters 46 . Evidence from a randomized, controlled human clinical trial demonstrated that bioactive folic acid supplementation in persons with mild cognitive impairment improves cognitive function, as measured by pre- and post-intervention full scale IQ, verbal IQ, verbal comprehension index, and working memory index, and deceases dementia-related biomarkers including beta-amyloid, homocysteine, S-adenosylmethionine, and amyloid precursor protein mRNA expression 46 . 
     Phytochemicals in  Eucommia ulmoides  Oliver have neuroplasticity properties 47, 48 . Laboratory models demonstrate that phytochemicals in  E. ulmoides  inhibit acetylcholinesterase, the enzyme that degrades acetylcholine, in lesioned brain regions including the frontal cortex and hippocampus 47 . In the lesioned brain, via phytochemical modulation of different molecular pathways, mitochondrial structure and function is stabilized and neurotransmitter and brain-derived neurotrophic factor concentrations are increased 47 . Phytochemicals in  E. ulmoides  also regulate the ubiquitin-proteasome system to increase neuron survival and control neuron apoptosis 48 . 
     Nootropic and Systemic Effects 
     Human studies demonstrate that bioactive folate has clinical benefit in selective serotonin reuptake inhibitor (SSRI)-resistant major depression, analgesic effect in chronic pain syndromes, incidence of neural tube defects, and autism spectrum disorder as well as neuromodulatory properties in the primitive limbic system of persons with schizophrenia 49, 50, 51 . 
     Laboratory models demonstrate that active phytochemicals in  E. ulmoides  significantly improve short-term and working memory impairment and partially reverse acquired learning deficits 48 . Studies also demonstrate that phytochemicals in  E. ulmoides  have a calming effect via promotion of synapsin I, which increases serotonin release with resultant anti-fatigue and antidepressant effects 48 . In epilepsy-associated memory impairment, phytochemicals in  E. ulmoides  protect memory-associated CA-2 hippocampal neuron degeneration 48 . 
       E. ulmoides  also has potent anti-aging effects via augmentation of collagen synthesis and UV radiation-induced photo-aging 48 . Human and laboratory studies demonstrate non-neurological benefits of  E. ulmoides  in immunoprotection, hypertension, cardiovascular remodeling, hyperlipidemia, diabetes mellitus, erectile dysfunction, glaucoma, bone metabolism and diseases of the hepatic, renal, and pulmonary systems 48 . 
     Trans-Pterostilbene in the Telomere Mortality Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     In maladaptive brain aging, the production of abnormal neuronal substrates increases as a result of the erroneous integration, excision, and modification of genetic material originating from chromosomes 52, 53, 54 . Chromosomes change in morphology and decrease in length over time via the degeneration of telomeres, which are structures at the distal ends of chromosomes purposed to preserve chromosomal integrity 52, 53, 54 . The preservation of chromosomal integrity is due to telomerase, an enzyme produced in a subset of non-human animals to preserve telomeres and thereby maintain original chromosomal function 52, 53, 54 . 
     The genotype and phenotype of a subset non-human animals such as those in the Nephropidae family are not significantly altered over a lifespan secondary to sufficient production of telomerase 52, 53, 54 . The adult human has minimal telomerase reserves within highly sensitive, eloquent regions of the brain including the hippocampus 52, 53, 54 . 
     The substance in BrainTheory™ N o  12 that increases telomere length is Trans-Pterostilbene, the bioactive and neuro-available component of resveratrol. Trans-Pterostilbene restores RNA splicing ability and is associated with decreased human telomerase reverse transcriptase (hTERT) activity 55,56 . RNA splicing involves post-production protein modification to excise and integrate target protein regions 55 . Splicing errors have significant downstream effects that propagate deleterious changes in neurons and shortens telomeres 55 . Trans-Pterostilbene also has the capacity to regulate apoptosis to maintain systemic homeostasis 55 . The latter will be discussed in the ‘Dose-Dependent Substrates’ section. 
     Evidence from a randomized, double-blind, placebo-controlled human clinical trial demonstrated that Trans-Pterostilbene significantly improves function in and slows progression of neurodegenerative processes in amyotrophic lateral sclerosis (ALS) 55 . Quality of life factors including speech, salivation, swallowing, handwriting, feeding, dressing, hygiene, walking, climbing stairs, and pulmonary function as well as muscular strength and skeletal muscle:fat weight ratio were evaluated 55 . ALS an appropriate model neurodegenerative disease given that corticospinal motor neuron degeneration and neuromuscular unit atrophy are pathognomonic of of maladaptive brain aging. 
     Nootropic and Systemic Effects 
     Human studies demonstrate that Trans-Pterostilebene significantly improves mini-mental status cognitive examination and the ability to perform activities of daily living in persons with neurodegenerative disease 57, 58 . Human studies demonstrate that Trans-Pterostilebene mimics anti-aging benefits of caloric restriction through the critical aging pathway SIRT1 57 , a system involved in the onset of neurodegeneration. Trans-Pterostilebene demonstrates clinical benefit in neurological diseases such as ischemic stroke and non-neurological autoimmune diseases associated with its potent antioxidant, anti-inflammatory, and anti-immunosenescence properties 58 . Pre-clinical studies also demonstrate that Trans-Pterostilebene facilitates hippocampal regeneration at a low to moderate dose by enhancing neurogenesis and inhibiting apoptosis of normal cells 59, 60, 61 . 
     Cholecalciferol in the Immunosenescence Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The immunosenescence hypothesis posits that the immune system has innate and adaptive cell lines that become senescent, or senile, in maladaptive brain aging 62 . The innate immune response is non-specific and ubiquitously protects points of entry, namely the integumentary system and mucous membranes, whereas the adaptive immune response is targeted, specific, with inherent memory capabilities 62 . The adaptive immune system is comprised of antibody-generating B cells and helper or killer T cells 63 . Helper T-cells trigger an immuno-inflammatory response and killer T-cells directly neutralize pathogens 63 . In a functional immune system, this hyperdynamic response is limited in space and time 63 . 
     In the hyperactive young, adaptive immune system, targets may be supra-specific and identify self-antigens as foreign, hence the propensity for autoimmune diseases in younger populations 62, 63 . As the same young persons age, autoimmune diseases defervesce as a result of loss of this hyperactive self-antigen response 62, 63 . In maladaptive brain aging, T-cells and molecular checkpoints become dysfunctional 63, 64 . As a result, multiple aberrant phenomena may occur. Chronic, dormant infectious agents can reactivate and previously targeted immuno-inflammatory responses may become non-specific and unregulated (not limited in space and time) 63, 64 . The dysfunctional aging immune system self-attacks as well and usually with a more fulminant course given there are less neurological reservese 63, 64 . 
     The substance in BrainTheory™ N o  12 that regulates immunosenescence is Cholecalciferol, the bioactive and neuro-available form of vitamin D 65 . Evidence from a randomized, controlled, double-blind human clinical trial demonstrated that high doses of bioactive vitamin D are associated with fewer new brain lesions, reduced disability, and improved mobility when used as adjunctive therapy in multiple sclerosis 65 . 
     However, doses greater than the recommended allowance should be reserved for hyperactive immune system responses in autoimmune diseases 62, 63, 64 . In the aging immune system, a low to moderate dose of Cholecalciferol, to be described in the ‘Dose-Dependent Substances’ section, optimizes T-cell function and reduces neuron microglial response 62, 63, 64 . Human and laboratory models demonstrate that bioactive vitamin D increases neuronal development, function, and survival via neurotrophins and promotes growth of axons and myelin 66, 67 . This has implications in the pathophysiology of a range of processes from neurodegenerative to neurorestorative 66, 67 . 
     Anti-inflammatory omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) down-regulate immunosenescence 66, 69 . ω-3 PUFAs balance existing T-cells subtypes, regulate T-cell production pathways, and effect the ability of antigen presenting cells to signal T-cells 68, 69 . Laboratory models of immunosuppression demonstrate immune system enhancement with ω-3 PUFA supplementation 68, 69 . Human studies additionally demonstrate positive effect on telomere length associated with enhanced healthspan 70 , preservation of brain volume, and mitigation of amyloid proteinopathy 71 . A meta-analysis of seven randomized controlled trials investigating the effect of ω-3 PUFAs on mild cognitive impairment demonstrated that supplementation improves cognition in older adults with mild cognitive impairment 70 . 
     Nootropic and Systemic Effects 
     Cholecalciferol deficiency plays a major role in a spectrum of neurological and psychiatric disorders, including painful peripheral nerve injury, depression, and epileptic seizures as well as medical conditions including rheumatoid arthritis, type 1 diabetes, lupus, and inflammatory bowel disease 66, 72, 73 . 
     ω-3 PUFAs plays role in attention, processing speed, and recall in persons with mild cognitive impairment as well as neurological processes including early neurodevelopment, depression, and attention deficit hyperactivity disorder 71 . ω-3 PUFAs also demonstrate benefit in non-neurological conditions including cardiovascular disease, age-related macular degeneration, rheumatoid arthritis, inflammatory bowel disease, childhood allergies, and pulmonary function in cystic fibrosis 71 . 
     Apigenin in the Proteinopathy Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Proteinopathy Hypothesis posits that, in maladaptive brain aging, protein misfolding and errors in protein post-processing result in the subsequent buildup of pathological protein compounds, known as proteinopathy 74, 75 . For context, neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer&#39;s disease (AD), cerebral amyloid angiopathy (CAA), Parkinson&#39;s disease (PD), and chronic traumatic encephalopathy (CTE) are primarily proteinopathies 75 . The former two are characterized by the abnormal accumulation of TDP-43 protein 75 . AD is characterized by the abnormal accumulation of tau and amyloid proteins 75 . CAA is characterized by the abnormal accumulation of amyloid protein 75 . PD is characterized by the abnormal accumulation of alpha-synuclein protein 75 . CTE is characterized by the abnormal accumulation of both tau and TDP-43 proteins 75 . Clinical presentations are based on the localization of pathological protein deposition. Clusters of differentiation, or CD, cells are critical in immune system signaling and protein expression 76 . CD-40 regulates microglial immuno-inflammation in the nervous system and is dysfunctional in neurodegenerative disease such as ALS, AD, PD, prion diseases, and HIV-associated dementia as well as immuno-inflammatory diseases such as multiple sclerosis 76 . 
     The substance in BrainTheory™ N o  12 that mitigates proteinopathy is Apigenin. Apigenin is a flavonoid that prevents the buildup of misfolded amyloid protein, tau protein, alpha-synuclein protein 77  and modulates CD-40 expression 76 . Evidence from critical appraisal of human studies demonstrates that Apigenin improves memory, learning, and sleep in both persons with Alzheimer&#39;s disease and healthy persons 77, 78 . Human studies show that Apigenin promotes brain-derived neurotrophic factors to augment long term memory, promote neurogenesis, and support angiogenesis 79, 80, 81 . 
     Nootropic and Systemic Effects 
     Apigenin is a non-addictive alternative to anxiolytic agents secondary to its GABA-nergic mimicry and antagonistic effects on stress-induced hypercortisolism 78 . Apigenin has antidepressant and anti-insomnia properties as well as anti-carcinogenic and anti-viral properties 78, 82 . 
     Luteolin in the Glymphatic Dysfunction Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Glymphatic Dysfunction Hypothesis posits that the brain has a glial-dependent clearance system for waste and metabolic byproducts termed the glymphatic system, as the brain itself lacks true lymphatic vessels 83, 84 . The glymphatic system transports waste and metabolic byproducts, such as those that buildup in proteinopathies, from the interstitial fluid 83, 84 . Waste is then transferred to the true lymphatic system outside of the brain via the meningeal dura, cranial nerves, and blood vessels that exit the skull base. This occurs through the blood brain barrier, the permeability and integrity of which is largely regulated by mast cells 85 . The glymphatic system is impaired in maladaptive brain aging as well as neurodegenerative diseases, traumatic brain injury, intra-cerebral hemorrhage, and ischemic stroke 83, 84, 85 . 
     The substance in BrainTheory™ N o  12 that facilitates neurotoxic waste product clearance is Luteolin. Luteolin is a flavonoid that regulates neuronal mast cells to facilitate the clearance of neurotoxic waste through the blood-brain barrier. Evidence from critical appraisal of human studies demonstrates that Luteolin improves ‘brain fog,’ cognition, concentration, and multitasking in the healthy brain 85 . 
     Luteolin promotes the production and distribution of different neurotrophins that stimulate neuronal survival, development, and function 85 . In both immunosenescence and the hyperactive immune system, Luteolin&#39;s potent anti-inflammatory properties have the capacity to alter transcription of microglial cells to a neuroprotective phenotype 86, 87, 88 . 
     Nootropic and Systemic Effects 
     Human studies demonstrate that Luteolin improves attention and sociability in autism spectrum disorder 89  and is protective in fibromyalgia, fatigue syndromes, celiac disease, and other diseases associated with increased mast cells 85 . 
     Melatonin in the Circadian Clock Epigenetics Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Circadian Clock Hypothesis posits that the circadian clock becomes desynchronized in maladaptive brain aging and becomes dysfunctional in the pre-clinical stages of neurodegenerative diseases such as Alzheimer&#39;s 90 . Circadian clocks are automated molecular feedback loops that coordinate time-based rhythms (circadian rhythms) of gene expression, neuronal function, neurophysiological processes, and behavior 90 . The circadian system is preserved across species from insects to humans, and the aging circadian system is well-understood in organisms with accelerated life stages such a fruit flies 90 . Mutations in clock genes, which are essential to facilitating automated molecular feedback loops, are associated with decreased lifespan and healthspan 90 . 
     The substance in BrainTheory™ N o  12 that resets the circadian clock is Melatonin. Melatonin is an endogenous substance produced in the brain&#39;s pineal gland and other specific brain regions 91 . Melatonin balances the production of clock proteins responsible for the brain&#39;s pituitary and hypothalamic functions dependent on automated time-based molecular feedback loops 91 . The pituitary gland and hypothalamus are responsible for neurological and non-neurological functions and behaviors, including but not limiting to muscle and bone growth, fat distribution, appetite, gastrointestinal motility, water retention, blood pressure, glycemic control, fertility, arousal, bonding, stress, metabolism, energy, temperature control, memory, and learning 90, 91 . Evidence from human studies demonstrate that after a battery of neurocognitive testing, Melatonin supplementation in persons with mild cognitive impairment is associated with significant improvement in cognition 91 . 
     Melatonin has potent anti-aging properties as a result of caloric restriction mimicry via the SIRT-1 pathway and telomerase activation 92 . Evidence also demonstrates efficacy in mitigating cerebral ischemia, neurodegenerative diseases and proteinopathies, reductive oxidative stress, inflammation, and immunosenescence 93 . Dose dependence will be discussed in the ‘Dose-Dependent Substrates’ section. 
     Nootropic and Systemic Effects 
     Human studies demonstrate that normalization of the circadian rhythm is critical in addiction treatment with application for Melatonin usage 94 . Human studies also demonstrate Melatonin is effective as an adjunctive treatment for migraine because of its anti-CGRP properties, similar in mechanism of action to pharmaceutical anti-CGRP agents 95 . 
     Magnesium L-Threonate in the Calcium-Dependent Synaptic Plasticity Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Calcium-Dependent Synaptic Plasticity Hypothesis posits that dysregulation of neuronal molecular calcium homeostasis occurs in maladaptive brain aging via impairment of synaptic plasticity 96, 97 . There are two main forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD) 96, 97 . LTP is critical in several afferent (pre-synaptic) pathways of the hippocampus, amygdala, visual, somatosensory, and prefrontal cortices 96, 97 . LTP increases synaptic transmission and memory formation, and LTD decreases synaptic transmission and results memory loss 96, 97 . In maladaptive brain aging, to achieve the same level of synaptic transmission in LTP, a higher induction threshold must be reached and is difficult to maintain 96, 97 . Conversely, in maladaptive brain aging, the LTD induction threshold is lowered and easier to attain 96, 97 . This low relative LTP/LTD ratio is associated with maladaptive age-related cognitive decline, deficits in memory and learning, and the ability to process, integrate, and consolidate new information 96, 97 . 
     Unregulated post-synaptic calcium release is the result of dysfunctional neurotransmitter-associated communication, namely glutamate. This occurs at AMPA receptors, NMDA receptors, L-dependent voltage-gated calcium channels (VDCC), plasma membrane ATPase, and within intracellular endoplasmic reticulum and mitochondria 96, 97 . Unregulated post-synaptic calcium release is also associated with aberrant phosphorylation, protease activation, and caspase-related apoptosis 96, 97 . Elevated calcium levels markedly increase the LTP induction threshold and down-regulate neural-selective activity-dependent genes responsible for longterm memory formation such as brain-derived neurotrophic factor (BDNF) 96, 97 . 
     The substance in BrainTheory™ N o  12 that modulates calcium-dependent synaptic plasticity is Magnesium L-Threonate. Magnesium L-Threonate is a bioactive, neuro-available form of magnesium with superior blood-brain-barrier penetration. Magnesium L-Threonate regulates molecular NMDA-receptor check points to induce neuroplasticity of synapses via lowering the LTP threshold. Evidence from a randomized, controlled, double-blind human clinical trial demonstrated that Magnesium L-Threonate functions as a synapse density enhancer that improves learning and memory in mild cognitive impairment 98  and moderate dementia 99 . Human studies demonstrate that Magnesium L-Threonate is neuroprotective in cerebral edema related to ischemic stroke, mitigates cerebral vasospasm, and has anti-epileptogenic properties 100 . 
     Nootropic and Systemic Effects 
     Human studies demonstrate Magnesium L-Threonate is an adjunctive treatment in migraines and has therapeutic potential in depression and chronic pain 100 . 
     Apple Pectin Prebiotic in the Gut-Brain Axis Hypothesis 
     Modulation of Biological Brain Aging to Enhance Healthspan 
     The Gut-Brain Axis Hypothesis, also known as the Second Genome Hypothesis, posits that the relationship between enteric, central, and peripheral nervous systems becomes asynchronous in maladaptive brain aging 101 . The gut-brain axis plays an integral role in critical phases of neurodevelopment (cognitive, emotional, and social neural networks) and neurodegenerative processes that are preceded by decreased microbial diversity and composition 101 . 
     The enteric nervous system (ENS) and microbiota engage in critical bidirectional communication with the central, autonomic, and peripheral nervous systems 101 . Functions of the ENS include modulation of small intestinal permeability to mitigate entrance of pro-inflammatory cytokines, production of short-chain fatty acids, and independent production of neurotransmitters including dopamine, serotonin, norepinephrine, and gamma-aminobutyric acid 101 . Functions of the hypothalamic-pituitary-adrenal axis, circadian rhythm, and sympathetic and parasympathetic nervous systems influence these processes 101 . Loss of symbiotic microbial diversity and the shift towards pathological microbiota is associated with loss of ENS function, cognitive deficits, and behavioral changes 101 . 
     The substance in BrainTheory™ N o  12 that balances the microbiota is Apple Pectin Prebiotic 102, 103 . Apple Pectin is a prebiotic that promotes the production of butyrate, a byproduct of symbiotic microbiota, preserves diversity of symbiotic microbiota, and prevents a shift towards pathological microbiota 102, 103 . Prebiotics differ from probiotics in that prebiotics provide nutritional substrates, such as butyrate, to existing microbiota, allowing intrinsic epigenetic changes to be made 102, 103 . Butyrate specifically plays a critical rule in neuroepigenetics 104 . Evidence from critical appraisal of 14 human studies in the literature demonstrate that prebiotics have a significantly positive effect on cognition and mood including affect, verbal episodic memory, immediate recall, and recognition 102 . 
     Nootropic and Systemic Effects 
     Prebiotics have clinical benefits in sleep organization, stress response and cortisol levels, alcoholism, cancer 103, 104 , autism spectrum disorder 105 , depression 106 , pain and fatigue syndromes, multiple sclerosis, inflammatory bowel disease, cardiovascular disease, and obesity 107 . Human studies also show that prebiotics strengthen immunity via mucosal defense, stabilize bioactive foods and supplements, optimize vitamin biosynthesis, mitigate immunosenescence, and enhance bone density metabolism 103, 104 . 
     Dose-Dependent Substances 
     Trans-Pterostilbene Dose-Dependent Efficacy 
     There is not a standard recommended or allowable dose of resveratrol or Trans-Pterostilbene. 
     At low doses (to be described), resveratrol has superior blood-brain-barrier penetration, promotes stability in reductive-oxidative stress, augments telomerase activity, and increases cell survival proteins 60 . At high doses (to be described), resveratrol inhibits RNA and DNA synthesis, causes structural chromosome damage, and is pro-apoptotic in self and non-self cells, hence the rationale for its usage in adjunctive cancer treatment 60 . 
     An upper limit dose of 2.5 mg/kg of resveratrol is shown to promote longevity pathways 60 . Bioavailability of the resveratrol derivative, Trans-Pterostilbene, is 80 to 95% whereas the bioavailability of resveratrol is 20% 108 . Based on an approximate bioavailability of 80% and a dose of 2 mg/kg in a 70 kg (154 lb) adult, BrainTheory™ N o  12 is composed of 100 mg of Trans-Pterostilbene 108 . 
     Cholecalciferol Dose-Dependent Efficacy 
     While calciferol deficiency (less than 20 ng/ml) is associated with maladaptive health and aging outcomes, serum calciferol levels above the higher range of normal (greater than 50 ng/ml) 109  are associated with accelerated aging, central thymus involution, and decreased naïve T cell production 109 . For example, the age-suppressor gene, Klotho, is a model of aging. When over-expressed, Klotho increases healthspan 110 . When knocked out in animal studies, it results in accelerated aging, short lifespan, accelerated atherosclerosis, skin atrophy, osteoporosis, and high plasma calcitriol levels 110 . For cholecalciferol supplementation, the tolerable upper intake level, defined as the highest average daily intake of a nutrient that is likely to pose no risk of adverse health effects for nearly all persons in the general population, is 4000 IU 109, 110 . This equates to 100 mcg or 500% of the recommended daily allowance 109, 110 . Given the efficiency of oral absorption of cholecalciferol is approximately 50%, BrainTheory™ N o  12 uses 50 mcg (2000 IU or 250% recommended daily allowance) 111 . 
     Melatonin Dose-Dependent Efficacy 
     There is not a standard recommended or allowable dose of melatonin. Melatonin has a dose-dependent effect on basal pituitary and hypothalamic hormone release 112 . A double-blind randomized crossover study demonstrated that neurohypophyseal hormone release is augmented by low dose melatonin 0.5 mg and inhibited by higher dose melatonin 5 mg. BrainTheory™ N o  12 is composed of 0.5 mg of melatonin 112 . 
     Dosing of Other Substances 
     (R)-Alpha Lipoic Acid 
     There is not a standard recommended or allowable dose of Alpha Lipoic Acid. A daily dose of 200 to 2400 mg/day of ALA is safe and well-tolerated 113 . Alpha Lipoic Acid is a racemic mixture where the R-enantiomer, which is bioactive and neuro-available, is present in equal portion to the non-bioactive S-enantiomer 9 . Based on this evidence, BrainTheory™ N o  12 is composed of 100 mg of (R)-Alpha Lipoic Acid. 
     Crocetin 
     There is not a standard recommended or allowable dose of Crocetin. In a human pharmacokinetics study, persons given different doses of saffron extract (56 mg and 84 mg) had undetectable serum concentration of saffron isomers including crocin, safranal, and picrocrocin 114 . However, the Crocetin isomer was detected at a significant concentration with a bioavailability of 40% (22.4 mg and 33.6 mg, respectively) 114 . Similarly, a related study demonstrated a maximum Crocetin serum concentration was achieved after administration of 22.5 mg 114 . To account for at least 50% loss during trans-cellular intestinal and blood brain barrier passage 115 , BrainTheory™ N o  12 is composed of 50 mg of Crocetin. 
     Curcumin 
     The Joint United Nations and World Health Organization Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA) have set an allowable daily curcumin intake of 0-3 mg/kg 116 . Therefore, BrainTheory™ N o  12 uses 100 mg of crocetin. For a 70 kg (154 lb) person, this equates to 1.5 mg/kg. 
     Methods to enhance oral bioavailability of Curcumin are under investigation, however, serum concentrations may be underestimated 117 . Curcumin undergoes gastrointestinal biotransformation to glucuronidated, sulfated, and methylated metabolites. This results in low levels of free bioactive Curcumin 117 . To simplify the quantification of serum concentration in research methodologies, samples are largely treated with β-glucuronidase to hydrolyze the bioactive Curcumin-glucuronide conjugate 117 . However, β-Glucuronidase incompletely hydrolyzes sulfate-containing conjugates that are also major metabolites 117 . 
     Of note, co-supplementation with piperine is suggested to significantly increase Curcumin bioavailablity 118 . This may be a result of piperine-associated glucuronidation inhibition, which results in more free bioactive curcumin as compared to bioactive conjugates 119 . 
     Methylcobalamin 
     The recommended daily allowance of cobalamin is 2.4 mcg 120 . However, studies have suggested that 4 to 7 mcg of bioactive cobalamin (Methylcobalamin) in persons 18 to 50 with normal absorption is optimal to mitigate molecular aging pathways 120 . Given insignificant evidence exists for cobalamin toxicity, a tolerable upper intake level does not exist 121 . Approximately 10 to 30% of adults over 50 years of age have cobalamin malabsorption and the presumed 50% bioavailability in the general population cannot be assumed 121 . To account for 10% bioavailability based on an intake of 5 mcg Methylcobalamin, BrainTheory™ N o  12 is composed of 50 mcg of Methylcobalamin. 
     5-Methyltetrahydrofolate 
     The recommended daily allowance of folate is 400 mcg and based on erythrocyte folate, plasma homocysteine, and plasma folate concentrations 121 . BrainTheory™ N o  12 uses 400 mcg dietary folate equivalents of 5-Methyltetrahydrofolate. Dietary folate equivalents adjust for 50 percent lower bioavailability of folate acquired from food compared with that of folic acid 121 . 
     Apigenin 
     There is not a standard recommended or allowable dose of Apigenin. An open-label clinical trial evaluated the effect of a combination therapy including Apigenin 100 mg in persons with Alzheimer&#39;s disease, Parkinson&#39;s disease, and multiple sclerosis 122 . A trend towards clinical stabilization and reduction of specific biomarkers including abnormal protein deposition was observed without adverse effects 122 . BrainTheory™ N o  12 is composed of 100 mg of Apigenin. 
     Luteolin 
     There is not a standard recommended or allowable dose of Luteolin. A prospective, open-label trial evaluating the tolerability and efficacy of a combination therapy including Luteolin 100 mg in children with autism spectrum disorder 85  demonstrated no adverse effects. Data analysis suggested improvement in adaptive functioning and reduction in Aberrant Behavior Checklist subscale scores 85 . BrainTheory™ N o  12 is composed of 100 mg of Luteolin. 
     Magnesium L-Threonate 
     The recommended daily allowance of magnesium is 310 to 420 mg 123 . The tolerable upper intake levels for supplemental magnesium in adults is 350 mg 123 . Data from the National Health and Nutrition Examination showed that average magnesium intake from diet alone is approximately 268 mg in males and 234 mg in females 123 . There is not a standard recommended or allowable dose specifically for Magnesium L-Threonate 123 . 
     High doses of supplemental magnesium are associated with diarrhea, most commonly with magnesium carbonate, chloride, gluconate, and oxide 123 . Studies have demonstrated that Threonate does accumulate in the periphery to the same extent as other forms of magnesium 124 . Studies demonstrate that Threonate concentration is approximately 20 μM in plasma and 100 μM in cerebrospinal fluid (CSF) 124 . In the brain, physiological extracellular magnesium concentration is 0.8 mM 124 . In maladaptive brain aging and early neurodegeneration, extracellular magnesium concentration is less than 0.6 mM 124 . 
     A study evaluating supplementation with L-Threonate 675 mg demonstrated a significant increase in CSF Threonate concentration by 54%, magnesium concentration by 15%, and synaptic density by 67% at 4 weeks 124 . Delayed analysis was due to prior studies demonstrating that adequate Threonate concentration takes at least 2 weeks to attain 124 . The relationship between extracellular magnesium and synapse density are bell-shaped 124 . When extracellular magnesium is increased beyond 0.8 mM, intracellular magnesium and synapse density are decreased 124 . In consideration of these findings, BrainTheory™ N o  12 is composed of a conservative dosage of 11.7 mg of Magnesium L-Threonate. 
     Apple Pectin 
     There is not a standard recommended or allowable dose of Apple Pectin Prebiotic. Although Apple Pectin Prebiotic stimulates endogenous production of short chain fatty acids (SCFAs) including butyrate, dose-dependence and pharmacokinetics are not well-studied 125 . As such, BrainTheory™ N o  12 uses 50 mg of Apple Pectin, a conservative dosage based on current pectin-only containing supplements. 
     Product Manufacturing 
     BrainTheory N o  12 is sourced and manufactured in the USA, contains USDA organic ingredients, and is produced in an FDA-registered and GMP-certified facility (NutraCap Labs, Norcross, Ga.).