Therapeutic efficacy of the Ginkgo special extract EGb761 within the framework of the mitochondrial cascade hypothesis of Alzheimer s disease

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1 The World Journal of Biological Psychiatry ISSN: (Print) (Online) Journal homepage: Therapeutic efficacy of the Ginkgo special extract EGb761 within the framework of the mitochondrial cascade hypothesis of Alzheimer s disease Walter E. Müller, Anne Eckert, Gunter P. Eckert, Heidrun Fink, Kristina Friedland, Serge Gauthier, Robert Hoerr, Ralf Ihl, Siegfried Kasper & Hans- Jürgen Möller To cite this article: Walter E. Müller, Anne Eckert, Gunter P. Eckert, Heidrun Fink, Kristina Friedland, Serge Gauthier, Robert Hoerr, Ralf Ihl, Siegfried Kasper & Hans-Jürgen Möller (2017): Therapeutic efficacy of the Ginkgo special extract EGb761 within the framework of the mitochondrial cascade hypothesis of Alzheimer s disease, The World Journal of Biological Psychiatry, DOI: / To link to this article: The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 02 May Submit your article to this journal Article views: 369 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 09 January 2018, At: 09:43

2 THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY, REVIEW ARTICLE Therapeutic efficacy of the Ginkgo special extract EGb761 VR within the framework of the mitochondrial cascade hypothesis of Alzheimer s disease Walter E. M uller a, Anne Eckert b, Gunter P. Eckert c, Heidrun Fink d, Kristina Friedland e, Serge Gauthier f, Robert Hoerr g, Ralf Ihl h, Siegfried Kasper i and Hans-J urgen M oller j a Department of Pharmacology, Biocenter, Goethe-University, Frankfurt/M, Germany; b Neurobiological laboratory, Department of Psychiatry, Basel, Switzerland; c Department of Nutritional Sciences, Justus-Liebig University, Giessen, Germany; d Department of Pharmacology and Toxicology, Free University, Berlin, Germany; e Department of Molecular and Clinical Pharmacy, University Erlangen, Erlangen, Germany; f McGill Center for Studies in Aging, Montreal, Canada; g Dr.Willmar Schwabe GmbH & Co. KG, Karlsruhe, Germany; h Alexianer Hospital, Clinic of Geriatric Psychiatry, Krefeld, Germany; i Department of Psychiatry, Medical University, Vienna, Austria; j Department of Psychiatry, Ludwig-Maximilian University, Munich, Germany ABSTRACT Objectives: The mitochondrial cascade hypothesis of dementia assumes mitochondrial dysfunction as an important common pathomechanism for the whole spectrum of age-associated memory disorders from cognitive symptoms in the elderly over mild cognitive impairment to Alzheimer s dementia. Thus, a drug such as the Ginkgo special extract EGb 761 VR which improves mitochondrial function should be able to ameliorate cognitive deficits over the whole aging spectrum. Methods: We review the most relevant publications about effects of EGb 761 VR on cognition and synaptic deficits in preclinical studies as well as on cognitive deficits in man from aging to dementia. Results: EGb 761 VR improves mitochondrial dysfunction and cognitive impairment over the whole spectrum of age-associated cognitive disorders in relevant animal models and in vitro experiments, and also shows clinical efficacy in improving cognition over the whole range from aging to Alzheimer s or even vascular dementia. Conclusions: EGb 761 VR shows clinical efficacy in the treatment of cognitive deficits over the whole spectrum of age-associated memory disorders. Thus, EGb 761 VR can serve as an important pharmacological argument for the mitochondrial cascade hypothesis of dementia. The mitochondrial cascade hypothesis: Alzheimer s disease beyond b-amyloid Amyloid-containing plaques, no longer major pathomechanism or relevant target for therapeutic intervention Since the first clinical case described more than 100 years ago by Alois Alzheimer, Alzheimer s disease (AD) has been characterised by neurodegeneration (synaptic deficits and finally neuronal loss) and the presence of histopathological alterations (extracellular amyloidcontaining plaques and intracellular tangles of hyperphosphorylated tau-protein), as well as by severe cognitive deficits often clinically accompanied by neuropsychiatric symptoms, such as delusions, as already described in the first famous patient Auguste D at the Psychiatric Hospital of Frankfurt. ARTICLE HISTORY Received 5 January 2017 Revised 8 March 2017 Accepted 15 March 2017 KEYWORDS Dementia; neuropathology; pharmacotherapy; EGb 761 VR ; mitochondrial dysfunction If or if not one or both of the two histopathological hallmarks play a causative role remained unclear for many decades. The discovery of homozygotic risk genes in most of the very rare (probably less than 1%) cases of early onset Alzheimer s disease (EOAD), which share increased production of b-amyloid (Ab) as one (but probably not the only one) common property led to the hypothesis of Ab as the major causative factor not only for EOAD but also for late-onset AD (LOAD). These findings were supported by a large number of mainly preclinical data using transgenic cell and animal models finally leading to the amyloid cascade hypothesis (Hardy & Selkoe 2002), suggesting the slow accumulation of Ab-containing plaques as the major causative pathomechanism of AD, even if neurotoxic low-molecular-weight Ab aggregates (oligomeric Ab) were also seen to be relevant in the later years (Selkoe CONTACT Walter E. M uller w.e.mueller@em.uni-frankfurt.de Department of Pharmacology, Biocenter, Goethe-University, Frankfurt, Germany ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

3 2 W. E. M ULLER ET AL. 2008). To make a long story short, plaques were seen as the major bad guys and removing plaques by drugs was suggested to be the most relevant new diseasemodifying strategy to treat AD. This hypothesis was strongly driven by the many transgenic animal models of AD, which all show a substantial Ab plaque load, although cognitive deficits and signs of neurodegeneration were often only remote at best and cognitive deficits did not correlate with Ab levels (Pozueta et al. 2013; Foley et al. 2015; Grimm et al. 2016a). Aging as the major risk factor of LOAD was neglected as the fact that plaque density in the brain of AD patients did not correlate with presence and severity of clinical symptoms was neglected, while synaptic deficits were not (Pozueta et al. 2013), even in the first observations already published many years ago (Terry et al. 1991). Nevertheless, based on the amyloid cascade hypothesis, many drug treatments to remove Ab plaques were developed (inhibitors of aggregation, inhibitors of the secretases producing Ab from its precursor protein APP and antibodies to remove Ab, or the increased production of antibodies by vaccination). Even if all seemed to remove Ab to some extent, all strategies failed to improve the symptoms of dementia, some of the treatments made dementia even worse (Iqbal et al. 2014; Karran & Hardy 2014; Swerdlow et al. 2014; Herrup 2015). Moreover, recent advances in imaging techniques confirm that many patients die quite old with a large Ab plaque load without showing symptoms of dementia, and that accumulation of Ab-containing plaques already reaches its maximum decades before the first symptom of cognitive impairment or even dementia develop (Sperling et al. 2011; Jack et al. 2013, 2014). Thus, it became quite clear that the simple Ab cascade hypothesis has failed, especially as a basis for the development new AD-specific drugs. Accordingly, other aspects of AD pathology, more closely related to the clinical symptoms of the disease (Alzheimer s dementia) are currently investigated as targets for therapeutic improvement, such as the already-mentioned synaptic deficits and their relationship to mitochondrial dysfunction (Drachman 2014; Castello & Soriano 2014; Morris & Berk 2015). Mitochondrial dysfunction and synaptic deficits, now in focus Impaired cerebral glucose metabolism arising from memory-related brain regions such as the hippocampus and entorhinal cortex represents an early pathomechanism of AD, detectable long before its clinical manifestation (Kapogiannis & Mattson 2011). Accordingly, in cognitively impaired but not yet demented elderly, impaired glucose metabolism as determined by positron emission tomography using 18 F-fluorodeoxyglucose seems to be a sensitive very early biomarker for the later development of AD (Sperling et al. 2011; Jack et al. 2013). While other mechanisms also play a role, impaired mitochondrial function seems to be the major cause (Kapogiannis & Mattson 2011). This parallels many other observations of mitochondrial deficits in AD brains such as reduced activities of mitochondrial enzymes and of complexes of the respiratory chain and increased oxidative stress due to elevated reactive oxygen species (ROS) damage (Gibson et al. 2010; Friedland-Leuner et al. 2014). Mitochondrial dysfunction is also a common feature of all AD mouse models (Leuner et al. 2007; M uller et al. 2010; Friedland-Leuner et al. 2014; Grimm et al. 2016a). Accordingly, mitochondrial dysfunction is observed in FAD and LOAD brain and has been proposed as a fundamental component of AD (Swerdlow & Khan 2004: Swerdlow et al. 2010, 2014). Moreover, all known risk factors of AD (LOAD and EOAD) lead to mitochondrial dysfunction independent of each other (Figure 1). This is the case for elevated Ab levels as well as the presence of neurofibrillary tangles and for all other relevant risk factors such as brain aging, microvascular dysfunction, APOE4 genotype, mdna polymorphisms and gender, which all converge at the level of impaired mitochondrial function (Leuner et al. 2007; M uller et al. 2010; Gibson et al. 2010; Grimm et al. 2016a, 2016b). Moreover, as synaptic function and synaptic plasticity strongly depend on energy (adenosine triphosphate, ATP), mainly provided by the mitochondria, mitochondrial dysfunction is closely associated with synaptic deficits in aging and AD (Reddy et al. 2005; Reddy 2007; Cheng et al. 2010; Reddy et al. 2010; Du et al. 2012; Cavallucci et al. 2013; Wang et al. 2016). These observations led to the hypothesis that impaired mitochondrial function, associated with reduced energy metabolism and enhanced oxidative stress as well as synaptic dysfunction represents a common final pathway of all specific (genetic) and non-specific risk factors for the development of AD in general but specifically for LAOD, a scenario which is shown in Figure 1. While this concept originally assumed that the synergistic impairment of mitochondrial and synaptic function by all those factors goes to some extent parallel with the cognitive symptoms of AD and becomes relevant at a time point where the clinical diagnosis becomes possible, a more complex picture emerges in respect to the development of the individual biomarkers and the cognitive symptoms (Sperling et al. 2011; Jack et al. 2013). Most recent

4 THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 3 Figure 1. Mitochondrial dysfunction and enhanced oxidative stress represent a common final pathway of genetic and other risk factors for the development of early- and late-onset Alzheimer s disease. Adapted and modified form Leuner et al. (2007), M uller et al. (2010), and M uller et al. (2016). studies demonstrated the presence of Ab deposits very early in the development of AD, long before functional deficits become detectable. This led to the concept of preclinical AD, characterising a situation where AD is only characterised by Ab deposits and may develop over time to Alzheimer s dementia (Jack et al. 2013). The concept of preclinical AD still assumes Ab as the major player getting deposited years or decades ahead of cognitive symptoms and as the major driving force for the later development of AD (Sperling et al. 2011; Jack et al. 2013). It fails to give a simple explanation why Ab deposits already reach maximum levels before cognitive symptoms develop and why Ab deposits do not correlate with cognitive symptoms or even the development of cognitive symptoms over time. Thus, other concepts to explain the very longlasting development of biomarkers, pathomechanisms and clinical symptoms of AD over time are needed. Mitochondrial dysfunction a cascade of events representing the interface between aging and AD Probably the most important example for such an alternative concept is the mitochondrial cascade hypothesis put forward more than 10 years ago by Swerdlow and co-workers (Swerdlow & Khan 2004; Swerdlow et al. 2010, 2014) assuming mitochondrial dysfunction as the major pathomechanism of AD, which slowly develops through aging (Figure 2). The most important aspect of this mitochondrial cascade hypothesis likely relates to the very early stages of mitochondrial dysfunction, caused by the combined effect of oxidative stress due to aging, and slightly elevated Ab levels caused by genetic and individual risk factors, long before Ab deposits begin to form. Driven by genetic, environmental and individual factors, mitochondrial dysfunction associated with elevated ROS production cumulates in susceptible patients over many years. This process is self-accelerating as ROS will further damage mitochondria, which respond with further elevation of ROS. At some point, elevated ROS production will reach a level where Ab production increases due to b-secretase and c-secretase activation (Leuner et al. 2012a, 2012b). Ab in turn will further impair mitochondrial function (Figure 1) and will aggregate to fibrils and finally to plaques (Figure 1). This scenario suggests that Ab still has a causative role but it is probably not the major player. It also seems to be a side product once aggregated to plaques without major functional relevance. This can easily explain the fact that Ab deposits themselves do not correlate with early signs of neurodegeneration or impaired cognition (Sperling et al. 2011; Jack et al. 2014). The major aspect of this concept relates to mitochondrial dysfunction as the major pathomechanism directly driving neurodegeneration and psychopathology

5 4 W. E. M ULLER ET AL. Figure 2. The mitochondrial cascade hypothesis assumes mitochondrial dysfunction as a major driving force behind the slowly developing decline of cognitive function within the continuum from aging over MCI to dementia. This hypothess assumes that, driven by genetic (e.g., mdna, ApoE4), environmental and individual factors, mitochondrial dysfunction associated with elevated free radical production cumulates in susceptible patients over many years and will at some point lead to synaptic deficits, cognitive impairment, Tau pathology, neuroinflammation and enhanced b-amyloid production, which in turn can lead not only to additional mitochondrial deficits but can also trigger other pathomechanisms independent of mitochondrial function. While this concept assumes that impaired mitochondrial function plays a critical role in triggering all these functional deficits it does not suggest that it is the only underlying cause. For example, once elevated b-amyloid increases its own production not only by elevated ROS production (Leuner et al. 2012a, 2012b) but also by its effects on membrane fluidity (Peters et al. 2009), and also further impairs neuronal function by a large number of mechanisms besides disturbed mitochondrial function Adapted and modified by Swerdlow et al. (2010, 2014). independently of Ab deposits, from the initial phase of the disease, long before a clinical diagnosis becomes possible, to the later phases of mild-to-moderate dementia (Figure 2). Accordingly, mitochondrial dysfunction can lead to early signs of neurodegeneration or synaptic deficits as well as distinct cognitive deficits without Ab deposits being present (Figure 2). This concept differs substantially from the concept of preclinical AD (see above) but is supported by several most recent findings in a substantial number of patients having early signs of neurodegeneration and cognitive decline or even the clinical diagnosis of mild AD without any evidence for Ab pathology (Wirth et al. 2013; Castello & Soriano 2014; Salloway et al. 2014; Caroli et al. 2015; Edmonds et al. 2015; Burnham et al. 2016; Jack et al. 2016). The notion that cortical hypometabolism, signs of neurodegeneration, as well as distinct cognitive deficits can be present independent of Ab in individuals at risk for AD is supported by studies in patients with the APOE4 genotype, the most relevant genetic risk factor for LOAD (Huang 2010; Valla et al. 2010; Risacher et al. 2013; Bangen et al. 2016; Perkins et al. 2016; Seo et al. 2016). As mentioned above mitochondrial dysfunction and elevated ROS levels might slowly increase over years (Figure 2) and lead to further neurodegeneration and cognitive deficits, and may independently increase Ab levels due to b-secretase and c-secretase activation (Leuner et al. 2012b) up to the limit of solubility leading to aggregation and deposits. This may explain the fact that many cognitively healthy individuals show substantial Ab deposits at greater age. The question whether mitochondrial dysfunction alone can lead to more severe cognitive impairment as in AD is difficult to answer in man, since elevated ROS will automatically lead to elevated Ab and Ab deposits regardless of whether Ab plays a causative role. In non-transgenic animals (mice, rats) where, in agreement with the cascade model (Figure 2), non-toxic mouse or rat Ab increases with aging (Chiu et al. 2002; Church et al. 2014), substantial deficits of cognition (learning, memory or orientation) can sometimes be observed as a consequence of aging only (Gallagher & Rapp 1997). Moreover, AD-like neurodegeneration and/or cognitive deficits can be seen in patients without Ab deposits Jack et al. (2014, 2016). In summary, the scientific evidence for the mitochondrial cascade hypothesis of dementia are quite consistent and supported by many data from cell models, animal studies and from patients. Additional proof for this concept could come from drug treatment findings, if drugs which improve mitochondrial dysfunction also improve cognitive impairment over the whole spectrum, from aging over mild cognitive impairment (MCI) to AD. Pharmacological strategies to improve mitochondrial function While the concept of mitochondrial dysfunction as a major pathomechanism of AD has received substantial support over the last decade, improving mitochondrial function as a strategy for new drug development has

6 THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 5 not. Preclinical data about improvement of mitochondrial dysfunction and associated deficits of synaptic function and neuronal plasticity as well as cognitive deficits have been reported for several antioxidants, for many polyphenols and other natural compounds and for some drugs. However, very few of these compounds have subsequently been investigated in welldesigned clinical trials, although in some cases preliminary evidence for a possible clinical benefit has been reported (Leuner et al. 2007; M uller et al. 2010; Eckert et al. 2012; Andreux et al. 2013; Picone et al. 2014; Caldwell et al. 2015; Kumar & Singh 2015; Onyango et al. 2016). In nearly all cases the data and especially the clinical data are not sufficient to recommend the substances for further drug development. Moreover, in nearly all cases the data are not consistent and convincing enough to serve as pharmacological proof of the mitochondrial cascade hypothesis. EGb 761 VR, a special extract made from the leaves of Ginkgo biloba is the only exception. Evidence from preclinical and clinical studies for its broad efficacy are therefore given below. With respect to other anti-dementia drugs, only acetylcholinesterase inhibitors also significantly improve cognition in aging and dementia but by enhancing acetylcholine availability which is impaired over the whole aging spectrum (see the classic paper by Bartus et al. 1982). However, these drugs have no general effect on energy metabolism and mitochondrial function as well as neuronal plasticity as the major pathomechanisms of cognitive decline in dementia (Wesson Ashford 2015). Improvement of mitochondrial dysfunction by EGb 761 VR across the aging spectrum Preclinical evidence EGb 761 VR is a special dry extract of Ginkgo leaves, made with acetone 60% (w/w) as the extraction solvent, developed by Schwabe (Germany) and Ipsen (France) more than 40 years ago. Relative to the original composition of the leaves, pharmacologically active components (flavonoids, terpene lactones) are enriched, and possible toxic components (ginkgolic acids) are downgraded. The final standardised extract is adjusted to % Ginkgo flavonoids and % terpene lactones consisting of % ginkgolides A, B and C, and % bilobalide, with a content of ginkgolic acids below 5 ppm. Most of the clinical studies and the majority of preclinical studies published for Ginkgo over the last decades used this standardised extract (Christen & Maixent 2002; De Feudis 1998). The extract shows pronounced antioxidant properties and additionally improves mitochondrial function, especially when it is impaired. Most of its beneficial effects on cognition, neuronal plasticity, energy metabolism and neuroinflammation seem to be secondary to these properties (Leuner et al. 2007; Montes et al. 2015; M uller et al. 2016). Only few effects seem to be independent of improvement of mitochondrial function. An increase of blood viscosity, probably due to antagonistic effects at the platelet activation factor receptor has been associated with effects on microcirculation, but requires rather high doses of the extract (Koch 2005). Small effects on dopaminergic and noradrenergic neurotransmission (Fehske et al. 2009; Yoshitake et al. 2010) are probably relevant for the modest cognition-improving properties in rather healthy individuals (Beck et al. 2016), but may not play a role in the beneficial effects in aging and dementia. With respect to the mitochondrial cascade hypothesis of dementia, it is important to note that EGb 761 VR has been shown in a large number of studies to improve mitochondrial dysfunction in aging or other situations of impaired brain function (Sastre et al. 1998; M uller et al. 2012; Leuner et al. 2007; Eckert et al. 2012; Kumar & Singh 2015). As one mechanism, EGb 761 VR directly scavenges free ROS mainly by its flavonoid fraction. This agrees with many other findings about the antioxidant properties of flavonoids (Schaffer et al. 2012). Moreover, many experimental studies have clearly shown that EGb 761 VR reduces mitochondrial ROS production and protects mitochondria and the mitochondrial complexes of the respiratory chain from further damage by ROS and enhances the availability of ATP (Janssens et al. 1995; Eckert et al. 2003; Smith & Luo 2003; Eckert et al. 2005; Abdel-Kader et al. 2007; Rhein et al. 2010; Eckert et al. 2012; Baliutyte et al. 2014). As a consequence, neuronal function improves especially following previous impairment (aging, hypoxia, hypoglycaemia, elevated Ab or cerebrovascular pathology) (Bastianetto et al. 2000; Abdel-Kader et al. 2007; Shi et al. 2009; Rhein et al. 2010; Wan et al. 2014). Positive effects of EGb 761 VR on neuroinflammation may also be secondary to its effects on mitochondrial function (Liu et al. 2015; Wan et al. 2016). Moreover, EGb 761 VR protects neurons from toxic effects of Ab or the neurotoxic small aggregates (oligomers) leading to mitochondrial dysfunction, deficits of the respiratory chain complexes and apoptosis. It also seems to have some effects on Ab aggregation and production (Ahlemeyer et al. 1999; Schindowski et al. 2001; Luo et al. 2002; Colciaghi et al. 2004; Blasko et al. 2005; Longpre et al. 2006; Shi et al. 2009; Wan et al. 2014; Xie et al. 2014; Liu et al. 2015). As a consequence of its pronounced effects on

7 6 W. E. M ULLER ET AL. Table 1. Effects of EGb 761 VR or its constituents on different aspects of neuronal plasticity. Long-term potentiation (LTP) Williams et al. (2004) LTP is reduced significantly in the hippocampus of old mice by EGb 761 VR treatment but not in young mice. Wang et al. (2006) LTP is reduced in the hippocampus of old rat, e.g., in spatial memory. Both parameters are improved by EGb 761 VR treatment in old but not in young animals. Vitolo et al. (2009) Ab 1-42 inhibits LTP in hippocampus slices of mice. Improvement by ginkolide J. Spine morphology (number or shape) Lacour et al. (1991) Improved synaptic reoccupation by EGb 761 VR after vestibular lesion in the cat. Tchantchou et al. (2009) Elevated spine density in hippocampal neurons after treatment with bilobalide and quercetin. Synapto(neurito)genesis Bertoni-Freddari et al. (2002) EGb 761 VR treatment increases synaptic density in vitamin E-deficient rats. Xu et al. (2007) Elevated levels of pcreb and BDNF in Ab-treated neuroblastoma cells. Tchantchou et al. (2009) Increased dentritic growth and elevated levels of CREB and BDNF in hippocampal neurons. Garcia-Alloza et al. (2010) Increased neuritogenesis by EGb 761 VR in an AD mouse model. Wang et al. (2012) Increased axonal growth of retinal ganglion cells by ginkgolide B. M uller et al. (2012) Improved neuritogenesis of PC12 cells by EGb 761 VR and ginkgolides, flavonoids and bilobalides. Liu et al. (2015) EGb 761 VR treatment increases synaptic marker proteins in a mouse AD model. Neurogenesis and neuroprotection Tchantchou et al. (2007) Increased neuritogenesis in a mouse model of AD by EGb 761 VR Tchantchou et al. (2009) Improved neuritogenesis in a mouse model of AD by bilobalide and quercetin Rocher et al. (2011) Reduced neuronal loss in a gerbil model of vascular dementia by EGb 761 VR. Nada et al. (2014) Enhanced neurogenesins by EGb 761 VR after experimental stroke in mice. Wang et al. (2013) Improved neuritogenesis after experimental stroke in a rat. most measures of impaired mitochondrial function and reduced energy supply, brain functions which need large amounts of energy benefit most, like all situations which need extensive intracerebral communication, such as neuronal plasticity, cognition and balance control. These aspects are typically affected by aging and dementia. Effects on synaptic function and neuroplasticity Synaptic plasticity, the dynamic regulation of synaptic mechanisms such as long-term potentiation, spine density and form, number and length of dendrites and axons (neuritogenesis) and the number of neurons (neurogenesis and apoptosis) represents a major mechanism by which our brain can adapt to periods of pathologically enhanced or reduced function or to save information at the synaptic level. Mitochondria play an important role as they provide the cellular energy (ATP) for these adaptive responses or initiate apoptosis in the case of neuronal damage beyond the possibility of repair (Mattson et al. 2008; Zhang & Wang 2008; Du et al. 2012; Yu& Lu2012). Changes of synaptic function and plasticity play a major role for cognitive deficits in aging and dementia (Burke & Barnes 2006; Scheff et al. 2007; Selkoe 2008; Balietti et al. 2012; Marcello et al. 2012; Spires-Jones & Knafo 2012; Pozueta et al. 2013; Scheff et al. 2013; van der Zee 2015). In line with these findings and the positive effect of EGb 761 VR on mitochondrial function outlined above, EGb 761 VR improves synaptic function and plasticity in a large number of cell and animal models (M uller et al. 2012). Its effect is usually mainly seen when these parameters are impaired due to experimental conditions of reduced energy supply by aging, Ab overexpression, hypoglycaemia or hypoxia, all have in common enhanced oxidative stress and impaired mitochondrial function. A selection of relevant observations about effects of EGb 761 VR on synaptic function and plasticity is given in Table 1. Effects on cognitive impairment in aging and animal models of dementia After early studies with EGb 761 VR in patients with cerebral vascular disease had reported positive effects on cognition (De Feudis 1998), many experimental investigations in mice or rats confirmed improvement of cognitive functions (see the summary about older studies by M uller & Chatterjee 2003). These effects include improvements in many different cognitive domains such as learning, short-term memory and aspects of working memory. Many of these older studies already reported better effects on cognitive performance in aged than in young or adult animals (M uller & Chatterjee 2003). These initial observations have been confirmed in many of more recent subsequent studies, extending the better improvement of cognition from aging to overexpression of human Ab (AD mice), to hypoxia, and cerebral vascular impairment, situations typical for the aging continuum of the mitochondrial

8 THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 7 Table 2. Effects of EGb 761 VR or its constituents on cognitive functions in animals. Stoll et al. (1996) Young, middle-aged NMRI mice Improvement of passive avoidance learning in the aged mice only Hoffman & Stein (1997) Brain contusion (Cortes), rat Improved water maze learning Winter (1998) Aged rats Improved radial maze learning Hoyer et al. (1999) Intracerebral injection of stretozotoxin rat (AD model) Improved hole board and passive avoidance learning Wirth et al. (2000) Adult and aged rats Improved olfactory recognition Lin et al. (2003) Experimental stroke model, rat Improved radial arm maze learning Pardon et al. (2004) Chronic mild stress, aged mice improved decision making (Tmaze) Walesiuk et al. (2005) Restraint stress, chronic cortico-sterone treatment rat Improved learning water maze and novel object recognition Paganelli et al. (2006) Experimental stroke, rats Improved learning in the aversive radial arm maze Wang et al. (2006) Aged rats Improved water maze learning Walesiuk et al. (2006) Restraint stress, chronic corticosterone treatment, rats Improved passive avoidance and conditioned avoidance learning Takuma et al. (2007) Restraint stress, rats Improved novel object recognition Oliveira et al. (2009) Fear conditioning, rats Improvement Satvat & Mallet (2009) Double Y maze, rats Improved acquisition Blecharz-Klin et al. (2009) Aged rats Improve water maze learning Walesiuk and Braszko (2009) Restraint stress chronic corticosterone treatment Improved water maze and radial maze learning Rocher et al. (2011) Gerbil model of vascular dementia Improvement in object location test Li et al. (2013) Rat model of vascular dementia Improvement in water maze learning by bilobalide Liu et al. (2015) Mouse model of AD Improved learning in Barnes maze test Wan et al. (2016) Mouse model of AD Improved learning in water maze test Belviranlı and Okudan (2015) Female rat of different ages Improved water maze learning in aged rats only cascade hypothesis. Several important studies addressing this topic are summarised in Table 2. Preclinical effects of EGb 761 VR and the mitochondrial cascade hypothesis of dementia The pharmacological properties of EGb 761 VR focussing on neuronal and synaptic function, on neuronal plasticity and on cognitive functions, such as learning and memory as the major functional consequences of mitochondrial dysfunction in the brain, are definitively in line with the cascade hypothesis, as beneficial effects are seen over the whole spectrum of agerelated cognitive disturbances from aged animals, transgenic AD animals expressing high levels of Ab or animals with vascular impairment. Clinical improvement by EGb 761 VR across the whole spectrum of age-related cognitive disorders, from aging over MCI to AD and vascular dementia EGb 761 VR has been widely used since its introduction into the market to improve deficits of cognition over a large range of conditions from aging to dementia. However, scientific proof for this use was always seen very critical because of the large range of cognitive disturbances investigated (it is not so long ago that mild cognitive dysfunction in aging was considered completely different from mild stages of AD), the different and sometimes poor design of some of the studies and of the rather low doses used in some of the older studies. The last Cochrane analysis (Birks & Grimley Evans 2009) is a good example for the dilemma, as 36 trials were included for its effect on people with acquired cognitive impairment, including dementia, of any degree or severity. It is understandable, that (meta-analysing the same outcomes for different stages of impairment (MCI, AD or vascular dementia (VD)) and including studies with unspecific dementia diagnosis, with low-dose, low-quality extracts in addition to those of EGb 761 VR ) no predictable and clinically significant benefit for people with dementia and cognitive impairment could be shown. Furthermore, the scientific community was rather sceptical of accepting a drug for AD which also was effective in VD and even in MCI. Even more so because, at a time when any specific AD drug had to show efficacy in removing Ab load in the brain (which is not a major case for EGb 761 VR ), many would just not accept a drug which reduces oxidative stress in the brain as an effective treatment of AD. This negative perception changed over recent years as drugs became available for clinical trials which did affect Ab but which did not show clinical efficacy at all. Mitochondrial improvement became an accepted concept for treatment of AD. Moreover, many additional clinical trials with EGb 761 VR with positive outcome have been published over the last 10 years. Accordingly, all recent meta-analyses about the efficacy of EGb 761 VR in dementia demonstrated relevant positive effects on cognition and the activities of daily living (Janssen et al. 2010; Wang et al. 2010; Weinmann et al. 2010; Brondino et al. 2013; Gauthier & Schlaefke 2014; Tan et al. 2015; von Gunten et al. 2015). Patients with Alzheimer s and VD benefit similarly, as outlined in many of these metaanalyses. See also the secondary analyses by

9 8 W. E. M ULLER ET AL. Napryeyenko et al. (2009) and Ihl et al. (2012). Several of the meta-analyses mentioned above addressed the possible clinical relevance of the beneficial effects of EGb761 VR by analysing responder rates or effect sizes. When clinically relevant responder rates were calculated using standard criteria for anti-dementia drugs (e.g., improvement of cognition by at least 3.9 points on the ADAS-cog scale or 3.0 points on the SKT scale) superiority of EGb761 VR was seen for cognition and also for activities of daily living (Gauthier & Schlaefke 2014). Using similar criteria response rates of about 25% were reported leading to NNT values of 4 5 (von Gunten et al. 2015). The same authors also reported effect sizes ranging from 0.62 (cognition) to 0.25 (quality of life) for the effects of EGb761 VR. Comparable effect sizes were also reported by Weinmann et al. (2010). It is not the intention of the present work to summarise the clinical data about the efficacy of EGb 761 VR in dementia again, but we will critically use the clinical studies of EGb 761 VR as possible proof of concept of the mitochondrial cascade hypothesis of dementia. Thus, if mitochondrial dysfunction is a major cause and driving force for the cognitive decline over the continuum from aging to dementia, a drug which improves impaired mitochondrial function should show some clinical efficacy along that continuum. Moreover, with our increasing knowledge of mitochondrial dysfunction, as a common pathomechanism of AD type dementia and VD (de Deyn et al. 1999; Calabrese et al. 2016; Vijayan & Reddy 2016) a drug which improves mitochondrial function should have efficacy in both types of dementia. The evidence for this concept is summarised below. Several. reviews of clinical studies showed beneficial effects of add-on treatment with EGb761 VR on positive, negative and cognitive symptoms of schizophrenia (Singh et al. 2010; Brondino et al. 2013; Chen et al. 2015). Regarding the increasing knowledge about a role of oxidative stress and mitochondrial dysfunction in the pathophysiology of schizophrenia (Montes et al. 2015; Morris & Berk 2015; Rajasekaran et al. 2015), these data further support the concept of the relevant mitochondrial improving properties of EGb761 VR. Moreover, EGb761 VR also shows beneficial effects on vertigo of peripheral and central origin, especially in elderly patients (Hamann 2007; Sokolova et al. 2014). Impaired plasticity mechanisms within the vestibular pathways (vestibular compensation) (Lacour et al. 1991; Tighilet & Lacour 1995; Sokolova et al. 2014) seem to be the targets. Again, this fits well into the properties of EGb761 VR described in the present manuscript. From cognitive normal aging to MCI The mitochondrial cascade hypothesis assumes the appearance of MCI in the presence of small mitochondrial impairment around midlife (Swerdlow et al. 2010, 2014). One study addressed this population, where aged but cognitively not impaired volunteers (aged years) showed no improvement in most of the cognitive tests used after taking EGb 761 VR (240 mg daily) for several weeks. Only the quite demanding recalled an appointments test, which measures the recall of appointments to be done in the future, showed some significant improvement (Kaschel 2011). Two studies investigated the possible procognitive effects of EGb 761 VR in cognitively intact older (<60 years, mean 66 years) volunteers at a daily dose of 180 mg over 6 weeks (Mix & Crews 2000, 2002). Using a variety of cognitive paradigms, positive effects over baseline were found for tests indicative of delayed free recall, a recognition of non-contextual auditory verbal material as well as recognition of visual material. Another study, again in healthy volunteers over 60 years, giving the drug over 6 weeks at a daily dose of 120 mg did not report procognitive effects using other cognitive tests (Solomon et al. 2002). Beside the lower dose, the blinding of this study has been questioned as the drug was given as tablets and placebo as capsules. In a very recent study, again in middle-aged volunteers (50 65 years), the effects of EGb 761 VR (240 mg daily for 8 weeks) were investigated on the performance in several cognitive tasks as well as brain activity using fmrt (Beck et al. 2016). The subjects showed mild subjective memory impairment, but were otherwise cognitively not impaired. Cognitive improvement under EGb 761 VR was only observed for the switchcost outcome of the task-set-switching paradigm. No effect on the fmrt data was seen, indicating small positive effects on cognition without major effects on brain activation during the cognitive tasks. These more recent data in elderly subjects with rather normal cognitive performance all suggest only small effects of EGb 761 VR on cognitive functions. This very simply parallels that cognition is rather not impaired. By contrast, patients with MCI show much more pronounced deficits on cognition and therefore better respond to treatment with EGb 761 VR (Grass-Kapanke et al. 2011; Gavrilova et al. 2014). In a placebo-controlled double-blind study with 300 subjects with serious MCI (subjective complaints of impairment and at least one cognitive domain one standard deviation below appropriate norm), EGb 761 VR (240 mg daily) was given for 12 weeks (Grass-Kapanke et al. 2011). Positive effects of treatment on several measures were

10 THE WORLD JOURNAL OF BIOLOGICAL PSYCHIATRY 9 observed, indicative for attention and memory. Better treatment effects were associated with lower scores at baseline. Another placebo-controlled double-blind study included MCI patients older than 55 years who were treated with EGb 761 VR (240 mg daily) for 24 weeks (Gavrilova et al. 2014). Inclusion criteria were amci according to Winblad et al. (2004) and a total score of at least 6 or higher on the Neuropsychiatric Inventory (NPI). Significant improvement over placebo was found for the NPI and some cognitive scores. While the symptomatic improvement in MCI was still small it was more consistent in this preclinical stage than in the cognitively normal older individuals (see above). In general, the data are in agreement with the hypothesis that, with increasing mitochondrial dysfunction from healthy aging to the preclinical stages of MCI, the efficacy of EGb761 VR increases. Alzheimer s and VD: the role of enriched samples by the primary inclusion of patients with neuropsychiatric symptoms As already mentioned, the clinical efficacy of EGb 761 VR in Alzheimer s and VD has been addressed by several meta-analyses within the more recent years, all indicating relevant efficacy in measures of cognition and/or activities of daily living. At that stage of the progression of the disease substantial mitochondrial deficits will be present (Figure 1) and can be addressed by the beneficial effects of EGb 761 VR (Figure 2). Several of the more recent clinical trials with EGb761 VR in dementia primarily included patients with a certain level of neuropsychiatric symptoms (NPS) (Ihl 2013), as preliminary data showed a better response of cognitive symptoms to EGb761 VR in demented patients with NPS and a faster cognitive decline (Hoerr 2003; Schneider et al. 2005). NPS are typical symptoms of dementia and are present in most patients with AD or VD. The prevalence of NPS in different populations of patients with dementia is around 80 90% (Petrovic et al. 2007; Aalten et al. 2007, 2008; Apostolova & Cummings 2008; Steinberg et al. 2008; Di lulio et al. 2010; Herrmann et al. 2015). Thus, the presence of NPS is rather the norm than the exception. One major aspect of the presence of NPS in dementia seems to be a faster decline of cognitive functions over time (Schneider et al. 2005; Steinberg et al. 2008; Rabins et al. 2013; Peters et al. 2013; Herrmann et al. 2015; Peters et al. 2015). Accordingly, the three studies reviewed by Ihl (2013) only included patients with a certain level of NPS. All showed significant improvement of cognition and activities of daily living (Ihl 2013). Not only total improvement (placebo verum difference at endpoint) but also improvement over baseline were best in the patients showing the fastest decline during the study (Napryeyenko et al. 2007), followed by those with an intermediate decline (Ihl et al. 2011), and those with the smallest decline (Herrschaft et al. 2012). Even if the neurobiology of NPS in dementia is not yet completely understood, patients with NPS or symptoms typical for NPS seem to have pronounced mitochondrial dysfunction besides other neurobiological deficits (Meeks et al. 2006; Cerejeira et al. 2012; Sultzer et al. 2014; Morris & Berk 2015; Vavakova et al. 2015). This could easily explain the better response rates of EGb761 VR in this large subgroup of demented patients as well its positive effects on NPS (Bachinskaya et al. 2011; Brondino et al. 2013). Slowing down the progression of the disease In respect to the mitochondrial cascade hypothesis and the pharmacological properties of EGb 761 VR, one would expect that the drug slows down the progression of the disease and the progression from preclinical states to MCI or even dementia. Two epidemiological studies suggest a reduced risk for dementia in individuals taking Ginkgo over many years but both studies and especially the newer one can be criticised because of the design (Andrieu et al. 2003; Amieva et al. 2013). Accordingly, both give only hints but not proofs of efficacy. Therefore, two large-scale prospective placebo-controlled studies were designed to address this important topic. The GEM study (Ginkgo evaluation of memory) included more than 3000 rather old (80 years) subjects, otherwise fairly healthy in respect to cognition. Treatment was EGb 761 VR 240 mg daily for up to 6 years. Both primary endpoints, conversion to dementia or decline of cognitive function were not different for the Ginkgo and the placebo groups at study end (DeKosky et al. 2008; Snitz et al. 2009). Even if this study is quite impressive on first sight, there is increasing scepticism about the interpretation of the results. One major point is the recruitment of the patients (Fitzpatrick et al. 2006), a high selection of fairly old but reasonably healthy individuals. Consequently, the participants showed very little decline over time for the primary endpoints, which makes it difficult to demonstrate efficacy for a drug which works best in patients showing the typical decline of the disease. Even more important, compliance of medication was poor and no attempt was made to correct the analyses for this major drawback. This is even more strange, as a similarly designed but smaller study (about 120 patients) did show significant effects in favour of

11 10 W. E. M ULLER ET AL. Ginkgo biloba extract at endpoint (up to 42 months) after correction for compliance which is quite remarkable considering the small sample size (Dodge et al. 2008). In the GuidAge-study, more than 2500 patients were treated with EGb 761 VR (240 mg daily or placebo for up to 5 years). Primary endpoint was the conversion from spontaneous memory complaints (inclusion criterion) to a diagnosis of dementia (endpoint). No significant placebo verum difference was found at endpoint (Vellas et al. 2012). However, two assumptions of the original study plan relevant for the choice of the statistical evaluation were not confirmed by the data. First, the conversion rate was much lower than originally assumed for the statistical analysis and, second, the rate of conversions was not linearly dependent on time, but showed a curvilinear relationship with increasing rates 2 3 years after entering the study. The statistical power of the proportional hazards analysis specified by the protocol was thus considerably lower than expected. A prospectively defined subgroup analysis of subjects treated for at least 4 years Vellas et al. (2012) showed a significantly lower conversion rate (about 50%) in those treated with EGb 761 VR. This substantial effect, however, must be seen in respect of the absolute numbers of converters in the verum and placebo groups which were rather small (15 versus 28). A retrospective analysis applying a statistical method more appropriate to address the increasing hazards led to a significant result for the full study sample with a P value of (Scherrer et al. 2015). Taken together, both studies are not convincing final proof of no effects of EGb761 VR on the conversion rate from rather healthy aging to AD, but are also not final proof for a beneficial effect of the drug. Even if we assumed a positive effect, it might only be detectable after many years of improving mitochondrial function by EGb 761 VR. This is an important finding which has a major impact for future clinical studies with other drugs working by improving mitochondrial dysfunction (Andreux et al. 2013; Kumar & Singh 2015). Final conclusion The mitochondrial cascade hypothesis of dementia assumes mitochondrial dysfunction as one but certainly not the only important cause underlying the whole spectrum of age-associated cognitive disorders. In a large number of preclinical and clinical studies EGb 761 VR shows therapeutic efficacy over the whole spectrum, giving pharmacological support for the mitochondrial cascade hypothesis. While this is very much in favour of our initial concept, two important caveats have to be considered. First, while mitochondrial improvement certainly represents the major mechanism of action of EGb 761 VR, this phytomedical drug seems to have some additional pharmacological properties probably not related to its effects on mitochondrial function. However, these effects are mainly seen at rather high concentrations and cannot explain the broad spectrum of procognitive effects (De Feudis 1998; Christen & Maixent 2002; Shi et al. 2010; Montes et al. 2015). This has already been discussed. Second, the effects seen for EGb 761 VR on cognitive impairment are usually modest, which in one way goes parallel with its similarly modest effects on mitochondrial dysfunction. Moreover, although mitochondrial dysfunction seems to be an important common driving force for the cognitive decline over the aging spectrum (Figure 2), it might initiate other pathomechanisms that become relevant over time and which do not respond directly to EGb761 VR treatment. Acknowledgements The expert secretarial and graphical assistance of E. F orster und U. Hermanni and the substantial contribution of L. Bl umel to Table 2 are gratefully acknowledged. Statement of interest WEM received grant support from Schwabe and UCB as well as speakers or advisory honorarium from Lundbeck and Schwabe. AE received speakers honorarium from Schwabe (Switzerland). HT carried out contract research for Schwabe. SG has received speakers honorarium from from Schwabe. RH is a full time employee of Schwabe. RI has received grants/research support and honoratium as speaker, authors, or consultant from Eisai, Janssen, Lundbeck, Pfizer and Schwabe, SK received grants/research support, consulting fees and/or honoraria from Angelini, AOP, Orphan Pharmaceuticals AG, AstraZeneca, Eli Lilly, Jansen, KRKA Pharma, Lundbeck, Neuraxpharm, Pfizer, Pierre Fabre, Schwabe, and Servier. HJM received honoraria for lectures or for advisory activities by Lilly, Lundbeck, Servier, Schwabe, and Bayer. He was president or in the Executive Board of the following organisations: CINP, ECNP, WFSBP, EPA and chairman of the WPA-sextion of Pharmacopsychiatry References Aalten P, Verhey FR, Boziki M, Brugnolo A, Bullock R, Byrne EJ, Camus V, Caputo M, Collins D, De Deyn PP, et al Consistency of neuropsychiatric syndromes across dementias: results from the European Alzheimer Disease Consortium. Part II. Dement Geriatr Cogn Disord. 25:1 8.