The discovery and development of new potential antioxidant agents for the treatment of neurodegenerative diseases.

Review

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Biological mechanisms involved in the generation of free radicals

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Memorial University of Newfoundland on 08/01/14 For personal use only.

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Free radicals and OS in several neurodegenerative diseases

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Antioxidants and their role as free radical scavengers

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Development of antioxidant-based therapeutics for NDs

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Conclusion

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Expert opinion

The discovery and development of new potential antioxidant agents for the treatment of neurodegenerative diseases Chhanda Charan Danta & Poonam Piplani† Panjab University, University Institute of Pharmaceutical Sciences, Chandigarh, India

Introduction: Several neurodegenerative disorders (NDs) including Alzheimer’s and Huntington’s diseases have had associations with the oxidative process and free radical damage. Consequently, in past decades, several natural and synthetic antioxidants have been assessed as therapeutic agents but have shown limitations in bioavailability, metabolic susceptibility and permeability to the blood brain barrier. Given these issues, medicinal chemists are hard at work to modify/improve the chemical structures of these antioxidants, thereby improving their efficacy. Areas covered: In this review, the authors critically analyze several biological mechanisms involved in the generation of free radicals. Additionally, they analyze free radicals’ role in the generation of oxidative stress and in the progression of many NDs. Further, the authors review a collection of natural and synthetic antioxidants, their role as free radical scavengers along with their mechanisms of action and their potential for preventing neurodegenerative diseases. Expert opinion: So far, preclinical studies on several antioxidants have shown promise for treating NDs, despite their limitations. The authors do highlight the lack of the adequate animal models for preclinical assessment and this does hinder further progression into clinical trials. Further studies are necessary to fully investigate the potential of these antioxidants as ND therapeutic options. Keywords: antioxidants, free radicals, neurodegenerative diseases, oxidative stress Expert Opin. Drug Discov. [Early Online]

Biological mechanisms involved in the generation of free radicals

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Metabolic process Mitochondrion is the main organelle of the cell which continuously and abundantly produces free radicals like oxy-radicals and reactive oxygen species (ROS) through various metabolic processes including the enzymatic activity of mitochondrial cytochrome oxidase. Under hypoxic conditions, the mitochondrial respiratory chain produces nitric oxide (NO), which can generate reactive nitrogen species (RNS). It can further generate other reactive species, for example, reactive aldehydes -malondialdehyde and 4-hydroxynonenal -- by inducing excessive lipid peroxidation. Lipid peroxidation is a major source of free radicals. Xanthine oxidase also plays an important role in free radical-generation process. These are also generated during the metabolism of arachidonic acid, neutrophils, macrophages, platelets and smooth muscle cells. Upregulation of NADPH oxidase produces superoxide radicals through its catalytic subunit gp91Phox that damages neurons, and this enzyme is activated in the brain of patients suffering from Alzheimer’s disease (AD) and 1.1

10.1517/17460441.2014.942218 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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C. C. Danta & P. Piplani

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Drugs and toxic chemicals Drugs like adriamycin, bleomycin, mitomycin C, nitrofurantoin, chlorpromazine and aniline dyes, benzopyrene, carbon tetrachloride, toluene, paraquat and so on induce the generation of free radicals. 1.5

Article highlights. Neurodegenerative disease is a process of deterioration of the intellectual and cognitive faculties and characterized by progressive loss of the CNS which results in the cognitive, motor, and/or behavioral dysfunction and its etiology is still vividly unknown. Oxidative stress (OS) is an imbalance between production of free radicals and reactive metabolites that have been implicated in the progression of neurodegenerative disorders (NDs). A number of biological mechanisms are involved in the generation of free radicals which leads to commencement of OS that can be reversed by antioxidant therapy. Preclinical studies of several antioxidants for the prevention of NDs have been found to be successful despite their therapeutic limitation which is now a great challenge to the medicinal chemists for finding new potent antioxidants.

This box summarizes key points contained in the article.

Parkinson’s disease (PD). Similarly, the biochemical conversion of arginine to citrulline by NO synthase leads to formation of peroxynitrite that can nitrate the proteins and is linked to b-amyloid toxicity in cultured neurons. The nitration of misfolded protein aggregates protein oxidation and is elevated in hippocampus and neocortex of patients with AD. Likewise, nitration of tyrosine residues within the asynuclein (a-syn) protein is found to be accumulated in Lewy bodies (LBs) in case of Parkinson’s disease (PD). Microglial oxidative stress (OS) causes the cell activation that leads to occurrence of neuroinflammation where oxidative injury increases the expression of genes that are involved in synthesis of both NO and cytokine. Activation of the p38 MAPK has been correlated with amyotrophic lateral sclerosis (ALS) disease progression. These agents are responsible for neurodegeneration. Inflammation During inflammation, mast cells and leukocytes are recruited to the site of damage, which leads to a ‘respiratory burst’ due to an increased uptake of oxygen and, thus, an increased release and accumulation of ROS at the site of damage. Along with this, cytokines are released in inflammation that mediates the production of free radicals by neutrophils and macrophages. 1.2

Stress Generation of free radicals is triggered by mental stress along with the hormones that mediate the stress reaction in the body like cortisol, and catecholamine themselves degenerate into destructive free radicals. 1.3

Radiations Gamma rays, X-rays, ultraviolet radiations and microwave radiation are also involved in the generation of free radicals. 1.6

Pollution Air pollutants (asbestos, automobile exhausts fumes, benzene, carbon monoxide, chlorine, formaldehyde, ozone, toluene and smoking of tobacco products), chemical solvents (cleaning products, glue, paints, paint thinners, perfumes and pesticides) and water pollutants (chloroform and other trihalomethanes) are all potent generators of free radicals. Burning of organic matter during cooking, forest fires and volcanic activities can also generate free radicals. 1.7

Dietary factors Foods containing high levels of lipid peroxides from animal origin, highly fried or cooked at high temperatures, foods that have been browned or burned, and foods that have been processed with herbicides, pesticides, hydrogenated vegetable oils and sugar can produce free radicals [1-3]. 1.8

Free radicals and OS in several neurodegenerative diseases

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OS is defined as an imbalance between production of free radicals and reactive metabolites, usually called ROS or oxidants and their elimination by protective mechanisms, referred to as antioxidants or the imbalance between generation of ROS/RNS and antioxidant defenses leading to a negative condition known as oxidative/nitrosative stress in which cell antioxidants are insufficient to keep ROS/RNS below a toxic threshold due to excessive production of ROS/RNS and/or loss of cell antioxidant defenses [4,5]. This imbalance feebly damages the important macrobiomolecules (proteins, lipids, carbohydrates and nucleic acids), other cellular components or the whole organism [6-8]. However, cells are equipped to counteract this oxidative attack with numerous cellular antioxidant defenses such as glutathione (GSH), superoxide dismutases (SOD) and catalase (CAT) [7,9]. The consequences of increased oxidant levels and decreased antioxidant defenses lead to oxidative damage that have been implicated in the progression of many neurodegenerative disorders (NDs) as discussed below. Alzheimer’s disease Alzheimer’s disease (AD) is a progressive neurodegenerative disease. It is the major cause of dementia in elderly people with a typical age of onset as early as 60 years of age. It is 2.1

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Immune system

In response to pathogens, immune cells generate ROS and oxy-radicals. 2

Expert Opin. Drug Discov. (2014) 9(10)


The discovery and development of new potential antioxidant agents

the fourth-leading cause of death in developed nations and its etiology remains unknown. OS has also been implicated in the pathogenesis of AD [10], which has been indicated by enhanced lipid peroxidation, in specific areas of the brain in postmortem studies [11]. It has been found by different researchers that there is an increase in the level of activity of CAT, SOD, GSH peroxidase and GSH reductase in the hippocampus and amygdala of AD brain. [12,13]. A research study has strongly suggested that OS causes oxygen radical formation with resultant neurodegeneration and possibly plaque formation in the CNS [14]. Another study hypothesized that b-amyloid is neurotoxic and this toxicity is mediated by free radicals in vitro and in a transgenic mouse model of AD [15]. Although the participation of OS as a cause of AD is still a topic of discussion, the brain of AD patients is associated with many markers of OS and it increases the severity of symptoms [16,17]. Huntington’s disease Huntington’s disease (HD) is relatively rare but fatal autosomal dominant mode of inheritance ND. It affects 5 -- 10 per 100,000 people in Europe and North America. HD is caused by an increased repetition (‡ 35 repeats) of a CAG trinucleotide sequences in exon 1, encoding a polyglutamine tract at the N terminus of the huntingtin (htt) gene, encoding a protein with unknown function, called htt [18,19]. Most of the hypotheses have included the role of oxidative damage to describe the pathogenesis of HD [20]. ROS is produced by the excessive glutamate activation of the excitatory receptors [21]. The association of OS to the pathogenesis of HD has also been studied by measuring the levels of F2-isoprostanes in the cerebrospinal fluid (CSF) of 20 patients in the early phase of the disease and found that its concentration was moderately but significantly higher in HD patients than in the control group (35% increase) [22]. The theory of impaired energy metabolism with concomitant OS has been supported by animals and human postmortem studies [23]. Postmortem striatum and cerebral cortex tissue of HD individuals and mouse models of HD demonstrated signs of oxidative damage [24]. At the early stage of the disease, no signs of neuronal cell loss were visible, thus suggesting that increased oxidative damage is an early event in HD pathogenesis and might significantly contribute to neurodegeneration. The observation that antioxidant treatment is neuroprotective in HD mice further points in the direction of ROS as an important player in neurodegeneration in HD pathogenesis [25]. 2.2

(SNpc) and striatum along with the appearance of intraneuronal proteinaceous eosinophilic inclusions of spheroids called LBs whose basic components consist of fibrillar a-syn and ubiquitin [26-28]. In the several past decades, a lot of research work has been attempted but the real etiology of PD is still unidentified. It has been proposed that several pathogenic mechanisms are involved in PD, that is, oxidative and nitrative stress, mitochondrial dysfunction, apoptosis, excitotoxicity, protein misfolding and aggregation and inflammatory responses [27-30]. Neurochemically, mitochondrial complex I dysfunction and increased indices of OS have been observed in PD [27,28]. Although, none of these reported mechanisms have proved to be the exact cause leading to the damage of SNpc DAergic neurons in PD, in recent years, research has provided strong evidences supporting the hypothesis about involvement of free radicals and its consequence of OS which plays a pivotal role in the pathogenesis of PD. This evidence is by the observation of the oxidation of DA, which produced toxic semiquinones, and the metabolism of DA by monoamine oxidase-B, which induced the formation of excessive hydrogen peroxide, hydroxyl radicals and superoxide anions, studies on selective toxicities against substantia nigra by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, which induced Parkinson’s-like symptoms in primates acted through its active metabolite 1-methyl-4-phenylpyridine, inhibiting mitochondrial complex I [31,32]. Numerous postmortem studies on brain tissues of PD patients have suggested that ROS and RNS are involved in the degeneration of DAergic neurons which is confirmed by the presence of excess level of protein oxidation, lipid peroxidation, depletion of GSH and DNA damage [33]. Immunochemical staining on surviving DAergic nigral neurons in the midbrains of PD patients showed the presence of 4-hydroxy-2-noneal (HNE)-modified proteins, which are formed by HNE, a reactive a, b unsaturated aldehyde, that is one of the major products formed during the oxidation of membrane lipid polyunsaturated fatty acids (PUFAs) and forms stable adducts with nucleophilic groups on proteins such as thiols and amines. This HNE modification of membrane proteins forms stable adducts that are used as biomarkers of cellular damage due to OS [34]. Cholesterol lipid hydroperoxide, a marker of lipid peroxidation increased 10 times as compared to control in substantia nigra of PD patients [35]. Increased oxidative damage to DNA in PD was found with a marked enhancement in 8-OHdG in caudatum and substantia nigra. Amyotrophic lateral sclerosis ALS is a most frequent rapidly progressive adult-onset lethal ND whose real cause is yet unknown. It is a motor neuron disease and its pathology is characterized by the degeneration of upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord [36,37]. Recent findings have suggested that OS is the main implication in the pathogenesis of all forms of ALS and it has been strongly supported by the discovery of mutations in the gene (on 2.4

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Parkinson’s disease

PD is the second-leading neurodegenerative impairment disorder with a prevalence of approximately 160 per 100,000 in the Western world but a typical sporadic PD has a prevalence of 0.6% at 65 years of age and its developing risk increases with age with a prevalence of 4 -- 5% by the age of 85. It occurs as a consequence of dopamine (DA) depletion due to neurodegeneration in substantia nigra pars compacta


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chromosome 21) encoding the antioxidant enzyme Cu/Zn SOD1 in the familial ALS [36,38-40]. This mutation leads to generation of hydroxyl free radicals that results into oxidative damage of neurons through mutant SOD1 along with mitochondrial dysfunction, protein misfolding and aggregation [36,41]. As a result, the normal physiological functions of the SOD1 enzyme, that is, binding to Zn and Cu ions, with the Cu atom playing the active role in the free radical scavenging activity, the removal of superoxide and prevention of further generation of ROS, are altered due to these mutations [38,42]. The role of ROS-mediated OS in ALS has been reported by many researchers that major oxidation products like malondialdehyde, hydroxynonenal, membrane phospholipids, DNA and oxidized proteins are found to be elevated both in sporadic ALS and familial ALS patients as well as in several model systems [38,43-45]. It has also been suggested that free radical damage is more prominent in the mitochondria which is attributed to intracellular OS and result into apoptotic cell death of motor neurons in ALS [46,47].

symptoms. These research findings further support that free radical-mediated OS may be involved in the pathophysiology of schizophrenia.

Cognitive dysfunction in the elderly Cognitive dysfunction or mild cognitive impairment in the elderly is a very common problem in the over-65-year age group and can be defined as a transitional stage between normal aging and dementia. It creates a symptomatic clinical situation that a person has complaints about memory loss. It subsequently progress toward the most devastating form of clinical dementia, that is, Alzheimer dementia, shown by around 5% of the population. It has been reported that memory or cognitive dysfunctions are due to the consequences of ROS [48,49]. Excessive ROS oxidize the proteins in the brain, and due to the accumulation of oxidized modified proteins, neurodegeneration and impairment of cognitive functions occurs [48,50]. Again excessive ROS activities lead to severe mitochondrial decay and it plays a major role in the deficit of cognitive functioning abilities which is most commonly associated with advanced age [51,52].

Tardive dyskinesia Tardive dyskinesia (TD) is a delayed extrapyramidal neurological syndrome and is considered to be a serious movement disorder associated with the long-term clinical exposure to antipsychotic drugs. It is estimated that the prevalence of TD is in the order of 10 -- 15% in young population, 12 -15% in more chronic patients and 25 -- 45% in very chronic patients [69]. The pathophysiological basis of TD is obscure; however, more recently, it has been postulated with strong evidences that free radical-induced OS is the probable mechanism of neuronal damage in TD which is supported by the findings of elevated levels of lipid peroxidation products in the CSF of dyskinetic patients and a possible role of vitamin E on dyskinetic symptoms [70].

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Schizophrenia Schizophrenia is a chronic, severe, disabling and deteriorating psychiatric illness that affects about 1% of the population worldwide [53,54]. Although the pathophysiological mechanisms of schizophrenia remain unknown, there is evidence that an excessive free radical production or OS affecting the CNS may be involved in the pathophysiology of schizophrenic patients. OS has repeatedly been shown in schizophrenic patients [55,56]. It has been reported that high level of lipid peroxidation products was found in plasma [57], red blood cells [58] and in CSFs [59] of schizophrenic patients with significant correlations between psychopathology and levels of antioxidant enzymes and lipid peroxidation products [60]. Currently, it has been reported that antioxidants such as vitamins [61], extract of Ginkgo biloba [62] and essential PUFAs (EPUFAs) [63] show improvement in some of the schizophrenia 2.6

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Multiple sclerosis Multiple sclerosis (MS) is a T-cell-mediated autoimmune chronic inflammatory and demyelinating disease of the CNS affecting both white and gray matter [64]. Neuropathologically, it is characterized by infiltration of inflammatory cells, such as T cells and monocytes-derived macrophages into the CNS which play a central role in MS pathology because phagocytes’ myelin causes damage to and breakdown of myelin sheaths, oligodendrocytes and axons along with inflammatory mediators, including chemokines, cytokines, NO and ROS, which contribute to the development and progression of the MS [65,66]. Although the etiology of MS is yet enigmatic, it has been recently found that OS has a major role in the pathogenesis of MS and it has been confirmed by several studies that antioxidant therapy is beneficial in MS and hence it may be an attractive approach for its treatment [67,68]. 2.7

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Ataxia Ataxia is a wider group of NDs characterized by degeneration or malfunctioning of the cerebral cortex. It has been classified as congenital, hereditary and non-hereditary. Among ataxias, ataxia telangiectasia (AT) and Friedreich’s ataxia (FRDA) are considered to be more prominent [71]. AT is an autosomal recessive, genetic childhood disease characterized by a pleiotropic phenotype which includes progressive cerebellar degeneration, immunodeficiency, sterility, genomic instability and increased risk of cancer along with high susceptibility to ionizing radiations and radiomimetic chemicals. Hitherto no treatment has been reported and patients have a life span of only about 20 years [72]. Mutations in the AT-mutated (ATM) gene, a protein kinase known to be an ‘early responder’ to double-stranded DNA breaks has been identified as the physiological and molecular basis of the AT. This kinase protein which is expressed by the ATM gene plays a key role in the cellular responses to DNA damage and in the activation of cell cycle checkpoints [73]. However, recently, it 2.9




has been reported that OS with high levels of ROS contributes to the neurodegeneration in AT [74]. The evidences reported for this are the use of natural antioxidants such as vitamin C and vitamin E [75] and a-lipoic acid (LA) [74] as potential antioxidants for the treatment of AT. FRDA is an autosomal recessive inherited neurodegenerative disease characterized by progressive gait and limb ataxia, areflexia, dysarthria, loss of vibratory and position sense, scoliosis, diabetes, cardiomyopathy and a progressive motor weakness of central origin. The gene responsible for FRDA encodes a mitochondrial protein of unknown function called frataxin. Evidence of OS which includes disabled early antioxidant defense systems that result in oxidative insult to the highly sensitive iron-sulfur proteins aconitase and three mitochondrial respiratory chain complexes (I -- III) has been reported. Again, in vitro studies have demonstrated that frataxin-deficient cells not only generate more free radicals but also show a reduced capacity to mobilize antioxidant defenses. As a result, antioxidant-based therapy appears to be an attractive approach for its treatment [76].

Antioxidants and their role as free radical scavengers

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Antioxidants are defined as the substances which at low concentration significantly inhibit or delay the oxidative process, while often being oxidized themselves. Both endogenous and exogenous antioxidants are capable of neutralizing the fastreacting activities of free radicals and hence protect the human body by minimizing oxidative damage which contributes to the development and pathology of several diseases by maintaining redox balance [1,77]. Superoxide dismutase It is an important metalloprotein endogenous antioxidant enzyme mostly found in four forms in humans. They can contain copper and zinc (Cu-ZnSOD/SOD1), or manganese (MnSOD/SOD2), nickel (NiSOD) or iron (FeSOD) as their metal part. They act as the first-line defense system against ROS which scavenge superoxide free radicals by dismutating . the superoxide radical (O2 -), into hydrogen peroxide (H2O2) and molecular oxygen (O2) [78]: (1) 3.1

SOD

i−

O2 + 2H + ⎯⎯⎯→ H2 O2 + O2 Catalase It is a tetrameric hemeprotein endogenous antioxidant enzyme and has four porphyrin heme groups in its structure that allow reaction with hydrogen peroxide. It decomposes millions of toxic hydrogen peroxide molecules into water and oxygen per second [79]: (2) 3.2

CAT

H2 O2 ⎯⎯⎯ → H2O + 1 / 2O2

GSH (g-L-glutamyl-L-cysteinylglycine) It is a soluble tripeptide and a powerful nonenzymatic antioxidant present in higher concentrations in cytosol (1 -11 mM), nuclei (3 -- 15 mM) and mitochondria (5 -- 11 mM). It efficiently scavenges the singlet oxygen and hydroxyl radical and detoxifies hydrogen peroxide and lipid peroxides [1,80]. 3.3

LA (1, 2-dithiolane-3-valeric acid) LA is a widely occurring sulfur-containing coenzyme. LA and its reduced form dihydrolipoic acid (DHLA) (6, 8-dimercaptooctanoic acid or 6, 8-thioctic acid) are considered as ‘universal antioxidants’ because they can efficiently quench the free radicals in both lipid and aqueous domains. LA or DHLA scavenges the ROS such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals and singlet oxygen. DHLA exerts prooxidant actions through reduction of iron and inhibits the lipid peroxidation [81]. 3.4

L-arginine It is the most metabolically versatile basic natural amino acid. It is an efficient antioxidant and capable of scavenging superoxide free radicals and reducing the lipid peroxidation product malondialdehyde. It also reduces the glycoxidation product N-e-carboxymethyllysine and free oxygen radicallinked fluorescence. It remarkably reduces the vascular release of superoxide anions and restores NO production [82]. 3.5

Essential fatty acids The long-chain PUFAs can be categorized into two main families. They are omega-3 (w-3) essential PUFAs (w-3EPUFAs) and omega-6 (w-6) essential PUFAs (w-6EPUFAs). The w-3 PUFAs are mainly obtained from fish oils and some plants, whereas w-6 PUFAs are derived from vegetable oils. Both series of fatty acids cannot be synthesized endogenously from carbohydrates, and hence need to taken in diet by human beings. They act as potential antioxidants. The w3EPUFAs include eicosapentaenoic acid, docosahexaenoic acid and a-linolenic acid. Supplements of PUFAs are very helpful in OS along with other antioxidants like vitamin C and vitamin E [83]. 3.6

Carotenoids These are a family of pigmented compounds that are synthesized by plants and microorganisms but not by animals. Plant products are the major sources of carotenoids. In plants, they are involved in photosynthesis and protect them against photodamage. Chemically, all carotenoids possess polyisoprenoid units joined by tail-to-tail bond, a long chain of conjugated double bonds and a nearly bilateral symmetry around the central double bond. However, most carotenoids have one or two ring structures (five or six membered) formed by the cyclization of the end groups. They may also contain oxygen atom along with carbon and hydrogen atoms. They are 3.7


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regarded as effective powerful antioxidants. The antioxidant behavior of a carotenoid molecule (whether mediated by direct or indirect means) depends on its molecular structure and also the chemical nature of the oxidizing species itself. It includes mainly not only the length of the conjugated C=C chain but also the shape, nature, position and the number of substituent groups, and physical form (aggregated or monomeric, cis/trans configuration etc.) of the carotenoid molecule. They interact with the free radicals by the following three mechanisms: (3)

ROOi + CAR ⎯⎯ → ROO− + CAR i + (4)

ROOi

+ CAR ⎯⎯ → ROOH +

CAR i (5)

ROOi

CAR)i

+ CAR ⎯⎯ → (ROO − Among carotenoids, a-carotene, b-carotene, b-cryptoxanthin, lutein and lycopene are considered as potent antioxidants [84]. Flavonoids They are a class of widely occurring natural compounds and represent the single-most group of phenolic phytochemicals. They have benzo-g-pyrone system containing a pyrane heterocyclic and phenolic aromatic rings representing a diphenylpropane (C6C3C6) skeleton. They are regarded as potential prototypic chain-breaking antioxidants and are described as hydrogen-donating antioxidants by virtue of the reducing properties of the multiple hydroxyl groups attached to the aromatic systems, along with their ability to delocalize the resulting phenoxyl radical within the structure. In flavonoids, the 3¢- and 4¢-hydroxyl groups of catechol, hydroxyl group at position 3, carbonyl group at position 4 and 2,3 C=C unsaturation have been found to be essential for antioxidant activity; however, the presence of a single hydroxyl group at position 5 is not essential. These polyphenolic flavonoids are capable of scavenging different oxygen and nitrogen radicals, such as hydroxyl, superoxide, peroxyl, alkoxyl and peroxynitrite radicals, respectively, by the following mechanism: (6) 3.8

Flavonoid(OH) + R i ⎯⎯ → Flavonoid(Oi ) + RH where is a free radical and is an oxygen-free radical. Till date > 4000 flavonoids have been identified; however, quercetin is considered as the best antioxidant in comparison to others [85,86]. Vitamins (A, C and E) They are considered as very effective powerful antioxidants. Vitamin A is mainly obtained from green and yellow vegetables, dairy products, fruits and meats. Carotenoids are the main dietary sources of vitamin A as it is structurally similar to them. Carotenoids such as a- and b-carotene, and b-cryptoxanthin are able to convert into vitamin A in the body and they are regarded as pro-vitamin A compounds. 3.9

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Within the body, vitamin A can be found as retinol (vitamin A1), retinal or retinoic acid. It acts as a chain-breaking antioxidant by combining with peroxyl radicals. Its antioxidant activity is conferred by the hydrophobic chain of the polyene units that can quench singlet oxygen, neutralize thiyl (RS.) radicals, and combine with and stabilize peroxyl radicals. It can auto-oxidize, when O2 tension increases, and thus acts as a more effective antioxidant at low O2 tensions that are typical of physiological levels found in tissues. The antioxidant activities of retinoids can be ranked as retinol ‡ retinal >> retinoic acid [85-87]. Vitamin C (L-ascorbic acid) is mainly obtained from fruit juices, vegetables and beverages. It is commonly recognized as a major, naturally occurring nutrient and antioxidant in our daily diet. It is highly bioavailable and acts as a most important water-soluble potent antioxidant in the cells. It efficiently scavenges several different ROS such as hydroxyl, superoxide, peroxyl radicals and singlet oxygen [88]. Vitamin E (a-tocopherol [a-TOH]) is a major lipophilic, phenolic compound having potent antioxidant property obtained mostly from plant sources. It includes eight different related homologues and those can be grouped as TOHs and tocotrienols, designated as a-, b-, g- and q-forms. TOHs have a phytyl side chain, while tocotrienols have a similar chain but with three double bonds at positions 3¢, 7¢ and 11¢. All the naturally occurring vitamin E forms, and synthetic all-rac-a-TOH, have relatively similar antioxidant activities; however, among them. a-TOH is the most abundant, active and having higher antioxidant potential. In addition to this, human body prefers only a-TOH as it is a form of vitamin E and hence in this account vitamin E refers to only aTOH. Its antioxidant activity relies on the H-atom donating ability of the hydroxyl group of the chromanol ring to the reactive chain propagating radicals, to yield a stabilized phenoxyl radical (PhO ) [89].

Development of antioxidant-based therapeutics for NDs

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Antioxidants have emerged as potential therapeutic agents for the treatment of neurodegenerative diseases. Those that are found to be efficacious in animal models and clinical studies [1,90] are enlisted below. Vitamin E Vitamin E is the most potent antioxidant that has shown some promise in the treatment of AD [91]. A large, 2 years, double-blind, placebo-controlled, randomized multicenter clinical trials with 2000 IU/day of vitamin E in moderately impaired 341 AD patients have shown delayed functional deterioration [92]. Studies with cocktails of vitamin E have been shown to prevent or slow the progression of PD and both animal models and clinical studies have suggested that vitamin E deficiency contributed to nigral neurodegeneration and the onset or progress of PD [93]. In a rat animal model induced with 6-hydroxydopamine (6-OHDA), a neurotoxin 4.1




known to induce unilateral nigrostriatal lesions, treatment with different types of vitamin E for a period of 1 month has shown a significant reduction in the apomorphineinduced rotational behavior. Vitamin E has also shown attenuation of the toxic effects of 6-OHDA and its metabolites on striatal DA. Thus, two data suggested the preventative action of the vitamin on 6-OHDA-induced DA depletion [94]. In another study, pretreatment of rats with vitamin E caused significant attenuation of the toxic effects of 6-OHDA on GSH and SOD levels in most brain regions. These results show that vitamin E can spare the antioxidant scavenging system from the injurious effects of 6-OHDA [95]. It has also been reported that consumption of foods rich in vitamin E early in life may decrease the risk or delay the onset of PD [96]. One clinical study has suggested that a-TOH treatment given early in the course of HD may slow the rate of motor dysfunction [97]. Before 1996, of the 12 studies performed on the use of vitamin E for the treatment of TD, 9 studies showed some improvement in subjects who had milder symptoms at the onset; however, other studies reported no effect [98]. Vitamin C Vitamin C is a strong antioxidant. It is found in 10-fold higher concentration in brain than in serum [99]. It can cross the blood brain barrier (BBB) in its oxidized form, is retained in the brain tissues in the form of ascorbic acid and is efficacious only when given in combination with other vitamins [100]. One study has suggested that the intake of high-dose of vitamin C and E supplements may reduce the risk of AD [101]. In another study, cognitive function showed a significant correlation with the intake of both vitamin C and E supplements for a 4-year duration [102]. 4.2

Vitamin A and analogs Brain generally does not require vitamin A. However, it has been found that the brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid in special structures of the BBB which suggests that vitamin A and the analogs can significantly cross the BBB [103]. A study showed that peroxidation in rat brain mitochondria was inhibited by the fat-soluble vitamins especially retinol and retinol acetate, retinoic acid, retinol palmitate and retinal at concentrations of 0.1 -- 100 mmol/l [104]. Even vitamin A is a potent antioxidant; hitherto, its clinical value has not been determined clearly. 4.3

a-LA It is a metabolic antioxidant that can be absorbed from the diet and can cross the BBB. It has shown potential for the treatment of age-related behavioral decline that is associated with AD. In an open-field memory test and 24 h after the first test, treatment of aged mice (20 -- 23 months) with oral a-LA (100 mg/kg for 15 days) has shown improved performance with better results than untreated young animals [105]. Another study demonstrated that intraperitoneal administration of 4.4

a-lipoate or dihydrolipoate (10 mg/kg for 10 days), decreased rat striatum lesions induced by excitotoxins, which affect NMDA receptors and which may lead to calcium influx, as well as the generation of free radicals. Reports suggested that in animals that received NMDA, striatal lesion size was reduced by 49% with a-lipoate treatment and by 41% with dihydrolipoate treatment. However, in animals receiving malonic acid, lesion size was reduced by 45% with a-lipoate treatment and by 68% with dihydrolipoate [106]. A study has suggested that a-LA may potentially correct metabolic abnormalities in PD, and the lesions induced by 6-OHDA in rats were partially prevented by pretreatment with a-LA for 5 days [107]. Flavonoids and other polyphenolics Flavonoids are naturally occurring polyphenolic compounds having potent antioxidant abilities. They have been proposed to exert beneficial effects in NDs [108]. Recently, several studies have shown that they are capable of protecting neurons against OS more effectively than ascorbate, even used at 10-fold higher concentrations [109]. In the recent past, it has been identified that polyphenolic compounds have neuroprotective effects and their molecular structures are identified to explore the mechanisms behind the neuroprotective action. Some of the polyphenolic compounds have been in routine clinical applications despite their little beneficial effect in experimental treatment of neurodegeneration [110]. It has been reported that epigallocatechin gallate (EGCG) postponed the onset of neurological symptoms and prolonged the life span in a mice model of ALS [110] and it has shown that long-term treatment with EGCG increased the life span and enhanced movement abilities in a transgenic Drosophila melanogaster model of PD [111]. EGCG has shown strong anti-acetylcholinesterase (AChE) activity and was accompanied by improvement of cognitive functions, like learning and memory [112]. Resveratrol blocked acetylcholine release from adrenal chromaffin cells [113]. Other polyphenols such as huperzine A, quercetin, Kuwanon U, E, and C, kaempferol, tri- and tetra-hydroxyflavone have shown anti-butyrylcholinesterase effects in addition to their anticholinesterase activity [112,114]. Huperzine A is found to be a potent, reversible and selective inhibitor of AChE and its potency is similar or superior to other AChE inhibitors (AChEIs) [115] and has shown higher potency than tacrine, galantamine, physostigmine and rivastigmine but less potent than donepezil [116]. It has shown longer duration of AChEI activity, better BBB penetration ability and higher oral bioavailability as compared to other AChEIs [117]. It has also shown promising clinical reports on cognitive and functional impairments of AD and schizophrenia and increase in memory performance of normal individuals. Phase IV clinical trials conducted in China revealed that huperzine A has a significant role in the cognition improvement in elderly people, patients with AD and patients with vascular dementia [118]. Recently, polyphenols such as catechin, curcumin, tannic acid, EGCG, resveratrol, trans-resveratrol, honokiol, tea polyphenol, mangiferin and 4.5


7


morin have shown protective effects against NMDA neurotoxicity [119,120]. In another study, polyphenolics including alvidin, curcumin, dihydroguaiaretic acid, EGCG, ellagic acid, exifone, myricetin, oligonol, piceid, resveratrol, salvianolic acid B and tannic acid are reported to have protective effects against b-amyloidopathy and protein aggregation in different cell lines or animal models [121,122].


5.

Conclusion

Neurodegenerative diseases in humans are strongly associated with OS generated by ROS. Although antioxidants can effectively counteract the detrimental role of ROS, they are better considered as neuroprotective agents. Hitherto, several antioxidants of widely varying chemical structures have been investigated for use as therapeutic agents. However, a very few of them have shown effective results in preclinical and clinical trials. Some antioxidants used as drugs in practice failed to be better neuroprotectives. Likewise, with belief of lower risk of unexpected side effects and safety profiles in the use of dietary sources of antioxidants have also shown dissatisfied results. Thus, antioxidant-based clinical trials and therapeutic intervention approaches have disappointed the researchers regarding the neuroprotective nature of the agents. Therefore, there is an urgent need for discovery of novel antioxidants which can be better utilized as therapeutic agents with minimal side effects and having no or less biopharmaceutical problems. 6.

Expert opinion

Free radical is a very fast-reacting chemical species having high energy and free electron(s). Due to environmental damage, stress, change in lifestyle and food habits, generation of free radical has become a very easy process in human body. They play a major role in the onset of threatening neurodegenerative diseases. Human beings are highly susceptible to ROSinduced OS despite the presence of endogenous antioxidant defense system against it due to relative depletion of endogenous antioxidant enzymes. OS damages the nerve cells and their functions and subsequently leading to the diseases progress. Now the question arises: how these free radicals can be stopped? The answer seems to be very easy -- by using antioxidants. They act as free radical scavengers by accepting the free electron(s) and subsequently neutralizing the free radicals. Dietary antioxidants from fruits could be a better approach for the treatment of NDs. However, antioxidants from food sources are not completely available to the biosystem. Vitamin E and vitamin C supplements have shown lack of improvement in AD prevalence, although a high dietary intake of the same shows remarkable decrease in the risk of developing the disease but is also associated with the increased risk of cardiovascular problems and mortality. Hence, a necessary balance in dosage and synergistic effect with other antioxidants (either endogenous or given as exogenous) must be kept in mind when treating with antioxidants. The dose of 8

antioxidants reduces ROS or RNS and simultaneously can halt cellular functions where a high dose scale up the unfavorable conditions that lead to more OS. Antioxidants like vitamin C, vitamin E, a-lipoate, carotenes, flavonoids and so on have shown very less positive results from preclinical and clinical trials for the treatment of neurodegenerative diseases. They are evaluated either as monotherapy or in combination. Combination of vitamins C, E and A and b-carotene, flavonoids and vitamin C are found to be inefficacious in PD. Vitamin E is found to be inefficacious in PD but efficacious in AD. Combination of vitamins C and E is found to be efficacious in both AD and cognitive disorders. However, they are not proved to be up to the mark. They are also unable to cross the BBB. Flavonoids are considered as potent antioxidants but they are highly polar molecules due to presence of many phenolic groups and restricted to blood brain permeability. Even some of the drug candidates like allopurinol, 5-aminosalicylic acid, omeprazole, cimetidine, diltiazem, glibenclamide and so on are reported as antioxidants but still not vividly investigated in human or animal models. Currently available antioxidants lack efficacy in large-scale controlled studies. A collection of molecular structure of some potential antioxidants is given in Table 1 which can be valuable for the designing of new antioxidant molecules to produce efficient drugs for the treatment of neurodegenerative diseases. Hence from this partial positive but successful information, the following questions arise: what could be a better antioxidant? What is needed in the molecule for the discovery of a new potent antioxidant? The research should aim toward the discovery and development of new potential antioxidants that overcome the above-discussed problems. This may be a direct challenge to the medicinal chemists that they should apply their knowledge that what could be the basic moiety of the molecule, side substituents and the demand of functional group(s) so that the molecule can capture the free electron(s) of the free radicals very nicely, should be stable itself after capturing the free electron(s), able to cross BBB and showing no or less biopharmaceutical problems and side effects. Some original research articles discussed the structure--activity relationships of quercetin as a potential antioxidant, importance of phenolic groups in flavonoids and importance of conjugated double bonds in carotene in capturing free electrons and so on. Hence, the ultimate goal in this field is to construct the molecules in such a way that at least a single successful molecule could be effective in both ways as a drug candidate for the neurodegenerative diseases and a potential antioxidant which has the ability of neuroprotection. The exhaustive list of molecular structures of potential antioxidants listed in Table 1 can be utilized to develop better therapeutics. They can be used as a lead for the development of new analogs as well as dimerized and hybrid antioxidants. They can be further explored clinically or preclinically as mitochondria-targeted compounds as single or in combination. Almost all the antioxidants currently used are polar in nature that pose the major problem of penetration into the



Table 1. Chemical structures of some potential antioxidants. Name of antioxidant a-lipoic acid

Chemical structure S

OH

S O

Vitamin A

H3C

CH3

CH3

CH3


OH CH3 OH

Vitamin C O

O

OH H

HO OH

Vitamin E (a-tocopherol)

HO O OCH3

Melatonin

H N

CH3

HN O

Coenzyme Q10 (Ubiquinone)

O H3CO

CH3

H3CO

H CH3 6-10

O

Glutathione

O

HS

O

HO

N H

NH2

N-acetyl cysteine

O H3C

H N

O OH

O

SH H N H

O OH O

Uric acid

H N

NH

O N H

N H

NH

L-arginine H2N

N H

O O OH NH2

b-carotene

Lycopene


9


Table 1. Chemical structures of some potential antioxidants (continued). Name of antioxidant

Chemical structure OH

Lutein H

HO O


Allopurinol N

NH N H

N

O

Penicillamine

OH

HS NH2

O

5-Aminosalicylic acid H2N

OH OH

Apomorphine

HO HO

H CH3 N

Selegiline

N H

Omeprazole

O

N

N

S N H

H3CO

CH3 H3C

OCH3

4-Hydroxytamoxifen H3C

CH3 N CH3

HO

Captopril

O

HS O O

N

OH

Ramipril O

O

O

HO

N

H

10


HN

H

O




Quinapril O O

N


HN O O

OH

Losartan

HN N

N

Cl

N N

N

N

Ketoconazole

N O

O O

N

N

O H Cl

Cl

OH

Propofol

Carvedilol OH HN

O

O H N

OH

Metoprolol O

H N

CH3 CH3

H3CO HN

Cimetidine S

HN N

N HN

Diltiazem

O

N

N

N

O O CH3 O

S

O CH3


11



Nicardipine

Chemical structure

O2N O

O


N

O

O N H

Phenylbutazone

O N N O

O

Nitecapone HO O

HO NO2

O

Entacapone O2N

N

HO

N

OH

Idebenone

OH

O O

O O

Troglitazone

O O

HO

S

O N H O

Tacrolimus HO

HO O O

O

N O O HO

12


O H O

O




Epigallocatechin gallate

OH O

HO

OH O


OH

OH

O

OH OH

trans-Resveratrol

OH

HO OH

Huperzine A NH O H2N OH

Quercetin

OH O

HO

OH OH

O

Kuwanon C HO

OH

O

HO

OH

O

OH

Kaempferol O

HO

OH OH

O OH

Catechin O

HO

OH OH

OH O

Curcumin

O OCH3

H3CO HO

OH

OH

Mangiferin HO HO

O

HO O OH

OH HO


O

OH

13



Chemical structure O

Ellagic acid O

HO

OH

HO OH

O


O

Terbinafine

N

OH

Myricetin

OH O

HO

OH OH

OH NH

Metformin N

Glibenclamide

O NH

N H

NH2

H N

O O

Cl

Repaglinide

O N H

HN HN S O O O

COOH OC2H5

N

CNS. Therefore, there is a need to work on this very aspect that can conventionally be done by substituting the bulky groups that include alkyl groups, aromatic rings, aromatic alkyl groups or heterocyclic aromatic ring systems in order to increase the lipophilicity of the molecule so that it can easily penetrate the CNS. Rather recent advances in drug formulation and drug delivery include conjugation to a lipophilic cation, orally bioavailable mitochondria targeted antioxidants like Mito Q, Mito Vit E and Mito TEMPOL, SS tetra peptides, “Sk”compounds, XJB-peptides or polymer based nano-carriers like liposomes and solid lipid nanoparticles to a lipophilic cation. The compounds should be vividly investigated preclinically before entering to the clinical development phase in terms of neuroprotective dose--response relationships, pharmacokinetics--pharmacodynamics correlations, therapeutic windows, optimum dosing regimens and treatment durations. But the unavailability of the adequate animal models for the preclinical 14

assessment hinders the way for clinical trials. In addition, more preclinical studies in rodent and other mammalian models are necessary. Endogenous antioxidant capacity which is found to be reduced in patients with NDs can be augmented by using natural or synthetic antioxidants that are proved to be a useful therapeutic strategy. Current antioxidant therapy may aim at site-specific delivery, regulated dosing and cocktails of dietary or pharmacological antioxidants that work synergistically for better response. Synthesis of dimerized products from clinically successful antioxidants and preclinical trials of these could be a better option for the discovery and development of new antioxidant molecules. Finally, application of mitochondria-targeted antioxidant therapy using nanomaterials could be an attractive strategy for development of disease-modifying drugs for the better treatment of NDs. It is a new field of biomedical research that has led to innovative therapeutic molecules, some of which are under clinical Phase II trials.



Declaration of interest The authors are both employees of Panjab University. The authors have no other relevant affiliations or financial Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers.


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Affiliation

Chhanda Charan Danta1 & Poonam Piplani†2 † Author for correspondence 1 Research Scholar, Panjab University, University Institute of Pharmaceutical Sciences, Chandigarh-160014, India 2 Professor in Pharmaceutical Chemistry, Panjab University, University Institute of Pharmaceutical Sciences, Chandigarh-160014, India E-mail: [email protected]

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