**4. Juxtacanalicular tissue**

The corneoscleral meshwork is separated from the endothelium of Schlemm´s canal by a thin tissue, the juxtacanalicular tissue [29].

The juxtacanalicular tissue is the outermost portion of the meshwork in contact with the inner wall of Schlemm`s canal. This tissue consists of a layer of connective tissue (types III, IV and V collagen, fibronectin) and ground substance (glycosaminoglycans and glycoproteins), and it is lined on either side by endothelium [31,32]. There is evidence that the juxtacanalicular tissue contains elastic fibers that provide support for Schlemm`s canal and that these fibers are attached to the tendons of the ciliary muscle.


Static Power Conversion

Chapter 4

Abstract

1. Introduction

55

Rectifiers

Mahmoud I. Masoud

Five-Phase Line Commutated

multiphase line commutated rectifiers and specifically five phase.

Keywords: five phase, multiphase generators, wind turbine, controlled and uncontrolled rectifiers, AC-DC converters, line commutated rectifiers

Nowadays, energy generation using renewable energy sources is considered as contemporary issue. Many countries are experiencing economic pressures because of the reduction in oil prices. Moreover, energy that is produced by burning fossil fuels is mainly responsible for the global air pollution problem, and consequently, it is not environmentally friendly. As a consequence, many countries are impelled to concentrate on renewable energy sources including generation, management, and planning. Renewable energy share, of the total energy consumption by the end of 2016, is 24.5% where it was 3.3% in 2010 [1]. This number is tremendously increasing each year. Accordingly, most research institutes foster research in the energy sector to go to clean and green energy generation. From renewable energy sources, wind energy is considered to be a pillar using appropriate generator. By the end of 2016, 29 countries have more than 1 GW in operation from wind energy, and the total capacity generated for the world is 487 GW. Only in 2016 a 23.4 GW is added due to wind energy generation [1]. Incontrovertibly, wind power becomes a big industry and a big topic for research. It includes mechanical parts and electrical parts. Accordingly, a generator that converts wind mechanical power to electrical power is a must. But, the power generated using wind turbines is not constant and could fluctuate. Also, the load profile fluctuates with respect to time. To accomplish

Multiphase systems including multiphase generators or motors, especially fivephase, offer improved performance compared to three-phase counterpart. Fivephase generators could generate power in applications such as, but not limited to, wind power generation, electric vehicles, aerospace, and oil and gas. The five-phase generator output requires converter system such as AC-DC converters. This chapter introduces the basic construction and performance analysis for uncontrolled/controlled five-phase line commutated rectifier guided by numerical examples. This rectifier is suitable for wind energy applications to be the intermediate stage between five-phase generator and DC load or inverter stage. The filtration for AC side and effects of source inductances are detailed in other references. Here, this chapter gives the reader a quick idea about the analysis and performance of

## Chapter 4


**2. Pathophysiology**

Despite increasing diagnosis and awareness of TTS, the exact mechanism remains unknown. The most accepted etiologic theory of Takotsubo syndrome is one of excess catecholaminergic-induced myocardial stunning. The association between stressful emotional states and development of TTS has been well-documented. Studies have measured the plasma concentration of catecholamines (dopamine, epinephrine, norepinephrine) present in patients with TTS, finding them higher (two to threefold) than both the general population and patients with ACS [2]. The mechanism by which supraphysiologic catecholaminergic state causes myocardial injury is a continued debate. Multivessel coronary artery spasm, cardiac microvascular dysfunction, and direct cardiomyocyte injury are proposed mechanisms [3].

Direct myocyte injury is thought to occur via beta-2 inhibitory effects of G-protein [guanine nucleotide-binding protein] coupled receptors resulting in negative

inotropy with resultant LV dysfunction. Normal circulating levels of catecholamines bind to Beta-2 adrenergic receptors of ventricular cardiomyocytes resulting in stimulation of Gs protein-adenylyl cyclase-protein kinase A pathway and thus a positive inotropic effect. The inhibitory effect happens with *supra*physiologic levels of catecholamines, though to take place to protect myocytes from apoptosis occurring with strong stimulation of Gs, a phenomenon known as 'stimulus trafficking' [4].

The gender disparity in disease presentation remains a point of ambiguity. Men have higher basal levels of sympathetic hormone than women, they produce higher amounts of catecholamines when presented with stressors and have a higher degree of catecholamine-mediated vasoconstriction. Yet, women seem to have a higher susceptibility to adrenergic myocardial stunning as exampled in LV dysfunction after subarachnoid hemorrhage. The cardioprotective effects of estrogen including protection against atherosclerosis and endothelial dysfunction is well known. There is the additional proposed benefit that estrogen downregulates beta-adrenergic receptors. Studies performed on ovariectomized rats subjected to stress demonstrated a higher deleterious cardiovascular response compared to groups provided with estrogen supplementation [5]. The overwhelming majority of patients with TTS are post-menopausal, further correlating that estrogen deficiency may predispose women to developing TTS. This may be the rationale behind the higher mortality of TTS seen in men. Men with TTS have an approximate mortality rate of 4.4%, comparable to the mortality rate of men with ST elevation myocardial infarction treated with primary percutaneous coronary intervention [6].

A hallmark feature of TTS is underlying inflammatory injury and edema of myocardial cells. In TTS, edema is generally diffuse and transmural in nature but worsened in areas with regional wall motion abnormalities and not limited to a vascular territory. Endomyocardial biopsies show mononuclear infiltrates, contraction-band necrosis, and myocardial inflammation-mediated edema.

Recent studies have demonstrated that elevated neutrophil/lymphocyte ratios, inflammatory markers, and even elevated neoplastic markers as a surrogate for systemic inflammation are independent risk factors for increased in-hospital complications, long-term adverse events, and death [7].


**6. Conclusions**

Recent commitments to sustainable development and mitigating climate changes made by most of the world's governments also found their way into the energy policy of the European Union, becoming the basis of its national energy efficiency targets for 2020 embedded in the Europe 2020 strategy. The Europe 2020 strategy and the 3 × 20 climate and energy package envisage the reduction of greenhouse gas emissions in EU Member States as well as increasing the share of renewable energy for enhancing energy efficiency. Due to the varying level of economic development, different objectives were set for different EU Member States, with the Czech Republic and Slovakia, which constitute the case studies employed in this paper, facing targets that were lower than that in the case of the more economically developed EU countries.

With regard to the above, one has to understand that climate targets were set in order to slow down or even reverse (albeit in the long run) the depletion of natural resources and preventing environmental degradation. In no way were these targets set with a purpose of halting the economic growth (especially when it comes to the economies in transition) but rather to help the countries in question to develop in accordance with the principles of energy efficiency and sustainable economic growth.

Both countries selected for our case study, the Czech Republic and Slovakia, experienced deep system transformation after the fall of Communism in 1989 that are apparent in the GDP, gross inland energy consumption and GHG emissions that constitute the measures of sustainable development used in our research. Our results indicate that it is quite unlikely that the planned increase in renewable energy is going to reach its targets for the Czech Republic and Slovakia (which is similar to the case of other EU Member States that joined after 2004) but it will be possible to reduce energy consumption and greenhouse gas emissions. This is, among other things, due to the fact that gross inland energy consumption and greenhouse gas emissions in the Czech Republic and Slovakia appear to be correlated. Greenhouse gas emissions are going down in both countries in question a little faster than energy consumption, but this pace is more rapid in the Czech Republic than in Slovakia.

In addition, it becomes apparent that implementation of GHG emissions in the Czech Republic and Slovakia may be at risk in case the proper energy policy is not maintained by the stakeholders and governments of the respective countries. Moreover, our findings show that regardless of the mix of fossil and renewable energy, the state of economic development and the geographical location of any EU Member States, a proper energy policy is required for e ffectively reducing energy consumption and greenhouse gas emissions. The energy intensity of Czech and Slovak economies increased in the early 2000s and then stabilized at a level about twice of the EU average. Our analysis of the energy intensity for the both countries in question shows that in the forthcoming years its value is likely to remain the same.

Overall, our results also demonstrate that maintaining a proper balance between economic development and environmental protection should be kept at all cost regardless of the position of the country. The cases of the Czech Republic and Slovakia scrutinized in this paper confirms that. Both countries have (common) Communist pasts but both underwent a spectacular economic transition and became Member States of the European Union. However, their story might be used by other EU Member States, both constituting the "core" EU and those that joined in 2004 or after. It might be also interesting to study the implications of Brexit and the shift of energy policies during and after the transition period for the United Kingdom. Further progress in maintaining a proper balance between economic development and environmental protection might be ensured by the decisive steps of the Czech and Slovak (as well as other EU) stakeholders and policymakers in terms of investments into renewable energy sources, modernizing the old energy sector and seeking for new solutions for sustainability and energy e fficiency.

**Author Contributions:** Conceptualization, J.B., W.S. and A.F.; methodology, J.B. and N.N.; validation, J.B. and A.F.; formal analysis, J.B. and W.S.; resources, N.N. and A.F.; writing—original draft preparation: J.B., W.S., A.F., and N.N.; project administration, W.S. and J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** The paper received no funding.

**Conflicts of Interest:** The authors declare no conflict of interest.


In contrast to OxR1 antagonists, fewer studies have assessed the effects of selective OxR2 antagonists on addiction-related behaviors. This is likely due to the more recent development of these compounds and a predicted increase in the likelihood of sedation (Zhou et al., 2011). Selective OxR2 antagonists include JNJ-10397049 (McAtee et al., 2004; Dugovic et al., 2009), EMPA (Malherbe et al., 2009a), and TCS-OX2-29 (Hirose et al., 2003), all of which have at least a 250-fold selectivity for OxR2 (Scammell and Winrow, 2011; Zhou et al., 2011). Studies exploring the effects of these OxR2 SORAs have reported less consistent effects on drug-motivated behaviors compared to the OxR1 SORA SB-334867. For example, Shoblock et al. (2010) showed that JNJ-10397049 dose-dependently deceased ethanol self-administration, as well as the acquisition, expression and reinstatement of ethanol CPP. In contrast, Brown et al. (2013) showed that TCS-OX2-29 attenuated ethanol self-administration but had no effect on cue-induced reinstatement of extinguished ethanol seeking. Interestingly, these authors identified the nucleus accumbens core (NACc) as an important site for OxR2 signaling, as infusions of TCS-OX2-29 into the NACc, but not shell, reduced ethanol self-administration. In contrast, OxR2 SORAs have been shown to have no effect on cocaine self-administration or reinstatement (Smith et al., 2009) or the expression of nicotine withdrawal symptoms (Plaza-Zabala et al., 2012). Thus, regarding SORAs, the case for OxR1-based therapies in treating addiction is better developed than for antagonists targeting OxR2.

With respect to DORAs, a large number of these compounds have been developed, prompted largely by their potential as a novel treatment for insomnia. Indeed, at least three pharmaceutical companies (GSK, MERCK, Actelion) have initiated clinical trials investigating the utility of DORAs in modulating the sleepwake cycle.  Only a limited number of studies have examined the effects of DORAs on addiction-related behaviors, with this work focusing almost exclusively on almorexant. For example, systemic and intra-VTA almorexant treatment was shown to attenuate ethanol self-administration (Srinivasan et al., 2012). Similarly, systemic almorexant blocked nicotine selfadministration behavior (LeSage et al., 2010). Interestingly, whilst almorexant attenuated the expression of CPP to high doses of cocaine and amphetamine, it had no effect on morphine CPP (Steiner et al., 2013c). This study also showed that almorexant reduced the expression of behavioral sensitization to morphine but not to cocaine or amphetamine (Steiner et al., 2013c).

#### **EFFECT OF SORAs AND DORAs ON FOOD AND NATURAL REWARD-SEEKING BEHAVIOR**

An important consideration with respect to the potential clinical application of orexin receptor antagonists is the effect of these compounds on other appetitive behaviors, including food seeking. Indeed, studies carried out immediately following the discovery of the orexin peptides firmly implicated orexin in feeding behavior, with intracerebroventricular (i.c.v.) infusions of orexin shown to increase food consumption (Sakurai et al., 1998) whereas systemic treatment with SB-334867 was found to block feeding behavior (Haynes et al., 2000).

Since these initial demonstrations, subsequent studies have sought to investigate whether doses of orexin receptor antagonists that are required to block drug seeking also affect feeding behavior. For example, Martin-Fardon and colleagues recently showed that systemic administration of SB-334867 (1–10 mg/kg, i.p) attenuated reinstatement of cocaine seeking, but not sweetenedcondensed milk seeking, elicited by discriminative cues (Martin-Fardon and Weiss, 2014). Similarly, Jupp et al. (2011) showed that systemic injections of SB-334867 (5 mg/kg) were sufficient to reduce responding for both ethanol and sucrose under an FR3 schedule of reinforcement. However, SB-334867 (5 mg/kg) attenuated responding for ethanol but not sucrose under a PR schedule. The authors suggested that the contribution of orexin A to motivation for alcohol is independent of non-specific effects on appetitive drive. Likewise, Hollander et al. (2012)showed that systemic injections of SB-334867 (2–4 mg/kg) reduced cocaine selfadministration, but had no effect on responding for food rewards under an FR5 schedule. Comparable effects have also been shown with doses of SB-334867 that attenuate responding for nicotine (LeSage et al., 2010). Further, we have previously reported that intra-VTA infusions of SB-334867, at doses that suppress cueinduced cocaine seeking, had no effect on reinstatement for a natural reward (sweetened condensed milk; James et al., 2012). With respect to OxR2 antagonists, Brown et al. (2013) showed that central infusions of 100µg TCS-OX2-29 reduced self-administration of ethanol, but had no effect on sucrose self-administration. In contrast however, LeSage et al. (2010) showed that systemic treatment with the DORA almorexant attenuated responding for both food pellets and nicotine. Similarly, systemic almorexant suppressed ethanol self-administration, and responding for sucrose (Srinivasan et al., 2012).

Taken together, these data indicate that a putative therapeutic window exists in which SORAs could be used to treat addiction-relevant behaviors, including 'relapse,' without producing 'off-target' effects on natural reward-seeking behavior. Conversely, it appears likely that the use of DORAs in the treatment of addiction may be associated with a risk of interfering with natural appetitive processes or promoting sedation. It is also important to acknowledge that there are very few studies that have assessed the effect of subchronic or chronic orexin receptor antagonism on drug or food-motivated behavior (discussed below).

#### **RECENT PROGRESS IMPLICATING THE OREXIN SYSTEM IN STRESS-RELATED NEUROENDOCRINE RESPONSES**

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is an important component of the adaptive response to stress (Dayas et al., 2001; Day and Walker, 2007; Ulrich-Lai and Herman, 2009). In this regard it is noteworthy that the neuroendocrine paraventricular nucleus (PVN), the apex of the HPA axis, contains both OxR1 and OxR2 and that both receptor sub-types are expressed in the anterior and intermediate lobe of the pituitary gland (Trivedi et al., 1998; Date et al., 2000). Consistent with this anatomical evidence, several reports have suggested that orexins can modulate the HPA axis. For example, i.c.v. injections of orexin have been shown to increase Fos-protein expression in PVN corticotropinreleasing factor (CRF) neurons (Sakamoto et al., 2004), provoke adrenocorticotropin-releasing hormone (ACTH) release from the anterior pituitary, and increase the release of corticosterone from the adrenal glands (Jászberényi et al., 2000; Kuru et al., 2000; Russell et al., 2001; Moreno et al., 2005).

Importantly, researchers using systemic administration of orexin receptor antagonists have reported less consistent effects on HPA axis activity as would have been predicted from studies assessing HPA axis activity after orexin peptide infusions. For example, systemic injections of the OxR1 antagonist GSK-1059865 did not alter corticosterone responses to the pharmacological stressor yohimbine in rats (Gozzi et al., 2011, 2013). Further, oral treatment with almorexant had no effect on basal, social interaction, novelty or restraint stress-induced corticosterone release (Steiner et al., 2013b). Systemic SB-334867 administration also did not attenuate withdrawal-induced increases in plasma corticosterone release (Laorden et al., 2012) despite reducing the physical symptoms associated with morphine withdrawal in Wistar rats.

These equivocal effects of orexin on HPA axis function are somewhat surprising given the abundance of orexin receptors in the PVN and pituitary (Trivedi et al., 1998). Interestingly, i.c.v. administration of TCS-OX2-29, a selective OxR2 antagonist, attenuated swim stress-induced increases in plasma ACTH release (Chang et al., 2007) and, intra-PVT infusions of SB-334867 attenuated the ACTH response to restraint but only following repeated swim stress (Heydendael et al., 2011). Thus, any role of orexin in HPA axis control might depend on prior stress exposure, the category of stressor e.g., physical vs. psychological, its intensity and duration, or whether in repeated stress experiments, homotypic or heterotypic stressors are applied.

Despite the above data, exposure to psychological and physical stressors increase surrogate indices of orexin system function. For example, increased Fos-protein expression is observed in orexin neurons following exposure to acute footshock (Harris and Aston-Jones, 2006), fear-associated contexts and novel environments (Furlong et al., 2009). Interestingly, similar effects are not observed following acute immobilization stress (Furlong et al., 2009), however this form of stress does increase orexin mRNA levels in the LH (Ida et al., 2000). An explanation for these contrasting findings remains to be determined, however, one interpretation is that only sufficiently intense or salient stimuli recruit the orexin system and that prior arousal state strongly influences the likelihood of orexin system recruitment. With respect to pharmacological stressors, increased Fos-expression is observed in orexin neurons following systemic injections of the anxiogenic drug FG-7142 (Johnson et al., 2012), caffeine (Johnson et al., 2012) and intravenous administration of sodium lactate (Johnson et al., 2010). Additionally, systemic administration of OxR1 antagonist GSK-1059865 has demonstrated functional inhibition in stress-relevant brain regions such as the NAC, dorsal thalamus, amygdala, and ventral hippocampus following the administration of yohimbine (Gozzi et al., 2013).

Acute stress also appears to have long lasting effects on orexin gene expression. For example, 2 weeks following a single session of footshock stress, increased prepro-orexin mRNA levels were observed in both the medial and lateral divisions of the hypothalamus (Chen et al., 2013). Elevated levels of orexin-A peptide in the cerebrospinal fluid (CSF) of Wistar rats have also been demonstrated following a short-term forced swimming paradigm (Martins et al., 2004). In addition, increased orexin mRNA was observed in rats immediately following morphine withdrawal (Zhou et al., 2006). Consistent with this Fos-activity mapping of orexin cell reactivity to stress, acute orexin peptide infusions evoke anxiety-like behavior. Specifically, i.c.v. administration of orexin-A produced anxiogenic-like effects i.e., increased the time spent in the closed arms of the elevated plus maze and the time spent in the dark compartment of the light-dark test (Suzuki et al., 2005). Orexins have also been shown to modulate the activity of extrahypothalamic CRF systems with i.c.v. injections of orexin-A found to increase the percentage of CRF cells that express Fos in the central amygdaloid nucleus (Sakamoto et al., 2004). Together, this evidence forms the basis for the hypothesis that manipulation of the orexin system using SORAs and DORAs will likely also impact on stress–induced anxiety-like behavior.

#### **EFFECT OF SORAs AND DORAs ON ACUTE BEHAVIORAL STRESS REACTIVITY AND ANXIETY-LIKE BEHAVIOR**

Several recent preclinical studies indicate that SORAs and DORAs have a limited effect on basal/non-stress evoked behavioral responses including anxiety-like behavior. For example, systemic treatment with SB-334867 in both rats and mice had no effect on activity in the elevated plus maze and social interaction task, two common tests of anxiety-like behavior (Johnson et al., 2010; Rodgers et al., 2013). However, intra-PVT SB-334867 infusions resulted in decreased anxiety-like behavior in the elevated plus maze (Heydendael et al., 2011). This could indicate an important role for orexin signaling in the PVT in regulating basal arousal or anxiety state.

Importantly, SORAs reliably attenuated anxiety-like behavior evoked by an acute psychological or physical stressor. For example, systemic SB-334867 administration reduced the expression of anxiety-like behavior induced by acute nicotine (Plaza-Zabala et al., 2010) and sodium lactate injections (Johnson et al., 2010) as well as cat odor (Staples and Cornish, 2014). Administration of SORAs also reduced physiological responses such as increased heart rate and body temperature produced by exposure to acute i.p injection stress (Rusyniak et al., 2012). With regards to OxR2 antagonists, intra-PVT infusions of TCS-OX2-29 reduce anxietylike behavior elicited by footshock stress (Li et al., 2010) suggesting a role for orexin signaling through the OxR2 in stress-relevant behaviors.

There are also several recent animal studies that have explored the role for the orexin system in anxiety-like behavioral responses to conditioned cues that predict the presentation of a fearful stimulus, such as footshock. This approach has been used to explore brain mechanisms responsible for generating panic disorder and post-traumatic stress disorder in humans (Grillon, 2008). In male Sprague-Dawley rats, Sears et al. (2013) administered SB-334867 i.c.v. before fear conditioning and demonstrated an impairment in conditioned stimulus (CS)-induced freezing behavior 24 h after CS/unconditioned stimulus (US) presentation. Further, systemic injections of TCS-1102 decreased conditioned and generalized fear and anxiety-like behavior in an open field in rats previously exposed to footshock stress (Chen et al., 2013). Moreover, OxR1 knockout mice demonstrated a reduction in freezing behavior in both cued and contextual fear conditioning. Interestingly, OxR2 knockout mice also showed reduced freezing behavior to contextual but not conditioned fear (Soya et al., 2013). Consistent with a role for both receptor subtypes in aspects of conditioned fear responses, Steiner et al. (2012) reported that oral administration of almorexant reduced fearpotentiated startle in response to a CS but found no change in elevated plus maze behavior. Interestingly, human genetic studies have linked a polymorphism in Orx-2 receptor gene with panic disorder (Annerbrink et al., 2011).

Together these data indicate that exposure to acute stress and the expression of anxiety-like behavior is generally associated with increases in orexin system activity. Further, orexin antagonists appear to be potential candidates for the suppression of anxietylike behavior and maladaptive stress-reactivity. Importantly, as is outlined below, recent evidence suggests that chronic suppression of the orexin system may be associated with the development of depression-like symptomology. These findings have significant implications for the potential use of orexin antagonists in the treatment of neuropsychiatric disorders.

#### **EFFECT OF CHRONIC STRESS ON THE OREXIN SYSTEM AND IMPLICATIONS FOR DEPRESSION**

In recent human studies, a polymorphism in Orx-1 receptor gene has been associated with major mood disorders and increased orexin peptide levels were correlated with positive emotions and social interaction (Rainero et al., 2011; Blouin et al., 2013). Non-genetic factors, such as chronic stress, have also implicated orexin in the etiology of depression (Katz et al., 1981; Kendler et al., 1999; Charney and Manji, 2004; Russo and Nestler, 2013). This link has been studied in animal models typically involving extended periods of psychological stress exposure (Lutter et al., 2008). Increasing evidence from these types of studies links chronic stress exposure to a downregulation of orexin system activity. For example, mice exposed to the well characterized social defeat model of chronic stress, which evokes symptoms thought to mimic depression in humans, display reduced orexin mRNA expression, lowered orexin cell number, and diminished levels of orexin A and orexin B peptide (Lutter et al., 2008; Nocjar et al., 2012). Similarly, Wistar-Kyoto (WKY) rats, which exhibit a depressive-like behavioral phenotype, express lower numbers of orexin neurons and a smaller orexin soma size compared to Wistar rats (Allard et al., 2004). The WKY rat strain also exhibits reduced prepro-orexin mRNA levels and orexin A immunoreactivity is reduced in the hypothalamus, thalamus, septum and amygdala (Taheri et al., 2001).

Early life stress (ELS) is a known risk factor for the development of stress-related mood disorders in adulthood (Graham et al., 1999). Surprisingly though, work has shown that ELS in fact increases frontal cortical OxR1 and hypothalamic orexin A levels in adulthood (Feng et al., 2007). Given these results, we recently investigated the effects of ELS on the reactivity of the orexin system to a second psychological stressor in adulthood. Both male and female rats exposed to neonatal maternal separation displayed a hypo-active orexin cell response to stress in adulthood. These animals also displayed reduced open field behavior but notably no overt anxiety-like behavior was observed on an elevated plus maze. Our interpretation of these data is that the ELS procedures evoked behaviors more akin to a depression-like profile after stress however further behavioral tests will be necessary to further explore this hypothesis (Campbell et al., 2013).

What effect a second stressor, such as restraint in adulthood, might have on LH-orexin circuits is unknown. This would seem important given the behavioral effects of this "two-hit" paradigm outlined above (Campbell et al., 2013). It is possible that increased drive to this system reflects an attempt to enhance orexin activity and prevent the expression of depression-like behavior. It may be that in response to a significant subsequent stressor, the reduced functional integrity at a synaptic and molecular level is unmasked, which may then develop into a depressive-like state. Regardless, taken together with the Fos data outlined above, our findings suggest that ELS can have long-term impacts on the normal functioning of orexin cells.

Consistent with a process where ELS can induce orexin cell dysfunction and depression-like behaviors, clinical evidence supports the possibility that decreased orexin signaling might promote depression-like behaviors. For example, reduced concentrations of orexin A were reported from CSF samples of adults with major depressive disorder and chronic, combat-related posttraumatic stress disorder (Brundin et al., 2007a,b, 2009; Strawn et al., 2010). Additionally, reduced orexin A mRNA has been correlated with increased scores on the Hamilton rating scale for depression (Rotter et al., 2011). Interestingly, infusions of orexin peptides into the ventricles of rats have been shown to have antidepressant-like effects. For example, i.c.v. administration of orexin reduces the duration of immobility in the forced swim test, and this effect is blocked by the administration of the OxR1 antagonist, SB-334867 (Ito et al., 2008). Thus, it is possible that in some forms of depression, increasing orexin system signaling may have therapeutic benefit. In line with this interpretation, we recently found that a period of exercise in adolescence prevented reductions in orexin system function and the expression of stress-related behavior in rats exposed to ELS (Campbell et al., 2013).

Despite the above work, it is important to acknowledge that there are both preclinical animal and human data that do not support a link between a hypoactive orexin system and depression. For example, Mikrouli et al. (2011) demonstrated that the Flinders Sensitive Line, considered a genetic rat model of depression, displayed elevated, not depressed, levels of orexin neurons compared to controls. Further, Nollet et al. (2011) demonstrated that mice subjected to unpredictable chronic mild stress displayed increased depressive-like behavior following the tail suspension test but presented with elevated orexin neuron activity in the DMH and PFA subregions of the hypothalamus. Interestingly, these authors were able to reverse this elevation in orexin cell activity with 6 weeks of fluoxetine treatment. Similarly, exposure to unpredictable chronic mild stress produced depressivelike behaviors in the tail suspension test, elevated plus maze and resident-intruder task; and 7 weeks exposure to the DORA almorexant produced an antidepressant-like behavioral effect in these tasks (Nollet et al., 2012). And, in a recent study, OxR1 mRNA expression in the amygdala was reported to be positively correlated with increased depressive-like behavior in the forced swim test (Arendt et al., 2013) - however it is possible that this effect might be caused by downregulated orexin system function. Finally, a recent study reported that decreased depressive-like behavior is observed in OxR1 knockout mice, whereas OxR2 knockout mice exhibit *increased* depressive-related behavior, possibly pointing to a differential role for OxR1 vs. OxR2 in the regulation of these behaviors (Scott et al., 2011). These authors highlight the fact that behavioral pharmacology studies typically use the non-selective OxR agonist orexin-A, along with SORAs at doses that are potentially non-selective *in vivo*, therefore making it difficult to differentiate roles for OxR1 vs. OxR2 in the regulation of depression-like behavior.

In human studies, Salomon et al. (2003)reported that orexin A CSF levels were higher in depressed patients and treatment with sertraline, an antidepressant drug, resulted in an attenuation of CSF orexin levels. Furthermore, a positive correlation between orexin plasma concentrations, depressive symptoms and global distress indices on the brief symptom inventory is seen following alcohol withdrawal (von der Goltz et al., 2011). Finally, Schmidt et al. (2011) failed to find any association between CSF orexin A levels and depression.

Taken together, these results suggest that acute stress may activate the orexin system in order to enhance an animal's ability to cope or adapt appropriately to a potential threat (**Figure 1C**). If these stressors persist, chronic or repeated exposure to stress may downregulate orexin system function (**Figure 1D**). Hypoactivity of the orexin system may impair an animal's ability to adapt to stress and lead to the expression of depressive-like behavior. Data from human and animal studies not supporting this link may reflect the heterogeneous nature of depression—i.e., depression presenting with and without anxiety or anxiety presenting with or without depression. Supporting this conclusion, Johnson et al. (2010) found that patients exhibiting panic anxiety displayed increased CSF orexin levels compared to patients exhibiting panic anxiety with comorbid major depressive disorder. It will be important for human and animal studies exploring the link between the orexin system dysfunction and neuropsychiatric conditions to consider the heterogeneous nature of these conditions.

#### **POTENTIAL PITFALLS FOR APPROACHES TO TREAT NEUROPSYCHIATRIC DISORDERS USING OREXIN RECEPTOR ANTAGONISTS**

As outlined above, significant progress has been made in our understanding of the contribution of the orexin system to normal and "pathological" behavior. In the case of addiction, there seems sufficient evidence to conclude that the orexin system is important for drug-seeking behavior, particularly for relapselike behavior provoked by drug-cues and stress but not by drug itself. Further, several studies indicate that orexin antagonists, and in particular selective OxR1 antagonists, can reduce drugseeking at doses that have minimal effects on natural rewardseeking behavior (Jupp et al., 2011; Hollander et al., 2012; James et al., 2012; Brown et al., 2013). Comparison of orexin's role in drug taking vs. seeking behavior also highlights that orexin receptor antagonists have effects on rewarded self-administration, but importantly these effects generally emerge only under high effort schedules of reinforcement. These data combine to produce a compelling case that orexin receptors represent promising targets for the treatment of addiction. This is particularly true given the long-established interaction between addiction and maladaptation of natural reward seeking brain pathways. For example, increased excitatory drive to the orexin system is thought to heighten orexin signaling in key reward-seeking regions such as the VTA and may contribute to the persistent plasticity within dopamine neurons seen after long-term cocaine self-administration and increased relapse vulnerability (Chen et al., 2008; James et al., 2012)

It is important given the clinical promise of SORAs and DORAs for neuropsychiatric disorders that studies continue to assess the potential for off-target effects, tolerance to prolonged orexin receptor blockade and differential or counter-regulatory effects on OxR1 vs. 2, as well as any possible compensatory adaptations that may occur in other hypothalamic neuropeptide systems in response to chronic orexin receptor blockade. Studies to date indicate a limited profile of chronic SORA and DORA tolerance. For example, Steiner et al. (2013c) showed that 12 days of chronic almorexant treatment had no effect on the maintenance of CPP or locomotor sensitization in animals exposed to cocaine, morphine or amphetamine. However, as mentioned above, Nollet et al. (2012) exposed mice to chronic almorexant treatment (7 weeks), which produced an antidepressant-like effect in the tail suspension test, elevated plus maze and residentintruder task following exposure to unpredictable chronic mild stress. Interestingly, chronic treatment with the OxR1 antagonist ACT-335827 (4 weeks) did not alter total energy intake in cafeteria diet fed rats compared to controls (Steiner et al., 2013b). In contrast, chronic SB-334867 treatment (14 days) had anti-obesity effects in a model of genetically obese mice by reducing food intake and body weight gain over the 14 day period (Haynes et al., 2002).

In one of the few examples where repeated doses of an orexin antagonist have been studied for addiction and relapse prevention, Zhou and colleagues demonstrated that chronic SB-334867 exposure resulted in a complex pattern of effects (Zhou et al., 2011). Specifically, they found that repeated SB-334867 exposure prior to extinction sessions resulted in reduced cocaineseeking behavior in rats during extinction; however, repeated SB-334867 treatment during extinction increased cue-induced reinstatement and had no effect on cocaine-primed reinstatement. Importantly, McNally and colleagues have also shown that cocaine and alcohol-seeking do not necessarily evoke a specific drug context-related activation of the orexin system, rather, recruitment of this pathway is necessary but not sufficient for drug-seeking behavior (Hamlin et al., 2007, 2008). It is also interesting to note that the OxR1 antagonist, SB-334867, has been shown to reduce cue-induced reinstatement of cocaine seeking behavior in male rodents yet no effect was seen in female rats (Zhou et al., 2012). Future work should focus on differentiating arousal vs. reward related function of the orexin neurons in the context of addiction. Sex-specific effects of stress and drug exposure on orexin circuitry also warrant further scrutiny.

With respect to the potential use of orexin receptor antagonists for anxiety and depression, recently, Johnson et al. (2010, 2012) proposed the use of OxR1 antagonists in the treatment of panic disorder. Certainly, the available data appears to support an important role for orexin signaling in ameliorating anxietylike states in animal models. Studies employing chronic stress paradigms, however, suggests that a more complicated picture exists and that the effects of repeated SORA or DORA treatment may be unpredictable (Zhou et al., 2012). Emerging data indicates that ELS and chronic stress can downregulate the activity of the orexin system in response to chronic stress (Lutter et al., 2008; Nocjar et al., 2012; Campbell et al., 2013). There is also a developing clinical literature indicating that depression may be associated with decreased orexin system function (Brundin et al., 2007a,b, 2009). These studies raise potential concerns for the long-term use of orexin antagonists in the treatment of addiction and anxiety disorders, as long-term suppression of the orexin system may precipitate depressive-like symptoms. Similarly, the emergence of anxiety or depression under conditions of augmented orexin system function will need to be carefully considered. Therefore, a greater understanding of the changes to orexin receptor expression in relevant brain regions will be necessary to confidently predict the potential outcomes of therapeutic manipulation of orexin signaling under these conditions. Further we propose that a thorough evaluation of chronic and subchronic orexin receptor antagonism in preclinical animal models of anxiety and depression is necessary. Together, such approaches may be able to identify therapeutic dosing regimens with preferential effects on the different aspects for the orexin system that influence mood, as well as drug- and natural-reward behaviors. Finally, the evidence that drugs of abuse or stress can rewire inputs onto orexin neurons indicates that targeting these changes within the LH might offer an alternative strategy. This would negate the mixed downstream effects of SORAs and DORAs in mood, addiction and reward by reducing aberrant orexin cell activity at the site of dysfunction, rather than simply masking the downstream effects using antagonists. For example, our group is currently investigating the changes in LH-orexin circuits responsible for the increased excitatory drive observed after cocaine or ELS.

In conclusion, significant progress has been made toward an understanding of the role of the orexin system in normal and pathological behaviors. Unsurprisingly, given the widespread projections of the orexin system, the role for these neurons crosses many domains including basic physiological responses to more complex functions. Our view is that only a comprehensive dissection of the changes in LH-circuit function, both within the hypothalamus and in target regions of these neurons, will reveal appropriate therapeutic avenues to augment or suppress dysregulated orexin function in neuropsychiatric and neurological disease states.

#### **REFERENCES**

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and 2 receptor antagonist, in humans. *Drug Metab. Dispos.* 39, 215–227. doi: 10.1124/dmd.110.035386

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 November 2013; accepted: 10 February 2014; published online: 25 February 2014.*

*Citation: Yeoh JW, Campbell EJ, James MH, Graham BA and Dayas CV (2014) Orexin antagonists for neuropsychiatric disease: progress and potential pitfalls. Front. Neurosci. 8:36. doi: 10.3389/fnins.2014.00036*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Yeoh, Campbell, James, Graham and Dayas. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Orexin 1 receptor antagonists in compulsive behavior and anxiety: possible therapeutic use

#### *Emilio Merlo Pich1 \* and Sergio Melotto2*

*<sup>1</sup> Neuroscience DTA, F. Hoffman – La Roche, Basel, Switzerland <sup>2</sup> Legnago, Italy*

#### *Edited by:*

*Michel A. Steiner, Actelion Pharmaceuticals Ltd., Switzerland*

#### *Reviewed by:*

*G. Koob, The Scripps Research Institute, USA Jonathan Hollander, The Scripps Research Institute, USA William A. Truitt, Indiana University School of Medicine, USA*

#### *\*Correspondence:*

*Emilio Merlo Pich, Neuroscience DTA, F. Hoffman – La Roche, Granzacherstrasse 126, Basel 4070 Switzerland e-mail: emilio.merlo\_pich@ roche.com<EMAIL_ADDRESS>
Fifteen years after the discovery of hypocretin/orexin a large body of evidence has been collected supporting its critical role in the modulation of several regulatory physiological functions. While reduced levels of hypocretin/orexin were initially associated with narcolepsy, increased levels have been linked in recent years to pathological states of hypervigilance and, in particular, to insomnia. The filing to FDA of the dual-activity orexin receptor antagonist (DORA) suvorexant for the indication of insomnia further corroborates the robustness of such evidences. However, as excessive vigilance is also typical of anxiety and panic episodes, as well as of abstinence and craving in substance misuse disorders. In this review we briefly discuss the evidence supporting the development of hypocretin/orexin receptor 1 (OX1) antagonists for these indications. Experiments using the OX1 antagonist SB-334867 and mutant mice have involved the OX1 receptor in mediating the compulsive reinstatement of drug seeking for ethanol, nicotine, cocaine, cannabinoids and morphine. More recently, data have been generated with the novel selective OX1 antagonists GSK1059865 and ACT-335827 on behavioral and cardiovascular response to stressors and panic-inducing agents in animals. Concluding, while waiting for pharmacologic data to become available in humans, risks and benefits for the development of an OX1 receptor antagonist for Binge Eating and Anxiety Disorders are discussed.

**Keywords: drug addiction, relapse, binge eating, repetitive behavior, emotion, OX1 receptor antagonist, GSK1059865**

#### **INTRODUCTION**

Hypocretin/orexin is an hypothalamic neuropeptide consisting of two forms, A and B, containing 33 and 28 amino acids respectively, and binding to two G-protein coupled receptors, OX1 (or hcrt-1) and OX2 (or hcrt-2) (de Lecea et al., 1998; Sakurai et al., 1998).

The hypocretin/orexin peptide is produced in a small population of neurons located in the dorsomedial—perifornical hypothalamic area (DMH/PeF) and in the lateral hypothalamic nucleus (LH). Positive nerve fibers and terminals can be found in several areas of the central and peripheral nervous system, including vagus nerve, spinal cord, brainstem, hypothalamus, thalamus, limbic system, and some cortical regions (Peyron et al., 1998; Heinonen et al., 2008). OX/hcrt receptors show a similarly extended distribution (Marcus et al., 2001), suggesting a potential relevant role of hypocretins/orexins as regulatory peptides for several adaptive and limbic system-controlled functions (Johnson et al., 2012a,b; Mahler et al., 2012; Boutrel et al., 2013; Tsujino and Sakurai, 2013).

The OX1 receptor contains 425 amino acids while the OX2 receptor contains 444 amino acids with a sequence similarity between the two receptors of 68% (Sakurai et al., 1998). While both receptors are coupled with Gq-protein, only the OX1 receptor is additionally coupled with Gs-protein. Receptor activation increases intracellular calcium levels via a Gq –dependent phospholipase C (PLC)-mediated increase of inositol-1,4,5-triphosphate (Lund et al., 2000) and diacylglycerol, resulting in the activation of the δ form of the protein kinase C, eventually engaging the ERK phosphorylation pathway (Ekholm et al., 2007).

OX1 and OX2 receptors are differentially distributed throughout the mammalian brain (Marcus et al., 2001), matching the distribution of hypocretin/orexin terminals. Evidence for a functional segregation of the two receptors was recently obtained using pharmacological magnetic resonance imaging (phMRI) in rats. The activating effects of amphetamine were differentially attenuated by pre-treatment with the selective OX2 receptor antagonist JNJ-1037049 or with the novel OX1 receptor antagonist GSK1059865: JNJ-1037049 attenuated the amphetamine effects in frontal cortex and thalamus, areas involved in arousal, while GSK1059865 reduced the activation in the extended amygdala, BNST and ventral striatum; all brain areas involved in stress and motivation (Gozzi et al., 2011). These differences in functional maps, together with differences in behavioral profiles support a rationale for exploring different indications for novel therapeutics that selectively target either OX1 or OX2 receptors. However, while the role of the OX2 receptor in sleep and arousal is strongly supported by the available experimental evidence (Gatfield et al., 2010), the therapeutic potential of selective antagonism of the OX1 receptor is still under evaluation (Gotter et al., 2012).

The better understanding of the biology of the hypocretin/orexin system has promoted drug discovery programs in several pharmaceutical companies, resulting in a series of patents and compounds with different selectivity and *in vitro* characteristics (Faedo et al., 2012; Lebold et al., 2013). As shown above, some compounds were used as pharmacologic tools to explore OX1- and OX2-dependent neurotransmission *in vivo*. Few compounds were successfully progressed in humans, in particular the dual OX1-OX2 receptor antagonist (DORA) almorexant (Hoever et al., 2012), SB-649868 (Bettica et al., 2012), and suvorexant (Herring et al., 2012). Only suvorexant went successfully through Phase 3 development and it was filed in USA as new treatment for insomnia in 2013.

The first pharmacological tool used as OX1 receptor antagonist was SB-334867 (Jones et al., 2001; Smart et al., 2001). Recently, other compounds have been proposed: GSK1059865 (Alvaro et al., 2009; Gozzi et al., 2011), 2,5 di-substituted piperidines (Jiang et al., 2012) and ACT-335827 (Steiner et al., 2013).

In this review we address the evidence, mostly collected with pharmacologic tools, for a preferential role of the OX1-mediated neurotransmission in compulsive behavior, particularly in relation to addiction and binge eating, and in anxiety.

#### **HYPOCRETIN/OREXIN AND THE OX1 RECEPTOR IN DRUG ADDICTION-LIKE AND COMPULSIVE EATING BEHAVIORS**

Several preclinical findings indicated the involvement of the hypocretin/orexin system in compulsive and repetitive behavior as well as in the control of goal-oriented behavior. Recent excellent reviews summaries the evidence collected in more than hundred articles indicting that the hypocretin/orexin system in the lateral hypothalamus (Harris et al., 2005) is involved in the behavioral addiction-like dysregulations associated with exposure to cocaine, amphetamine, morphine, heroin, nicotine, ethanol and cannabinoids in rodents (Espana et al., 2011; Mahler et al., 2012; Boutrel et al., 2013; Flores et al., 2013), as well as in the excessive intake of palatable food associated with binge eating (Tsujino and Sakurai, 2013).

Data supporting the hypocretin/orexin involvement in the effects of addictive drugs was initially obtained in mice carrying a null mutation (KO) of the hypocretin/orexin peptide, showing reduced signs of withdrawal from morphine (Georgescu et al., 2003). Subsequently, impaired conditioned place preference for morphine (Narita et al., 2006) and for nicotine (Plaza-Zabala et al., 2012) was demonstrated in rodents. More recently, studies in KO mice with deletion of the OX1 receptor showed reduced cocaine and cannabinoid self-administration and the blockade of reinstatement of drug taking after abstinence (Hollander et al., 2012; Flores et al., 2013), indicating a critical role for OX1 receptors in mediating reinstatement of drug seeking.

In rodents SB-334867, a preferential OX1 receptor antagonist, reduced sensitization, drug seeking behavior and withdrawal syndrome in rodents exposed to ethanol, nicotine, morphine, and cocaine. These and other findings were extensively described in recent reviews (Mahler et al., 2012; Boutrel et al., 2013). Of particular interest is the fact that SB-334867 consistently attenuated the compulsive behavior associated with the reinstatement of drug seeking, induced by either acute stress or cues associated previously with drug taking, a phenomenon observed for ethanol, nicotine, cocaine, cannabinoids and morphine.

Recently, the highly selective OX1 receptor antagonist GSK 1059865 (5-bromo-N-[(2S,5S)-1-(3-fluoro-2-methoxybenzoyl)- 5-methylpiperidin-2-yl]methyl-pyridin-2-amine) was characterized within the GSK collection (Alvaro et al., 2009). GSK1059865 at the dose of 25 mg/kg i.p. (estimated to fully occupy the OX1 receptors in the brain of the rat) only marginally modified the physiological sleep of rats, indicating a weak hypnotic effect (Gozzi et al., 2011; Piccoli et al., 2012) and confirming difference vs. OX2 receptor blockade (Mieda et al., 2011). Conversely, at 10 and 30 mg/kg i.p. doses, GSK1059865 significantly antagonized the cocaine effect in a conditioned place-preference paradigm (Gozzi et al., 2011). These results are in line with the proposed role of selective OX1 receptor antagonism in preventing relapse to drug seeking but not inducing sleep.

OX1 receptors were also recently involved in mediating the binge episodes of compulsive eating (Avena and Bocarsly, 2012), also defined as "food addiction," another compulsive behavior increasingly common among obese individuals (Volkow and Wise, 2005; Pedram et al., 2013). Although it was initially shown that the acute central administration of orexin-A stimulates feeding behavior by acting on specific hypothalamic circuits (Friederich et al., 2013), hypocretin/orexin-induced food intake appears to be influenced by several other factors, including the palatability of the food, the energy balance, the level of arousal and the emotional status (Yamanaka et al., 2003; Zheng et al., 2007; Choi et al., 2010; Tsujino and Sakurai, 2013). This suggests that the hypocretin/orexin system can activate more complex behavioral patterns than the sole increase of food intake (Mahler et al., 2012). Indeed, recent studies indicate a possible involvement of hypocretin/orexin dysregulation in compulsive intake of palatable food (Smith and Robbins, 2013).

Compulsive eating can be elicited in rodents by alternating periods of regular access to food with periods of food restriction over a few weeks, a specific chronic stressful condition that can produce binge episodes when a large amount of palatable food becomes suddenly available. The model was pharmacologically validated in rats, showing an inhibitory effect of topiramate on the compulsive food intake (Cifani et al., 2009) similar to that observed in human binge eaters (McElroy et al., 2003). Although data regarding the involvement of the hypocretin/orexin pathway in this experimental procedure of binge eating were not available, we studied the effect of GSK1059865 as a tool to assess the relevance of the OX1 receptors (Piccoli et al., 2012). Interestingly, GSK1059865, at the doses of 10 and 30 mg/kg, was not able to inhibit highly palatable food intake in control animals (not exposed to cyclic food restriction), confirming the minor effect of OX1 receptor blockade on natural reward when it occurs under physiological conditions. On the contrary, GSK1059865 potently inhibited the compulsive eating behavior in rats exposed to chronic stress/food restriction (Piccoli et al., 2012). Interestingly, regular food intake was also inhibited by the OX1 antagonist SB-334867 in rats genetically prone to obesity but not in control rats (White et al., 2005). These findings confirmed the role of the OX1 receptor-mediated transmission in attenuating the excessive drive produced by craving associated to distress that was also observed with addictive drugs.

Interestingly binge eating was also inhibited by the DORA SB-649868, but not by the selective OX2 receptor antagonist JNJ-10397049, suggesting that the effects of SB-649868 are probably due to the OX1 component of its mechanism of action (Piccoli et al., 2012). Intriguingly, the lack of effect of almorexant in animals exposed only to acute stress suggested that the procedure of alternating periods of food restriction is critical for the engagement of the hypocretin/orexin system in promoting compulsive eating of highly palatable food (Funabashi et al., 2009; Pankevich et al., 2010). This observation suggests that in this paradigm DORA and OX1 antagonists do not work primarily via an antistress effect. This is not surprising, given the complex role of hypocretin/orexin in the maintenance of energy balance and the sensitivity of hypocretin/orexin neurons to directly respond to the changes of circulating glucose levels and to endocrine signals (Tsujino and Sakurai, 2013).

Overall, the results obtained with GSK1059865 confirm that selective OX1 receptor antagonism is not directly affecting the reward pathways involved in hedonic eating, but rather support a role in the compulsive aspect of food intake, those that probably account for the development and persistence of abnormal eating behaviors in binge eaters and, possibly, in bulimic patients. In addition, these data highlight the need to re-evaluate the putative OX1 receptor antagonism profile, so far based mostly on SB-334867 (Haynes et al., 2000), a compound whose selectivity at high dose and stability have been under discussion (Hollander et al., 2012; McElhinny et al., 2012).

To date a limited number of human biomarker studies support a role for the hypocretin/orexin system in the behavioral dysregulation that characterizes addiction, and none of them used pharmacologic agents. Changes of hypocretin/orexin levels in blood were observed in alcoholics during alcohol withdrawal, showing positive association with distress scores (von der Goltz et al., 2011), while negative association was observed with craving scores in abstinent smokers (von der Goltze et al., 2010). Increased expression of hypocretin/orexin levels was also found in the in peripheral blood of cigarette smokers and cannabis abusers (Rotter et al., 2012). While the interpretation of these finding is still unclear, subjects affected by narcolepsy were studied for their liability to addiction with the hope to get more informative results. Accordingly, narcolepsy is commonly associated with mutations in the hypocretin/orexin gene (Peyron et al., 1998), resulting in a constitutive deficiency of the peptide, similar to that obtained in hypocretin/orexin KO mice. Subtle differences in reward processing and risk-taking behavior were reported in narcoleptic subjects, but the prevalence of tobacco smoking in these patients was not different from that of the normal population (Bayard and Dauvilliers, 2013). There results can be seen at variance with evidence of reduced effects of addictive drugs in hypocretin/orexin KO mice (for review see Mahler et al., 2012; Boutrel et al., 2013). Interestingly, narcoleptic subjects declared using cigarette smoking and nicotine patches as self-medication to reduce sleepiness and increase arousal (Ebben and Krieger, 2012). Altogether, the findings indicate complexity in the relationship between addictive behaviors and dysfunctional hypocretin/orexin system in humans, supporting the need of additional, more focused translational studies.

#### **HYPOCRETIN/OREXIN AND THE OX1 RECEPTOR IN ANXIETY**

Textbooks of physiology describe the posterior and perifornical regions of the hypothalamus as the part of the limbic circuit that controls "fight-or-flight" reactions in response to an imminent threat (Hess and Akert, 1955). As mentioned above, neurons that produce hypocretin/oirexin are located in the perifornical region (Peyron et al., 1998) and project to the majority of the limbic brain structures involved in the fear, stress and anxiety circuit (Shin and Liberzon, 2009), suggesting a possible role of hypocretin/orexin in controlling not only wakefulness and arousal, but also fear, anxiety, and stress responses (Johnson et al., 2012a; Sears et al., 2013).

This hypothesis was explored over the years and summarized in recent articles and reviews (Bisetti et al., 2006; Mathew et al., 2008; Johnson et al., 2010, 2012a). In these contributions interested readers can find the evidence supporting the role of hypocretin/orexin neurons in orchestrating the autonomic, respiratory, cardiovascular and behavioral responses to stressful and panic-inducing stimuli.

The working hypothesis implies that stressful stimuli (or high endogenous levels of anxiogenic mediators) would increase the activity on hypocretin/orexin neurons, which in turn will release more hypocretin/orexin in their terminal fields located in brain limbic regions that regulate emotion and stress response. Then hypocretin/orexin will be shifting the level of activation of the fear, stress and anxiety circuit toward a higher level of arousal, which includes vegetative, endocrine and behavioral phenomena typical of anxiety and panic states. An abnormal persistence of excessive release of hypocretin/orexin could be seen as a critical factor in the maintenance of high arousal and anxiety, as well as a liability to relapse into panic episodes in predisposed individuals, suggesting a potential critical pathophysiological role for anxiety.

Data in humans showed an increased release of extracellular hypocretin/orexin driven by emotional stimuli in the amygdala of subjects suffering from treatment-resistant temporal lobe epilepsy that were implanted with microdialysis probes (Blouin et al., 2013). In this study the levels of hypocretin/orexin increased during wake and decreased during sleep, but the highest peaks were observed during acute emotional activations of both positive and negative valence. The amygdala is considered a key structure for processing salience and negative emotion, which is associated with pathologic anxiety. In rodents microinjection of hypocretin/orexin into the amygdala increases anxiety-like behavior (Avolio et al., 2011). Interestingly, abnormal high levels of hypocretin/orexin were found in the cerebro-spinal fluid (CSF) of patients with Panic Anxiety Disorders, suggesting a possible hyperactivity state (Johnson et al., 2010). In another study increased levels of hypocretin/orexin were measured in the peripheral blood of subjects with chronic obstructive pulmonary disorder (COPD), a condition associated with hypercapnia, acidosis and a 10-fold increased risk of panic attacks (Zhu et al., 2011).

Converging findings suggest that the anxiogenic properties of hypocretin/orexin are primarily mediated by the engagement of OX1 receptors. In rodents the autonomic and behavioral responses to stress were attenuated by pre-treatments with OX1 receptor antagonists, such as SB-334867 (Johnson et al., 2010, 2012b), GSK 1034865 (Gozzi et al., 2011) and ACT-335827 (Steiner et al., 2013), or with a DORA, such as almorexant (Steiner et al., 2012). Significant attenuation of anxiety-like responses was observed in paradigms including fear conditioning (Sears et al., 2013; Steiner et al., 2013), panicogenic lactate infusion (Johnson et al., 2010), hypercapnia (Li et al., 2010; Johnson et al., 2012b), administration of FG-7142 (Johnson et al., 2012a), high dose nicotine (Plaza-Zabala et al., 2010), and yohimbine (Richards et al., 2008). Recent evidence using OX1 and OX2 KO mice showed a critical role of OX1 receptors located in the locus coeruleus in mediating cued fear-conditioning learning and threat memory formation (Sears et al., 2013; Soya et al., 2013).

Evidence that hypocretin/orexin-containing neurons receive inputs from other neurons producing the anxiogenic peptide corticotropin releasing factor (CRF) and that hypocretin/orexin neurons projects to CRF-rich brain regions suggested the possibility that these two peptidergic systems are functionally entangled in the control of the stress response (Ida et al., 2000; Pañeda et al., 2005). However, if this hypothesis is correct, the anxiolytic properties of CRF-1 antagonists (Zorrilla and Koob, 2004) and those of OX1 antagonists would overlap, showing similar profiles. Interestingly, a recent study using phMRI in rats suggests that the involvement of CRF-1 and OX1 in stress responses can be functionally segregated. In this experiment a phMRI activation brain map was elicited in rats with yohimbine at doses known to produce anxiogenic effects. Pre-treatments with either CP-154,526, a selective CRF-1 antagonist (Seymour et al., 2003), or the selective OX1 receptor antagonist GSK1059865 (Gozzi et al., 2011) were performed. The yohimbine activation brain map was attenuated by CP-154,526 in the motor, cingulate, retrosplenial, dorsal prefrontal cortex, dorsal portions of the caudate-putamen, and in the amygdala. Differently, GSK1059865 attenuated the yohimbine activation map in the nucleus accumbens, septum, dorsal thalamus, amygdala, ventral hippocampus, orbitofrontal, prefrontal, insular, cingulate retrosplenial, and piriform cortex (Gozzi et al., 2013). Overall, the OX1 receptor antagonist exerted a more extensive effect on the fear, stress and anxiety circuit than the CRF1 antagonist, attenuating the activations of regions of the dopaminergic mesolimbic system. In line with the latter observation, a dissociation was found between the effects of OX1 and CRF-1 antagonists on the rat mesolimbic dopamine system in stress-induced cocaine (Wang et al., 2009) and nicotine seeking (Plaza-Zabala et al., 2012).

Interestingly, in some studies both OX1 antagonists and DORAs did not show anxiolytic effects in specific behavioral tests (e.g., the elevated plus maze) (Steiner et al., 2012; Rodgers et al., 2013). These data are in line with the hypothesis that hypocretin/orexin receptor antagonists do not alter basal anxiety levels in rodents, but exert anxiolytic-like properties when anxiety levels are transiently exacerbated by potent stimuli such as acidosis/hypercapnia.

#### **LIMITATIONS AND CONCLUSION**

The rationale for considering OX1 receptors as a possible target in conditions such as Anxiety Disorders, Drug Addiction, and Binge Eating was reviewed. Based on the current knowledge on mechanism of action, OX1 receptor antagonists should have fewer development risks than DORAs. As highlighted by Scammell and Winrow (2011) and Boutrel et al. (2013) for DORAs, the chronic simultaneous blockade of both hypocretin/orexin receptors can potentially: (1) induce narcoleptic-like symptoms, including catalepsy; (2) impair goal oriented decisionmaking; (3) reduce pleasure associated with rewarding activities; (4) induce sedation, sleepiness and impaired coping responses under acute emergency or stress; (5) impact on basal metabolism with increased body weight. However, so far the human experience with the DORAs suvorexant, SB-649868 and almorexant is very encouraging, showing minimal occurrence of the above mentioned adverse events and, in particular, no induction of narcoleptic eoisodes or impairment of decision making performance when tested within the dose range currently proposed. However, more data on larger populations and higher doses are required to reach a final conclusion.

Interestingly, treatments with selective OX1 antagonists are not expected to share the same risks of DORAs since only the OX2 receptor is primarily associated with narcolepsy which was demonstrated for dogs carrying a disruptive genetic mutation of this receptor (Wu et al., 2011). The additional potential benefits of an OX1 antagonist include: (1) A preclinical anxiolytic profile that differentiates it from benzodiazepines, serotonin-uptake inhibitors, and CRF-1 antagonists; (2) The capacity of reducing hyper-arousal states associated to acute and severe anxiety episodes with relevant physical symptoms, such as panic attacks or withdrawal from addictive drugs; (3) The attenuation of compulsive and repetitive behaviors associated with sensitization, stress-relieve, or drug seeking; (4) The lack of effects on desire of natural rewards, and (5) the lack of sleep-inducing effects.

These considerations are based on a limited dataset of preclinical studies, only recently conducted with the new generation of selective compounds. Chronic dosing studies are missing and therefore information of long term risks and benefits are not available yet. In addition, no selective OX1 receptor antagonists have been tested in humans so far and the translational barrier is still unclear. However, the promising therapeutic potential in the modulation of anxiety and compulsive behaviors is stimulating further basic research and is encouraging active investments of pharmaceutical companies.

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**Conflict of Interest Statement:** Emilio Merlo Pich is full time employee of F. Hoffman-La Roche. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 August 2013; accepted: 27 January 2014; published online: 13 February 2014.*

*Citation: Merlo Pich E and Melotto S (2014) Orexin 1 receptor antagonists in compulsive behavior and anxiety: possible therapeutic use. Front. Neurosci. 8:26. doi: 10.3389/ fnins.2014.00026*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Merlo Pich and Melotto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*


**4. Conclusions**

Subatmospheric pressures occur during the emptying process in pipelines with undulating profiles, which is a typical and common operation that engineers have to face. A rigid two-phase flow model was developed for analyzing it, considering *n* possible air pockets, *d* air valves, *p* pipes, and *o* drain valves. The proposed model is validated using an experimental facility of an irregular profile of 7.3 m long and nominal diameter 63 mm (*DN*63), with an air valve located at the high point and several drain valves along the pipeline. The main hydraulic variables could be measured such as flow (both phases), pressure and air-water front position. Comparisons between computed and measured values of the absolute pressure, water velocity and the length of the emptying columns show that the proposed model can predict accurately not only the extreme values but also their patterns. According to the results, the following conclusions can be drawn:

1. The proposed model can be used for planning the emptying operations in pipelines with undulating profiles, considering their limitations.

In real hydraulic pressurized systems, the proposed model can be used for: (i) checking the risk of a system collapse considering factors such as stiffness class, the soil in natural conditions, the type of backfill and the cover depth; and (ii) selecting the air valve sizes depending on the characteristics of each hydraulic system.

**Supplementary Materials:** The following are available online at www.mdpi.com/2073-4441/9/2/98/s1: The following videos show the emptying process for emptying column 1. Videos: Test No. 1, Test No. 2, Test No. 3, Test No. 4, Test No. 5, Test No. 6, Test No. 7, Test No. 8, Test No. 9, and Test No. 10. The behaviour for emptying column 2 is similar to emptying column 1.

**Acknowledgments:** The authors acknowledge: (1) the financial support for the first author covered by Fundación Centro de Estudios Interdisciplinarios Básicos y Aplicados (CEIBA)—Gobernación de Bolívar (Colombia); and (2) the experimental facilities for conducting the tests provided by IST (Instituto Superior Técnico), Civil Engineering Research and Innovation for Sustainability (CERIS), of the University of Lisbon (Portugal).

**Author Contributions:** Helena M. Ramos and Vicente S. Fuertes-Miquel conceived and designed the experimental facility and type of tests; Oscar E. Coronado-Hernández and Mohsen Besharat performed the experimental tests; Oscar E. Coronado-Hernández wrote the first draft paper; and Helena M. Ramos and Vicente S. Fuertes-Miquel revised the paper and completed the submission.

**Conflicts of Interest:** The authors declare no conflict of interest.


**1. Introduction**

An analytical device that provides information about a biological process through a transducer is called a biosensor. The information can both be semi-measurable and measurable. The biochemical receptor is used to collect information about the biological process and then transferred to the transducer. An ideal biosensor should be temperature and pH-independent, recyclable, and specific. It was Cammann who first invented the term "biosensor" and the definition of the biosensor was introduced by IUPAC [1]. Biosensor fabrication is a multidisciplinary field as it involves chemistry, biology, physics, electronics, material science, engineering, etc. [2]. Recently, various new elements, such as molecularly imprinted polymers, aptamers, nanomaterials, etc., have been developed in the field of biosensors [3–7]. The growth of nanotechnology has provided strong benefits to the biosensor field as the nanomaterials have a large surface-to-volume ratio that helps in achieving higher sensitivity and efficiency [8]. In most of the biosensors developed so far, electrochemical detection has been used in one way or another, i.e., amperometric, voltammetric, and impedimetric detection [9]. Moreover, the catalytic biosensor that uses

**Citation:** Singh, A.; Sharma, A.; Ahmed, A.; Sundramoorthy, A.K.; Furukawa, H.; Arya, S.; Khosla, A. Recent Advances in Electrochemical Biosensors: Applications, Challenges, and Future Scope. *Biosensors* **2021**, *11*, 336. https://doi.org/10.3390/ bios11090336

Received: 24 July 2021 Accepted: 31 August 2021 Published: 14 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

some kind of enzymes covers half of the production of biosensors, whereas the affinity biosensors using antibodies represent the other half [10]. The affinity biosensors are mostly used in monitoring the quality and safety of food products along with DNA (de-oxyribose nucleic acid) detection in reactions (hybridization) [11]. The electrochemical biosensors consist of three electrodes, i.e., reference, counter, and working. The electron generated or consumed during the catalysis of enzymes is used for producing a signal that helps in the detection process [12]. These are further classified as potentiometric, amperometric, voltammetric, and impedimetric biosensors. Recently, computers have made a walk-in in every field of science and technology. Machine learning is an important aspect of the computer language program and has been used in monitoring and refining various data outputs. While sensing complex structures and entities, large data are obtained, and it becomes difficult to manually interpret all the data. Machine learning helps in interpreting large sensing data and has been used in various applications. Moreover, the data obtained sometimes are not clear and possess some noise and disturbance. Machine learning can help in analyzing such data and obtain the required results. In the case of biosensors, the presence of impurity affects the performance of the sensor and machine learning helps in removing the signals obtained from the contaminants to obtain a high sensitivity. Moreover, the interpretation of the data becomes quite easy and effective by using machine learning in the biosensing process [13–15]. In this review, various machine learning tools and techniques that are used for biosensing are discussed briefly along with their applications and limitations.

#### **2. Types of Biosensors**

## *2.1. Catalytic Biosensors*

The development of classical first-generation enzyme electrodes has started some five decades ago. The procedure involves depositing enzyme on the surface of the transducer and the signal produced depends upon the products produced on the transducer surface after the reaction and diffusion of the target on the substrate. To improve efficiency and lessen the enzyme electrode's cost, different types of electrodes are specifically modified using nanomaterials and sometimes replaced by carbon electrodes. Cinti et al. [16] developed a cholesterol biosensor using screen-printed electrodes that were modified using nanoparticles of Prussian blue. The hydrogen peroxide produced due to the oxidase enzyme can be detected with such a setup. Sannini et al. also explained this by developing a lactate biosensor that can be used to monitor fermentation in an interferent-rich matrix-like wine [17]. There has been the detection of spermine and spermidine, which are polyamines, which can help in monitoring food safety and human health. This method employs newly designed oxidase, i.e., spermine and polyamine. The oxidase is captured in the gel of polyvinyl alcohol, and carbon-based electrodes that are modified with Prussian blue are used for carrying out the reduction process [18]. Another method is to create a barrier on the electrode surface and entrapping the enzyme on the layer of the electrode surface by physical entrapment or cross-linking. For a selective membrane, an ideal contender is overoxidized polypyrrole. A Pt electrode having lysine oxidase entrapped on it is also used as a biosensor for detecting maturation [19]. The biosensors based on glucose oxidase have been mostly used for glucose sensing. Strambini et al. developed a microneedle-shaped biosensor that can monitor glycaemia present in the intestinal fluids [20]. They mounted a glucose biosensor on the back of the needle that can detect glucose present in the intestinal fluid with an accuracy of ±20% only in 30 s. The biosensor worked on the capillary action and can be used for real samples [21]. Figure 1a shows the design of the biosensor where the glucose sensor is mounted on the back of a needle-shaped chip. The glucose sensor comprises graphite electrodes having glucose oxidase entrapped into the pores. Figure 1b shows the calibration curves and Figure 1c,d shows the experimental data depicting that the sensor can be used to detect concentrations as low as 0 to 35 mM with a sampling time of 5, 30, and 100 s.

**Figure 1.** (**a**) Representation of the biosensor having glucose sensor attached on the back of the microneedle; (**b**) Calibration curves for the concentration between 0–35 mM; (**c**) Calibration curves of developed biosensors and stand-alone biosensors in ISF, and (**d**) Calibration curves for developed biosensor for concentration 0–35 mM at 5 s, 30 s, and 100 s of sampling times. Reproduced from Reference [20] with permission. Copyright 2015, Elsevier.

The second-generation enzyme electrodes that have been developed by depositing gold on PC membranes have also been used in manufacturing electrochemical biosensors. By using the mediator as (Ferrocenylmethyl)trimethylammonium, the limit of detection (LOD) of the glucose was 36 µM. The fouling of the electrode surface can also be recognized due to oxidation which does not exist in the first-generation electrodes. Thus, first-generation electrodes can be used to prevent their fouling. Malvano et al. [22] used alcohol dehydrogenase and entrapped it into an SPE electrode customized with poly(2 acrylamido-2-methyl-1-propane) sulfonic acid polymer doped in polyaniline which helped in preventing the fouling. Functional parameters can also be calculated using enzyme electrodes, as the food contains antioxidants (produced by chemicals in the food). The enzyme-based biosensors have been used for detecting the antioxidants produced from yogurt and berry juices [23]. Moreover, these biosensors have also been used to monitor antioxidants and ascorbic acid in orange juice, blueberry, and kiwi using modified graphite and fullerenes as shown in Figure 2a [24]. In Figure 2b, the CSEM (Cryo scanning electron microscopic) images of the biosensor are shown, whereas Figure 2c,d represents the schematic diagram and working of the biosensor, respectively. Due to the oxidation of the enzyme, there was a decrease in the current which helps in monitoring and measuring the ascorbate present in the sample.

**Figure 2.** (**a**) Pseudoreference, working, and auxiliary electrodes; (**b**) CSEM images of biosensor surface modified with different materials. (**c**) Diagrammatic representation of the sensors with the modified surface; (**d**) working of the sensor. Reproduced from Reference [24] with permission. Copyright 2015, Elsevier.

It must be noted that enzyme-based electrodes have been used for sensing pesticides, but with advancement and technology, they can be used for monitoring food safety. Atrazine is an important component of pesticides. The activity of the enzyme tyrosinase can be inhibited by the atrazine chemical. The electrode-based on tyrosinase has been used for detecting atrazine with 0.3 ppm LOD in the water bodies [25]. Similarly, an enzyme-based electrode of bilirubin oxidase has been used to monitor the oxygen level of a microbial fuel cell having a 0–0.08 mW power range [26]. The enzymes such as urease, d-lactate dehydrogenase, acetylcholinesterase, and β-galactosidase have been used in catalytic biosensors for optical detection of milk quality and safety [27].

#### *2.2. Affinity Biosensors*

The term affinity biosensor in this article refers to a device incorporating immobilized biological receptor molecules that can reversibly detect receptor–ligand interactions with a high differential selectivity and in a non-destructive fashion. In affinity biosensors, antibodies are used as biochemical agents for detecting the biological process. Immunosensor is an important class of affinity biosensors. In the last four decades, the focus in the development of immunosensors has been reducing incubation time, better design, signal amplification, label-free detection, and control over antibody placement. When it comes to the sensitivity and production cost of such immunosensors, the enzyme-linked immunosorbent assay (ELISA) technique has been used most widely. A label-free immunosensor based on optical fiber was fabricated using a thin film of titania–silica dip-coated over the sensing area of the fiber [28]. The analyte sensing was done by measuring the wavelength at the initial and endpoint of the fibre. The fabricated immunosensor was successfully used for detecting Immunoglobin (IgG) and anti-IgG in human serum with very low LOD. In another example, Tardivo et al. [29] reported a sensor having thousands of micropillars and columnar resonators arranged in an area of 1 mm square. The sensing was done using image and software analysis capturing images at 15 fps. Since there are various sensors incorporated, thus the variation obtained in the frequency measurement by a single sensor

is neglected. Figure 3a,b shows an optical image and the process of measuring the intensity of the image as a function of frequency, whereas Figure 3c represents 53 traces obtained for 53 pillars, and Figure 3d,e shows the tracking of the parallel pillars at a resonant frequency in real-time.

**Figure 3.** (**a**) T-shaped pillar array and its optical image; (**b**) Process of measuring the intensity as a function of frequency; (**c**) 53 traces corresponding to 53 different pillars as a function of actuation frequency. Actuation mode, (**d**) single-mode, and (**e**) multiple modes. Reproduced from Reference [29] with permission. Copyright 2015, Elsevier.

For detecting a prostate antigen called interleukin, which is an inflammatory cytokine, an immunosensor with a detection range from 0.3 to 100 nM was fabricated using an electrolyte-gated organic field-effect transistor (EGOFET) [30]. Moreover, ELISA which is a complete and simple detection format, can be easily used with mobile phones, with mobile phones acting as a detector. In Figure 4, Zhao et al. proposed an improved screenprinted carbon electrode (SPCE) for multiplexed label-free amperometric immunosensor.

It is observed that the limit of detection was 5.5 pg mL−1 and the sensitivity of this immunosensor was 0.83 µA (lg(ng ml−1 ))−1 [31].

**Figure 4.** Multiplexed label-free amperometric immunosensor based on SPCE modified by Aupd NPs and some polymeric matrices for the detection of some biomarkers. Reproduced from Reference [31] with permission. Copyright 2018, Elsevier.

Aptamers can also be used for detecting antibodies. The aptamers are ribonucleic acid (RNA) and DNA molecules (single-stranded) that are highly selective and bind to particular antibodies, thus helping in their efficient detection. Different aptamers have different affinities based on which they can be used in developing biosensors with low LOD value. Castillo et al. [32] used a dendrimer structure for developing a voltammetric biosensor for sensing a mycotoxin called AFB1 (aflatoxin B1) in food products. The biosensor had a LOD of 0.40 nM for concentrations of 0.1–10 nM of AFB1. For the case study, the biosensor was tested on polluted peanut extract and peanut snacks. Scarano et al. [33] fabricated a piezoelectric material-based biosensor to detect this protein in human blood serum. Tintore et al. [34] used two aptamers with gold-coated magnetic nanoparticles for detecting single methylation in the DNA of aptamer using α-thrombin protein and plasmonic detection. The protein usually causes the agglomeration of nanoparticles resulting in the variation of color and UV of the solution. However, in this case, one of the aptamers prevented the agglomeration of the nanoparticles via single methylation of the DNA. Thus, the biosensor developed can be used to detect DNA modifications. Ravalli et al. [35] developed a biosensor based on an impedance study for detecting human epidermal growth factor receptor 2 (HER2). The antibody used for the detection of analyte was a protein produced using biotech engineering. The biosensor can detect a 0 to 40 µg/L concentration of HER2 with an LOD of about 6 µg/L. The albumin of the rat and human serum can bind with bicalutamide. In one such work, a transistor was developed where the gate of the transistor was modified with an odorant protein [36]. Pietrantonio et al. [37] deposited an odorant protein such as porcine, bovine, and double-mutant bovine protein on the surface of a wave resonator using the LIFT technique. The biosensor successfully distinguished between the carvone and octanol molecules that are present in the molds. Thus, the biosensor can be used for monitoring food products against molds. Cennamo et al. [38] detected butanal using an odorant protein called porcine. The biosensor selectively detected the butanal with concentrations ranging from 20–1000 µM. Compagnone et al. [39] developed a biosensor using gold-modified quartz crystal arrays. The biosensor was able to detect 95% of the off-flavored chocolate sample using the PLS-DA analysis technique.

Zuccaro et al. [40] developed a graphene-based biosensor that can monitor the behavior of the enzyme topoisomerase IB present in the human DNA. The interaction between the enzyme and the graphene substrate was analyzed by determining the field-effect characteristics of the biosensor. The biosensor provided real-time analysis and can be used for screening drugs in the future. The hybridization of DNA is an important feature whose detection is very helpful in various fields of biology. The biosensors with high selectivity and sensitivity can be used for detecting DNA hybridization. Mariani et al. [41] reported a method for detecting genomic human DNA (obtained from lymphocytes) without using the polymerase chain reaction (PCR) amplification process. The nanostars of gold were arranged in a sandwich-type model depending upon the analyte to be detected in the DNA and surface Plasmon resonance (SPR) imaging tool was used as a detector. A LOD of 10 pM was obtained with this method, which is low as compared to other biosensors developed, i.e., 6.1 nM. In another published work, Karel Lacina et al. [42] observed decrease in the impedimetric response (as shown in Figure 5) due to the binding of positively charged proteins. These assumptions were confirmed using gel electrophoresis which seems promising as a simple tool for such applications.

**Figure 5.** (**A**) Schematic visualization of the processes on the electrode. (**B**) Corresponding gel electrophoresis proving the charge of utilized compounds upon experimental pH. Reproduced from Reference [42] with permission. Copyright 2018, Elsevier.

Electrochemical methods can be used for sensing the hybridization of DNA. In one work reported, the electrochemical biosensor with an enzyme was used to detect the modified and unmodified DNA in PCR samples [43]. In another work, gold nanoparticles were incorporated as dots on a reduced graphene oxide electrode to produce an aptamerbased electrochemical biosensor [44].

#### **3. Electrochemical Biosensors**

The electrochemical biosensor is a special type of biosensor where a biological entity is detected by converting the information into an electrical signal, i.e., voltage, current, impedance, etc. [45,46]. The first electrochemical biosensor was developed by Clark to monitor the glucose level in human blood serum. The electrochemical biosensors have been developed to detect various biological entities such as enzymes, proteins, viruses, antibodies, etc. [47]. The electrode is the most vital component of an electrochemical biosensor as it controls the flow of electrons and bioagents. Various electrochemical biosensors that have been developed will be discussed in this section.

#### *3.1. Amperometric Biosensor*

An amperometric biosensor is a self-contained incorporated device based on the amount of the current ensuing from the oxidation offering exact quantitative analytical information. Generally, these Biosensors have reaction times, energetic ranges, and sensitivities comparable to the Potentiometric biosensors. In amperometric biosensors, the output current of the sensor is analyzed and used for the sensing process. The sensitivity of the amperometric biosensor is determined by comparing the current obtained for the different analyte concentrations. Such biosensors utilized only two electrodes, one for applying the voltage and the other for measuring the current flowing through the device. The amperometric biosensors do not utilize optical or electrochemical devices, but rather depend only on the current measurements. The development of biosensors started from glucose sensing and most of the glucose-sensing involves the catalytic reaction of the enzyme with the glucose oxidase [48,49]. The sensitivity of such glucose sensors is affected by the variation in temperature and pH. In amperometric glucose sensors, cyclic voltametry (CV) and electrochemical impedance spectroscopy (EIS) are the two electrochemical techniques that are used during the sensing process. The sensitivity of an amperometric glucose sensor is measured by the change in current per mM concentration in an area of one-centimeter square, i.e., µA mM−1 cm−2 . The sensitivity of an amperometric glucose sensor ranges from 1 to 15,000 µA mM−1 cm−2 . This is another important parameter while detecting glucose that determines its minimum and maximum concentration to detect the glucose successfully. The range of glucose in the blood (clinical) is between 4.4 and 6.6 mM. Tucci et al. [50] developed an amperometric biosensor for detecting herbicide using Anabaena variabilis cyanobacteria. Diuron and atrazine were used as target herbicides to be detected. The photocurrent produced in the biosensor due to the oxidation of water was found to be dependent upon the concentration of the herbicides. The current starts to decrease with an increase in the concentration of the herbicides. The sensitivity of the biosensor towards atrazine was found to be around 24.6 mA mM−1 cm−2 with an LOD of 0.56 mM. The mechanism of the inhibition is shown in Figure 6.

**Figure 6.** A diagrammatic representation of the photosynthesis inhibition by herbicides. Reproduced from Reference [50] with permission. Copyright 2019, Elsevier.

The biosensor was selectively used to detect glucose in uric acid, fructose, lactose, ascorbic acid, and mannose. The uric acid in the blood is in the range of 140–430 µM, and any changes in this level lead to kidney stones and gout [51]. The monitoring of uric acid in human blood is very important and biosensors are used effectively for this purpose [52]. Urea is another waste product produced in the kidneys and again variation in its level leads to serious kidney-related diseases [53,54]. The nanorods of ZnO were used to monitor the concentration of urea [54]. An array of ZnO nanotubes was deposited

on a silver/silicon substrate to detect cholesterol levels [55]. The sensor showed a sensitivity of about 79.40 µAmM−1 cm−2 with 0.5 nM LOD. Similarly, ZnO nanorods in a FET transistor, ZnO and multiwalled carbon nanotube (MWCNT) coated Pt/Au electrode, and a composite of CuO and ZnO have been successfully used to detect the cholesterol with good sensitivity and low LOD [56–58]. Fang et al. [59] successfully detected the presence of creatine kinase (an enzyme produced during muscle damage of brain or other body muscle) using an electrochemical biosensor made of gold. Cai et al. [60] reported an electrochemical immunosensor with gold and polythionine (PTh-Au) acting as an electrode. The immunosensor had good sensitivity and an LOD of 2.2 pg/mL towards the analytes. Both Fe3O4 and Ag@Au show good reducing properties towards the hydrogen peroxide. Zhang et al. [61] fabricated an electrochemical biosensor using nanospheres of Fe3O4 with nanorods of Ag@Au as an electrode. The biosensor demonstrated exceptional and superior behavior towards the reduction of hydrogen peroxide as compared to Fe3O4 and Ag@Au. Xu et al. [62] developed an amperometric biosensor for the detection of glucose in blood serum. In their work, they self-assembled glucose oxidase and Pt nanoparticles (encapsulated with dendrimers) on a multi-wall carbon nanotube (MWCNT). The biosensor displayed good sensitivity and a low LOD towards glucose. Using a nanocomposite of reduced graphene oxide and gold nanoparticles (rGo/AuNP), Han et al. [63] reported a label-free electrochemical biosensor with enhanced conductivity.

#### *3.2. Voltammetric Methods*

Voltammetry belongs to a category of electro-analytical methods, through which information about an analyte is obtained by varying a potential and then measuring the resulting current. The electrochemical biosensors in which voltammetric techniques such as differential pulse voltammetry (DPV), CV, SWV, and LSV are used for sensing are known as voltammetric biosensors. The voltammetric methods are widely used in sensing platforms due to their low cost, good selectivity, and high sensitivity. Gupta et al. [64] developed a biosensor based on DPV and CV methods to detect anti-iTG antibodies in humans. The biosensor was built by modifying gold nanoparticles with GQD/PAMAM and then embedding them into MWCNT. The biosensors detected successfully the concentration of the antibody as low as 20–50 fgmL−1 and are shown diagrammatically in Figure 7 below.

**Figure 7.** Diagrammatic representation of the biosensor based on DPV and CV to detect anti-tTG in the human celiac disease. Reproduced from Reference [64] with permission. Copyright 2017, Elsevier.

In another published work, Neves et al. [65] fabricated a nano immunosensor working on the CV technique to detect anti-tTG antibodies in the human serum. A carbon electrode that was modified with Au nanoparticles and CNT was taken and onto which the anti-tTG antibodies were printed using screen printing. The sensor showed excellent performance

and sensitivity towards the detection of antibodies and can be used for clinical trials. Hianik et al. [66] developed the first aptasensor that worked on voltammetric methods. In this sensor, the DNA aptamers were used to detect thrombin using the DPV technique. The aptamers were deposited on an Au electrode and methylene blue was used as an indicator. The aptasensors can be based on amperometric, potentiometric, voltammetric, conductometric, and impedimetric techniques [67,68]. The voltammetric aptasensors mostly use DPV, CV, and square wave voltammetry (SWV) techniques to detect the antibody. The voltametric biosensor is based on the detection of analyte using the variation in current according to the applied potential. Such biosensors detect both variations in potential and current. The potential is scanned over a wide range and the corresponding result in current and potential are measured. The current produced is directly proportional to the concentration of the target in the electrolyte [69,70]. The voltammetric methods produce low noise and can be used to detect multiple species with different peak potentials in a single scan [71]. The CV technique is used to study the stability, repeatability, and reproducibility of the biosensor [72]. Ly et al. [73] used a CNT electrode modified with bismuth to detect the DNA of Helicobacter pylori present in the gastric tissue of a patient. The biosensor successfully detected the DNA of Helicobacter pylori with a LOD of 6 µg/mL. The response time of the sensor was just 2s and can be used for clinical trials.