The Renaissance in Psychedelic Research: What Do Preclinical Models Have 2 to Offer

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The renaissance in psychedelic research: What do preclinical models have 2 to offer

Kevin S. Murnane1 Department of Pharmaceutical Sciences, Mercer University College of Pharmacy, Mercer University Health Sciences Center, Atlanta, GA, United States 1Corresponding author: Tel.: +1-678-547-6290; Fax: +1-678-547-6423, e-mail address: [email protected]

Abstract Human research with psychedelics is making groundbreaking discoveries. Psychedelics mod- ify enduring elements of personality and seemingly reduce anxiety, depression, and substance dependence in small but well-designed clinical studies. Psychedelics are advancing through pharmaceutical regulatory systems, and neuroimaging studies have related their extraordinary effects to select brain networks. This field is making significant basic science and translational discoveries, yet preclinical studies have lagged this renaissance in human psychedelic research. Preclinical studies have a lot to offer psychedelic research as they afford tight control of experimental parameters, subjects with documented drug histories, and the capacity to elucidate relevant signaling cascades as well as conduct invasive mechanistic studies of neurochemistry and neural circuits. Safety pharmacology, novel biomarkers, and pharmacokinetics can be assessed in disease state models to advance psychedelics toward clinical practice. This chapter documents the current status of psychedelic research, with the thematic argument that new preclinical studies would benefit this field.

Keywords Psychedelic, Preclinical, Serotonin, Neuroimaging, Alcoholism, Anxiety, Depression, Substance dependence

1 INTRODUCTION The term psychedelic has come to be associated with a broad class of drugs with diverse chemical, pharmacological, and psychoactive effects. Alternative nomencla- tures have used hallucinogen, entheogen, psychotomimetic and other appellations to

denote specific concepts related to these drugs. Hallucinations are an obvious and profound drug effect that can be produced by these drugs, but as has been noted, this is not an effect that is reliably produced at typical doses (Nichols, 2016), and the term hallucinogen has fallen out of favor. The term psychedelic has been used to denote the idea that these compounds elicit an experience related to the mind, inward reflection, and growth in the boundaries of the mind. The belief is that this term captures a better sense of the core effects of these drugs than other appellations it has replaced. There are many compounds that could potentially fall under the term psychedelic. Cannabinoid agonists (e.g., Δ9-tetrahydrocannabinol), N-methyl- D-aspartate receptor antagonists (e.g., phencyclidine; PCP), muscarinic receptor antagonists (e.g., scopolamine), and substrate-based releasers of monoamines (e.g., 3,4-methylendioxymethampheamine; MDMA or “ecstasy”) all engender psychoactive effects that could be included in an expansive view of psychedelics. How- ever, the core classes of psychedelics have often been restricted to phenethylamines such as 3,4,5-trimethoxy-phenethylamine (mescaline), tryptamines such as N,N- dimethyl-4-phosphoryloxytryptamine (psilocybin), and ergolines such as lysergic acid diethylamide (LSD). This chapter largely focuses on these core or “classic” psychedelics, with limited comparisons to other compounds to highlight specific concepts. Psychedelics have been used by humans in traditional indigenous rituals for thousands of years (Meyer and Quenzer, 2005). Mescaline was incorporated into the indigenous religious ceremonies of the native people of North America, and ayahuasca was incorporated into the indigenous religious ceremonies of the native people of South America (Carhart-Harris and Goodwin, 2017). Modern research with psychedelics largely commenced with the serendipitous discovery of LSD by Albert Hofmann, while he worked on novel analeptics derived from ergot alkaloids for Sandoz Laboratories (now a subsidiary of Novartis). Dr. Hofmann initially synthesized LSD in 1938, but discontinued work with the compound for several years as it showed little promise as an analeptic. In 1943, Dr. Hofmann decided to reexamine the effects of LSD, and while carrying out a new synthesis, he was overcome with a series of strange sensations and had to vacate his laboratory and return home. His description of this event illustrates some the core effects of pschedelics, and he wrote, “At home, I lay down and sank into a not unpleasant intoxiceded-like condi- tion, characterized by an extremely stimulanted imagination. In a dreamlike state, with eyes closed (I found the daylight to be unpleasantly glaring), I perceived an uninterrupted stream of fantastic pictures, extraordinary shapes with intense, kalei- doscopic play of colors” (Hofmann, 1959). As can be gleamed from Dr. Hofmann’s account, the interoceptive effects of psychedelics include elements of reflective lucid dream-like states and changes in sensation and perception, but not necessarily frank hallucinations (Kraehenmann, 2017). Despite the inherent difficulty in defining and describing such amorphous effects, extensive research has led to the development of validated questionnaires, such as the Hallucinogen Rating Scale (Tancer and Johanson, 2003) and the Dimensions of Altered States of Consciousness Scale and Mystical Experience Questionnaire (Liechti et al., 2017), to objectively 1 Introduction 27

measure the subjective effects of the psychedelics. Psychedelic-like effects on these measures include changes in somathesia (changes in the body such as feeling physically detached), affect (changes in emotions such anxiousness or closeness to others), cognition (changes in thoughts such as new insights or feelings of insanity), perception (changes in somatosensory, auditory, and visual detection and/or processing), volition (changes in attention and self-control), self (e.g., ego dissolution and oceanic boundlessness), and mystical-type experiences. Following this initial report of LSD, it was clear that such drugs could elicit powerful psychoactive effects, spurring great interest in their research. Over the next three decades, additional naturally occurring psychedelics were identified and new compounds synthesized, and hundreds of studies were completed. His discovery ushered in an era of intense LSD research, with nearly 1000 articles appearing in the medical literature by 1961 (Dyck, 2005; Grinspoon and Bakalar, 1986). This research was largely divided into two distinct areas. One body of research examined the psychedelics as a model for acute psychosis (Gouzoulis-Mayfrank et al., 1998). Although perhaps somewhat of an oversimplification, a general conclusion that can be drawn from this literature is that there is some overlap in the symptoms of acute psychosis and high dose administration of psychedelics, yet there are distinct differences between psychedelic effects and the nosological entity known as schizo- phrenia (Hollister, 1962). The occurrence of psychotic symptoms related to LSD, psilocybin, and ayahuasca in controlled and ritual settings is rare and can be further minimized by psychiatric screening and collecting family histories before administration of these drugs (Dos Santos et al., 2017). As this literature has been extensively discussed elsewhere (Gouzoulis-Mayfrank et al., 1998) and represents a distinct branch of psychedelic research, it will not be a major topic of this chapter. Another body of research examined the use of psychedelics as an adjunct to psychotherapy. Between 1950 and the mid-1960s over 1000 clinical trials were completed, several dozen books were published, and six international conferences were held providing data on approximately 40,000 patients that had undergone these “psychedelic” therapy sessions (Grinspoon and Bakalar, 1986; Riedlinger and Riedlinger, 1994). These approaches were primarily used in the treatment of alcoholism, depression, anxiety, neuroses, compulsive behavior, and psychosomatic disorders. Although advocates strongly argued that this was both safe and effective, the conclusions that can be garnered from these studies are limited due to the anecdotal nature of much of the work, lack of appropriate experimental control, and antiquated methodologies. However, it seems reasonable to hold that these compounds have shown the potential for therapeutic value. Unfortunately, the anti-establishment movement of the 1960s found commune with the use of psychedelics, and LSD became increasingly associated with student riots, antiwar demonstrations, and the counterculture (Dyck, 2005). Perhaps most troublingly, many of the leaders of the movement proposed the use of these compounds for a variety of non-medical purposes and in the absence of medical supervision. This led the government to tightly regulate the availability of the psychedelics and subsequently place many into the most restrictive level of the Controlled 28 CHAPTER 2 Preclinical psychedelic research

Substances Act (i.e., Schedule 1). Despite their great promise for advancing clinical care and elucidating our understanding of neurobiology and psychiatry, human research with the psychedelics largely terminated by the 1970s. In the context of this chapter, it is important to note that important basic science studies continued through the 1970s and 1980s, as will be discussed later.

2 RENAISSANCE IN PSYCHEDELIC RESEARCH The beginning of the 21st century has witnessed a rebirth of psychedelic research in humans as exemplified by the other chapters in this compilation. Largely commencing in the late 1990s with several notable studies examining the safety, pharmacokinetics, and neuropharmacological effects of psilocybin (Gouzoulis-Mayfrank et al., 1999; Vollenweider et al., 1997), there has been an explosion of research on the pharmacological and physiological effects of psychedelics in healthy volunteers as well as the therapeutic effects of psilocybin in a variety of disease states, most notably depression, anxiety, compulsion, and substance-use disorder (Carhart-Harris and Goodwin, 2017). These studies have documented an increasingly strong basis for the clinical use of psychedelics. In some of the first peer-reviewed publications in the modern era, it was reported that psilocybin, under medically and environmen- tally controlled conditions, occasioned mystical-type experiences associated with deep prayer, meditation, and other religious and non-religious experiences (Griffiths et al., 2006). At a 2-month follow-up, approximately 70 of the participants rated the psilocybin experience as among the most personally meaningful of their lives. A subsequent study documented that administration of psilocybin led to increases in the openness domain of personality that was stable for at least a year (MacLean et al., 2011). This was notable because few, if any, previous studies had demonstrated that any discrete experimental manipulation was capable of yielding long-lasting changes in personality. Psilocybin treatment in heavily tobacco-dependent smokers—who at intake were 51 years of age on average, had a mean of six previous lifetime quit attempts, smoked a mean of 19 cigarettes per day, and had smoked for a mean of 31 years—resulted in approximately 80% abstinence rates at 6 months (Johnson et al., 2014) and 67% abstinence rates at 12 months (Johnson et al., 2017). A recent clinical trial in 51 patients with a life-threatening cancer diagnosis reported that psilocybin decreased clinician- and self-rated measures of depression and anxiety, increased quality of life, life meaning, and optimism, and decreased death anxiety in a robust manner that was sustained over a 6-month follow-up period (Griffiths et al., 2016). Another recent study, reported significant decreases in alcohol consumption after 1–2 session of psilocybin in 10 alcohol-dependent subjects using an open-label design (Bogenschutz et al., 2015). 2016 saw the publication of the first modern brain imaging study of LSD (Carhart-Harris et al., 2016b). Initial and successful clinical studies of psilocybin (Carhart-Harris et al., 2016a) and ayahuasca (Sanches et al., 2016) for treatment- resistant depression and LSD for anxiety associated with terminal illness (Gasser et al., 2014) have been completed. Acute administration of psilocybin has been 3 What do preclinical models have to offer? 29

documented to enduringly change core personality domains, such as emotional empathy (Lewis et al., 2017) and openness to experiences (MacLean et al., 2011). The development of this second wave of psychedelic research has been breathtaking to observe, both in terms of its speed of advancement and in terms of the clinical efficacy observed.

3 WHAT DO PRECLINICAL MODELS HAVE TO OFFER? In this chapter, the term preclinical studies is used to refer to cellular and animal studies that do not involve human subjects. Animal models are a critical component of drug development programs and have greatly shaped our understanding of neuroanatomy and neuropharmacology. Cellular studies are a critical component of understanding drug binding, receptor signaling mechanisms, and the downstream mechanisms engaged by pharmacological agents. It seems improbable that a full understanding of the psychedelics can be achieved in the absence of preclinical studies. Nevertheless, in some ways, and largely in contrast to the expected order of research advancement, preclinical research has lagged behind human studies with psychedelics. There are numerous ways in which preclinical animal studies offer significant potential benefits for the study of the neuropharmacology and behavioral pharmacology of psychedelics. It is well established that the history of the organism (including behavioral and drug history) and the current environment in which a drug is administered can influence the effects of the administered drug (Young and Woods, 1981). This is certainly true for the psychedelics and has been referred to as “set and setting” (Cummins and Lyke, 2013; Liechti et al., 2017; Nichols, 2004). The use of laboratory animals provides exquisite control over the history of the organism and its current environment, which can be harnessed through the experimental analysis of behavior to provide important insights into the in vivo pharmacology of psychedelics. Laboratory animals can be exposed to psychedelics starting in an initially drug-naı¨ve state and followed through longitudinal within-subject designs to rigorously characterize aspects of the neurobiology and neuropharmacology associated with psychedelics. Psychedelics under investigation can be evaluated in subjects with well-documented drug histories. The capacity to engage in higher experimental control is significantly augmented by the ability to engage in invasive studies that would not be possible in human subjects given the necessary restrictions imposed in human clinical research. The twofold ability to tightly control critical experimental parameters and to conduct invasive studies affords laboratory animals the capacity to elucidate the mechanisms underlying the profound effects of psychedelics. For example, site specific injections of psychedelics into the brain (Gresch et al., 2007) can determine the critical neurobiological substrates mediating their effects. In vivo microdialysis (Murnane et al., 2010, 2012) can be used in laboratory animals to directly examine the effects of psychedelics on brain monoamine neurochemistry, as well as other neurotransmitters and brain signaling systems. 30 CHAPTER 2 Preclinical psychedelic research

Optogenetic (Deisseroth et al., 2006) and chemogenetic tools (Smith et al., 2015) can be used to discover the brain circuits activated by psychedelics, as well as elucidate the neurophysiological basis for their influence on the dynamics of brain activity. Cellular and molecular studies can characterize the molecular targets of the psychedelics as well as the signaling cascades that they initiate at the cellular level to induce their profound psychoactive effects. As such, preclinical studies offer significant promise for expanding our basic science understanding of psychedelics. Laboratory animal research as well as cellular and molecular studies also offers significant promise for advancing psychedelics toward safe and effective clinical practice. Disease state models for disorders ranging from substance dependence to anxiety to neurodegeneration can greatly aid in understanding the efficacy and safety of psychedelics. Mouse models, in particular, afford the opportunity to study the efficacy and safety of psychedelics in psychiatric and neurological disorders with a significant genetic component. Safety pharmacology and toxicology studies acceptable to regulatory agencies can be conducted in laboratory animals, and sup- plemented with cellular and molecular studies. Novel biomarkers for the clinical effectiveness of psychedelics can be developed and validated in laboratory animals and cellular studies. Laboratory animals allow for the repeated and controlled study of the pharmacokinetics and metabolism of psychedelics, an area where species may have particular import as there are important differences in the pharmacokinetics and metabolism of MDMA and related compounds between rodents and primates (Banks et al., 2007; Weerts et al., 2007). Lastly, nonhuman primates exhibit complex cognitive and social behaviors that provide unique opportunities for examining the effects of psychedelics (Morgan et al., 2002; Nader and Czoty, 2005; Nader et al., 2008). Recent human neuroimaging studies have greatly expanded our understanding of the neurobiology and neuropharmacology of psychedelics, as will be discussed later. Such studies would greatly benefit from preclinical studies in which neuroimaging can be conducted in tightly controlled subject populations, using high field strength magnets, and with sufficient access to subjects to provide replication across many brain regions, and coupled to invasive methodologies such as electrophysiology to clearly delineate mechanisms. Despite the significant potential of preclinical studies to expand our understanding of psychedelics and advance their clinical use, the last two decades have seen a robust renaissance in psychedelic research in humans without a concomitant expan- sion of the preclinical study of psychedelics. This chapter will document the studies that have been conducted in this area, but the thematic argument is that more preclinical research is needed, and preclinical studies need to catch up to the rapid advances occurring in the clinical literature, if we are develop a comprehensive and deep understanding of the scientific basis for psychedelic effects and if they are to achieve their clinical potential. It is important to note that preclinical approaches do have some important limitations in the context of psychedelic research. Some of the effects of psychedelics, such as distorted time perception and ego dissolution, may be difficult to study or may not occur at all in laboratory animals. Many characteristic psychedelic effects (such as distortions and mixing of the senses, 4 Mechanism of action 31

alterations in the perception of the passage of time, etc.) are largely unobservable and are especially challenging for study in laboratory animals. Although there are a paucity of studies, to date, psychedelics do not reliably maintain self-administration in laboratory animals (Poling and Bryceland, 1979), which limits the arsenal of preclinical tools available for their study, and presents potential false negatives with clinical findings. Several human studies have indicated that mystical-type experiences are important for the therapeutic effects of psychedelics (Garcia-Romeu et al., 2014). It is speculative, at best, whether mystical-type experiences can be recapitulated or modeled in laboratory animals.

4 MECHANISM OF ACTION Preclinical studies have contributed to our understanding of the mechanisms of action of psychedelics. They have informed our understanding of the molecular targets of psychedelics, the nature of their subjective effects, and their capacity to induce tolerance, as examples. Elucidating the receptor mechanism of action of psychedelics has been a particularly fruitful and bountiful area of research. With the discovery of serotonin as a biologically active substance (Rapport et al., 1948) it was immediately obvious to chemists that LSD and serotonin were structurally similar. This similarity led researchers to investigate serotonin systems as the critical mediators of psychedelic effects. Seminal early studies focused on suppression of serotonergic activity (Gaddum, 1953; Gaddum and Hameed, 1954). However, this early hypothesis of inhibiting the activity of serotonin fell by the wayside when the serotonin antagonist 2-bromo-LSD was shown to not share the subjective effects of LSD (Cerletti and Rothlin, 1955) and to block those subjective effects (Ginzel and Mayer-Gross, 1956). These findings, among others, lead to general acceptance that the subjective effects of LSD were in fact mediated through serotonin receptor agonist activity. Following the hiatus in clinical research with psychedelics in the 1960s, important preclinical studies continued to examine the effects of the psychedelics and the mechanisms underlying those effects. A series of studies were published from the Aghajanian laboratory at Yale investigating suppression of dorsal raphe firing as a key component of psychedelic effects (Aghajanian and Haigler, 1974; Aghajanian et al., 1968). The dorsal raphe nucleus is the major site of cells sending forebrain projections of terminal fields that release serotonin (Halliday and Tork, 1989). Their work led to a prominent hypothesis that psychedelic effects, and particularly low doses of psychedelics, were mediated by inhibition of the activity of the dorsal raphe nucleus through activation of presynaptic serotonin receptors. This hypothesis was developed as the scientific team led by Aghajanian observed that several tryptamines as well as LSD suppressed firing of neurons in the dorsal raphe using microiontophoretic and electrophysiology techniques, primarily in rats (Aghajanian and Haigler, 1974; Aghajanian and Hailgler, 1975; Aghajanian et al., 1968, 1970). This hypothesis eventually fell out of favor when it was subsequently discovered that 32 CHAPTER 2 Preclinical psychedelic research

the putatively non-psychedelic ergoline lisuride also suppressed dorsal raphe firing (Rogawski and Aghajanian, 1979) and psychedelic phenethylamines appear to be devoid of this effect (Aghajanian et al., 1970; Haigler and Aghajanian, 1973). More- over, research conducted in cat models revealed that behaviors such as limb flicks, abortive grooming and suppression of raphe firing were temporally correlated at peak doses of LSD, low doses of LSD marginally suppress raphe firing yet had robust effects on behavior, the raphe suppression lasted longer than the behavioral changes at peak doses, and LSD continued to suppress activity in animals made tolerant to the specific behavioral effects. Furthermore, phenethylamines elicited behavioral changes in cats while having little overall effect on raphe activity and increasing the activity of subpopulations of raphe neurons (Trulson et al., 1981). There are some interesting areas of note with these findings of Aghajanian and colleagues. That a psychedelic induces any particular behavioral effect should not be accepted as prima facie evidence of a direct relationship between that behavioral effect and psychedelic effects in human. Even in humans, psychedelics elicit off-target effects that are unrelated to their effects on the mind. It is therefore questionable that an imperfect relationship between suppression of raphe activity and any particular behavioral effect necessitates its abandonment as a critical component of psychedelic effects. Also, although its lack of consistency across different classes of psychedelics means that it cannot completely explain all aspects of psychedelic effects, there are likely to be subjective and therapeutic differences between the tryptamines, ergolines, and phenethylamines (Canal, 2018). Perhaps suppression of raphe firing could be revisited as a biomarker for therapeutic effectiveness in different disease states or patient populations. Modern research recognizes 7 different serotonin receptors (5-HT1–7) and 14 different serotonin receptor subtypes (Barnes and Sharp, 1999; Hannon and Hoyer, 2008). Consistent with the modern understanding of expression and function of this receptor, the suppression of raphe firing was later related to 5-HT1A receptors (Sprouse and Aghajanian, 1987, 1988), and it became clear that the tryptamines and LSD have activity at 5-HT1A receptors whereas some phenethylamines largely do not (Fantegrossi et al., 2008). As will be discussed later, in vivo preclinical studies have shown that 5-HT1A receptors may contribute to the behavioral effects of psychedelics, and in humans, the subjective effects (Pokorny et al., 2016) and attention-disrupting effects (Carter et al., 2005) of psilocybin can be mod- ulated by the 5-HT1A receptor. The precise role of 5-HT1A receptors in the effects of the psychedelics remains to be fully understood, and this is an area ripe for additional research that would greatly benefit from new preclinical studies. Research largely commencing in the 1980s began to focus on the second type of serotonin receptor (5-HT2 receptors) as well as its specific subtypes (2A,2B, and 2C) as a new focus for the molecular targets of psychedelics. Much of this preclinical research used assays of drug-elicited effects to study the mechanism of action of psychedelics. Drug-elicited behavior is a widely established approach to the study of the in vivo pharmacology of many psychoactive drugs (Ator and Griffiths, 2003). Basi- cally, an assay of drug-elicited behavior involves the administration of a drug and the measurement of some simple unconditioned behavior that is known to be mediated 4 Mechanism of action 33

by a discrete number of receptor subtypes. Such procedures have achieved notable advances through the study of stimulants using elicited locomotor and stereotyped behavior, straub tail responses for opioids, tetrad responses for cannabinoids, and sedative effects for alcohol and hypnotics, as examples. In this regard, the drug- elicited head twitch response (Corne and Pickering, 1967; Corne et al., 1963) has been of great utility in psychedelic research. Head twitching occurs in rodents spontaneously, but importantly is selectively increased in frequency by the administration of various psychedelics (Colpaert and Janssen, 1983; Darmani et al., 1990; Fantegrossi et al., 2004a; Goodwin and Green, 1985; Green et al., 1983; Peroutka and Snyder, 1981). While this assay has low face validity for psychedelic effects, its capacities to discriminate psychedelic from nonpsychedelic drugs and to reveal the neurophysiological and neuropharmacological mechanisms through which psychedelic drugs are acting are high. Head twitch studies using the selective antagonist M100907 (Fantegrossi et al., 2005, 2006) or genetic ablation of the 5-HT2A receptor have implicated that it has a critical role in psychedelic effects. The potency with which a 5-HT2A receptor antagonist blocks head twitching induced by psychedelics is highly correlated with the antagonist’s affinity for 5-HT2A receptors (Ortmann et al., 1982; Peroutka and Snyder, 1981). Substrate-based releasers of 5-HT, which can thereby indirectly agonize the 5-HT2A receptor, can have psychedelic-like effects in humans (Johanson et al., 2006; Tancer and Johanson, 2003). Such preclinical studies have been supported by seminal clinical research in which an incredibly tight correlation (r ¼0.97) between affinity at 5-HT2 receptors and psychedelic potency in humans was established (Sadzot et al., 1989). For the first time, these studies provide a common molecular target for all classes of psychedelics. A largely separate body of research used pre-pulse inhibition of the acoustic startle response as another drug- elicited effect of psychedelics (Halberstadt and Geyer, 2018), but mostly in the context of psychosis, which is beyond the scope of this chapter. These studies have greatly expanded our understanding of the psychedelics and provided a compelling mechanism of action to be more thoroughly explored by future research. It is important to note that much of the research on receptor subtypes was made possible only when selective pharmacological tools became available. In recent years compounds have been synthesized that are much more selective for the 5-HT2A receptor over other 5-HT2 receptor subtypes than compounds that have been workhorses of psychedelic research, such as the mixed 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). An example of these new pharmacological tools is 2-([2-(4-cyano-2,5-dimethoxyphenyl)ethylamino]methyl)phenol (25CN-NBOH), which is a full efficacy psychedelic agonist of 5-HT2A receptors that is much more selective than DOI (Hansen et al., 2014). These compounds afford the opportunity to generate new discoveries in the mechanism of action of psychedelics, and surprisingly, 25CN-NBOH is less effective at inducing head-twitch behavior than DOI and even attenuates the DOI-induced head twitch response (Fantegrossi et al., 2015), suggesting new complexity in the idea that 5-HT2A receptors are the principal molecular target of the psychedelics. This has been supported by the finding that central depletion of neurotrophic factors decreases 5-HT2A receptor 34 CHAPTER 2 Preclinical psychedelic research

expression in frontal cortex and suppresses DOI-induced ear scratch behavior but does not affect DOI-induced head twitch responses (Klein et al., 2010). Likewise, differences in the pattern of molecular targets engaged by different classes of psychedelics remain of interest, with recent research showing serotonin transporter en- gagement by the tryptamines (Rickli et al., 2016), yet genetic ablation of the serotonin transporter producing minimal effects on behaviors elicited by LSD (Kyzar et al., 2016). These new findings, and especially those documenting potential limitations of the 5-HT2A receptor hypothesis of psychedelic action, further bolster the argument for examining the role of both 5-HT1A and 5-HT2A receptors in their action. An interesting synthesis was recently penned arguing that 5-HT1A receptors may be critical for passive coping whereas 5-HT2A receptors may be critical for active coping (Carhart-Harris and Nutt, 2017). This suggests that further exploration of the role of 5-HT1A and 5-HT2A receptors in the effects of the psychedelics may afford opportunities to tailor treatment strategies for different disease states or patient populations that need a different balance of augmentation of active and passive coping. New preclinical studies that use the tight control of laboratory animal and cellular studies to unravel the balance of the 5-HT1A and 5-HT2A receptors in the psychoactive and therapeutic effects of the psychedelics would help to inform and guide the clinical development of the psychedelics.

4.1 RECEPTOR SIGNALING Preclinical molecular and cellular studies have been enormously helpful in determin- ing the signaling pathways induced by the psychedelics. As the 5-HT2A receptor hypothesis of psychedelic activity has come to dominate the literature, the prepon- derance of this research has focused on signaling through 5-HT2A receptors. Agon- ism of 5-HT2A receptors has been classically linked to activation of the Gαq/11-PLC signaling cascade, which leads to increases in intracellular inositol-triphosphate and subsequent Ca+2 mobilization, as well as increases in diacylglcerol. Together, these intracellular second messengers activate downstream protein kinases, for example protein kinase C, facilitating many of the agonist-mediated cellular responses. It +2 is also well known that brain 5-HT2A receptor agonism also activates Ca - dependent cytosolic PLA2, leading to increased breakdown of fatty acids and formation of arachidonic acid (Nichols, 2004). It has been documented that 5-HT2A agonism also stimulates phospholipase D activity in a manner independent of G-protein signals, demonstrating signaling diversity via functional selectivity of this receptor (Barclay et al., 2011). 5-HT2A receptor agonism has also been shown to signal through β-arrestin (Bhatnagar et al., 2001; Schmid and Bohn, 2010), which act as molecular scaffolds that link the receptor to downstream transducers, such as the extracellular-signal regulated kinases ERK1/2 (Kurrasch-Orbaugh et al., 2003), protein kinase B/AKT (Schmid and Bohn, 2010), and PI3 Kinase (PI3K). Importantly, functional selectivity and biased agonism have been demonstrated for some of these 5-HT2A receptor pathways including β-arrestin/AKT (Schmid and Bohn, 2010), 4 Mechanism of action 35

PLC/IP3 (Kurrasch-Orbaugh et al., 2003), and PLA2/AA (Kurrasch-Orbaugh et al., 2003; Moya et al., 2007). Functional selectivity is a relatively novel concept in pharmacology that posits that while two drugs may be agonists at the same receptor, they may nonetheless selectively activate different intracellular functional cascades. Developed over the last few decades, this phenomenon has been demonstrated in a number of in vitro and in vivo assays in several laboratories (Nichols, 2004). Research by Gonzales-Maeso and colleagues has extended this work to further explain the mechanism of psychedelics. They have shown that activity of psychedelic and non-psychedelics agonists at the 5-HT2A receptor engenders coupling of the receptor to different intracellular proteins, activation of different down-stream second messenger cascades, and upre- gulation of a different set of genes (Gonzalez-Maeso et al., 2003, 2007). Another major caveat to the 5-HT2A hypothesis of psychedelic effects has been the existence of agonists at the 5-HT2A receptor that are not psychedelic in people, such as lisuride and ergotamine. The idea that different agonists of the 5-HT2A receptor can activate distinct post-receptor signaling cascades may reconcile the existence of non- psychedelic 5-HT2A receptor agonists with the 5-HT2A hypothesis of psychedelic effects, and it may lead to a better understanding of the molecular underpinnings of psychedelic effects. It was shown that psychedelics could be distinguished from purportedly non-psychedelic 5-HT2A receptor agonists in vivo by the mouse head twitch assay and that they may engender different post-receptor events. This further supported the predictive validity of the head twitch assay by demonstrating it could discriminate psychedelic from non-psychedelic 5-HT2A receptor agonists in vivo. Using a series of toxins (Gonzalez-Maeso et al., 2007), they demonstrated in vitro that psychedelic 5-HT2A agonists may induce a conformation whereby the receptor can couple to a Gi protein in addition to the Gq/11 protein that is classically considered to be coupled to the 5-HT2A receptor. The concept of functional selectively presents an interesting opportunity for the therapeutic potential of psychedelics. This idea suggests that it is possible to develop compounds that have similar direct pharmacological effects to the psychedelics while engendering distinct psychoactive effects. It has been demonstrated in recent human studies that mystical-type experiences correlate with the therapeutic effects of psychedelics (Garcia-Romeu et al., 2014), leading to the hypothesis that they may be necessary for these therapeutic responses. The administration of non-psychedelic 5-HT2A receptor agonists may be an avenue for testing this hypothesis. Recent advances have increased the probability of developing novel non-psychedelic 5-HT2A receptor agonists. The crystal structure of the 5-HT2B receptor, which shows substantial homology with the 5-HT2A receptor, was recently reported in complex with ergotamine (Wacker et al., 2013) or LSD (Wacker et al., 2017), along with the structure of the 5-HT1B receptor in complex with ergotamine (Wang et al., 2013). Molecular dynamics simulations have revealed the second intracellular loop of the 5-HT2A receptors as a critical determinant of the conformations that agonist binding induces, and presumably the downstream consequences of these conformational states (Perez-Aguilar et al., 2014). The compound TCB-2 has been developed, which 36 CHAPTER 2 Preclinical psychedelic research

is a psychedelic 5-HT2A receptor agonist but which induces reduced head-twitch behavior and activates a distinct set of post-receptor signaling pathways than DOI (Fox et al., 2010). In our laboratory, we have used ligand-based pharmacophore modeling of LSD and lisuride to identify critical structural features of ligand binding to 5-HT2 receptors that predict psychedelic versus non-psychedelic activity in combination with in vivo and molecular pharmacology techniques, and discovered that non- psychedelic 5-HT2A receptor activity may be a novel mechanism of action for the beta blocker carvedilol (Murnane et al., n.d.). It should be noted when it comes to lisuride that its status as a non-psychedelic 5-HT2A receptor agonist is controversial as there appears to be some generalization in the subjective effects of lisuride and LSD in laboratory animals (Appel et al., 1999; Callahan and Appel, 1990; Fiorella et al., 1995) and high toxic doses of lisuride may induce reactions in humans that include visual and auditory hallucinations, reduced awareness, delusions, and paranoid ideation (Critchley et al., 1986; Lees and Bannister, 1981; Parkes et al., 1981). Nevertheless, such effects are not representative of typical experiences with lisuride administration, or psychedelic administration, and it would be hard to argue for substantial overlap in the effects of lisuride and psychedelics at typical doses. As such, we remain hopeful that lisuride and related compounds can be used to elucidate the critical signaling pathways of psychedelics and establish novel non-psychedelic 5-HT2A receptor agonists. Recent exciting studies have provided even more impetus to this undertaking. It has been shown that DOI induces profound anti-inflammatory effects both in tissue (Yu et al., 2008) and in vivo (Nau et al., 2013), and at doses below those required to induce head-twitch behavior. If non-psychedelic agonists that share the anti-inflammatory effects of DOI could be developed, this could have significant therapeutic implications and is an area that preclinical research can be of tremendous value.

4.2 IMAGING/ELECTROPHYSIOLOGY The last two decades have seen an extraordinary explosion in neuroimaging studies with psychedelics (Canal, 2018). In early studies, the binding of an LSD analog was shown to localize to frontal and temporal cortices (Wong et al., 1987). This distribution of binding is similar to the distribution of the 5-HT2A receptor (Burnet et al., 1995) and the distribution of binding of the selective 5-HT2A receptor antagonist M100907 (Hall et al., 2000; Kristiansen et al., 2005). Furthermore, this distribution of binding has extensive overlap with the distribution determined for radiolabeled psychedelics (in this study LSD and DOI), using autoradiography (McKenna et al., 1989b). Using a radioactive analog of glucose, it has been shown that an acute bolus of psilocybin increases brain metabolism in frontal, temporal, and cingulate cortex, and that prefrontal hypermetabolic activity correlates with psychedelic effects such as ego dissolution (Vollenweider et al., 1997). These findings were subsequently corroborated in a study using double-blind and placebo controls and positron emission tomography (PET) (Gouzoulis-Mayfrank et al., 1999). More recent research using magnetoencephalography has shown that psilocybin reduces 4 Mechanism of action 37

oscillatory power in posterior and frontal association cortices with large decreases in areas related to the default-mode network, a circuit or network that is activated when the brain is rest (Muthukumaraswamy et al., 2013). Electroencephalographic techniques have resulted in the similar finding that psilocybin-induced spiritual experiences and insightfulness correlate with changes in oscillatory activity in anterior and posterior cingulate cortices (Kometer et al., 2015). This may mean that desynchronization of ongoing oscillatory rhythms in the cortex is the neurophysiological basis for key elements of the psychedelic experience. A functional magnetic resonance imaging (fMRI) study has revealed that intravenous infusion of psilocybin increased brain signal variability and connectivity in higher brain systems such as the default mode, executive control, and dorsal attention networks, which may account for the lucid dream-like state of consciousness and alterations in the perception of time, space and selfhood that occur during the psychedelic experience (Tagliazucchi et al., 2014). Psilocybin-induced loss of functional connectivity between the medial temporal lobe and high-level cortical regions has also been associated with ego dissolution (Lebedev et al., 2015), and modulation of the default-mode network has also been observed following ayahuasca administration using fMRI (Palhano-Fontes et al., 2015). A combined fMRI and magnetic resonance spectroscopy study showed that psilocybin decreases the neural response to social exclusion and aspartate content in the cingulate cortex (Preller et al., 2016). An arterial spin labeling fMRI study reconciled previous disparate findings by using adjustments for global perfusion to demonstrate that psilocybin induces a mixture of hypoperfusion in parietal, left temporal, and subcortical regions and hyperperfusion of frontal, right temporal, and insular brain regions (Lewis et al., 2017). Additional magnetoencephalographic analysis resulted in the finding that the altered states of consciousness induced by psilocybin, ketamine and LSD are associated with higher spontaneous brain signal diversity (Schartner et al., 2017). These studies have dramatically expanded our understanding of the neuropharmacology and neurophysiology of psychedelic effects. They have clarified the role of serotonin systems in the human and provided new findings and hypotheses related to brain networks, brain neurochemistry, functional connectivity in signaling dynamics, and the effects of psychedelics on brain activity and perfusion. Although the human neuroimaging literature has provided important advances in our understanding of the neuropharmacological and neurophysiological mechanisms mediating psychedelic activity (Dos Santos et al., 2016), such studies are most likely to fully delineate these processes if they are coupled to imaging studies in laboratory animals. As described earlier, the ability to rigorously control critical experimental parameters and to conduct invasive studies affords laboratory animal research the capacity to elucidate the mechanisms underlying the profound effects of psychedelics, especially when conducted in conjunction with human brain imaging studies. Although preclinical studies in this area have lagged their human counterparts, there have been some recently reported findings. Single unit and local field potential extracellular recordings in rats have been used to show that DOI disorganizes network activity in frontal cortex, reduces low-frequency oscillations, and desynchronizes 38 CHAPTER 2 Preclinical psychedelic research

pyramidal discharges (Celada et al., 2008). Consistent with both the clinical literature and the effects of DOI, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a constituent of ayahuasca, disrupts cortical activity and low frequency cortical oscillations in the frontal cortex of rats, which was reversed by haloperidol, clozapine, risperidone, and the mGlu2/3 agonist LY379268 (Riga et al., 2014). Pharmacolog- ical magnetic resonance imaging (phMRI) in rats led to the findings that psilocin induced brain signal increases in olfactory and limbic areas and brain signal decreases in somatosensory and motor cortices. The investigators used local field potential recording to demonstrate that psilocin-induced phMRI responses reflect changes in both neuronal activity and neurovascular coupling, creating a more nuanced understanding of the effects of psychedelics measured using fMRI (Spain et al., 2015). Additional local field potential, multi-unit recording analysis of the effects of 5-MeO-DMT on oscillatory activity in frontal, visual and thalamic brain regions has been conducted in wild-type and 5-HT2A receptor knockout mice at different frequency bands and to examine coherence between brain areas. The authors report a possible association between alternating activity in frontal and visual areas and psychedelic effects, that many effects of 5-MeO-DMT are retained in 5-HT2A receptor knockout mice, and that the selective 5-HT1A receptor antagonist WAY-100635 pre- vented most of the effects of 5-MeO-DMT on oscillatory activity in 5-HT2A receptor knockout mice, suggesting redundancy in psychedelic activity between 5-HT1A and 5-HT2A receptors (Riga et al., 2017). These studies highlight the value of preclinical studies for the understanding the human clinical literate and support the case for more studies of this kind.

5 INTEROCEPTIVE EFFECTS Drug-discrimination procedures have also been and continue to be an important component of preclinical psychedelic research. The drug-discrimination assay compares a novel drug with another drug of known pharmacology in terms of the internal state produced by those drugs (Ator and Griffiths, 2003). In other words, a drug- discrimination procedure would be used to determine whether or not animal subjects report (on an operant lever) that the internal state produced by a test drug is similar to the one produced by a known psychedelic, such as LSD. This is termed substitution. Alternatively, a novel drug can be trained to provide stimulus control over behavior, and a known psychedelic drug can be substituted to test for stimulus generalization. Although it is not possible to know the pharmacological effects driving the selection of the lever by the animal, there is a very tight correlation between results in drug- discrimination procedures and “subjective effects” in humans (Brauer et al., 1997; Schuster and Johanson, 1988). Furthermore, drug-discrimination procedures likely measure central nervous system effects as it has been shown the cocaine methiodide, a cocaine analog that does not cross the blood–brain barrier but shares peripheral effects with cocaine, does not substitute for cocaine (Terry et al., 1994; Witkin et al., 1991). Therefore, if an animal reports that a novel drug substitutes for or 5 Interoceptive effects 39

generalizes to a drug that is a psychedelic in humans, there is a very high probability that the novel drug would be psychedelic in people. This assay is not limited to psychedelics and has been widely implemented with stimulants (Martelle and Nader, 2009; Schama et al., 1997), opioids (Makhay et al., 1998; Walker et al., 1997), benzodiazepines (Licata et al., 2005; Rowlett and Woolverton, 2001), and other drug classes. Drug discrimination studies have largely paralleled the head-twitch literature described earlier in linking psychedelic effects to the 5-HT2A receptor. Much of this research has used DOI and LSD as the prototypical agents. A convincing series of studies has demonstrated that psychedelics appear to largely generalize with one another (Glennon et al., 1983; Winter, 1978) and they engender stimulus control through agonist activity at 5-HT2A receptors (Colpaert and Janssen, 1983; Colpaert et al., 1982; Fiorella et al., 1995; Schreiber et al., 1994). These experiments were instrumental in the development and acceptance of the 5-HT2A receptor hypothesis of psychedelic effects. It is interesting, however, that drug discrimination studies also provided support for the 5-HT1A receptor hypothesis developed from the seminal work of Aghajanian and colleagues. Consistent with their typically higher levels of agonist activity at 5-HT1A receptors compared to the phenethylamines (Fantegrossi et al., 2008), discrimination studies reporting a role for 5-HT1A receptors have most often utilized tryptamines. The tryptamine 5-MeO-DMT exemplifies this phenomenon. This compounds can be trained to engender stimulus control in typical discrimination procedures, but the rank order of potency for compounds that substitute for 5-MeO-DMT tracks affinity for 5-HT1A receptors better than affinity for 5-HT2 receptors (Spencer et al., 1987), only antagonists that block 5-HT1A receptors attenuate its stimulus effects (Winter et al., 2000), and agonists of 5-HT1A receptors occasioned greater substitution for 5-MeO-DMT than agonists of 5-HT2A receptors (Winter et al., 2000). These results paradoxically suggest that the stimulus effects of 5-MeO-DMT are primarily mediated through the 5-HT1A receptor, even despite the finding that 5-MeO-DMT substitutes for LSD, at least in primates (Nielsen, 1985). Furthermore, antagonism of 5-HT1A or 5-HT2A receptors can attenuate the stimulus effects of the structurally similar tryptamine 5-MeO-DIPT (Fantegrossi et al., 2006). A role for the 5-HT1A receptor in the stimulus effects of psychedelics has not been limited to 5-MeO-DMT, or even only the tryptamines. It has been reported that the 5-HT1A receptor agonist 8-hydroxy-2-(N,N-di-n-propylamino)tetralin (8-OH-DPAT), buspirone, gepirone, and ipsapirone all enhance stimulus control by the ergoline LSD (Reissig et al., 2005) and the phenethylamine 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) (Khorana et al., 2009), and this effect could be blocked by specific 5-HT1A receptor antagonists (Khorana et al., 2009; Reissig et al., 2005). Moreover, enhancement of stimulus control by 5-HT1A receptor agonism may be specific to psychedelics as 8-OH-DPAT significantly attenuated the discriminative stimulus effects of the cannabinoid rimonabant (McMahon, 2016). In one of the few drug discrimination studies that have examined psychedelics in non-human primates, the 5-HT2 receptor antagonists ketanserin and pirenperone did not effectively attenuate the stimulus effects of LSD, mescaline and 40 CHAPTER 2 Preclinical psychedelic research

lisuride did not substitute for LSD, and 5-MeO-DMT fully substituted for LSD (Nielsen, 1985). In an elegant study conducted in both rats and primates trained to discriminate DOM, 5-HT2A but not 5-HT1A agonists substituted for the DOM cue in both species and the 5-HT2A agonists enhanced the discriminative stimulus effect of DOM substituted for itself, but the 5-HT1A agonists attenuated the discriminative stimulus effect of DOM substituted for itself in primates but not in rats, suggesting some important species differences in the stimulus effects of DOM (Li et al., 2010). On the contrary, buspirone and other 5-HT1A agonists have been administered to humans and do not occasion mystical-type experiences or psychedelic effects. The most plausible hypothesis is that 5-HT1A and 5-HT2A receptors can interact in vivo, leading to modification of one another’s stimulus effects, as well as other behavioral effects, as has been demonstrated in preclinical research (Reissig et al., 2008). It seems reasonable to suggest that understanding the role of both 5-HT1A and 5-HT2A receptors in psychedelic activity would be an important consideration for their clinical development, particularly when psychedelics can show marked differences in their affinities and efficacies at these receptors. Compounds could be tai- lored to disease states and patient subpopulation based on their balance of activity at these receptors. Efficacy and safety could be considered in the context of both of these receptors. This is again an area where more preclinical research would be likely to produce meaningful results that likely could not be obtained in human studies, and which would be of great value to those human studies. Discrimination studies seem especially well positioned to provide valuable preclinical findings that inform human studies with psychedelics. They can be used to develop our understanding of the neuropharmacology of the subjective effects of psychedelics in ever more precise ways. Careful control of the chemistry and pharmacology of the test agents can increase the specificity of the outcome of discrimination studies (Murnane et al., 2009). Variants of discrimination such as three lever discrimination (Callahan and Appel, 1990) or the training of a discriminable cue between a psychedelic and a set of alternative drugs, rather than between a psychedelic and vehicle, can also greatly increase the specificity of the discrimination (Appel et al., 1999). Moreover, it has been noted that many of the characteristic psychedelic effects (such as distortions and mixing of the senses, alterations in the perception of the passage of time, etc.) are largely unobservable and are especially challenging for study in non-verbal species. Given the profound effects of psychedelics on perception and other unobservable processes, a procedure capable of allowing an animal to report sensory and perceptual changes in a non-verbal manner would seem to be especially useful for their study. A key component of discrimination procedures is that they afford a powerful means to use behavior that an animal can emit (i.e., lever pressing) rather than a behavior that it is incapable of emitting (i.e., verbal behavior) when presented with a particular stimulus. Variants of discrimination assays can use interoceptive stimuli such as drug induced stimuli, hunger, and the passage or time as examples, or exteroceptive stimuli such as lights, tones, and other external cues. Re- search in this area has shown that DOI disrupts the temporal discrimination of 6 Psychedelic abuse 41

exteroceptive light cues that were presented for less than or more than 25s, which was likely 5-HT2A receptor dependent as it was reversed by ketanserin and the highly selective 5-HT2A receptor antagonist M100907 (Asgari et al., 2006). Subsequent study showed that DOI selectively impairs temporal discrimination of an exteroceptive light stimulus and has no effect on a light intensity discrimination, which was in contrast to amphetamine-induced disruption of both discriminations (Hampson et al., 2010). These findings were largely corroborated in a recent study using an interoceptive stimuli to track time periods lasting less than or more than 6.5s, as both DOI and the more selective 5-HT2A receptor agonist 25CN-NBOH disrupted temporal discrimination in mice, and this was reversed by 5-HT2A but not 5-HT2C receptor antagonism (Halberstadt et al., 2016). This research has begun to be extended into other novel procedures for studying temporal perception such as the five-choice serial reaction time task (Cope et al., 2016). Preclinical drug discrimination research has informed our understanding of neuropharmacology and interoceptive effects of psychedelics (Baker, 2018). Especially if this technique is extended to the study of observable stimuli specifically related to psychedelic effects, as well as used in combination with site specific injections of psychedelics into the brain, in vivo microdialysis, optogenetic and other mechanistic techniques, it has the potential to greatly shape our understanding of neuroanatomy and the psychedelics. In this line, it has been shown that LSD locally infused into anterior cingulate cortex dose-dependently substituted for rats trained to discriminate systemically administered LSD from saline (Gresch et al., 2007). More studies of this kind would dramatically enhance our understanding the extraordinary effects of these drugs and have the potential to significantly shape our ideas regarding the brain, the self, and psychiatric disease.

6 PSYCHEDELIC ABUSE The preclinical study of the abuse liability of novel drugs has largely relied on the use of self-administration procedures because these procedures have well documented utility in predicting the abuse liability of novel compounds (Ator and Griffiths, 2003; Brady et al., 1987; Mello and Negus, 1996; Schuster and Johanson, 1981). Moreover, drug self-administration in laboratory animals is a validated technique to model the etiology, maintenance, and consequences of human drug abuse (Everitt and Robbins, 2000; O’Brien and Gardner, 2005), and there is strong agree- ment between clinical and preclinical assessments of the reinforcing effects of drugs (Brady et al., 1984; Griffiths, 1980; Griffiths et al., 1978, 1980). In one of the earliest studies to establish this concordance, rhesus monkeys self-administered drugs such as cocaine, amphetamine, codeine, morphine, and ethanol that have substantial abuse liabilities. In contrast, monkeys did not self-administer drugs such as nalorphine and chlorpromazine that are not substantially abused by humans. An import caveat to these findings was that humans abuse mescaline but, under the conditions employed, the monkeys did not self-administer mescaline (Deneau et al., 1969). Based on these 42 CHAPTER 2 Preclinical psychedelic research

and the results of a few other studies, it has been long held that the psychedelics represent a false negative of self-administration procedures (Poling and Bryceland, 1979) as it is clear that there is some level of recreational abuse of psychedelics (Johnston et al., 2006). The development of self-administration procedures appropriate for studying psychedelics would greatly aid their clinical advancement because the mechanism mediating their abuse-related effects could be established, the compounds most likely to be abused could be assessed, and their relative propensity for abuse relative to other drug classes could be established. It is well recognized that the results of self-administration studies are determined by an array of variables including the dose, pharmacokinetics, and neurochemistry of the drug, the history of the organism, and the schedule of reinforcement under which the drug is available (Howell and Murnane, 2011). In regards to the neurochemical effects of the drug, reinforcement of behavior by a variety of stimuli has generally been associated with the neurotransmitter dopamine in both humans and laboratory animals (Ritz et al., 1989; Volkow et al., 1999; Wilcox et al., 2002). There is an established literature that the 5-HT2A receptor can modulate dopamine neurotransmission. 5-HT2A receptors are widely expressed in the brain (Pompeiano et al., 1994) and are localized to midbrain dopamine neurons that innervate striatal regions (Bubar and Cunningham, 2007; Bubar et al., 2005; Ikemoto et al., 2000; Nocjar et al., 2002). 5-HT2A receptor antagonists attenuate the facilitatory effects of cocaine, amphetamine, MDMA, and methamphetamine on dopamine neurotransmission as well as their locomotor stimulant and interoceptive effects (Auclair et al., 2004; Broderick et al., 2004; Murnane et al., 2013a,b). Systemic administration of the phenethylamine DOI increases the firing rates of dopamine neurons in the ventral tegmental area and induces dopamine release in the prefrontal cortex, effects that were attenuated by selective antagonism of the 5-HT2A receptor (Bortolozzi et al., 2005; Pehek et al., 2006). Central administration of DOI significantly increases dopamine levels in the nucleus accumbens (Bowers et al., 2000; Yan, 2000). The tight interplay between 5-HT2A receptors and the dopamine system provides a plausible basis for abuse-related effects of psychedelics, further strengthening the case that the human literature could benefit from the development of effective procedures to study psychedelic self-administration. Drug-taking behavior can be influenced by not only direct pharmacological variables, such as neurochemical effects, but also by the history of the organism and the environment in which the drug is available. For example, the pharmacological history of the organism is a key determinant of self-administration has been supported by the finding that monkeys would acquire PCP self-administration when they had previously self-administered ketamine but not when they had previously self- administered codeine (Young and Woods, 1981). In another such example, monkeys readily acquired diazepam self-administration following a history of pentobarbital self-administration but not following a history of cocaine self-administration (Bergman and Johanson, 1985). In this same regard, in human subjects, diazepam is an effective reinforcer in individuals with an extensive history of taking sedatives (Roache and Griffiths, 1989) but not in those who lack such a pharmacological 6 Psychedelic abuse 43

history (Johanson and de Wit, 1992). Other work has shown that monkeys will self- administer MK-801 following ketamine but not cocaine self-administration, and that MK-801 has similar interoceptive effects to ketamine but not cocaine (Beardsley et al., 1990). Another key determinant of self-administration is the current environmental in which the drug is available for consumption. One of the earliest described and most widely accepted environmental determinants of self-administration is the operant schedule under which the drug is available. Early studies showed that administration of a test compound could increase or decrease behavior depending on its ongoing rate, which was related to the schedule of reinforcement (Kelleher and Morse, 1968). More recent work showed that behavior can be reinforced by termi- nation of naloxone infusions under simple schedules of reinforcement but it can also be maintained by delivery of naloxone infusions under complex second-order schedules of reinforcement (Downs and Woods, 1975, 1976). An informative example of this phenomenon comes from the study of nicotine self-administration. In this regard, numerous studies have shown that nicotine maintains very low rates of responding under conditions that allow for robust reinforcement with other drug classes (Deneau and Inoki, 1967; Goldberg and Gardner, 1981; Le Foll and Goldberg, 2005). This is somewhat surprising because nicotine ranks among the most commonly abused drugs in the world. However, under a complex second-order schedule of reinforcement that spaces drug infusions and extensively incorporates drug-paired stimuli, nicotine maintains robust rates of responding (Goldberg and Gardner, 1981; Goldberg et al., 1981; Le Foll and Goldberg, 2005). This may be because under these schedules drug-associated stimuli take on conditioned reinforcing effects that can control behavior in the same way as the drug itself (Goldberg et al., 1976). Especially given the pronounced effects of the psychedelics on perception and associative learning (Gimpl et al., 1979; Gormezano and Harvey, 1980; Gormezano et al., 1980; Harvey et al., 1982), it is possible that similar procedures may be effectively reveal and assess their reinforcing effects. Indeed, these studies of pharmacological history and environmental variables collectively provide a compelling framework for developing a model of psychedelic self-administration. Based upon the previously described studies, it is reasonable to suggest that psychedelics may exhibit reinforcing effects under appropriate conditions. Consistent with this hypothesis, in a classic study, monkeys were allowed access to self- administration of n,n-dimethyltryptamine (DMT) (Siegel and Jarvik, 1980). Under baseline conditions, monkeys sampled DMT but did not engage in significant or persistent self-administration of DMT. However, following several days of environmental deprivation (i.e., the absence of light and sound), two of the three monkeys consistently self-administered DMT for up to 20 days, which was the a priori imposed endpoint of the experiment. Moreover, when various psychedelics were substituted in monkeys that were on a baseline of MDMA self-administration, none of the subjects consistently self-administered any of the compounds tested, but all of the subjects transiently and sporadically self-administered psilocybin, mescaline, and DMT at response rates that were comparable to MDMA and up to the maximum number of infusions that were available (Fantegrossi et al., 2004b). A more recent 44 CHAPTER 2 Preclinical psychedelic research

study in baboons used a daily access procedure to space drug availability and an intermittent access procedure with discriminative stimuli to signal drug availability. Two doses of LSD were self-administered at higher rates than saline in the intermittent access procedure, and more infusions of LSD were taken during intermittent access then during daily access (Goodwin, 2016). Another study examined self- administration of 5-methoxy-alpha-methyltryptamine in rats with no history of exposure to other psychedelics using a fixed-ratio 1 schedule of reinforcement and unsurprisingly reported that it appears to have minimal abuse related effects (Abiero et al., 2018). The results of these studies indicate that there may be conditions under which laboratory animals will reliably self-administer some psychedelics. Future experiments should manipulate both the pharmacological history of the subjects and the underlying schedule or reinforcement to determine whether these manipulations will also unmask the reinforcing effect of the psychedelics. Develop- ing a laboratory animal model of psychedelic self-administration would greatly enhance the preclinical study of this important class of psychoactive compounds and facilitate their safe and thoughtful advancement toward the clinic.

7 FIRST IN CLASS TREATMENTS FOR ADDICTIVE DISORDERS The first wave of psychedelic research focused on the treatment of alcoholism, depression, anxiety, neuroses, compulsive behavior, and psychosomatic disorders. A meta-analysis (Krebs and Johansen, 2012) of the six randomized trials conducted with LSD for alcohol dependence during that era (Bowen et al., 1970; Hollister et al., 1969; Ludwig et al., 1969; Pahnke et al., 1970; Smart et al., 1966; Tomsovic and Edwards, 1970) described consistent treatment effects and positive odds ratios sup- porting the efficacy of LSD. Today, there is growing evidence that psychedelics are effective treatments for substance-use disorders. Small scale clinical studies have been completed and published. As mentioned previously, psilocybin treatment in heavily tobacco-dependent smokers—who at intake were 51 years of age on average, had a mean of six previous lifetime quit attempts, smoked a mean of 19 cigarettes per day, and had smoked for a mean of 31 years—resulted in approximately 80% abstinence rates at 6 months (Johnson et al., 2014) and 67% abstinence rates at 12 months (Johnson et al., 2017). Another recent study, reported significant decreases in alcohol consumption after 1–2 session of psilocybin in 10 alcohol-dependent subjects using an open-label design (Bogenschutz et al., 2015). This may be related to 5-HT2A receptors as meta-analysis has revealed an rs6313 single nucleotide polymorphism of the 5-HT2A receptor that is strongly related to vulnerability to substance abuse in general, and most strongly with alcoholism (Cao et al., 2014). An additional study showed that psychedelic therapy is associated with reduced criminal recidivism, which is highly comorbid with substance abuse (Hendricks et al., 2014). This is a vitally important area as millions of adults in the United States have an alcohol- use disorder (AUD) and excessive drinking is directly responsible for many tens of thousands of deaths per year in the United States. Current FDA-approved 7 First in class treatments for addictive disorders 45

treatments for AUD include naltrexone (mu opioid receptor antagonist), disulfiram (enzymatic inhibitor designed to induce a sickness reaction), and acamprosate (a drug of unknown mechanism of action that reduces the desire to consume alcohol). While these therapies have been a tremendous advance for patients suffering from AUD, many individuals remain refractory to such therapy. Meta-analyses of the effects of each of these treatment options have documented significant deficiencies in their clinical efficacy. Disulfiram may reduce the frequency of drinking or induce short-term, transient abstinence (Garbutt et al., 1999; Jorgensen et al., 2011), a clinical profile that must be weighed against the psychological and medical consequences of inducing an untoward, and potentially severe, sickness reaction. Naltrexone reduces heavy and drinking days but does not increase abstinence (Rosner€ et al., 2010b). Acamprosate has marginal effects on the abstinence promo- tion and has no effect on heavy drinking (Rosner€ et al., 2010a). There is a major pub- lic health demand for new targets in the treatment of AUD, which could potentially be met by psychedelics such as psilocybin (de Veen et al., 2017). Preclinical analysis of the potential of psychedelics for AUD is starting to emerge. Self-infusion of alcohol directly into brain reward regions is attenuated by co-infusion of the non-selective 5-HT2 receptor antagonist R-96544 (Ding et al., 2009). In a two-bottle choice oral ethanol self-administration model, infusion of an anti-sense oligonucleotide to down-regulate the 5-HT2A receptor decreased ethanol self-administration when infused into the lateral ventricle (for widespread dis- persion) or the amygdala, had no effect on ethanol self-administration when infused into the hippocampus or raphe, and increased ethanol self-administration when infused into the frontal cortex (Blakley et al., 2001). Systemic administration blocks ethanol-induced behavioral sensitization (Oliveira-Lima et al., 2015) and the expression of an ethanol-induced conditioned place preference (Cata-Preta et al., 2018). In our laboratory, we have recently found in a two-bottle choice oral ethanol self- administration model that systemic administration of DOI significantly suppresses alcohol drinking, without concomitant effects on fluid consumption. These studies were conducted using continuous availability of ethanol in Swiss-Webster mice. Interestingly, a median split of the subjects by ethanol consumption revealed distinct subpopulations of low and high ethanol consuming mice. DOI had no effect in the mice that engaged in little consumption of ethanol, but significant reduced ethanol consumption in high consumption mice, which may represent a vulnerable phenotype (Fig. 1). The underlying pharmacological effects involved in this remain to be established. As mentioned above, the 5-HT2A receptor is a likely candidate. However, 5-HT2A receptor antagonism attenuates the dopamine releasing and behavioral effects of amphetamine (Murnane et al., 2013a), MDMA, and cocaine (Murnane et al., 2013b), as well as drug- and cue-induced reinstatement of extinguished cocaine self- administration in rats (Fletcher et al., 2002; Nic Dhonnchadha et al., 2009) and primates (Murnane et al., 2013b). As psychedelics induce robust downregulation of 5-HT2A receptors (Buckholtz et al., 1985; Leysen et al., 1989; McKenna et al., 1989a; Smith et al., 1999), their therapeutic effects could potentially be mediated 46 CHAPTER 2 Preclinical psychedelic research

FIG. 1

5-Dimethoxy-4-iodoamphetamine (DOI) is a mixed action 5-HT2A/2C receptor agonist as well as a phenethylamine psychedelic. In this study, Swiss-Webster mice were allowed to choose between drinking ethanol (20%) or water 7 days per week under a continuous access schedule. Mice were divided by a median split (50:50) into high ethanol drinkers and low ethanol drinkers. Acute administration of DOI significantly reduced drinking in the high ethanol drinkers over a 24h period. In contrast, DOI has no significant effect in the low ethanol drinkers. All values represent the mean + SEM. *P <0.05 relative to baseline.

by acute agonism of 5-HT2A receptors followed by sustained receptor downregulation. 5-HT2C receptor agonist activity could be an important part of the reductions in the abuse related effects of alcohol by psychedelics as DOI and many other psychedelics have relevant agonist affinity for 5-HT2C receptors and 5-HT2C receptor agonists are known to reduce self-administration of ethanol (Kasper et al., 2013; Tomkins et al., 2002), and 5-HT2C receptor agonism may be preferentially effective in alcohol sensitized animals, another potential vulnerability phenotype (Yoshimoto et al., 2012). Indeed, it has been recently argued that 5-HT2C receptor agonism by psychedelics limits their own potential for addiction (Canal and Murnane, 2017). Alternatively, perhaps the therapeutic effects of psychedelics are medicated by acute 5-HT2C receptor stimulation followed by sustained receptor downregulation of 5-HT2A receptors. It is also unclear whether there is any role for 5-HT1A receptors in these effects. These initial studies strongly warrant a definitive determination of the role of serotonin receptor subtypes in ethanol reward. Moreover, they provide a platform for unmasking the neuroanatomical or neuropharmacological systems critical for therapeutic effects of psychedelics for AUD. Future studies could begin to tie the preclinical models and endpoints to those that would be of most interest and benefit to the researchers studying psychedelics in humans suffering from alcohol 8 First in class treatments for anxiety and depression 47

addiction. The potential therapeutic effects of the psychedelics should also be extended to other drugs of abuse as, for example, the phenethylamine DOM reduced heroin self-administration in three of four nonhuman primates examined in a recent study (Maguire et al., 2013), consistent with treatment responses to LSD in opioid- dependent patients (Savage and McCabe, 1973).

8 FIRST IN CLASS TREATMENTS FOR ANXIETY AND DEPRESSION Mental health problems are a major worldwide source of morbidity and mortality. Anxiety related disorders can be largely categorized into post-traumatic stress disorder, panic disorders, phobias, and generalized anxiety disorder. This spectrum of disorders is diverse but shares common features of threat-relevant responding (e.g., anxious apprehension, fear, and avoidance). Significant risk factors have been identified, including heredity, temperament, cardiac tone, cognitive/discrimination disruptions, and associative learning (Craske and Waters, 2005). These disorders are some of the most common and debilitating mental health disorders in our society. Current pharmacotherapies have limited effectiveness and significant adverse effect profiles fueling the need to develop novel approaches to treating these disorders (Christmas and Hood, 2006). The prototypical classes of medications for anxiety disorders are the benzodiazepines and the barbiturates. The barbiturates have largely been abandoned in favor of the benzodiazepines due to the risk of lethal over-dose. However, both classes of compounds have significant abuse liabilities, sedating effects, carry a significant risk of tolerance or physical dependence developing, and can have dangerous withdrawal symptoms. The novel compound buspirone, which is a 5-HT1A receptor agonist, has achieved significant use in recent years despite a therapeutic lag time and reduced effectiveness compared to the prototypical compounds because of reduced adverse effects. Major depressive disorder is among the most common and debilitating psychiatric conditions in the western world. Although the precise cause of depression is unknown, it is well established that both the noradrenergic and serotonergic systems play a role in both its etiology and treatment. The involvement of these monoaminergic systems became widely accepted with the development of the first antidepressants of the modern age. Introduced in 1957, iproniazid and imipramine worked by inhibiting function of the monoamine degradation enzyme monoamine oxidase. Be- cause of drug–drug interactions and dietary restrictions necessitated by their use, the monoamine oxidase inhibitors were replaced by the selective serotonin reuptake inhibitors (SSRIs), such as fluoxetine, sertraline, and citalopram, as well as mixed serotonin norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine (Ban, 2001). The SSRIs and SNRIs are now mainstay agents for depression, yet they have some significant drawbacks as medications. They have a therapeutic lag time on the order of 2–6 weeks, upwards of 40% of patients are refractory to therapy, and they have a compliance limiting adverse effect profile, including disruption of appetite, sexual 48 CHAPTER 2 Preclinical psychedelic research

dysfunction, nausea, vomiting, irritability, anxiety, insomnia, hyperprolactinemia, cognitive dysfunction, galactorrhea, mammary hypertrophy, gynecomastia, and headache (Nelson, 1999). There is a medical imperative to develop safer, faster, and more effective pharmacotherapies for depression (Pilc et al., 2013). In addition to alcoholism, the first wave of human research with LSD and other psychedelics focused extensively on the treatment of anxiety and depression (Grinspoon and Bakalar, 1986; Riedlinger and Riedlinger, 1994). Initial and successful clinical studies of psilocybin (Carhart-Harris et al., 2016a) and ayahuasca (Sanches et al., 2016) for treatment-resistant depression and LSD for anxiety associated with terminal illness (Gasser et al., 2014) have been completed in the modern age. A recent systematic review of all clinical trials from 1960 to 2017 revealed 11 eligible clinical trials involving a total number of N ¼445 participants, of which 7 investigated LSD, 3 investigated psilocybin, and 1 investigated n,n- dipropyltryptamine (DPT). It was concluded that the more recent trials are of higher quality and support the consistent efficacy of psychedelics for anxiety and depression (Reiche et al., 2018). In an open-label feasibility trial of two oral doses of psilocybin (10 and 25mg, 7 days apart) administered in a supportive setting in patients with moderate-to-severe, unipolar, treatment-resistant major depression, psilocybin was well tolerated and induced marked and sustained improvements in anxiety and an- hedonia (Carhart-Harris et al., 2016a). A survey of 190,000 adult respondents in the United States revealed that lifetime psychedelic use was associated with reduced past month psychological distress, past year suicidal thinking, past year suicidal planning, and past year suicide attempts, in contrast with lifetime illicit use of other drugs, which was associated with higher a prevalence on these measures (Hendricks et al., 2015). A single treatment of psilocybin (0.2mg/kg) occasioned marked and sustained reductions in psychological distress and anxiety symptoms in patients with advanced stage cancer (Grob et al., 2011). Such successful clinical studies warrant the conduct of preclinical research to maximize the potential of psychedelic therapy for anxiety and depression. Pre-clinical studies of anxiety often use the elevated plus maze assay as a predictive test of anxiolytic or anxiogenic effects (Masse et al., 2007). In this assay, a rodent is placed on a plus sign shaped maze that is typically raised two to three feet above the floor. This maze contains one set of arms that is confined by walling off its pe- rimeter whereas the other set of arms is entirely open. Rodents will typically spend far more time in the closed arms than the open arms. However, anxiolytic compounds increase entry into and time spent in the open arms with anxiogenic compounds having the opposite effect. This is a common and widely accepted pre-clinical test of anxiolytic effectiveness. In 1977, Roger Porsolt reported that rodents placed in an inescapable tank of water will eventually cease swimming and begin to float. These results seemed to parallel earlier work with inescapable punishers and the corresponding phenotype of “behavioral despair” or “learned helplessness.” Antide- pressant compounds administered prior to testing decreased immobile behavior without simply increasing gross locomotor activity (Porsolt, 1979; Porsolt et al., 1977, 1978a,b). This test was eventually termed the “forced swim test,” and over 8 First in class treatments for anxiety and depression 49

the next few years it was established that the vast majority of drugs with known antidepressant effects in humans all induced similar behavioral results in this assay (Bourin, 1990). Subsequently, Steru and colleagues developed a similar paradigm based on inescapable stress, in which rodents were suspended by the tail some dis- tance above a platform, and mobile versus immobile behaviors were quantified over some duration (Steru et al., 1987). The utility of what has come to be known as the “tail suspension test” is predicated on the fact that antidepressants also decrease immobile behavior in this assay. Although these assays have fairly similar theoretical underpinnings, there are some compounds which are active in one test but not the other, with the forced swim test generally less sensitive to serotonergic antidepressants (Borsini and Meli, 1988). These assays were instrumental in the development of the SSRIs and are sensitive to the monoamine oxidase inhibitors, noradrenergic compounds, mixed action compounds, and novel compounds such as ketamine, and have since been used in hundreds of studies across numerous industrial, aca- demic, and governmental research programs. There is substantial preclinical support for the anxiolytic and antidepressant effects of psychedelics. In response to chronic corticosterone exposure, mice devoid of 5-HT2A receptors show a more pronounced anxious and depressive-like phenotype in these assays than wild-type mice (Petit et al., 2014). Central administration of brain derived neurotrophic factor produced prolonged reductions in depressive-like behavior in a mouse model of hereditary behavioral disorders, accompanied by an increase in 5-HT1A and 5-HT2A receptor gene expression and 5-HT2A receptor function (Naumenko et al., 2012). Psychedelics have demonstrated efficacy in these preclinical models as DOI produces anxiolytic-like effects in the elevated plus maze test (Nic Dhonnchadha et al., 2003a,b; Onaivi et al., 1995), this effect is blocked by a 5-HT2A selective antagonist (Nic Dhonnchadha et al., 2003b), this effect is elimi- nated by genetic removal of the Gαq protein (Garcia et al., 2007), and DOI poten- tiates the effects of benzodiazepines in this assay (Masse et al., 2007). Furthermore, ketanserin produces an anxiogenic-like effect (Nic Dhonnchadha et al., 2003a). Likewise, low doses of psilocybin hastened the extinction of a conditioned fear memory and increased neurogenesis in the hippocampus, suggesting possible mechanisms for psychedelic inducted reductions in anxiety (Catlow et al., 2013). Ayahuasca also has been reported to engender anxiolytic effects in the elevated plus maze and antidepressant effects in the forced swim test. These effects are comparable to those of fluoxetine and are present in the absence of locomotor stimulant effects or any neurotoxicity, and are related to increased activity in brain regions that receive serotonin projections (Pic-Taylor et al., 2015). DMT, the principle constituent of ayahuasca, induces an initial acute anxiogenic response in the rat, followed by a sustained anxiolytic and antidepressant response, associated with extinction of cued fear memories (Cameron et al., 2018). Consistent with these findings, we have observed that the closely related compound DPT has comparable effects to citalopram in the tail suspension assay (Fig. 2). Corroboration for these findings has been derived from several other classes of compounds. Several tryptamine-like beta-carbolines decrease immobility in the 50 CHAPTER 2 Preclinical psychedelic research

FIG. 2 The tail suspension test is a widely used preclinical assay predictive of antidepressant efficacy in humans. Reduced immobility is indicative of an antidepressant-like effect in this procedure. In this study, Swiss-Webster mice were given an acute administration of the tryptamine psychedelic n,n-dipropyltryptamine (DPT) or the selective serotonin reuptake inhibitor citalopram across a range of doses. Both compounds significantly decreased immobility and appeared to be roughly comparable in efficacy, but with DPT being more potent than citalopram. All values represent the mean + SEM. *P <0.05 relative to vehicle treatment.

forced swim test (Aricioglu and Altunbas, 2003; Farzin and Mansouri, 2006). A variety of natural product constituents have been shown to engender antidepressant effects by stimulating 5-HT2A receptors, at least as far as can be interpreted by antagonism with ketanserin, which notably is a mixed 5-HT2A/2C receptor antagonist (Abbasi-Maleki and Mousavi, 2017; Martinez et al., 2014; Pinto Brod et al., 2016; Pytka et al., 2015). These findings warrant additional study of the mechanism mediating psychedelic induced reductions in anxiety and depression that can inform the clinical literature. One area that appears ripe for investigation appears to be regulation of the 5-HT2A receptor. It is believed that inhibition of neuronal 5-HT2A receptors contributes to reductions in depressive symptoms in humans (Howland, 2016) and laboratory animals (Pandey et al., 2010), which lies in contrast to the agonist effects of the psychedelics. Paradoxically, mice devoid of 5-HT2A receptors show a more (Petit et al., 2014) or a less anxious (Weisstaub et al., 2006) phenotype. Agonist activation of 5-HT2A receptors can enhance both acquisition and extinction of fear memory and fear conditioning (Catlow et al., 2013; Zhang et al., 2013). This is further complicated by the finding that phenethylamines induce rapid tolerance and selective downregulation of 5-HT2A receptors (Buckholtz et al., 1985; Leysen et al., 1989; McKenna et al., 1989a; Smith et al., 1999), that repeated exposure to various stressors that could lead to an anxious phenotype increases the density of 5-HT2A receptors in the cortex (Berton et al., 1998; McKittrick et al., 1995; Metz and Heal, 1986; Ossowska et al., 2001; Takao et al., 1995; Torda et al., 1988), References 51

and that tryptamines may have a much lower propensity to induce tolerance and downregulation of 5-HT2A receptors than the phenethylamines and ergolines (Smith et al., 2014). These findings suggest a complex interplay between vulnerability factors for anxiety disorders and the chronic effects of psychedelic treatment, which may be different across specific psychedelics, disease states, and patient populations. It seems highly likely that additional preclinical studies in this area conducted under tightly controlled conditions are necessary to unravel this complexity, and such studies would be of great value to the field human psychedelic research.

9 SUMMARY This chapter documents the current status of psychedelic research. There are many other interesting topics that had to be excluded for brevity. There are additional bod- ies of research related to the effects of psychedelics on compulsive behavior (Matsushima et al., 2009) as well as spatial learning and memory (Rambousek et al., 2014), as examples. An elegant series of studies by Harvey and colleagues consistently showed that LSD and DOM enhances acquisition of associative learning (Gimpl et al., 1979; Gormezano and Harvey, 1980; Gormezano et al., 1980; Harvey et al., 1982) with aversive (e.g., shock) or appetitive (e.g., water delivery) unconditioned stimuli. A very recent study demonstrated that the atypical psychedelic TCB-2 prevents loss of hippocampal BDNF levels, formation of Aβ plaques, the development of hippocampal neurodegeneration, and the presentation of cognitive impairments in a streptozotocin rat model of Alzheimer’s disease (Afshar and Shahidi, 2018). MDMA is advancing through clinical trials for post-traumatic stress disorder (Doblin, 2002; Mithoefer et al., 2011), and preclinical studies are revealing potentially even safer forms of MDMA (Curry et al., 2018; Pitts et al., 2018). This is an exciting time to be conducting research with psychedelics. It feels as though the field is on the cusp of ground breaking basic science discoveries and game changing advances in clinical practice. Diseases that have remained refractory to therapy throughout the development of modern medicine may be finally becoming treatable. Preclinical research offers tremendous value to this field, and the hope is that it will begin to keep pace with the clinical literature.

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