Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research

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PNEC-2317; No. of Pages 14 Psychoneuroendocrinology (2012) xxx, xxx—xxx

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Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research Adam J. Guastella a,*, Ian B. Hickie a, Margaret M. McGuinness a,b, Melissa Otis a,b, Elizabeth A. Woods a,d, Hannah M. Disinger a,c, Hak-Kim Chan e, Timothy F. Chen e, Richard B. Banati a,f,g a

Brain & Mind Research Institute, University of Sydney, Sydney 2006, Australia Faculty of Arts and Sciences, Boston University, Boston 02215, USA c University of Minnesota, Crookston 55455, USA d College of Pharmacy, University of Kentucky, Lexington 40506, USA e Faculty of Pharmacy, University of Sydney, Sydney 2006, Australia f Faculty of Health Sciences, University of Sydney, Lidcombe 2141, Australia g Australian Nuclear Science and Technology Organisation, Kirrawee 2232, Australia b

Received 3 September 2012; received in revised form 25 November 2012; accepted 26 November 2012

KEYWORDS Social cognition; Nasal spray; Bioavailability; Hormone; Peptide; Human; Blood brain barrier; Absorption; Clinical trials; Methods

Summary A series of studies have reported on the salubrious effects of oxytocin nasal spray on social cognition and behavior in humans, across physiology (e.g., eye gaze, heart rate variability), social cognition (e.g., attention, memory, and appraisal), and behavior (e.g., trust, generosity). Findings suggest the potential of oxytocin nasal spray as a treatment for various psychopathologies, including autism and schizophrenia. There are, however, increasing reports of variability of response to oxytocin nasal spray between experiments and individuals. In this review, we provide a summary of factors that influence transmucosal nasal drug delivery, deposition, and their impact on bioavailability. These include variations in anatomy and resultant airflow dynamic, vascularisation, status of blood vessels, mode of spray application, gallenic formulation (including presence of uptake enhancers, control release formulation), and amount and method of administration. These key variables are generally poorly described and controlled in scientific reports, in spite of their potential to alter the course of treatment outcome studies. Based on this review, it should be of no surprise that differences emerge across individuals and experiments when nasal drug delivery methods are employed. We present recommendations for researchers to use when developing and administering the spray, and guidelines for reporting on peptide nasal spray studies in humans. We hope that these recommendations assist in establishing a scientific standard that can improve the rigor and subsequent reliability of reported effects of oxytocin nasal spray in humans. Crown Copyright # 2012 Published by Elsevier Ltd. All rights reserved.

* Corresponding author. E-mail address: [email protected] (A.J. Guastella). 0306-4530/$ — see front matter. Crown Copyright # 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

Please cite this article in press as: Guastella, A.J., et al., Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research. Psychoneuroendocrinology (2012), http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

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The hormone and peptide oxytocin (OT) is a key modulator of social behavior across mammalian species. In non-human mammals, research shows that central administration of OT agonists enhances social recognition, memory for peers, the development of partner preference, and partner bonding (for a review see (Young and Wang, 2004)). This work provided the foundation for a heated research field administering and studying OT nasal sprays effects on humans (Bartz et al., 2011). This research has shown that, for example, OT nasal spray enhances social cognition by improving social recognition, the encoding of social memories, the retrieval of social cues, and the accurate and positive appraisal of social information at various levels of processing (see Bartz et al., 2011; Guastella and MacLeod, 2012 for thorough reviews of the psychological effects of OT nasal spray in humans). Substantial variation in response to OT nasal spray has also been reported, across individuals, groups, and experimental laboratories. Bartz and colleagues (Bartz et al., 2011) presented a compelling argument for consideration of context and individual difference factors to account for this variation. We recently argued (Guastella and MacLeod, 2012) for a model that located the source of potential variance at four stages of the experimental design, including: (1) the composition of the nasal spray itself, methods of administration, spray deposition, dose and exposure, (2) the methods employed to operationalise hypotheses, including unintended, extraneous factors, (3) the identified individual difference factors that moderate dose response and direction of this response, and (4) the different intended manipulated experimental contexts that moderate response. The existent literature has paid little attention to the first stage of this model, the composition and administration methods of the nasal spray. This is despite numerous published studies using different compositions, different doses, and undefined administration methods. Moreover, the field is constantly challenged about the impact of the various doses employed and, subsequently, the ideal method for conducting dose finding experiments. Perhaps of greatest concern are growing numbers of failures to replicate. These may increase in coming years, potentially undermining hope that OT-based nasal medications can provide therapeutic benefit to treat psychopathology. Failure to adequately ensure effective nasal administration of therapeutic drugs could cause incorrect conclusions that a drug does not have therapeutic benefit. Inability to replicate studies could also result in the proliferation of false positive outcomes. The absence of an appropriate description of scientific method could, therefore, waste millions of dollars in research and community funds and set the field back years in advancing treatment. It is, therefore, of paramount importance that we consider factors that influence absorption and subsequent bioavailability of nasally administered products.

1. Aim of this review We focus this review on the transmucosal nasal drug delivery of OT and the major factors associated with delivery that may contribute to variations in systemic, CNS, and local bioavailability and, thus, the net-effects of the drug. Four pharmacokinetic steps influence nasal administration bioavailability. These include absorption, distribution,

metabolism and elimination, with the first two of these topics the subject of the review here. These include examination of different uptake routes, anatomical variations of the nasal cavity and its capacity, functional state of the nasal mucosa and methods of physical application. Following this review, and in order to improve the quality of methodology in future clinical trials, we propose a standardized protocol for nasal spray administration, as well as questionnaires and check-lists that could be used to record intra- and interindividual variations in nasal function. We finally suggest a set of experimental studies that are required as a priority to advance this research field.

2. Oxytocin in clinical practice In 1909, Sir Henry Dale discovered that extracts from posterior pituitary gland cause contractions of the pregnant feline uterus (Dale, 1908) due to the neuropeptide OT. Nearly 50 years on, it was the biochemist Vincent du Vigneaud who first described the OT molecule structure as a polypeptide. He did this through a nonapeptide derivative to synthesise an octapeptide amide with the hormonal activity of OT (du Vigneaud et al., 1954). Oxytocin is primarily synthesized by, and secreted from, magnocellular cells in the supraoptic and paraventricular nuclei, with receptors spread across the central nervous system, and released into circulation by the posterior pituitary (Gimpl and Fahrenholz, 2001). Its roles in key regulatory systems have been well documented elsewhere, particularly in labor, parturition, milk ejection, and sexual behavior (Gimpl and Fahrenholz, 2001). Therapeutically, OT administration has been used widely in obstetric applications (Stephens and Bruessel, 2012), under the commercial name of Pitocin or Syntocinon. The mode of action of OT in this context is believed to be a peripheral one, where it is used to stimulate the smooth muscle of the uterus to facilitate rhythmic contractions. These effects increase particularly toward the end of pregnancy, in labor, after delivery, and in the puerperium, when there is an increase in the number of OT receptors in the myometrium.

3. Nasal spray administration The principal method of OTadministration for labor induction is intravenous, where one has capacity to tightly control amount and timing of dosing. Such invasive methods are, however, not ideal for researchers and clinicians using OT for experimental and community treatment. Oral administration is also not suitable due to extensive metabolism in the gastrointestinal tract or liver of proteins, peptides (e.g., OT, arginine vasopressin (AVP), desmopressin, calcitonin), and steroid hormones (estradiol, progesterone, and testosterone). Transmucosal nasal drug delivery provides a non-invasive alternative route for drugs with poor systemic bioavailability after oral administration. Intranasal administration causes minimal discomfort for the patient, is simple to use, and typically results in high compliance. No specific skill is required to self-administer and it can be easily used at home. This is particularly important when specific populations, such as children and elderly patients, are recruited or when needle infection may be of concern.

Please cite this article in press as: Guastella, A.J., et al., Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research. Psychoneuroendocrinology (2012), http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

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Oxytocin Nasal Administration Historically, intranasal delivery has been used in applying low molecular weight and hydrophobic drugs for local treatment, such as the management of nasal congestion with sympathomimetic agents (e.g. oxymetazoline, xylometazoline). Here, drug absorption through a mucosal surface is relatively efficient (Zempsky, 1998). A major driving force underlying the widespread use of nasal sprays is the belief that nasal spray methods may provide a more efficient target for CNS mechanisms than local injection of molecules, where CNS target site distribution can be insufficient due to slow rates of brain diffusion relative to elimination (Dhuria et al., 2010; Bahadur and Pathak, 2012). We note this assumption is not without critics (Merkus and van den Berg, 2007). The nonhuman animal research has shown that direct injection of OT to specific brain targets enhances social behavior (Young and Wang, 2004), leading to assumptions that OT administration in humans also causes effects through direct central action. The nasal mucosa is the only location in the body that provides a direct connection between the central nervous system and the environment. Thus, nasal delivery may result in similar systemic effects that are produced by peripheral delivery methods (Landgraf, 1985), and potentially superior absorption for delivery to central CNS targets (Stevens et al., 2011). A significant limitation of nasal spray administration is capacity to control dosing and absorption, and therefore drug response (Bradfield, 1965). For example, while one study showed a 40 International Unit (IU) dose of OT nasal spray substantially improved milk-let down in women who recently gave birth (Ruis et al., 1981), a larger and more recent study failed to replicate these strong findings (Fewtrell et al., 2006). Thus, nasal spray administration methods have not been used widely for therapeutic purposes. While OT is widely available and used for intravenous administration, the nasal sprays are only commercially available in some European countries (e.g., Switzerland). Many researchers outside of these countries are restricted to importing the spray across international borders or engaging manufacturers to compound the spray for research purposes. It does, however, remain unclear which route of absorption of OT nasal spray is associated with the observed effects on social cognition and behavior. While low molecular weight molecules, therapeutic peptides, and proteins are capable of crossing the blood brain barrier (Costantino et al., 2007), OT is a nonapeptide which consists of nine amino acid subunit peptides with a molecular mass of 1007 Daltons (Gimpl and Fahrenholz, 2001). This constitutes a large-sized molecule (Bahadur and Pathak, 2012), making it unclear whether OT molecules sufficiently and directly cross the blood brain barrier to cause the documented impact on CNS function. Interspecies differences in nasal cavity structure limit the ability to extrapolate findings of nasal spray peptide absorption studies in non-humans (e.g., monkeys, rats; (DeSesso, 1993; Chang et al., 2012)) onto humans. For example, the olfactory epithelium makes up 50% of the nasal cavity in rats, but in humans it represents only 3% of the nasal cavity (Merkus and van den Berg, 2007). To date, only one study in humans has examined whether OT-like peptides deliver peptides directly to the brain compartment without peripheral delivery (Born et al., 2002). Born and colleagues administered AVP (which is similar to OT in structure and has a molecular mass of 1042 Daltons) intranasally and found AVP

3 elevated in the CSF but not in peripheral blood. The authors suggest direct diffusion of the peptide molecules into the subarachnoid space through patent intercellular clefts in the olfactory epithelium as the most plausible explanation. There was, however, no data to support the view that this CNS penetration was related to physiological and behavioral effects of the peptide, or that this method of administration resulted in greater absorption than other forms of administration (e.g., intravenous methods). Moreover, other studies have suggested peripheral increases following OT administration (Andari et al., 2010). Further research is also needed to show that the spray consistently causes impact across individuals, formulations and contexts (as discussed below).

4. Absorption and the anatomy of the nose A central goal of drug administration is to deliver an intended dose to a specific biological site of action. The degree to which a drug preparation is capable of being delivered to the site is referred to as the drug’s bioavailability. At this point, however, we cannot determine the bioavailability of OT, since it is not clear which site is being targeted, nor have a set of markers been identified to indicate response of the critical site, or sites, associated with the improvement in social cognition or behavior. Indeed, as noted above, a primary reason OT nasal delivery methods are not widely used in obstetric therapeutic practice relates to difficulties in obtaining consistent responses across individuals and contexts. Transmucosal absorption refers to the uptake of a compound into the circulation after application onto mucosa (e.g., nasal, buccal, or rectal mucosa) (Ghilzai and Desai, 2004). Transmucosal absorption includes drug release, penetration (entry into a layer, permeation, and transition of a layer), and absorption (uptake into the vascular system) (Suter-Zimmermann, 2008).

5. Anatomy of the nose The upper airway is comprised of the nasopharynx, oropharynx, and laryngopharynx; all of which are capable of mucosal resorption to variable degrees (Zempsky, 1998). The human nose is divided by the nasal septum into two compartments; each containing three cavernous structures, the inferior, middle, and superior concha, into which drain the sinuses of the facial skull (see Figure 1). Overall, the internal surface area of the nose is about 150 cm2 and internal nasal volume is approximately 15 ml (Mygind and Anggard, 1984). The main arteries include the maxillary artery, the nasal branches of the facial artery, and the ethmoidal arteries in the area of the cribriform plate. The vasculatures of both the nose and the lower airways have a dense subepithelial capillary network and a system of venules and sinuses or sinusoids. In the anteroinferior part of the nasal septum, in particular, four arteries form a vascular plexus and Kiesselbach’s plexus. The nose has an effective system of vascular countercurrents. For example, arterial blood streams forward along the nasal mucosa are cooled by the venous systems at the front of the nose exposed to cold air. In mammals, the function of this vascular counter-current has been shown to operate differently depending on whether breathing is through the nose or the mouth (Widdicombe, 1993). While

Please cite this article in press as: Guastella, A.J., et al., Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research. Psychoneuroendocrinology (2012), http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

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Figure 1

The human nasal cavity.

the neural control of human nasal vasculature is still not completely understood, the sensory innervation of the nose occurs through the branches of the 5th cranial nerve, the trigeminal nerve (Riederer et al., 1996). It is also known that classic neurotransmitters are the main regulators of nasal vasculature. Larger vessels tend to have a mixed autonomic innervation, while veins are mostly under sympathetic control. The stronger innervation of arteries and cushion veins appears to relate to the control of nasal air flow. Each of the two nasal compartments contains three distinct epithelial layers known as the squamous epithelium, respiratory epithelium, and olfactory epithelium. Embedded in the epithelium are cilia, hair-like structures that beat and cause movement in the direction of the throat. Within the nasal cavity are goblet cells which produce mucus in the posterior segment of the nasal cavity. Nasal mucus is composed of approximately 95% water, 2% mucus glycoproteins, salts, and lipids, and is cleared every 10 to 15 min (Anderson and Proctor, 1983; Ghilzai and Desai, 2004).

5.1. Absorption from nasal spray administration Absorption from nasal spray delivery first occurs through passage of the mucus. The middle section of the nasal cavity, the respiratory region, provides a prominent area for drug absorption due to blood flow and larger surface area (Arora et al., 2002). The anterior section of the nasal cavity is characterized by poor systemic drug absorption compared to the middle section, but this is also where much of the spray is deposited (Southall and Ellis, 2000). Importantly, nasal spray deposition pattern correlates to biological effects (Suman et al., 1999), and some suggest the olfactory region provides the absorption route to the central nervous system (Suman et al., 1999; Southall and Ellis, 2000; Bahadur and Pathak, 2012). A drug administered intranasally may be taken up by one or more of these routes (see Figure 2) with interindividual variation in relative contribution to the net uptake. While not all of these routes have been shown in humans to result in uptake following nasal spray administration, potential pathways we identify here include uptake via (1) nasal vasculature into the systemic circulation, (2) oral mucosa and gastroenterally, (3) olfactory bulb pathways directly into the CSF and brain, including through surrounding lymphatic fluid (Weller et al., 2010; Xia et al., 2011; Bahadur and Pathak, 2012), (4) trigeminal nerve pathways to the brain

stem (Thorne et al., 2004; Qingfeng et al., 2012) and (5) the paravascular spaces that connect into the interstitial spaces of the brain parenchyma (Iliff et al., 2012). The first route (see Figure 2, Route 1) involves the transfer of the nasally administered drug through the epithelium to the network of blood vessels located underneath the basement membrane. Here, the drug is absorbed into the surrounding blood vessels and into the systemic circulation. The second route (see Figure 2, Route 2) is caused by mucociliary clearance. The nasally administered drug is transported with the mucus to the nasopharyx, where it is swallowed. It can then be absorbed on its way across the oral cavity, transferred to the lungs by the trachea, or to the stomach via the oesophagus. The third route (see Figure 2, Route 3) represents one of the most interesting candidates for the effect of nasally administered OT on central mechanisms. The nasally administered drug may be taken up by the olfactory sensory neurons in the olfactory epithelium. It is then transferred through the epithelium and to the olfactory bulb, and transferred to cerebrospinal fluid or brain tissue (Bahadur and Pathak, 2012). The spray may also pass through the nasal epithelium into the perineural space around the olfactory nerve bundles (Weller et al., 2010), and then into the CSF surrounding the brain (Qingfeng et al., 2012). Of particular note for this route the olfactory slit is only 1 mm wide, and is in a location that is difficult for deposition by standard nasal spray bottles (Merkus and van den Berg, 2007). Many studies show that standard nasal spray devices barely deliver to the olfactory cleft (Scheibe et al., 2008), with variation of deposition to the area influenced by a range of factors (formulation, device variables, etc. (Gao et al., 2007)). One study showed that only 1 in 15 individuals who use a standard nasal spray bottle will show deposition on the olfactory cleft (Scheibe et al., 2008). This means that only a small percentage of any given nasal spray dose is likely to pass via this route, if at all, and this amount is likely to be variable between doses, individuals, and experimental contexts. More recently, there has been growing recognition of another potential delivery pathway allowing for direct CNS effect, the trigeminal nerve pathway (see Figure 2, Route 4; (Thorne et al., 2004; Qingfeng et al., 2012), although research in humans documenting nasal spray absorption via this pathway is currently lacking. The trigeminal nerve widely innervates the respiratory and olfactory epithelium and enters the brain stem and the pons. Some animal studies

Please cite this article in press as: Guastella, A.J., et al., Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research. Psychoneuroendocrinology (2012), http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

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Figure 2 Potential OT absorption routes following nasal spray administration in humans. This includes (1) nasal vasculature into the systemic circulation, (2) oral mucosa and gastroenterally, (3) olfactory bulb pathways directly into the CSF and brain, including through surrounding lymphatic fluid, the (4) trigeminal nerve pathways to the brain stem and (5) through the paravascular spaces that connect into the interstitial spaces of the brain parenchyma. It should be noted that only pathway 3 is restricted to deposition via the olfactory epithelium.

using low weight molecules (Thorne et al., 2004; Qingfeng et al., 2012) have shown substantially higher radioactivity in trigeminal nerve branches than in the olfactory bulb following intranasal administration. Subsequent activity associated with this pathway has been noted in the brain stem, pons, spinal chord and medulla. Interestingly, uptake via this pathway may not correlate with change in CSF (Qingfeng et al., 2012), suggesting that CSF has limited potential as a marker of brain delivery via this route. While no human studies suggest the importance of this route for OT administration, it is rather curious that OT nasal administration does impact on heart-rate variability (Kemp et al., 2012). Such an effect could be associated with close links of the trigeminal nerve with the vagus nerve and their associated regulation of heart rate variability. Finally, whilst the brain does not have histologically identifiable lymphatic vessels, there is substantial parenchymal solute transport throughout the CNS (see Figure 2, Route 5). Efficient fluid transport appears to occur along paravascular spaces (Iliff et al., 2012). Drug up-take via the nasal mucosa may occur by flux along paravascular spaces that connect into the interstitial spaces of the brain parenchyma.

6. Nasal cavity structure and environment Many factors influence transmucosal absorption including individual differences in nasal cavity structure and environment, formula, bottle design, and administration methods. Each of these factors will now be discussed.

6.1. Nasal cavity structure A substantial amount of research demonstrates that both nasal anatomy and environment influence absorption upon nasal spray administration. Studies have reported on human nasal models with variations in surface smoothness and geometry that significantly alter deposition (Schroeter et al., 2011). Similarly, small variations in nasal airways can significantly impact deposition (Guilmette et al., 1997; Kelly et al., 2004; Merkus et al., 2006b). For instance, size and volume of nasal cavities are highly variable across individuals and may be related to subject height and dorsoventral head width (Guilmette et al., 1997), age, and gender (Samolin et al., 2007). Males above the age of 16 show larger nasal cavity sizes, on average, in comparison to females (Samolin et al., 2007). Further, abnormally shaped nasal cavities can cause altered deposition patterns following nasal spray administration (Vidgren and Kublik, 1998; Merkus et al., 2006b) and also influence nasal patency (Jang et al., 2002; Korkut et al., 2012). Some degree of septal deviation (e.g., a crooked nose) likely effects the majority of individuals in a number of different ways (Gray, 1980; Cerkes, 2011). The actual prevalence of septal deviation is dependent on the criteria used to characterise it (Smith et al., 2010). Using a strict criteria for ‘severe’ deviation, rates of septal deviation drop to around 20% of individuals (e.g., (Calhoun et al., 1991; Smith et al., 2010)) across gender but increasing with age and physical trauma. Some researchers argue that individuals who exhibit major septal deviation should by routine be removed from nasal spray

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administration studies (Merkus et al., 2006b). Currently, there are few cost effective approaches to assess nasal cavity structure. An ear, nose, and throat specialist may inspect a participant’s nose by means of endoscopy prior to administration of nasal drugs (Wheeler and Corey, 2008). Another option for nasal cavity inspection would be to obtain images of the nasal cavity (Zhao et al., 2004; Smith et al., 2010). There is a need to develop easily administered techniques to characterise anatomical differences in the nasal cavity.

6.2. Nasal cavity environment Mucus tends to move at about 3—25 mm/min (average 6 mm/ min) towards the direction of the throat (Anderson and Proctor, 1983). There are two distinct mucus layers present in the nasal cavity. The bottom layer, the sol layer, is less resistant to flow or less viscous than the upper layer or gel layer (Dahl and Mygind, 1998). The mucus layers are transported by the cilia to the nasopharynx where they are swallowed (Dahl and Mygind, 1998). Any particles that are embedded in the mucus layer are transported out of the nasal cavity through this mechanism. This process involving the movement of the cilia and the mucus layers is known as mucociliary clearance. The rate of mucociliary clearance directly affects absorption. The faster the rate of clearance, the less amount of time the nasally administered drug has for absorption. Alternatively, the slower the clearance rate, the longer the drug has for absorption (Schipper et al., 1991). An autonomically-regulated alternation of nasal congestion and decongestion occurs in a majority of healthy individuals (Littlejohn et al., 1992) in time intervals from 30 min to 6 h. While one nasal cavity is congested, the other is decongested, keeping an almost uniform total nasal airway resistance (Littlejohn et al., 1992). This nasal cycle is not appreciated by most individuals as total nasal airway resistance remains constant. While its significance in healthy individuals is unclear, a congested nasal cavity generally has a slower mucociliary clearance rate than a decongested nasal cavity. In a common cold the reduction of mucociliary clearance rates is more obvious and substantially impairs drug absorption (Dahl and Mygind, 1998; Vidgren and Kublik, 1998). Some chronic health conditions such as cystic fibrosis, polyposis, Kartagener’s syndrome, and Sjogrens syndrome are known to alter mucociliary clearance (Vidgren and Kublik, 1998). It may be improper to administer nasal sprays to these patients due to alterations in capacity for drug absorption. Previous surgery on the nose, a deviated septum, and prolonged use of topical sympathomimetic vasoconstrictor (decongestants) has also been associated with a nonallergic, drug-induced form of rhinitis (Graf, 2005). Moreover, illicit drug users have long known about the fast and direct CNS effects of nasal administration, making it a common administration route for drugs of abuse. Evidence shows that illicit users (e.g., nicotine, opium, cocaine) are at greater risk of developing nasal diseases and infections, and their use may directly and permanently alter the structure of the cavity and nasal environment (e.g., by damaging the epithelium) through repeated inhalation (Trimarchi et al., 2003; Schubert et al., 2011). To illustrate, nicotine smoking has

been associated with cellular hyperplasia, mucosal edema, impaired ciliary function, and evidence of poor nasal patency (Hadar et al., 2009). Moreover, illicit drug abuse, including alcohol abuse, can damage central nervous sites important for nasal drug delivery that have the potential to reduce the efficacy of delivery (e.g., (Rupp, 2003)). Patients who have previously reported a serious history of substance misuse of significant and toxic drugs, such as cocaine, opiate, cannabis inhalation, and heavy nicotine use, should be routinely removed from nasal administration trials. Those with a history of use (e.g., alcohol) should be assessed and statistical methods used to control its potential influence.

6.3. Measurement of nasal environment Methods of assessing both nasal cavity structure and environment are needed. Quantifiable measures of these differences should be used within experimental studies as covariates, and to identify and exclude those patients who, due to more extreme abnormalities, may be highly unlikely to effectively respond to administration. Like nasal cavity structure, however, there exist few cost effective and reliable methods to precisely assess the environment of the nose. Air-flow is known to correlate with the rate of mucociliary clearance and the bioavailability of nasally administered drugs (Marttin et al., 1998). Methods of assessing mucociliary clearance include employing: a gamma-camera to obtain images of radioactively labelled particles as they transit through the nasal cavity (Sakakura et al., 1983), a fluoroscope image intensifier used in a similar manner to the gamma-camera (Yergin et al., 1978), dyes or sweet tasting substances to be placed in the nose (Marttin et al., 1998), rhinomanometry and acoustic rhinomanometry devices (Jones et al., 1991), and self-reports of nasal congestion (van Spronsen et al., 2008). The first of these options is largely impractical. A gammacamera or fluoroscope image intensifier is expensive and participants are exposed to radioactive material. The use of pharyngeal dyes involves depositing a droplet of dye in the anterior part of the nasal cavity (Marttin et al., 1998). The dye travels to the nasopharynx via mucociliary clearance where it can be viewed by pharyngeal inspection. The time it takes from the placement of the dye to viewing the dye in the back of the throat is recorded as the clearance time (Marttin et al., 1998). Alternatively, the experimenter may place a sweet-tasting substance, such as saccharin, in the anterior portion of the nasal cavity. The participant then notifies the test administrator as soon as they taste the saccharine. The time between placement and when they first taste the saccharine is recorded as the clearance time (Marttin et al., 1998). It may be useful to use both a dye and a sweet-tasting substance firstly to avoid the disadvantage of the taste threshold, and also in case of the event that the two layers of mucus have dissociated (Sakakura et al., 1983). Inert dyes are insoluble and will be found in the top layer, while saccharin is soluble and will be found in both layers (Schipper et al., 1991). Rhinomanometry may provide a reliable and sensitive method of air volume flowing through the nose. Outcomes provide measures of nasal airway resistance and patency and

Please cite this article in press as: Guastella, A.J., et al., Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research. Psychoneuroendocrinology (2012), http://dx.doi.org/10.1016/j.psyneuen.2012.11.019

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Oxytocin Nasal Administration involve assessment of both inspiration and expiration resistance (Jones et al., 1991). Determining the shifts in the nasal cycle also has capacity to show nostrils that are congested/ decongested, potentially allowing researchers ability to administer intranasal sympathomimetic agents if congested. Acoustic rhinometry (Szu ¨cs and Clement, 1998), in contrast, uses acoustic reflections to provide information about crosssectional area and nasal volumes within a given distance. Acoustic rhinometry gives an anatomic description of crosssectional areas and volumes of the nasal airway as a function of distance from the nostril. Subjective self-report measures are commonly used to assess nasal congestion, which may take the form of symptom scoring or visual analogue scales (van Spronsen et al., 2008). Some studies have found that nasal obstruction visual analogue scales are associated with objective measures of nasal obstruction using acoustic rhinometry and peak nasal inspiratory flow (Kjaergaard et al., 2009), however, other studies have found a relatively poor association (as reviewed in Schumacher, 2002). Ratings may be used to assess multiple symptoms associated with nasal congestion (e.g., congestion, sinus pain, sinus pressure, headache, sneezing, rhinorrhea, and postnasal drainage). These symptoms can also be summed for a total nasal symptom score (TNSS, e.g., (Bernstein et al., 2011)). Alternatively validated research questionnaires have been developed to assess nasal congestion and associated impact on quality of life (e.g., SNAQ-11, (Fahmy et al., 2002)). We, therefore, suggest the use of self-report measures, rhinomanometry and acoustic rhinometry devices in nasal administration studies when possible.

7. Formulation characteristics 7.1. Ingredients In addition to active compounds (e.g., OT), solutions for nasal spray delivery aim to improve stability, sterility and capacity of absorption. Each formulation includes different ingredients that can be described by its molecular weight, polarity, pH, chemical modification, lipophilicity, chemical form, polymorphism, pKa, solubility, dissolution rate, mucosal irritancy, osmolarity, and particle size (Southall and Ellis, 2000; Inhalation Drug Products Working Group of the Chemistry, 2002; Grassin-Delyle et al., 2012). Such characteristics impact on absorption capacity of nasal sprays through the mucosa, such that the FDA recommends the routine reporting of many of these factors when providing product specifications (Inhalation Drug Products Working Group of the Chemistry, 2002). The fluid itself may be described by its viscosity or resistance to flow. Highly viscous sprays are thick while low viscosity sprays are thin. Viscosity depends on and influences other factors such as droplet size and spray angle. Results of studies examining effects of viscosity on nasal drug delivery are somewhat contradictory (Guo et al., 2005; Foo et al., 2007; Kundoor and Dalby, 2010). Some researchers suggest that highly viscous sprays are more effective for nasal drug delivery through reduced clearance rate and more contact time in the nose to allow for greater absorption (Pennington et al., 1987; Merkus, 2006), while others suggest low viscosity sprays provide greater deposition area (Guo et al., 2005; Foo

7 et al., 2007; Kundoor and Dalby, 2010, 2011), and less unintentional dispersion past the middle region of the nasal cavity, (e.g. leading to swallowing). Surface tension may have a minimal direct effect on spray properties and deposition itself (Foo et al., 2007) but play an important role in determining spray and other properties such as droplet size (Vidgren and Kublik, 1998; Dayal et al., 2004; Guo and Doub, 2006). Compared to larger droplets, smaller particles may be more sensitive to administration conditions and deposit more posteriorly (Inthavong et al., 2006). Smaller droplets are, however, more vulnerable to breathing patterns during spray administration and reach further into the nasal cavity (Guo et al., 2005). Smaller particles or droplets (20 mm) may actually cause problems if they reach too far back into the nasal cavity and lung, thus causing ineffective deposition (Inthavong et al., 2006). Thus, droplet sizes that are either too small (<10 mm) or too large reduce absorption due to ineffective deposition.

7.2. Enhancers Bioavailability may depend largely on permeation enhancers, particularly for high molecular weight drugs such as proteins and peptides like OT (Costantino et al., 2007). For drugs to readily permeate the blood brain barrier from circulation, the drug must be capable of transport across tight junctions (cells with adjoining membranes). Several approaches can increase solubility of low-solubility compounds (Costantino et al., 2007). Absorption rates can be improved using mucoadhesive gels or emulsions or packaging of drugs and vaccines in liposomes, as well as conjugated nanoparticles (Vyas et al., 2006; Romero and Morilla, 2011; Grassin-Delyle et al., 2012; Perez et al., 2012), and the use of pro-drugs that bypass Efflux Transport systems (Dalpiaz et al., 2012). Technology is rapidly advancing, with new methods improving the molecule size and amount that may pass through these systems (Grassin-Delyle et al., 2012; Nance et al., 2012). Such advances have the potential to substantially improve dose response rates of OT nasal spray. Changing dosage forms to powders, drops, or gels and using bioadhesives or absorption enhancers may provide opportunity for an increased systemic uptake (Merkus, 2006). For small and medium-sized particles, using bioadhesives can also optimize the contact time between the formulation and the nasal cavity, thus allowing for more absorption (Turker et al., 2004). Unfortunately, absorption enhancers or promoters can sometimes irritate the nose, and harmfully affect the nasal cavity (Suman et al., 1999; Merkus, 2006).

7.3. Volume and number of sprays The amount of liquid and number of sprays delivered to each nostril has important effects on absorption. Nasal solutions are commonly delivered using metered-dose pump sprays that aim to deliver the same dose after each actuation. Nasal sprays tend to deposit the droplets in the front part of the nose, and it is mucociliary clearance that causes the drug to be transferred throughout the entire nasal cavity (Marx and Birkhoff, 2011). The nasal cavity has a limited capacity that can only absorb a specific amount of fluid. Volumes greater than 100 ml inevitably drip into the posterior pharynx to be

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swallowed by the patient (Harris et al., 1988). Improved absorption results when doses are split between two nostrils, as opposed to one, due to the greater surface area for absorption across both nostrils (Dalton et al., 1987). That is, two doses delivered to each nostril of the nose provides improved absorption over just one dose in one nostril (Merkus, 2006). The number of sprays delivered to the nostril on a given administration session may impact upon absorption. Repeated application to a single nostril likely saturates the nose when this amount exceeds 100 ml (Harris et al., 1988; Merkus and van den Berg, 2007), increasing clearance rate and reducing contact time (Merkus, 2006). In summary, ingredients of nasal spray formula are likely to substantially influence potential for absorption. Researchers may not be in the best position to identify when important formula modifications have been made and should routinely consult chemists to discuss these issues. For this reason, however, all formula ingredients need to be reported in scientific manuscripts for the benefit of this research field.

7.4. Bottle design The decision around use of a delivery device is a critical consideration (Wermeling, 2009). Squeeze bottles are not typically used because they have no metering capacity. The most commonly employed device is a metered multi-dose pump spray bottle and its composition will be described in more detail below. A standard syringe with a Luer fitting for a nasal atomizer may also be used to draw up and administer injection-based drug solutions into the nasal cavity. Interestingly, this delivery method may substantially improve delivery to the olfactory cleft when a squirt system is attached to it (Scheibe et al., 2008). Alternatively, other novel delivery devices have been developed for delivery to the olfactory cleft (Devender et al., 2005). For example, Craft and colleagues (Craft et al., 2012) recently describe using a novel device to deliver insulin over a two minute continuous period. The device used a chamber to cover both nostrils simultaneously. In evaluation, properties of nasal spray tips, dip tubes, and actuators have been identified as important for effective drug delivery (Inhalation Drug Products Working Group of the Chemistry, 2002). Conventional nasal drug delivery systems have been found, at times, to produce only 30—40% of an intended dose (Marx, 2009). A spray with a delivery mechanism that provides a more consistent dose per pump, by maintaining pressure with a sealed tip, may deliver an improved 80% dose (Marx, 2009). This can also prevent evaporation and clogging. If the formulation does not contain preservatives, a sterile filtration with a venting system may prevent microorganisms from entering the system (Marx, 2009). Similarly, more flexible dip tubes (the narrow tube that draws the formulation from the lower part of the bottle into the pump to be sprayed) increase the number of full sprays by directing towards the lowest point in the spray bottle regardless of orientation (Doughty et al., 2010). An actuator (the motor controlling the pump mechanism) can vary by degree of automation. More automated actuations provide less variability across administration doses and the length of the actuator affects the deposition of the spray in the nasal cavity (Vidgren and Kublik, 1998). The FDA

recommends the use of a fully automated actuator to eliminate the possible variability that arises from hand actuations (Inhalation Drug Products Working Group of the Chemistry, 2002). Pumps and actuators for the device may also depend on particle size and viscosity of the formulation (Chandira, 2009). Thus, researchers need to understand the relationship between bottle design and the associated formulation characteristics to choose optimum delivery devices. The spray angle (also called plume or cone angle/geometry) describes the angle of the spray mist as it exits the bottle (Merkus, 2006). Spray angle depends partly on droplet size and the administration angle, thus, the effect it has on deposition is variable across these factors. Consensus on the ideal spray angle is not possible due to the vastly different research methods described in the literature. Some studies report that narrower, smaller spray angles, around 35—458 improve deposition (Merkus, 2006) (CF: (Guo et al., 2005; Kimbell et al., 2007)). Several in vitro and in vivo studies show that deposition efficiency improves with reduced spray angle (Newman et al., 1988; Cheng et al., 2001; Foo et al., 2007). For example, Foo and colleagues found that sprays with small spray angles (less than 508) allowed for improved passing through the nasal valve, while wider, larger spray angles (greater than 708) deposit spray in the anterior region of the nasal valve (Foo et al., 2007). Other studies report no effect of spray angle on deposition (Guo et al., 2005; Kimbell et al., 2007). The issues here have important implications for scientific research. Effective delivery devices may be expensive or inaccessible to researchers. Much of the funding for research development of this technology has been provided by private industry and the transfer of associated knowledge, data, and devices into the public domain remains a key issue. This field offers potential for private-public partnerships. While identifying the optimum delivery device to target the olfactory epithelium is recommended, the reporting of the selected device and its properties should be required in any scientific report and a minimum standard established to ensure optimum delivery methods are routinely used.

8. Methods of spray administration: technique 8.1. Priming According to several studies, priming, or pumping the spray until there is a fine mist is important for some spray pumps when first using the spray and after long periods of non-use (Pennington et al., 1987; Southall and Ellis, 2000). Priming is necessary in order to displace air that may be present in the dip tube (Marx and Birkhoff, 2011). Importantly, preparing the spray for use should not include shaking it unless specified by the chemist or manufacturer, as this may negatively affect spray composition (Doughty et al., 2010).

8.2. Head position for nasal delivery Four positions have been typically identified (Merkus, 2006), the head upright, lying head back, lateral head low and head down and forward. Most studies show that head position has little influence on deposition at a group level (Merkus et al.,

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Oxytocin Nasal Administration 2006a). Ejection velocity of the spray may counteract some of the gravitational effects that could influence deposition of the spray (Aggarwal et al., 2004; Benninger et al., 2004). Head positions may be increasingly important to account for various individualised anatomical differences of the nasal cavity (Merkus et al., 2006b). The notion that head positions could be tailored to an individual’s nasal cavity structure is of significant relevance, yet protocols and methods around how to do this remain absent. A patient may not respond to the drug with one head position, but it may be possible when another head position is employed. Studies investigating these possibilities are required. In summary, in recognition of the state of the current literature, the most important factor in determining the appropriate head position is compliance, as positions that are more uncomfortable result in lower compliance (Dhuria et al., 2010). For this reason, we recommend in standard practice the head upright position as it typically results in the highest compliance.

8.3. Insertion depth and administration angle Several elements should be considered, including how to hold and insert the spray bottle relative to the nose. The spray should not have an insertion depth (how far the spray inserts into the nostril) greater than 15 mm (1.5 cm) to optimize compliance and is best, or highly deposited, at 10 mm (1 cm) inside the nose (Kimbell et al., 2007; Kundoor and Dalby, 2011). Higher administration angles (>608 from tip of spray bottle to horizontal plane) appear to result in improved deposition because lower angles lead to the pooling of the spray in only a small part of the nasal cavity (Kimbell et al., 2007; Kundoor and Dalby, 2011). For example, Weber and colleagues suggest a 458 administration angle from ground where the spray is directed parallel to the middle of the nose (Weber et al., 1999). This angle has been most widely recommended in administration guidelines for various drugs since. Other studies have, however, suggested smaller administration angles (308) may further increase deposition, especially if narrower or smaller spray (plume) angles are used (Foo et al., 2007). The decision of administration angle must therefore be considered in the context of the device chosen and associated plume angle of the associated device.

8.4. Breathing Several studies recommend sniffing while administering a nasal spray, although other studies suggest that breathing rates and patterns minimally affect deposition efficiency or area (Guo et al., 2005; Foo et al., 2007). Some researchers have suggested gentle, quiet, uniform breathing with minimal delay from spray release can reduce nostril drainage of the spray (Kimbell et al., 2007; Garlapati et al., 2009). Similarly, a ‘sniff-like’ inhalation after spraying may allow some ‘droplets’ to move further into the posterior nasal cavity, increasing deposition area (Mygind and Vesterhauge, 1978). Light breathing may be particularly helpful for deposition in patients who have a high amount of mucus (Garlapati et al., 2009). Blocking the opposing nostril while breathing in to administer has also been recommended in a number of pharmaceutical guidelines (PSA, 2012), although there is limited publically available scientific research on this.

9

9. Training and patient compliance: methods for assessing and increasing compliance Patient compliance is particularly important when self-administration occurs in the absence of direct monitoring by the experimenter. To enhance compliance, it is important to provide very clear instructions about how to administer nasal sprays. We provide a copy of our administration protocol and recommend that laboratories either use these instructions or report deviations in scientific reports (see Table 1, point 5). We recommend clear verbal instructions and modelling by the experimenter before any administration. This training should be followed by monitoring of participant’s administration technique at each assessment and correction of technique where necessary. In the case of children, and people who have great difficulty administering nasal sprays, we recommend the use of practice sprays before randomization. For example, in our trial of OT nasal spray treatment for children with autism, aged 3 to 8 years of age, we provided practice sprays to 39 patients at least a week before randomization. Carers were given lessons on how to administer the spray to their child and observed on administration both on the training day and on the actual drug administration day. They were provided with written information illustrating and verbally describing the administration process to refer to at home. Difficulties reported following the week were corrected before randomization to drug. Once patients are adequately trained in how to administer medications, it is then important to assess the rate of compliance to control for missed or incomplete administration. Researchers will need to agree on educational protocols describing what to do if there is a missed dose, an excessive dose is administered, how to store and maintain the intranasal device, and how to respond to any suspected adverse or unusual effect. Techniques must also be employed to monitor adherence. We recommend the use of a monitoring diary to record each administration, each missed administration, and any problems identified during administration. Further, a selfreport daily symptom report of nasal symptoms (VAS, TNSS, etc.) could be routinely included in the monitoring task. Bottles with dose counters of actuations may additionally enable researchers to identify the number of doses ejected by the participant or carer (this does not control for priming and compliance to administration technique). Some of these latest nasal spray devices also include lockout functions that disable the device for a period of time once the actuation has occurred, to reduce the possibility of overdosing or patients who conduct ‘make-up’ doses following a missed administration. In our laboratory, we now measure the remnant liquid in each bottle upon its return. Volume of fluid remaining in the bottle provides a measure of liquid ejected over the administration period. Such variables could be included in covariate analysis and to exclude patients who have insufficiently administered medication. Finally, researchers may wish to consider blood or urine sampling to increase the perception of patients that their use may be monitored for adherence.

10. Future research summary and conclusions Nasal spray technology for the administration of drugs has a long history, but its burgeoning use in experimental research

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Table 1

Factors influencing transmucosal absorption.

1

Anatomy of the nose and airways

2

Nasal cavity environment

3

Formulation

4

Bottle Design

5

Use standardised instructions across experiments.

6

Training and Safety

7

Monitoring Compliance

There is no cost-effective method to assess anatomical nasal cavity differences across patients. Ideally, an ear nose and throat specialist could inspect the nose. Individuals showing major septal deviation should either be removed or noted in any study and controlled for in analysis. As a result of anatomical differences, non-responders need to be identified and new methods (e.g., change of head position during administration) employed to improve response. Thorough histories of nasal congestion and disease should be taken before admission into nasal spray studies. This should include past use of decongestants, surgery and drug use history. Participants with evidence of previous nasal disease, surgery, and dependence on inhaled drugs should be removed from clinical trials. All aspects of drug dependence should be recorded. Individuals with common colds that result in significant nasal congestion should not, when possible, be administered nasal sprays. Assessments of the nasal environment primarily rely on tests of airflow. Researchers may consider the use of dyes, sacharrin, and, in particular, rhinomanometry and acoustic rhinometry devices to assess airflow, nasal patency and size. Self-report measures (e.g., VAS, TINS) should always be employed. All aspects of the nasal spray formulation need to be reported in scientific manuscripts and, if possible, the pH value. Differences in formulation across studies have the potential to interact with factors that influence bioavailability and conclusions between studies and individuals. Researchers need to carefully consider and report on the use of any enhancers. Ideally, liquid should be delivered to both nostrils as opposed to delivering to one nostril or across many sprays that will saturate the nostril. Identically matched placebos should always be employed. The bottle design, including the degree of automation by the actuator, dip tube, bottle type, amount of liquid delivered per spray to each nostril, and the associated plume angle should be described in scientific manuscripts. Bottle design must be seriously considered during protocol development and bottles that specifically enhance delivery to the olfactory epithelium encouraged. Where possible, an automated actuator should be employed. We provide an example of instructions that researchers could use: 1. Use the head upright position in standard practice but allow patients to choose the most comfortable position for themselves. This will likely ensure higher compliance rates. 2. Do not administer if the nose is heavily congested and clear the nose from obvious obstruction before administering. 3. Ensure the bottle is primed and complete a test spray if this is necessary for your bottle type. 4. Close one nostril with one finger while administering the spray to the other nostril1. Upon delivery of the medication, inhale and breathe in lightly. 5. Insert bottle 1 cm into the nostril and encourage an administration angle of 45 degrees into the nose (this instruction needs to be evaluated in the context of the given formulation and bottle type). 6. Alternate administrations between nostrils. Allow time between each re-administration to the one nostril- at least 15 seconds1. Provide active instruction, modelling, and regular monitoring to train participants in administration. Children and their carers, those with spray, nasal, and tactile sensitivities may need to use practice sprays before the experiment. Use written and illustrative instruction to participants. Provide take-home instructions when using nasal sprays at home to encourage standardised administration procedures. Develop a standardised set of protocols to respond to overdosing, missed and incomplete dosing, and strict written instructions on storage and maintenance of devices. Also include procedures for responding to adverse events and a side-effect checklist. Include diaries, dose counters, and, where possible, measure the left-over liquid when each bottle is returned. Self-reports of nasal congestion should be included in daily monitoring. Once identified, routine markers of response should be regularly employed to detect system engagement with the drug so that corrections may be applied as required.

1. We include this point as it is widely recommended in most nasal spray guidelines despite limited publically available data to support its use. In the absence of any known cost, we encourage the inclusion of this instruction at this point in time.

is relatively recent with acceleration of use over a wide variety of research disciplines. This technology provides a relatively safe and easy delivery method for novel interventions in multiple experimental contexts. There is no better example of this than OT nasal spray, which is currently being trialled as a

potential treatment in many countries for numerous psychopathologies and for a wide variety of research questions. Our own laboratory for instance, has trialled the medication for the treatment of autism, schizophrenia, early psychosis, substance misuse, anorexia, anxiety, prader-willi syndrome, and

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Oxytocin Nasal Administration distressed couples. Along with this international research effort, an enormous amount of public and private money, community hope, and time is being directed towards evaluating its therapeutic benefit. Most researchers using OT nasal spray, however, do not have a history of expertise in using this technology and our knowledge about how nasal sprays impact on the CNS and peripheral sites in humans is limited. Evaluation of the various pathways that are impacted upon following OT nasal administration is required (e.g., see Figure 2), along with how the various factors we review here influence this penetration. This evaluation needs to describe how any observed impact on pathways relates to subsequent changes in behavior, cognition, and physiology, since it is the ultimate goal to understand which pathways are responsible for the noted impressive effects. It also requires a critical examination of potential extraneous variables. The role of demand characteristics in experiments, such as the Hawthorne Effect (Roethlisberger and Dickson, 2003), is particularly concerning for a drug that is believed to heighten suggestibility (Bryant et al., 2012) and trust (Kosfeld et al., 2005). We have argued that through the identification of markers of response, and underlying circuitry, researchers will be better placed to identify who has been impacted by the spray and to quantify this impact (Guastella and MacLeod, 2012). This may provide a more accurate indication of drug response than the administered dose. Such research could shed light on whether the nasal administration method is the optimal approach to impact on targeted circuitry to cause the observed effects on social cognition and behavior. If the critical pathway that causes the observed effects on social cognition and behavior is initially peripheral (which then goes on to cause central action), as opposed to initially central, then nasal administration may not be the optimum method of increasing bioavailabilty for therapeutic purposes. Other methods (e.g., patches, injection) could provide greater control over dosing and outcome. It is interesting to note that the critical effects on social cognition have not only been observed following intranasal administration, but also through intravenous injection (Hollander et al., 2007). Further research is required to directly compare the subsequent cognitive, physiological, and behavioral effects of various OT administration methods. The identification of these critical pathways and markers may even suggest the use of alternate novel therapeutics to activate the central release of OT (e.g., AVP, melanocortin (Modi and Young, 2012), galanin (Izdebska and Ciosek, 2010), orexin and serotonin pathways (Ocsko ´ et al., 2012). Alternatively, if the critical pathway is mediated by deposition onto the olfactory cleft, it is almost certainly the case that standard spray bottles are not the ideal delivery device for such treatment. In this case, alternative delivery devices need to be identified. We have highlighted here how nasal anatomy, environment, formula, devices and administration methods all have important significant potential to also alter response to nasal delivery of OT. Difficulties in standardising administration protocols and dose-response have the potential to cause substantial impact on outcome and must be considered alongside dose selection, experimental design, individual variables, and contextual factors. Improved capacity to control delivery factors will also facilitate the study of

11 these other experimental variables, by controlling for a potential source of significant uncontrolled variance within these investigations. In recognition of this, we highlight a need to standardise and report the administration methods used by researchers to deliver nasal sprays to humans. Our recommendations (see Table 1) include considerations to reduce nasal administration volumes to prevent swallowing nasally applied preparations, instructions on how to maximally increase the potential to reach the olfactory epithelium situated in the superior conchae that common nasal sprays hardly moisten, and the systematic exploration of enhancers of transmucosal nasal absorption. We, therefore, suggest the use of these recommendations and guidelines in human intranasal research and emphasise the urgent need to advance effective and well-controlled drug delivery methods for the purpose of OT enhancement to the human brain.

Role of funding source Noted external grant funding was used to assist the development of this review. These sources had no influence on the ideas contained in the manuscript or had any role in its submission.

Conflict of interest Authors A.J.G., I.B.H., M.M., M.O., E.A.W., H.M.D., K.H.C., T.C., and R.B. report no conflicts of interest.

Contributors Authors A.J.G. initiated the review, A.J.G., M.M., M.O., H.M.D., E.W., and R.B. conducted the literature review and wrote the first draft. Authors I.B.H., T.C., and K.C., read and assisted with subsequent versions of the manuscript. Authors H.M.D. and A.J.G. contributed to the development of the figures. All authors contributed to and have approved the final manuscript.

Acknowledgements We would like to thank Daniel Wermeling (University of Kentucky), Anita Van Zwieten, and Lisa Whittle for comments on this manuscript, and Nigel Chen for assistance with manuscript preparation. This research was supported by NHMRC grants (No. 623624, No. 623625, No. 1043664), ARC Linkage grant (No. LP110100513), and a BUPA health foundation grant (No. 2012-00004) to Guastella. We also acknowledge a NHMRC Australian Fellowship (No. 511921) to Hickie.

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