Pharmacokinetics of intranasal amiloride in healthy volunteers

Abstract Anxiety and panic disorders are the most common mental illnesses in the United States and lack effective treatment options. Acid‐sending ion channels (ASICs) in the brain were shown to be associated with fear conditioning and anxiety responses and therefore are potential targets for treating panic disorder. Amiloride is an inhibitor of the ASICs in the brain and was shown to reduce panic symptoms in preclinical animal models. An intranasal formulation of amiloride will be highly beneficial to treat acute panic attacks due to advantages such as the rapid onset of action and patient compliance. The aim of this single‐center, open‐label trial was to evaluate the basic pharmacokinetics (PKs) and safety of amiloride after intranasal administration in healthy human volunteers at three doses (0.2, 0.4, and 0.6 mg). Amiloride was detected in plasma within 10 min of intranasal administration and showed a biphasic PK profile with an initial peak within 10 min of administration followed by a second peak between 4 and 8 h of administration. The biphasic PKs indicate an initial rapid absorption via the nasal pathway and later slower absorption by non‐nasal pathways. Intranasal amiloride exhibited a dose‐proportional increase in the area under the curve and did not exhibit any systemic toxicity. These data indicate that intranasal amiloride is rapidly absorbed and safe at the doses evaluated and can be further considered for clinical development as a portable, rapid, noninvasive, and nonaddictive anxiolytic agent to treat acute panic attacks.


INTRODUCTION
Anxiety disorders are the most common mental illnesses in the United States, and ~19.1% of adults in the United States experienced an anxiety disorder in the past year. 1 Panic disorder is a type of anxiety disorder characterized by physical symptoms, such as shortness of breath, smothering sensations, chest pain, palpitations, dizziness, and emotional-cognitive symptoms, such as fear of losing control. 2 Panic attacks are the core feature of panic disorder but may occur with other syndromes, such as social and generalized anxiety, depression, and suicidal behavior. 3 First-line pharmacological treatments for panic disorder include antidepressants, such as sertraline, paroxetine, and venlafaxine. 4 Unfortunately, antidepressants need weeks to achieve the clinical benefit and do not treat panic attacks acutely. [5][6][7] Benzodiazepines may provide some immediate relief from panic attacks but cannot interrupt an impending attack, and can lead to tolerance, addiction, and substance use disorder. [7][8][9] There is thus urgent and unmet medical need to identify more effective pharmacological approaches to panic attacks and panic disorders, and avoid the addictive potential of benzodiazepines.
Acid-sensing ion channels (ASICs) are proton-activated Na + -permeable channels that belong to the degenerin/ epithelial Na + channel superfamily. 10 ASICs are widely expressed in the brain, including the amygdala, brainstem, and periaqueductal gray. ASICs play a critical role in fear learning and emotional processing. 11,12 By exposing mice to the repeated cross-fostering (RCF) paradigm of early maternal separation, one can evoke stable respiratory and behavioral hypersensitivity to inhaled CO 2 , and replicate several features of human panic in the preclinical animal model. [12][13][14] Genomewide searches into the medulla oblongata (a critical brain structure for respiratory control, extracellular pH changes' detection, and nociception) of RCF mice showed increased expression of the asic1 gene and corresponding increase in medulla oblongata ASIC-1 channels. [15][16][17] A systematic review of animal studies investigating the role of ASICs in panic disorder concluded that most studies showed an association between panic symptoms and ASICs. 18 These data suggest that ASICs are potential targets to treat panic disorder.
In preclinical studies, we showed that inhalation of ASIC inhibitor amiloride prevents panic symptoms in mice that were exposed to anxiogenic stimuli. 19 In another study, acute treatment of intraperitoneal amiloride resulted in anxiolytic effects in mice. 20 Therefore, the current scientific evidence suggests that amiloride may be developed as a potential therapeutic to treat panic attacks.
Amiloride hydrochloride is a pyrazine-carbonylguanidine currently approved by the U.S. Food and Drugs Administration (FDA) as an adjunctive treatment with thiazide diuretics or other kaliuretic diuretic agents in congestive heart failure or hypertension. 21 Amiloride HCl is a potassium-sparing (antikaliuretic) drug that possesses weak (compared with thiazide diuretics) natriuretic, diuretic, and antihypertensive activity. Amiloride is not metabolized by the liver and is excreted unchanged by the kidneys. Therefore, amiloride does not undergo first-pass Email: venkata.yellepeddi@hsc.utah. edu

Funding information
Centre for Addiction and Mental Health Discovery Grant

WHAT QUESTION DID THIS STUDY ADDRESS?
This first-in-human study assessed the pharmacokinetics, and safety of ascending doses of amiloride after intranasal administration in healthy volunteers.

WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?
Amiloride after intranasal administration is rapidly absorbed into systemic circulation (within 10 min) and is safe and well-tolerated in humans at three doses (0.2, 0.4, and 0.6 mg) studied. Amiloride after intranasal administration exhibited dose-proportional increase in area under the curve and showed significantly higher relative bioavailability compared to oral and inhalational routes of administration.

HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?
Anxiety disorders are the most common mental illnesses in the United States. The current treatments need weeks to achieve the clinical benefit, do not treat panic attacks acutely, and may cause addiction and tolerance. Amiloride is an ASIC inhibitor that showed therapeutic efficacy to treat panic symptoms in preclinical models. Intranasal amiloride can be developed as a therapeutic alternative for portable, rapid, noninvasive, and nonaddictive anxiolytic agent to treat acute panic attacks. metabolism. 21 Amiloride is commercially available only as a tablet formulation for oral administration. However, to treat panic attacks, panic disorders, and other anxiety disorders, it would be beneficial to deliver drugs by intranasal route of administration due to advantages including rapid and extensive absorption, ability to bypass bloodbrain barrier, and increased patient adherence. 22 We previously developed an intranasal formulation of amiloride and evaluated its product characteristics, such as physical, chemical, and microbiological stability. 23 In this study, we evaluated the pharmacokinetics (PKs) of intranasal amiloride in healthy volunteers after administration of three single ascending doses (0.2, 0.4, and 0.6 mg). This study is the logical next step toward the clinical translation of intranasal amiloride. The primary goal of this exploratory study is to understand the rate and extent of amiloride absorption after intranasal administration across a dose continuum. In addition, the local and systemic safety of intranasal amiloride were also investigated in this study.

Study design
The study protocol was reviewed and approved by the FDA under an investigator-initiated investigational new drug (IND) application (IND #144445). The study protocol was also reviewed and approved by the Institutional Review Board (IRB), University of Utah (IRB #126328). This study was a prospective, single-center, open-label trial conducted at the Utah Clinical and Translational Science Institute (CTSI), Salt Lake City, Utah. The study was registered on clini caltr ials.gov (identifier: NCT04181008). 24 A total of 15 participants completed the study, with each dose group (0.2, 0.4, and 0.6 mg) consisting of five participants. The study was conducted in three phases starting with 0.2 mg cohort followed by 0.4 mg and 0.6 mg cohorts. As this was the first-in-human trial of intranasal amiloride, a data safety and monitoring committee met periodically to review safety and preliminary PK data before proceeding to the next higher dose.
In all three dosing groups, participants received a single dose of 0.2, 0.4, or 0.6 mg intranasal amiloride. All participants provided a written informed consent prior to the performance of any study-related procedures. After signing the informed consent, participants went through medical history review, which included review of current and previous medication use followed by physical assessment and examination of nasal cavity by an ear, nose, and throat (ENT) surgeon. During the same visit, blood samples were also collected for baseline biochemical, hematological, and kidney function safety assessments. Once the results were reviewed and participant's eligibility was confirmed, the participant was scheduled for the PK assessment part of the study. The schematic outline of the study design is provided as Figure 1.

Study participants
Eligible participants were healthy men or nonpregnant women aged 18-45 years. Participants had to have normal hepatic and renal functions. Participants were excluded from the study if they exhibited any of the following: (1) history of chronic drug, or narcotic abuse, including chronic use of tranquilizers, sedatives, aspirin, and antibiotics; (2) history or presence of major organ dysfunction, malignancy, stroke, or diabetes; cardiac, renal, liver, or severe gastrointestinal disease; or other serious illness; (3) history of conditions that might contraindicate or require caution be used in the administration of amiloride including hyperkalemia with elevated serum potassium levels (greater than 5.5 mEq/L), currently receiving other potassium-conserving agents, such as spironolactone or triamterene, currently receiving potassium supplementation in the form of medication, potassium-containing salt substitutes or a potassium-rich diet, history, or diagnosis of hypersensitivity to amiloride; (4) subjects with abnormal kidney function tests (estimated glomerular filtration <60, and albumin to creatinine ratio >30); (5) women who were pregnant or nursing at the time of screening; (6) subjects who underwent any kind of surgery of the nose and septum within the past year; (7) subjects diagnosed with chronic rhinosinusitis; (8) treatment with any other investigational drug during the 30 days prior to enrollment into the study; (9) subjects who smoke, have a history of smoking, or use nicotine-containing products; and (10) subjects who had donated blood within 30 days prior to study entry, including that withdrawn during participation in any other clinical study.

Intranasal amiloride formulation and administration
The study drug intranasal amiloride was prepared by the Investigative Drug Services Pharmacy, University of Utah Hospital, Salt Lake City, Utah, USA, using a previously published protocol. 23 The protocol for the preparation of formulation is provided as File S1. On the day of dosing, the intranasal amiloride (2 mg/mL) solution was dispensed to the clinical site and the participants self-administered the dose using an intranasal mucosal atomization device MAD Nasal, Teleflex. For participants receiving 0.2 mg dose, 50 μL of the intranasal amiloride solution (2 mg/mL) was atomized in each nostril. For participants receiving 0.4 mg dose, 100 μL of the intranasal amiloride solution (2 mg/mL) was atomized in each nostril. For participants receiving 0.6 mg dose, 100 μL of the intranasal amiloride solution (2 mg/mL) was atomized three times alternating each nostril for each atomization. To avoid leakage of the drug solution from the nostrils, participants were instructed to tilt their heads upward at 45 degrees angle before the intranasal administration and then continue keeping it in this position for 1 min after the prescribed dose was administered.

Pharmacokinetics
For PK analysis, a series of blood samples were collected at 0 (predose), 10, 15, 30, and 60 min, and 2, 4, 6, 8, and 24 h. The blood samples were collected in glass BD Vacutainer tubes with K 2 -EDTA as an anticoagulant. The plasma was separated from the blood samples immediately by centrifuging at 4°C at 5000 rpm for 15 min. The separated plasma was stored at −80°C until analyzed.

Safety
Safety assessments included a review of adverse events, clinical laboratory evaluations, vital signs, physical examinations, and 12-lead safety electrocardiograms (ECGs). The list of clinical laboratory evaluations performed for each participant before and after drug administration are provided in File S2. The local safety of intranasal amiloride was assessed 24 h after the study for each participant using the anterior rhinoscopy technique.

Measurement of amiloride concentrations in plasma samples
Plasma samples were analyzed at the Center for Human Toxicology, University of Utah, Salt Lake City, Utah. Sample analysis utilized solid phase extraction (SPE) followed by ultra-high-performance liquid chromatography -electrospray ionization -tandem mass spectrometry (UHPLC-ESI-MS/MS). The human plasma sample aliquot volume was 300 μL, and 5-H-amiloride was added as the internal standard. Reference materials for amiloride and the internal standard were obtained from Toronto Research Chemicals. Prior to F I G U R E 1 Schematic representing the clinical trial design to evaluate the pharmacokinetics of intranasal amiloride in healthy human volunteers. EKG, electrocardiogram; LC-MS/MS, liquid-chromatography tandem mass spectrometry. extraction, the pH of the human plasma was adjusted by addition of 0.5 mL of 2% (v/v) ammonium hydroxide. SPE was performed in a centrifuge using 60 mg (3 mL) Waters Oasis HLB cartridges. The SPE cartridges were conditioned with methanol and water prior to loading the samples. Cartridges were washed with 2% methanol in water, spun to dryness, and eluted with 2% formic acid in methanol. SPE eluents were evaporated to dryness, and the extracts were reconstituted in 10 mM ammonium formate (pH 4.0) for analysis.
The UHPLC-ESI-MS/MS system was a Waters Acquity UPLC autosampler and pumping system interfaced with a Waters Quattro Premier XE triple quadrupole mass spectrometer. Chromatographic separation was performed on a Phenomenex Luna Omega C18 column (2.1 × 50 mm, 1.6 μm particle size) using gradient elution with a cycle time of ~6 min per injection. The mobile phases were

Pharmacokinetic and statistical analyses
Descriptive statistics were used for baseline and demographic characteristics, safety data, and PK parameter estimates. Plasma PK parameters were calculated using the noncompartmental method in PKanalix version 2021R1 (Antony, France, Lixoft SAS, 2021, http://lixoft.com/ produ cts/PKana lix/). The area under the curve (AUC) was calculated using the Linear Trapezoidal Linear method of PKanalix. The elimination half-life was calculated using a linear regression equation of the line connecting the time points of the terminal elimination phase. Preparation and visual plots and statistical analyses were performed with Graph Pad Prism version 9.4.1 for windows, GraphPad Software using procedures appropriate for the analysis.

Study population
Overall, 17 subjects met inclusion/exclusion criteria and enrolled in the study. Of these, 15 participants completed the study, and two participants withdrew from the study prior to drug administration. The two participants withdrew from the study due to time constraints. The demographic characteristics of the participants are summarized in Table 1. We have not observed any leakage of the amiloride solution from the nostrils of all participants in this study.

PK analysis
Mean plasma-concentration versus time profiles for amiloride at three doses 0.2, 0.4, and 0.6 mg are shown in Figure 2. In all 15 participants, amiloride concentrations were detected within 10 min of administration indicating a rapid absorption of amiloride after intranasal administration (Figure 2b). The individual plasma concentration versus time curves for each participant are provided in File S3. The plasma concentration versus time data showed that amiloride after intranasal administration showed biphasic PKs with an initial rapid absorption phase around 10 min followed by a second absorption phase between 4 and 8 h. The AUC from zero to 24 h (AUC 0-24 ) values increased with increase in dose from 0.2 to 0.6 mg. The dose-proportionality as calculated using the linear equation fitted to dose versus maximum concentration (C max ) and AUC data showed an R 2 value of 0.19 and 0.5, respectively (R 2 = 0.9607). The dose proportionality curves for C max and AUC 0-24 are provided in Figure 3. The estimated values for clearance and volume of distribution varied from 19.1 to 21.7 L/h and 523 to 592 L, respectively, for all three doses tested. There were no dose-dependent changes in total clearance of amiloride from plasma or volume of distribution, indicating linear kinetics. The details of all PK parameters for three dosing groups 0.2, 0.4, and 0.6 mg are provided in

Safety and tolerability
In this single ascending dose PK study, intranasal amiloride was well-tolerated by healthy volunteers. No grade 2+ adverse events were reported in this study. The comprehensive metabolic panel, kidney profile, complete blood count with platelet count and auto differential, estimated glomerular filtration rate (EGFR), and cardiovascular tests before and after administration with intranasal amiloride did not report any serious adverse events. In a small number of participants (n = 3), the anterior rhinoscopy after intranasal administration showed mild redness in the nostrils. However, this was believed to be due to seasonal dryness in the weather and not an adverse event.
The detailed report on the adverse events reported in this study for each participant is provided as File S6. The ECG data for all 15 participants did not show any QTc prolongation or other ECG abnormalities. The QTc data for all 15 participants in this study are provided in File S7.

DISCUSSION
This study documents the first-in-human administration of intranasal amiloride at three doses 0.2, 0.4, and 0.6 mg. Amiloride is a potent inhibitor of ASICs and has shown promising responses in preclinical models of panic. 19,20 For the first time, we report the PKs of amiloride in humans after single-dose intranasal administration at three doses. Intranasal amiloride was well-tolerated systemically with no cardiovascular adverse events. However, in a few patients', mild redness in the nostrils was observed that was attributed to the seasonal allergies by the ENT. In all participants, the amiloride levels were seen in blood within 10 min of administration. The data obtained from this study will be useful to determine dosing for the future efficacy studies in patients with anxiety. The PKs of amiloride in humans were previously evaluated after oral and inhalational routes of administration. [25][26][27] Currently, there are no reports of PKs of amiloride after intravenous administration in humans. We calculated the dose normalized relative bioavailability of amiloride after intranasal administration compared to oral and inhalation routes by taking the ratios of AUC 0-24 for intranasal and each of oral and inhalational routes. The AUC 0-24 values for oral and inhalational routes were obtained from a human clinical PK study of amiloride after oral and inhalational routes reported by Jones et al. 25 The percent relative bioavailability of intranasal amiloride compared to oral route was 218% and compared to inhalation route was 520%, indicating that intranasal amiloride has significantly improved bioavailability compared to oral and inhalation routes. The detailed information of the equations and calculations of the percentage of relative bioavailability are provided as File S8. This is expected as intranasal route provides easy access of amiloride to systemic circulation via olfactory and nasal epithelia. Similar to the data obtained in this study, Jones et al. reported a double peak phenomenon after inhalational administration of amiloride. 25 This double-peak phenomenon after inhalation administration was attributed to direct pulmonary absorption first, followed by extrapulmonary absorption. Based on the individual PK data we could not conclude any baseline covariate from the covariates age, sex, race, weight, and height that  could explain the interindividual variability. Some of the sources for interindividual variability in this study include the type of intranasal device used and the technique of selfadministration. In our future comprehensive PK studies, we will perform covariate analysis using the population PK approach to identify potential sources of interindividual variability. Overall, these data suggest that intranasal administration of amiloride is superior to oral and inhalational routes in terms of optimal bioavailability and for providing rapid onset of action. It is reported that ASICs are enriched in human neurons and are distributed widely in the central nervous system. 28 The studies on membrane topology of ASICs have confirmed that ASICs are predominantly present as extracellular domains neurons and are activated by changes in the extracellular environment. 29 Therefore, the concentration of amiloride in the brain is the major determinant of amiloride's anti-anxiety efficacy and rapid absorption of amiloride via nose-to-brain pathway is highly beneficial for rapid onset of action to treat acute panic attacks. Intranasal administration of amiloride can treat the acute panic attacks by initial rapid absorption of amiloride via nose-to-brain pathway followed by providing a sustained anti-anxiety effect by absorption through nasal epithelial and non-nasal routes.
Perhaps the most important aspect of this study is the amiloride PK profile wherein amiloride was detectable with all three doses within the first 10 min after nasal administration. Furthermore, the PK profile at higher doses (0.4 and 0.6 mg) shows that amiloride concentrations peak rapidly (within 10 min) after intranasal administration followed by a quick decrease and then the concentrations slowly increase to the initial C max at 24 h. Therefore, with a single intranasal dose we can rapidly achieve concentrations to reduce the panic symptoms and then maintain the anti-anxiety effect for up to 24 h. This supports the strategy of using intranasal administration as a ready-to-use therapeutic to "nip in the bud" an impending panic attack in the real-world context. Interestingly, CO 2 hypersensitivity is common to panic disorder and post-traumatic stress disorder, 30 and provided ASICs mediate acute anxiety responses to CO 2 , 10 the amiloride spray-mediated ASIC inhibition may prove effective for immediate relief both from panic attacks and post-traumatic stress disorder symptoms.
The FDA approval of Spravato® (esketamine), 31 Narcan® (naloxone hydrochloride), 32 Nayzilam® (midazolam), 33 Lazanda® (fentanyl), 34 and Valtoco® (diazepam) 35 within a short timeframe of 3 years, marks the beginning of a new era in the development of intranasal therapies to treat central nervous system (CNS) disorders. All these drugs are intended for rapid onset of action to treat acute CNS disorders, such as sedation, pain, reversal of drug overdose, and depressive episodes. These approvals strongly support the nose-to-brain strategy for delivering drugs to the CNS with improved bioavailability and rapid onset of action. Our previous studies using a physiologically-based pharmacokinetic modeling approach has shown that amiloride can reach the brain after intranasal administration. 36 However, these predictions must be verified by future clinical studies. Based on the recent FDA approvals and data from our studies the chances for successful clinical translation of our intranasal amiloride are very high.
In this study, we provided proof-of-concept that amiloride can be delivered intranasally with rapid absorption (~10 min) into systemic circulation and without any local or systemic toxicities. The rapid absorption of amiloride into systemic circulation is due to passive diffusion across olfactory and nasal epithelia which are predominant routes for nasal drug absorption. 37,38 Our study has several limitations. First, is the use of the MAD Nasal device to deliver amiloride intranasally. Further PK studies involving use of a nasal spray device specifically designed to deliver amiloride must be conducted as the use of MAD Nasal devices is not feasible for long-term use by patients. Second, our first sample collection timepoint after intranasal administration of the drug is at 10 min. As our drug is intended for rapid onset of action, more frequent sample collection time points within first the 10 min, such as 2, 4, 6, and 8 min, must have been included for accurate determination of the absorption rate constant. In our future phase I PK study with the optimized nasal spray formulation, we will include the timepoints (1, 2, 4, 6, 8, and 10 min) closer to the drug administration. Third, we did not have enough timepoints in the terminal phase for accurate determination of elimination half-life. This was primarily due to our inability to house the participants overnight at the study facility. The future phase I study will include adequate timepoints to ensure appropriate estimation of the terminal half-life and AUC 0-∞ . Our future studies will include optimization of the amiloride nasal spray formulation and selection of a suitable nasal spray device to allow long-term amiloride use to treat panic attacks.
In conclusion, this study demonstrates that, for the doses tested, intranasal amiloride has rapid absorption and dose-dependent accumulation in humans. Intranasal amiloride after single-dose administration was welltolerated by the study subjects with no major systemic adverse events. Based on this study in healthy volunteers, it is evident that amiloride can be successfully delivered intranasally and can be developed as a therapeutic alternative for portable, rapid, noninvasive, and nonaddictive anxiolytic agent to treat acute panic attacks.