1521-009X/47/7/690–698$35.00
`DRUG METABOLISM AND DISPOSITION
`U.S. Government work not protected by U.S. copyright
`
`https://doi.org/10.1124/dmd.118.085977
`Drug Metab Dispos 47:690–698, July 2019
`
`Pharmacokinetic Interaction between Naloxone and Naltrexone
`Following Intranasal Administration to Healthy Subjects
`
`Philip Krieter, C. Nora Chiang, Shwe Gyaw, Phil Skolnick, and Rebekah Snyder
`
`National Institutes of Health, National Institute on Drug Abuse, Bethesda, Maryland (P.K., C.N.C., S.G., P.S.); and Sekisui XenoTech,
`LLC, Kansas City, Kansas (R.S.)
`
`Received December 18, 2018; accepted April 11, 2019
`
`ABSTRACT
`
`Naloxone (17-allyl-4,5a-epoxy-3,14-dihydroxymorphinan-6-one HCl),
`a m-opioid receptor antagonist, is administered intranasally to re-
`verse an opioid overdose but its short half-life may necessitate
`subsequent doses. The addition of naltrexone [17-(cyclopropylmethyl)-
`4,5a-epoxy-3,14-dihydroxymorphinan-6-one], another m-receptor
`antagonist, which has a reported half-life of 3 1/2 hours, may extend
`the available time to receive medical treatment. In a phase 1 phar-
`macokinetic study, healthy adults were administered naloxone and
`naltrexone intranasally, separately and in combination. When ad-
`ministered with naloxone, the Cmax value of naltrexone decreased
`62% and the area under the concentration-time curve from time zero
`to infinity (AUC0–inf) decreased 38% compared with when it was
`given separately; lower concentrations of naltrexone were observed
`as early as 5 minutes postdose. In contrast, the Cmax and AUC0–inf
`values of naloxone decreased only 18% and 16%, respectively, when
`
`given with naltrexone. This apparent interaction was investigated
`further to determine if naloxone and naltrexone shared a transporter.
`Neither compound was a substrate for organic cation transporter
`(OCT) 1, OCT2, OCT3, OCTN1, or OCTN2. There was no evidence of
`the involvement of a transmembrane transporter when they were
`tested separately or in combination at concentrations of 10 and
`500 mM using Madin-Darby canine kidney II cell monolayers at
`pH 7.4. The efflux ratios of naloxone and naltrexone increased to six
`or greater when the apical solution was pH 5.5, the approximate
`pH of the nasal cavity; there was no apparent interaction when
`the two were coincubated. The importance of understanding how
`opioid antagonists are absorbed by the nasal epithelium is magnified
`by the rise in overdose deaths attributed to long-lived synthetic
`opioids and the realization that better strategies are needed to treat
`opioid overdoses.
`
`Introduction
`
`Opioid overdose in the United States led to 750,000 emergency
`department visits and more than 49,000 opioid-related deaths in 2017
`(https://www.drugabuse.gov/related-topics/trends-statistics/overdose-
`death-rates). The use of the antagonist naloxone (17-allyl-4,5a-epoxy-
`3,14-dihydroxymorphinan-6-one HCl) has been endorsed by multiple
`government agencies to limit opioid-induced fatalities (https://
`www.surgeongeneral.gov/priorities/opioid-overdose-prevention/
`naloxone-advisory.html). Improvised naloxone kits for intrana-
`sal administration have been promoted for use by nonmedical
`personnel and the general public to counteract opioid overdoses
`(Carpenter et al., 2016); however, approximately one-half of subjects
`in a human use study could not assemble and use the device without
`proper training (Edwards et al., 2015). In 2015, the U.S. Food and
`Drug Administration approved Narcan, an intranasal device that
`
`This work was supported by the National Institutes of Health National Institute
`on Drug Abuse [Contracts N01DA-12-8905, N01DA-13-8920, and N01DA-14-
`8914].
`P.K. and S.G. are employees of the National Institutes of Health (NIH). C.N.C. is
`a former NIH employee. P.S. is an employee of Opiant Pharmaceuticals; he was
`employed by the NIH when this study was designed and executed. R.S. is an
`employee of Sekisui XenoTech LLC.
`https://doi.org/10.1124/dmd.118.085977.
`
`delivers 4 mg naloxone in a volume of 0.1 ml. Ninety percent of
`subjects were able to use it correctly without any training; it also
`produces plasma concentrations as rapidly as an intramuscular
`injection (Krieter et al., 2016). Due to its short half-life, naloxone
`may be effective for 1 hour or less, and depending on the quantity and
`nature of the opioid ingested the person could relapse into respiratory
`depression (Li et al., 2018) before trained medical personnel respond.
`Naltrexone [17-(cyclopropylmethyl)-4,5 a-epoxy-3,14-
`dihydroxymorphinan-6-one], a m-opioid receptor antagonist, has a
`reported half-life of approximately 3 1/2 hours (Yuen et al., 1999) and
`has a 5-fold higher affinity for the receptor compared with naloxone
`(Cassel et al., 2005). While the duration of occupancy of naloxone on the
`m-receptor has a half-life of 2 hours (Kim et al., 1997), naltrexone has a
`half-life duration of 72 hours (Lee et al., 1988). This is longer than the
`plasma half-life of naltrexone and its major metabolite 6b-naltrexol
`(Meyer et al., 1984), suggesting it remains on the receptor longer than
`indicated by the plasma concentrations. Combining it with naloxone
`may increase the window for response to an opioid overdose.
`An initial study demonstrated that naltrexone can be absorbed after
`nasal administration of 2 mg in 1 ml of water; the Cmax value was
`3.86 ng/ml at 0.38 hours (Brown et al., 2014). When a crushed extended-
`release oxycodone tablet containing 3.6 mg naltrexone was administered
`intranasally, the maximum concentration of the antagonist was 4.4 ng/ml
`at 0.3 hours (Setnik et al., 2015).
`
`ABBREVIATIONS: AUC0–inf, area under the concentration-time curve from time zero to infinity; D-PBS, PBS containing 0.2% bovine serum
`albumin; HEK293, human embryonic kidney 293; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MDCKII, Madin-Darby canine
`kidney II; m/z, mass-to-charge ratio; naloxone, 17-allyl-4,5a-epoxy-3,14-dihydroxymorphinan-6-one HCl; naltrexone, 17-(cyclopropylmethyl)-4,5a-
`epoxy-3,14-dihydroxymorphinan-6-one; OCT, organic cation transporter; Papp, apparent permeability; PK, pharmacokinetic.
`
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`PK of Naloxone and Naltrexone with Intranasal Administration
`
`691
`
`While naltrexone is indicated for the prevention of relapse to opioid
`dependence, it has not been evaluated for the reversal of an opioid
`overdose. A pilot study was conducted to assess the feasibility of
`combining naltrexone with naloxone as an intranasal formulation to
`lengthen the time for the reversal of opioid-induced respiratory de-
`pression in emergency situations. The results demonstrate reductions in
`plasma concentrations when the two drugs were combined relative to
`when they were given separately; the reduction in naltrexone concen-
`trations was more pronounced than for naloxone. In vitro studies were
`conducted to determine the nature of the interaction. This report details
`the results from both the clinical and in vitro studies.
`
`Pharmacokinetic Study
`
`Materials and Methods
`
`Study Participants. The clinical study was conducted by Vince & Associates
`Clinical Research (Overland Park, KS). The study was approved by the MidLands
`Independent Review Board (Overland Park, KS); all subjects gave written,
`informed consent before participation. The study was carried out in accordance
`with the 1996 International Conference on Harmonization for Good Clinical
`Practices Guidelines (http://www.ich.org/fileadmin/Public_Web_Site/
`ICH_Products/Guidelines/Efficacy/E6/E6_R1_Guideline.pdf). The study was
`registered on ClinicalTrials.gov as NCT03851731.
`Healthy male and female volunteers aged 18–55 years, with body mass index
`of 18–30 kg/m2, participated in the pharmacokinetic (PK) study. Participants were
`currently not taking either prescription or over-the-counter medications, and
`nonsmokers or those who smoked 20 or fewer cigarettes per day were enrolled.
`Screening procedures conducted within 21 days of study initiation included the
`following: medical history, physical examination, evidence of nasal irritation,
`12-lead ECG, complete blood count, clinical chemistry, coagulation markers,
`hepatitis and human immunodeficiency screening, urinalysis, and urine drug
`screen. Female participants were tested for pregnancy at screening and
`admission to the clinic. Participants were excluded if they had abnormal nasal
`anatomy or symptoms (e.g., runny nose, nasal polyps), an upper respiratory
`tract infection, used opioid analgesics for pain relief within the previous
`14 days, or in the judgment of the investigator had significant acute or chronic
`medical conditions. Participants were required to abstain from grapefruit juice
`and alcohol 72 hours prior to admission to the end of the last blood draw of the
`study. On days of dosing, a participant’s vital signs were required to be within the
`acceptable range before receiving naloxone, defined as: systolic blood pres-
`sure .90 and #140 mm Hg; diastolic blood pressure .55 and #90 mm Hg;
`resting heart rate .40 and #100 beats per minute; and respiratory rate .8
`and #20 respirations per minute.
`Study Design. This was an inpatient, double-blind, randomized, three-period,
`three-treatment, six-sequence, crossover study. Participants were randomly
`assigned to one of six possible sequences. On the day after clinic admission,
`participants were administered the study drugs in randomized order with a 4-day
`washout period between doses. Participants remained in the clinic for 13 days
`until all three treatments were administered; they received a follow-up phone call
`3–5 days after discharge. They fasted from midnight before each dosing day until
`1 hour after dose administration. Participants refrained from smoking and
`caffeine-containing drinks for 1 hour before until 2 hours after dosing. They
`received one of the following three treatments in one nostril:
`
`Treatment 1: Naltrexone (2 mg), intranasally; (one 0.1 ml spray of a
`20 mg/ml formulation);
`Treatment 2: Naloxone (4 mg), intranasally; (one 0.1 ml spray of a
`40 mg/ml formulation); or
`intranasally
`Treatment 3: Naltrexone (2 mg) and naloxone (4 mg),
`(one 0.1 ml spray of the 20 mg/ml naltrexone plus 40 mg/ml naloxone
`formulation).
`
`The study drugs were administered in the supine position, and subjects remained
`in this position for approximately 1 hour after dosing. Participants were instructed
`not to breathe when the drug was administered to simulate an opioid overdose
`with a patient in respiratory arrest. Twelve-lead ECGs were collected predose and
`at 1 and 6 hours postdose. Venous blood samples (4 ml) were collected for the
`analyses of plasma naloxone and naltrexone concentrations at predose; 2.5, 5, 10,
`
`15, 20, 30, 45, and 60 minutes; and 2, 3, 4, 6, 8, 12, 24, 30, 36, 48, 60, and 72 hours
`postdose using Vacutainer tubes containing sodium heparin. The plasma was
`stored at , 260°C until analyzed.
`Study Drugs. Naltrexone HCl and naloxone HCl powders were purchased
`from Mallinckrodt, Inc. (St. Louis, MO) and were current Good Manufacturing
`Practices grade (https://www.fda.gov/drugs/pharmaceutical-quality-resources/
`current-good-manufacturing-practice-cgmp-regulations). The formulations were
`made by the pharmacists at Vince & Associates Clinical Research; sterile water
`for injection was the vehicle for both compounds. The study drugs were
`administered using a LMA mucosal atomization device (Teleflex Medical Europe
`Ltd., Athione, Ireland) and a 1-ml disposable syringe. The syringes and devices
`were weighed before and after dose administration. Based on the dose analysis
`and weight of the dose administered, the mean 6 S.D. of milligrams administered
`were the following: treatment A, 2.24 6 0.03 mg naltrexone HCl; treatment B,
`4.58 6 0.05 mg naloxone HCl; and treatment C, 2.27 6 0.07 mg naltrexone HCl
`plus 4.62 6 0.15 mg naloxone HCl.
`Analytical Methods. Plasma naloxone concentrations were assayed as
`described previously (Krieter et al., 2016); the lower limit of quantitation was
`0.01 ng/ml. The interday precision of the calibration curves and quality control
`samples ranged from 3.22% to 9.05% and the accuracy ranged between 23.14%
`and 5.33% during the analysis of the samples.
`Plasma naltrexone and 6b-naltrexol concentrations were determined using a
`validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay
`by XenoBiotic Laboratories (Plainsboro, NJ). Plasma samples (0.15 ml) were
`mixed with 0.1 ml of 1% formic acid in water and 0.05 ml of acetonitrile:water
`(2:8) containing the internal standards (0.5 ng naltrexone-d3 and 0.25 ng
`6b-naltrexol-d3) and added to individual wells of a preconditioned 96-well plate.
`The plate was washed sequentially with 1% formic acid in water, water, methanol:
`water (1:1), and methanol. The analytes were eluted using 4% ammonium
`hydroxide in methanol. After evaporation, the residue was dissolved in 0.15 ml
`methanol:0.1% formic acid (8:92) and submitted to LC-MS/MS analysis. The AB
`MDS Sciex API-5000 LC-MS/MS system (Framingham, MA) with an atmo-
`spheric pressure chemical ionization source was operated in the positive ion
`detection mode. The mobile phase consisted of a gradient from 93% mobile phase
`A (10 mM ammonium formate, pH 4.0)/7% mobile phase B [acetonitrile:
`methanol (2:8)] to 80% mobile phase A/20% mobile phase B over 1.7 minutes at a
`flow rate of 0.5 ml/min through a 2.1  50 mm Kinetex EVO C18 2.6 mm column
`(Phenomenex, Torrance, CA). Naltrexone eluted at approximately 1.45 minutes;
`ions monitored had mass-to-charge ratios (m/z) 342.2 and 324.2 for naltrexone
`and 345.2 and 327.3 for its internal standard. 6b-Naltrexol eluted at approx-
`imately 1.60 minutes; ions monitored had m/z 344.2 and 326.2 for 6b-naltrexol
`and 347.1 and 329.3 for its internal standard. The interday precision of the
`calibration curves and quality control samples for naltrexone ranged from 2.92%
`to 7.87%, and the accuracy ranged between 23.50% and 0.75% during the
`analysis of the samples. The interday precision of the calibration curves and
`quality control samples of 6b-naltrexol ranged from 2.89% to 7.38%, and the
`accuracy ranged between 27.13% and 2.00% during the analysis of the samples.
`The lower limit of quantification for both naltrexone and 6b-naltrexol was
`0.02 ng/ml.
`Data Analyses. The safety population included all subjects who received at
`least one intranasal dose; the PK population included all participants who received
`at least one dose with sufficient data to calculate meaningful PK parameters. The
`PK parameters were calculated using standard noncompartmental methods and
`a validated installation of WinNonlin Phoenix, version 6.3 (Cetera, Princeton,
`NJ). Values of peak plasma concentrations (Cmax) and the time to reach Cmax were
`the observed values obtained directly from the concentration-time data. The
`terminal elimination half-life was estimated by linear regression analysis. The area
`under the concentration-time curve from time zero to the last quantifiable
`concentration was determined by the linear-up/log-down trapezoidal method
`and extrapolated to the area under the concentration-time curve from time zero to
`infinity (AUC0–inf) by adding the value of the last quantifiable concentration
`divided by the terminal rate constant. Since the extrapolated area under the plasma
`concentration-time curve was less than 20% for all participants, only AUC0–inf is
`reported. The apparent total body clearance was calculated as the dose divided by
`AUC0–inf. Within an ANOVA framework, comparisons of ln-transformed PK
`parameters were performed using a mixed-effects model, where sequence, period,
`and treatment were the independent factors. The 90% confidence intervals for the
`ratio of the geometric least-squares mean values of Cmax and AUC0–inf were
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`Krieter et al.
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`constructed for comparison of the two drugs administered in combination versus
`separately. The 90% confidence intervals were obtained by exponentiation of the
`90% confidence intervals for the differences between the least-squares mean values
`based on a ln scale. All analyses of demographic and safety data were performed
`using SAS statistical software, version 9.3 (SAS Institute, Inc., Cary, NC).
`
`In Vitro Transporter Studies
`
`Methods. Transporter studies with organic cation transporter (OCT) 1, OCT2,
`and OCT3 were conducted using human embryonic kidney 293 (HEK293) cells
`(ATCC, Manassas, VA) that had been transfected with vectors containing human
`transporter cDNA; control cells were transfected with the vector only. The culture
`medium was Dulbecco’s modified Eagle’s medium supplemented with 8.9%
`FBS, 0.89% antibiotic, and 1.79 mM L-glutamine. The incubation medium for the
`HEK293 cells was Hanks’ balanced salt solution containing 10 mM HEPES,
`pH 7.4. OCTN1 and OCTN2 transporter studies used S2 cells established by
`culturing microdissected S2 segments derived from transgenic mice harboring the
`temperature-sensitive simian virus 40 large T-antigen gene. Cells were transfected
`with vectors containing the human transporter cDNA. Control cells for all five
`transporters contained only the vector cDNA. The culture medium for the S2 cells
`was RITC80-7 supplemented with 4.7% FBS, 9.3 mg/ml epidermal growth factor,
`0.08 U insulin/ml, and 9.5 mg/l transferrin. PBS containing 0.2% bovine serum
`albumin (D-PBS), pH 7.4, was used as the incubation medium for the S2 cells. All
`cells were cultured with 5% CO2 and 95% relative humidity at 33°C (S2 cells) or
`37°C (HEK293 cells).
`The medium was removed by aspiration, the cells were rinsed with either 1 ml
`of Hanks’ balanced salt solution or D-PBS (this was replaced with medium
`containing the control inhibitor or solvent control), and then the cells were
`preincubated. After preincubation, the medium was removed and replaced with
`0.3 ml buffer containing either the test article or the positive control. The final
`concentration of naloxone and naltrexone was 1 mM in 0.2% DMSO, the solvent
`for the test articles and controls. Cells were incubated for 2, 5, 10, or 20 minutes at
`37°C, at which time the solutions were removed. Positive controls were 10 mM
`[14C]metformin (OCT1 and OCT2), 5 mM [14C]triethylamine (OCT3 and
`OCTN1), and 0.03 mM [3H]carnitine (OCTN2). All incubations were done in
`triplicate. HEK293 cells were washed one time with 1 ml of ice-cold D-PBS and
`twice with 1 ml of ice-cold PBS; S2 cells were washed three times with ice-cold
`D-PBS. After removal of the medium, 0.5 ml of purified water was added to each
`well to lyse the cells and samples were collected for analysis of naloxone and
`naltrexone and then analyzed by LC-MS/MS. Samples were mixed with 20 ml
`water with the internal standard (nalmefene). An AB Sciex API-4000 mass
`spectrometer was operated in the positive mode with an Acquity UPLC BEH C18
`analytical column (2.1  50 mm, 1.7 mm). The mobile phase consisted of a
`gradient from 95% mobile phase A (10 mM ammonium acetate)/5% mobile phase
`B (10 mM ammonium acetate in acetonitrile with 0.1% ammonium hydroxide)
`to 10% mobile phase A/90% mobile phase B in 2 minutes. The flow rate was
`0.5 ml/min. The ions monitored had m/z 328.2 and 310.0 for naloxone, m/z 342.2
`and 324.1 for naltrexone, and m/z 340.3 and 322.3 for the internal standard.
`Concentrations were calculated as the area under the curve compared with that of
`the internal standard using known concentrations of naloxone and naltrexone. For
`the positive controls, a 0.3 ml aliquot of the cell lysate was mixed with 5 ml of
`scintillation cocktail, and radioactivity was measured by liquid scintillation
`
`counting. Samples were collected for protein content using the BCA-Protein
`Assay Kit (Thermo Fisher Scientific, Waltham, MA). The uptake amount and
`cleared volume of naloxone and naltrexone were calculated as follows:
`Uptake  amount  into  cells  ðpmol=wellÞ ¼ pmol  in  cell  lysate
` 500  ml=1000  ml
`
`Cleared  volume  ðml=mg  proteinÞ ¼
`uptake  amount  into  cells  ðpmol=wellÞ
`mg  protein=well  initial  concentration  ðpmol=mlÞ
`
`For positive controls, disintegrations per minute were substituted for picomoles
`in the previous equations.
`Transport studies were also conducted using wild-type Madin-Darby canine
`kidney II (MDCKII) cells that had been transfected with vectors containing
`human transporter cDNA (Netherlands Cancer Institute, Amsterdam, Netherlands).
`Cells were plated and maintained on 24-well Transwell plates (Corning,
`Corning, NY) for 3–5 days prior to the experiment. Culture medium was
`removed and incubation medium (Hanks’ balanced salt solution supplemented
`with 25 mM HEPES and 25 mM glucose) was added to the cells. The pH of
`the basolateral buffer was 7.4 and that of the apical buffer was either pH 7.4
`or 5.5. Approximately 10 minutes after incubation medium was added, the
`transepithelial electrical resistance was recorded and cells were preincubated at
`37°C for 30–60 minutes. After preincubation, the medium containing naloxone,
`naltrexone, or control compounds ([3H]mannitol and [14C]caffeine) was added to
`the donor chamber. Samples were collected from the receiver side at 15, 60, and
`120 minutes and replaced by 0.1 ml of incubation medium. The transepithelial
`electrical resistance was also measured at the end of the incubation to determine if
`the cells were still confluent. Samples were mixed with 25 ml of methanol:water
`(1:1 v/v) and 75 ml of the internal standard (hydroxybuproprion-d6) in methanol:
`water (1:1 v/v) and analyzed by LC-MS/MS using an AB Sciex API-5500 mass
`spectrometer as described previously, except that the gradient changed from 70%
`mobile phase A/30% mobile phase B to 5% mobile phase A/95% mobile phase B
`over 3 minutes. Ions for the internal standard were monitored at m/z 262.0 and
`244.0. Concentrations of radioactivity were determined as detailed previously.
`The apparent permeability (Papp) was calculated as follows:
`
`
`1
`A0  C0
`where dQ is the amount of test drug transported (in picomoles); dT is the
`incubation time (in seconds); A0 is the surface area of the membrane (in squared
`centimeters); and C0 is the initial concentration of the test drug in the donor
`chamber (in picomoles per cubic centimeter). The efflux ratio was calculated as
`the Papp basal-to-apical/Papp apical-to-basal ratio. The sex of the cell lines used in
`the experiments is unknown.
`
`dQ
`dT
`
`Results
`
`Pharmacokinetic Study
`Subject Characteristics. All subjects initiating the study (Table 1)
`received at least one dose of naloxone and/or naltrexone; 11 subjects
`
`TABLE 1
`Pharmacokinetics of naloxone: subject demographics
`
`Demographics
`
`All
`
`Male
`
`Number
`Mean age, y (range)
`Race
`White
`Black/African American
`Ethnicity
`Hispanic or Latino
`Not Hispanic or Latino
`Mean Weight, kg (range)
`Mean BMI, kg/m2 (range)
`
`BMI, body mass index.
`
`12
`36.0 (22.0–48.0)
`
`6
`39.2 (26.0–48.0)
`
`4
`8
`
`1
`11
`74.7 (49.4–99.2)
`25.0 (19.2–29.3)
`
`1
`5
`
`0
`6
`82.7 (71.8–99.2)
`25.3 (23.6–27.2)
`
`Female
`
`6
`32.8 (22.0–48.0)
`
`3
`3
`
`1
`5
`66.6 (49.4–84.8)
`24.6 (19.2–29.3)
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`PK of Naloxone and Naltrexone with Intranasal Administration
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`693
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`When naloxone was added to the naltrexone intranasal formulation,
`the Cmax value decreased from 4.55 to 1.71 ng/ml, a decline of
`approximately 62% (Fig. 2; Table 2), and the AUC0–inf value decreased
`approximately 38%. The median time to reach Cmax value increased
`from 0.33 to 0.75 hours when the combination formulation was
`administered compared with naltrexone alone. However, the half-life
`of naltrexone was unchanged.
`The decreased concentration of naltrexone concentrations in treatment
`C was evident even at 5 and 10 minutes after dose administration; the
`concentrations were 83%–86% lower than when naltrexone was dosed
`alone. The concentrations of naltrexone continued to be considerably
`lower even at 6 hours postdose. In contrast, there was no change in the
`PK values of 6b-naltrexol when naltrexone was administered with or
`without naloxone (Fig. 3; Tables 2 and 3). The two formulations were
`bioequivalent for 6b-naltrexol, based on the 90% confidence intervals of
`Cmax and AUC0–inf (Table 3).
`There were minor differences in the PK parameters between males
`and females (Table 4). However, the small sample size of this pilot study
`precludes any definitive conclusions regarding sex-related differences
`following intranasal administration of either drug.
`Safety. Five subjects experienced at least one adverse event of any
`grade or attribution that was judged to be related to the test drugs; all
`were mild in severity. Headache was the single most frequent adverse
`event (three events in each of three subjects). There was one drug-related
`incident of mild inflamed mucosa (score of 1) that occurred 24 hours
`after dosing with 2 mg naltrexone. Vital signs, ECG, and clinical
`laboratory parameters did not reveal any clinically significant changes
`after any of the doses.
`
`In Vitro Transporters
`The ratios of naloxone and naltrexone uptake by the five transporter-
`expressing cell lines compared with the control cells were all less than 2,
`indicating that neither compound was a substrate for OCT1, OCT2,
`OCT3, OCTN1, or OCTN2 (Table 5). Positive controls had ratios of
`uptake that ranged from 6.4 for OCT3 to 75.4 for OCTN2 and
`demonstrated inhibition of uptake by their respective inhibitor.
`Permeability of naloxone and naltrexone across a polarized cell layer
`was tested using control MDCKII cells. Concentrations on the donor
`side were either 10 or 500 mM. Transporter studies normally use buffers
`that are pH 7.4 on both the apical and basolateral sides. Since the pH of
`the nasal passage is approximately pH 5.5–6.5,
`the studies were
`conducted also with the apical buffer at pH 5.5, while the basolateral
`buffer remained at pH 7.4.
`When the pH of the buffer was pH 7.4 on both sides, the efflux ratios
`of both naloxone and naltrexone were less than 2 at concentrations of
`
`Fig. 1. Mean (S.D.) concentrations of naloxone in healthy participants following
`intranasal administration of 4 mg naloxone alone and in combination with 2 mg
`naltrexone. Upper graph: concentrations to 12 hours postdose; Lower graph:
`concentrations to 1 hour postdose.
`
`completed the study. One female subject withdrew during the first period
`due to a moderate headache with mild nausea that occurred 28 hours
`after administration of 2 mg naltrexone.
`Pharmacokinetics. The geometric mean Cmax and AUC0–inf values
`of naloxone following a 4-mg intranasal dose were 4.30 ng/ml and
`8.13 ng·h/ml, respectively (Fig. 1; Table 2). The Cmax and AUC0–inf
`values decreased by approximately 18% and 16 %, respectively, when
`naloxone was administered in combination with 2 mg naltrexone
`(Table 3). The median time to reach Cmax value remained unchanged
`at 30 minutes, and the elimination half-life was also unchanged.
`
`TABLE 2
`Geometric mean pharmacokinetic parameters (%CV) of naloxone, naltrexone, and 6b-naltrexol
`
`Variablea (U)
`
`Naloxone
`
`Naltrexone
`
`6b-Naltrexol
`
`Alone (Treatment B)
`
`Plus Naltrexone (Treatment C) Alone (Treatment A)
`
`Plus Naloxone (Treatment C) Alone (Treatment A)
`
`Plus Naloxone (Treatment C)
`
`N
`Cmax (ng/ml)
`Tmax (h)
`AUC0–inf (ng·h/ml)
`lz (h21)
`t1/2 (h)
`CL/F (l/min)
`
`11
`4.30 (47.5)
`0.50 (0.25–0.75)
`8.13 (38.2)
`0.380 (32.1)
`1.83 (32.1)
`6.71 (38.2)
`
`11
`3.60 (36.5)
`0.50 (0.25–0.75)
`7.00 (32.5)
`0.355 (43.9)
`1.95 (45.7)
`7.79 (43.9)
`
`12
`4.55 (80.0)
`0.33 (0.17–1.0)
`9.61 (39.1)
`0.319 (18.2)
`2.17 (18.2)
`3.12 (39.1)
`
`11
`1.71 (35.1)
`0.75 (0.25–2.0)
`5.88 (25.2)b
`0.322 (10.3)b
`2.15 (10.3)b
`5.10 (25.2)b
`
`12
`2.09 (32.5)
`2.00 (0.75–4.0)
`30.8 (32.7)
`0.0433 (31.6)
`16.0 (31.6)
`NC
`
`11
`2.09 (26.2)
`2.00 (0.75–3.0)
`28.3 (28.4)
`0.0430 (24.5)
`16.1 (24.5)
`NC
`
`AUC0–inf, area under the plasma concentration-time curve from time zero to infinity; CL/F, apparent clearance; lz, terminal phase rate constant; NC, not calculated; t1/2, terminal phase half-life;
`Tmax, time to reach Cmax; Treatment A, 2 mg naltrexone, intranasally; Treatment B, 4 mg naloxone, intranasally; Treatment C, 2 mg naltrexone plus 4 mg naloxone, intranasally.
`aGeometric mean (%CV) for all except median (range) for Tmax.
`bN = 10.
`
`Nalox1225
`Nalox-1 Pharmaceuticals, LLC
`Page 4 of 9
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`694
`
`Krieter et al.
`
`TABLE 3
`Statistical summary of treatment comparisons
`
`Variable
`
`Analyte
`
`Comparison (Treatment
`C vs. Reference)
`
`Geometric Mean Ratio
`(Treatment C/Reference)
`
`Cmax (ng/ml)
`
`AUC0–inf (ng·h/ml)
`
`Naloxone
`Naltrexone
`6b-Naltrexol
`Naloxone
`Naltrexone
`6b-Naltrexol
`
`C vs. B
`C vs. A
`C vs. A
`C vs. B
`C vs. A
`C vs. A
`
`81.5
`38.4
`101
`84.6
`61.6
`94.4
`
`90% CI
`
`63.6–105
`25.7–57.3
`92.7–110
`70.3–102
`50.9–74.6
`89.3–99.8
`
`AUC0–inf, area under the plasma concentration-time curve from time zero to infinity; CI, confidence interval; Treatment A, 2 mg naltrexone, intranasally; Treatment
`B, 4 mg naloxone, intranasally; Treatment C, 2 mg naltrexone plus 4 mg naloxone, intranasally.
`
`10 and 500 mM (Tables 6 and 7). The addition of 50-fold higher
`concentration of naloxone to both the lower and higher naltrexone
`donor solutions did not reduce the efflux ratio to an appreciable
`amount. Similar results were observed when the higher concentration
`of naltrexone was added to the naloxone solutions.
`Lowering the pH of the apical buffer to pH 5.5, while maintaining the
`basolateral at pH 7.4, caused a 3- to 5-fold decrease in Papp values of
`naltrexone in the A-to-B ratio and 2- to 4-fold increase in the efflux
`direction (Table 6). The efflux ratios increased to between 12.1 and 18.2.
`Similar results were observed using naloxone (Table 7). The Papp values
`changed considerably regardless of whether naloxone and naltrexone
`were tested separately or in combination.
`The mean transepithelial electrical resistance values were above
`100 V  cm2 both pre- and postdose in all of the MDCKII studies. The
`Papp values of [3H]mannitol were in the range of 0.41–1.37  10–6 cm/s
`in the A-to-B and B-to-A directions, while they ranged between 15.5 and
`53.5  10–6 cm/s for [14C]caffeine in all of the incubations.
`
`Discussion
`
`Delivery of naloxone by the nasal route has been recognized by the
`medical community and public officials as an effective way to reverse
`opioid overdoses (Ryan and Dunne, 2018). However, the short half-life
`of naloxone and the increased incidence of overdoses linked to synthetic
`opioids with a longer duration of action may require more than one dose
`to be administered to prevent renarcotization (Klebacher et al., 2017).
`Therefore, the addition of a longer-acting opioid antagonist to the
`naloxone formulation was initially hypothesized as a means to increase
`the time to obtain proper medical attention.
`The large decrease in the nasal absorption of naltrexone in the
`presence of naloxone was unexpected. It was observed as early as
`5 minutes following administration of both opioid antagonists but was
`far more pronounced for naltrexone. The Cmax value of naltrexone was
`reduced to 1.7 ng/ml when the drugs were combined, i.e., less than
`2 ng/ml, which is generally regarded as a concentration that
`is
`sufficient to adequately block the effects of opioid agonists (Comer
`et al., 2006). The combination product of naloxone and naltrexone
`was not pursued further.
`Although the concentration of naltrexone was below the target of
`2 ng/ml, direct absorption into the brain via the olfactory nerves that
`protrude through the cribiform plate in the olfactory epithelium may lead
`to a higher concentration at the site of action (Illum, 2000). The cerebral
`spinal fluid/plasma ratio of zidovudine was higher after intranasal
`administration compared with an intravenous infusion at 15 minutes
`postdose in rats (Seki et al., 1994). Similar results were observed in the
`rat using cephalexin (Sakane et al., 1994). However, the olfactory
`epithelium accounts for only 3%–5% of the nasal cavity’s total surface
`area, which may limit its role in the efficient transfer of drugs directly to
`the central nervous system (Grassin-Delyle et al., 2012).
`
`Rabiner et al. (2011) hypothesized that 6b-naltrexol contributed to
`the long occupancy of the m-opioid receptor due to its long plasma
`half-life. Due to this possibility, the Food and Drug Administration
`draft guidance on new formulations of naltrexone hydrochloride requires
`the analysis of 6b-naltrexol (https://www.fda.gov/downloads/Drugs/
`GuidanceComplianceRegulatoryInformation/Guidances/UCM194641.pdf).
`This metabolite, while approximately 2-fold less potent than the parent
`compound on the m-receptor, is 100-fold less potent than naltrexone
`in vivo in nonhuman primates (Ko et al., 2006) and has no effect on
`either morphine-induced analgesia or pupil constriction in humans
`(Yancey-Wrona et al., 2011). Thus, 6b-naltrexol
`is peripherally
`restricted; therefore, its involvement in the central nervous system
`effects of naltrexone is minimal.
`
`Fig. 2. Mean (S.D.) conce