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`atomizers has substantially increased the accessibility to this
`opioid antidote (4,6,7). So far one naloxone IN product
`(Narcan® nasal spray) has obtained the FDA approval, while
`at least two other applications have received complete
`response letters (CRL) from the agency (8,9). One of the
`products that received a CRL was reported to exhibit
`insufficient early-stage drug uptake, suggesting that optimal
`IN absorption at an early time point could be challenging in
`developing IN formulations of naloxone (8,10).
`To ensure a sufficient uptake of naloxone through IN
`route, a more concentrated naloxone solution may be needed
`to enhance drug absorption and overcome the volume
`limitation of IN route (4,11). In addition, IN formulations of
`naloxone usually have more additives compared with paren(cid:173)
`teral formulations, which could potentially enhance the nasal
`absorption of naloxone. According to the package label of
`Narcan® nasal spray and the formulation example described in
`its corresponding patent ( owned by Adapt Pharma Ltd.,
`Table I), ethylenediaminetetraacetic acid (EDTA) and
`benzalkonium chloride (BKC) are employed in the formula(cid:173)
`tion as a stabilizer and preservative, respectively (13-15). Both
`of these additives are known to enhance the drug permeation
`across epithelia and thus could function as permeation
`enhancers (16). Interestingly, another patented IN formulation
`of naloxone reported in the literature (owned by AntiOp Inc.,
`Table I) also contains EDTA but uses benzyl alcohol (BA)
`instead of BKC as a preservative due to a stability issue (17).
`The latter patented formulation reported substantially lower
`relative bioavailability compared with intramuscular (IM)
`naloxone injection, implying that the composition of IN
`formulations may play a critical role in determining the rate
`and extent of nasal absorption of naloxone (4,12).
`While there may be many naloxone IN formulations under
`development, there is little scientific literature available on the
`effect of formulation variables including preservatives and stabi(cid:173)
`lizers on the absorption of naloxone from these IN formulations
`(18-20). The objective of the present study is to evaluate the effects
`of various formulation variables on the permeability of naloxone
`from IN formulations employing an in vitro nasal permeation
`model, EpiAirway™ tissue-mounted Ussing chamber (21,22).
`EpiAirway™ is a mucociliary tissue model consisting of normal,
`human-derived upper respiratory tract epithelial cells. This primary
`cell culture model exhibits relevant human tissue structure and
`cellular morphology and thus has been extensively used for in vitro
`testing of nasal bioavailability (23,24). In addition, since some of
`the formulation additives mentioned above were reported to affect
`the stability of naloxone (17), stability studies were conducted per
`ICH QlA guidance to evaluate the impact of formulation on the
`
`stability of naloxone. The methodology developed and knowledge
`gained from this study could be helpful in the selection of suitable
`ingredients for optimal permeation and early evaluation of
`naloxone IN formulations.
`
`MATERIALS AND METHODS
`
`Materials
`
`Naloxone HCI USP was purchased from RIA Interna(cid:173)
`tional LLC (East Hanover, NJ). The reference standard of
`naloxone was purchased from the United States Pharmaco(cid:173)
`peia (Lot No. R007WO, USP, Rockville, MD). The reference
`standards of noroxymorphone (Lot No. FE10141502),
`naloxone-N-oxide (Lot No. FE1203150), and 2,2-bisnaloxone
`(Lot No. NQS1405) were purchased from Cerilliant Corp
`(Round Rock, TX) and Noramco Inc. (Wilmington, DE).
`Ethylenediaminetetraacetic acid (EDTA) (USP grade), ben(cid:173)
`zyl alcohol (BA) (reagent grade), benzalkonium chloride
`(BKC) (NF grade), and sodium chloride (USP grade) were
`purchased from Spectrum Chemical MFG Corp (New Bruns(cid:173)
`wick, NJ). Citric acid (reagent grade) was purchased from
`Ricca Chemical Company (Arlington, TX); Krebs-Ringer
`bicarbonate buffer was purchased from Sigma-Aldrich (St.
`Louis, MO). Other reagents and solvents were purchased
`from Fisher Scientific (Norcross, GA). All materials were of
`analytical grade unless otherwise specified.
`
`Preparation of Formulations for Nasal Permeability and
`Stability Studies
`
`A full factorial design of experiment (DoE) was used to
`investigate the effect of various formulation variables on drug
`nasal permeation. The DoE included one continuous factor
`(drug concentration) and two nominal factors (stabilizers and
`preservatives) which resulted in eighteen naloxone IN
`formulations. The composition of each formulation is de(cid:173)
`scribed in Table II. Formulation variables for this study were
`selected based on the published literature on naloxone IN
`formulations (Table I). These factors included concentration
`of naloxone in the solution, pH, type of preservative (BKC or
`BA), and EDTA as stabilizer (14,15,17). Based on the results
`of a preliminary study, a concentration of 0.01 % wlv of BKC
`was found to significantly affect the integrity of EpiAirway™
`tissue in Ussing chambers (Fig. 1). Therefore, a lower
`concentration of 0.003% wlv was used in the DoE for
`permeability experiments while a concentration of 0.01 % wl
`v of BKC was used for stability studies. In addition, because
`
`Table I. Formulation Examples of IN Naloxone Described in the Literature (12)
`
`US patent number
`
`US 9,211,253 and US 9,775,838 (14,15)
`
`Assignee
`Concentration of naloxone
`Stabilizing agent
`Preservative
`Buffering agent
`Osmolality agent
`Solution pH
`
`Adapt Pharma Ltd.
`20 or 40 mg/mL
`EDTA 0.2% wlv
`BKC 0.01 % w/v
`
`Sodium chloride 0.74% wlv
`4.5
`
`us 9,192,570 (17)
`
`AntiOp, Inc.
`lOmg/mL
`EDTA 0.372% w!v
`BA 0.5% w!v
`Citric acid 0.48% w/v
`Sodium chloride (q.s.)
`4.25
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`60 min time points from the receiver side of the chambers,
`and the Jost fluid volume was immediately replaced by fresh
`Krebs-Ringer buffer. The samples were analyzed for nalox(cid:173)
`one content using a Waters ACQUITY H-class UPLC
`system. The apparent permeability (Papp) of naloxone across
`the EpiAirway™ tissues was calculated using the following
`equation (25).
`
`evaluated. These naloxone solutions were prepared by
`dissolving naloxone (10 mg/mL) in purified water with or
`without EDTA (0.35% wlv). The pH was adjusted by 1 N
`sodium hydroxide or 1 N hydrochloric acid. The formulations
`were filled in 2-mL vials as described earlier and placed on
`stability at 60°C for 7 days. The samples were analyzed using
`a chromatographic method.
`
`1
`dC
`Papp = Vr X dt X ACo
`
`(1) Analytical Method
`
`Whereas Vr is the volume of the solution in the receiver
`chamber, dC/dt is the slope of the cumulative concentration of
`naloxone in the receiver chamber at the steady state (10 to
`45 min), A is the surface area of the insert (1.2 cm2
`), and C0 is
`the initial concentration of naloxone in the donor chamber.
`To evaluate the effects of pH on the permeability of
`naloxone, the naloxone solutions (4 mg/mL) were prepared in
`Krebs-Ringer buffer with final pH adjusted to 4.0, 4.5, 5.0, 5.5,
`and 6.0. The permeability of above naloxone solutions was
`measured using the method described above.
`
`Evaluation of the Buffering Ability of In-House Prepared
`Patented Naloxone IN Formulations
`
`For assessing the buffering ability, the formulation
`examples described in the patents were prepared along with
`a buffer-free naloxone aqueous solution in-house. Briefly, in(cid:173)
`house formulation A was prepared to represent the formula(cid:173)
`tion example described in Adapt's patent, which contains
`naloxone HCl, EDTA, and BKC (Table I) (14,15). The pH of
`the solution was adjusted to 4.5 using 1 N sodium hydroxide.
`In-house formulation B was prepared to represent the
`formulation example described in AntiOp's patent, which
`contains naloxone HCl, EDTA, BA, and citric acid (Table I)
`(17). The pH of the solution was adjusted to 4.25 using 1 N
`sodium hydroxide. The buffer-free naloxone solution (0.4 mg/
`mL) was prepared in purified water, and the pH was adjusted
`to 4.0 using 1 N hydrochloric acid, representing naloxone
`injections used with the improvised IN device (26,27). To
`determine the buffering ability, 15 mL of each naloxone IN
`formulation was titrated with 0.1 N sodium hydroxide to raise
`the pH to 6.0. The buffering ability is equivalent to the
`amount (nmol) of sodium hydroxide required to raise the pH
`of 100 µL of IN solution to pH 6.0.
`
`Stability Studies on Naloxone IN Formulations
`
`The stability of all formulations listed in Table II was
`evaluated. The stability samples were prepared by filling
`1 mL of the formulation solution in a 2-mL USP type I amber
`vial under nitrogen purge and sealed with PTFE faced 14B
`rubber lined caps (Wheaton, Millville NJ). The samples were
`placed in upright position with the cap down in stability
`chambers (ES2000 Series, Bahnson ES, Clemmons, NC)
`under accelerated (40°C/75% RH), intermediate (30°C/75%
`RH), and long-term (25°C/60% RH) conditions. The samples
`were analyzed at 0, 3, 6, and 12 months using a chromato(cid:173)
`graphic method described below. In addition, the stability of
`naloxone solution with pH adjusted to 4.0 and 6.0 was also
`
`A stability-indicating UPLC method was developed and
`validated for analyzing stability samples of naloxone IN
`formulations and samples from in vitro permeation studies.
`The method is also capable of separating and quantifying the
`two naloxone-related compounds (noroxymorphone and 2,2-
`bisnaloxone listed in the USP monograph of naloxone HCJ)
`and a potential degradant (naloxone-N-oxide) in nasal spray
`formulations. The method employed a Waters ACQUITY H(cid:173)
`class UPLC system with photodiode array detector (PDA).
`The output signal was processed using Waters Empower 3
`software. An ACQUITY UPLC BEH C18 2.1 x 100 mm
`column with 1.7-µm particle size and with a 2.1 x 5 mm pre(cid:173)
`column containing the same packing materials was used. The
`separation was achieved using a gradient method. Solvent A
`is an aqueous solution containing 1.4 g Sodium 1-
`octanesuHonate, 1.0 g sodium chloride, and 1.0 mL phospho(cid:173)
`ric acid per liter. Solvent B is a methanol/acetonitrile (4:1)
`mixture. The flow rate of the mobile phase was 0.5 mL/min.
`The gradient program (min/%B) was set as 0.50/25%, 4.0/
`50%, 6.0150%, 6.05/25%, and 10.00/25%. The injection
`volume was 2.0 µL. The column temperature was set at
`50°C and the PDA detection was at 229 nm. The method was
`validated per USP <1225> and deemed suitable for intended
`use per the results in Table III. The short run time of this
`method, 10 min, helps in high-throughput analysis of large
`sets of samples during in vitro permeation and stability
`studies.
`
`Statistical Analysis
`
`The permeation data obtained in this study was analyzed
`using JMP software (ver. 13) to evaluate the effect of
`formulation variables on the permeability (Papp) and %
`change in TEER The formulation variables (independent
`variables) included naloxone concentration, EDTA, and the
`type of preservative (BA, BCK, or none). The response
`factors were the Papp of naloxone from the formulations and
`the % change in TEER of the tissues. Data was fitted in a
`three-way full factorial linear model using least squares fit.
`Analysis of variance (ANOVA) was utilized to determine the
`significance of the model and the effect of each variable.
`
`RESULTS AND DISCUSSION
`
`Effect of Formulation Variables on Nasal Permeation of
`Naloxone
`
`The results in Table IV and Fig. 2a describe the transport
`rate and Papp of naloxone from IN formulations. Figure 2b
`describes the % change in TEER of tissues when exposed to
`various IN formulations for 30 min. Papp values of
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`Table m. Validation of the Stability-Indicating UPLC Method for the Analysis· of Naloxone
`
`Parameter
`
`Naloxone (for assay) Naloxone (for related substances) Noroxymorphone Naloxone-N-0xide 2,2-Bisnaloxone
`
`LOO (µg/mL)
`LOD (µg/mL)
`Regression equation"
`3609.9
`Slope
`13,194.02
`Intercept
`Y.. interceptb
`1.88%
`? value
`0.9992
`1.04%
`Precision (% RSD)"
`Accuracy(% recoveryt 99.20%
`
`0.40
`0.15
`
`3727.4
`105.00
`<0.01%
`0.9998
`3.13%
`101.83%
`
`0.25
`0.09
`
`4806.6
`-87.70
`< 0.01%
`0.9996
`3.86%
`109.00%
`
`0.30
`0.10
`
`3974.2
`-4.27
`<0.01%
`0.9999
`3.38%
`104.56%
`
`0.20
`O.o?
`
`7085.2
`-208.48
`<0.01
`0.9997
`2.34%
`105.33%
`
`• Linearity range is 50-600 µg/mL for assay and LOQ to 6.4 µg/mL for related substances
`b Compared with 100% level (200 µg/mL)
`c Six determinations using 100% level sample (200 µg/mL) for assay and LOQ for related substances
`
`formulations that do not contain any excipient (formulations
`F2, F8, and F14) range from 1.17 x 10~ to 2.41 x 10-6 cm/s.
`These Papp values for naloxone are in line with those reported
`in the literature (23). The data in Table IV and Fig. 2 were
`fitted to linear regression models using a least squares
`method. The ,:Z between experimental and model predicted
`values were found above 0.9 for both models (Papp and %
`change in TEER). The ANOVA on the observed means of
`the responses shows that the formulation factors significantly
`affect the P •PP of naloxone and the % change in TEER of
`tissues (p < 0.05). Figure 3 describes the main effect and
`significance of each formulation factor.
`The results in Table IV and Fig. 3a show that an increase
`in naloxone concentration reduced the Papp, and the results in
`Fig. 3b show that an increase in the concentration of naloxone
`did not significantly affect the % change in TEER of tissues.
`Although the Papp of naloxone from more concentrated
`naloxone solutions were lower, the greater concentration
`gradient still resulted in significantly higher drug transport
`
`rates across the tissue inserts. For instance, the Papp of
`naloxone formulation containing 40 mg/mL of naloxone is
`around 50% lower than that of formulation containing 4 mg/
`mL of naloxone (Table IV), but the transport rate of
`naloxone from formulation containing 40 mg/mL is nearly 5
`times higher. These results confirm that a higher concentra(cid:173)
`tion of naloxone solution would increase the amount of
`naloxone absorbed (4,11). This approach may be useful in
`formulating IN solutions where the drug has limited residence
`time for absorption in the nasal cavity.
`The results in Figs. 2a and 3a show that addition of BKC
`at a concentration of 0.003% wlv and BA at 0.5% wlv both
`resulted in increased Papp of naloxone across the
`EpiAirway™ tissue, As shown in Fig. 3b, BKC and BA
`produced significant reduction in TEER of tissues, suggesting
`compromised tissue integrity and a consequential increase in
`naloxone permeation. Among the two preservatives, BKC is
`clearly a more effective permeation enhancer since it
`produced a comparable increase in Papp of naloxone at a
`
`Table rv. The Naloxone Transport Rate and Papp Across EpiAirway™ Tissues
`
`Formulation no.
`
`Naloxone cone. {mg/ml)
`
`Transport rate (ng·cm2·s)
`
`Papp (x 10-6 emfs)
`
`F-1
`F-2
`F-3
`F-4
`F-5
`F-6
`F-7
`F-8
`F-9
`F-10
`F-11
`F-12
`F-13
`F-14
`F-15
`F-16
`F-17
`F-18
`
`4
`4
`4
`4
`4
`4
`22
`22
`22
`22
`22
`22
`40
`40
`40
`40
`40
`40
`
`The exposure area of EpiAirway™ tissue is 1.2 cm2
`
`1.70±0.13
`9.63±0.66
`3.07±0.49
`11.16 ± 0.11
`5.28± 1.69
`14.44±0.90
`8.51 ±0.85
`33.71 ±5.39
`26.28± 1.84
`47.18 ± 1.80
`20.44±4.01
`46.72±3.98
`10.84±3.07
`46.65±2.45
`76.28±5.12
`73.70±5.38
`48.43±21.01
`59.01 ±4.55
`
`0.42±0.03
`2.41±0.16
`0.77±0.12
`2.79±0.03
`1.32±0.42
`3.61 ±0.23
`0.39±0.04
`1.53 ±0.25
`1.19±0.08
`2.14±0.08
`0.93 ±0,18
`2.12±0.18
`0.27±0.08
`1.17±0.06
`1.91 ±0.13
`1.84±0.13
`1.21 ±0.53
`1.48±0.11
`
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`
`4. Lewis CR, Vo HT, Fishman M. Intranasal naloxone and related
`strategies for opioid overdose intervention by nonmedical
`personnel: a review. Subst Abus Rehabil. 2017;8:79-95. https://
`doi.org/10.2147/SAR.SlOl 700.
`5. Wolfe TR, Bernstone T. Intranasal drug delivery: an alternative
`to intravenous administration in selected emergency cases. J
`Emerg Nurs. 2004;30(2) :141-7. https://doi.org/10.1016/
`j.jen.2004.01.006.
`6. Robertson TM, Hendey GW, Stroh G, Shalit M. Intranasal
`naloxone is a viable alternative to intravenous naloxone for
`prehospital narcotic overdose. Prehosp Emerg Care.
`2009;13(4):512-5. https://doi.org/10.1080/10903120903144866.
`7. Merlin MA, Saybolt M, Kapitanyan R, Alter SM, Jeges J, Liu J,
`et al. Intranasal naloxone delivery is an alternative to intrave(cid:173)
`nous naloxone for opioid overdoses. Am J Emerg Med.
`2010;28(3):296-303. https://doi.org/10.1016/j.ajem.2008.12.009.
`8. Press release: lndivior receives complete response letter from
`FDA not approving naloxone nasal spray new drug application
`for opioid overdose 2015. Available from: http://indivior.com/
`wp-con ten t/uploads/2015/11 /N as al-N aloxone-Fin al(cid:173)
`Release_1124151.pdf. Accessed 16 Apr 2019.
`9. Press Release: Amphastar announces the receipt of a CRL for
`intranasal naloxone for the emergency treatment of opioid
`overdose 2017. Available from: http://ir.amphastar.com/static(cid:173)
`files/l9b13l50-7ff8-4d3b-8e3f-452578083dbb . Accessed 16
`Apr 2019.
`10. Hertz S., Narcan nasal spray summary review for regulatory
`action US Food and Drug Administration. 2015. Available from:
`https://www.accessdata.fda.gov/drugsatfda_docs/nda/2015/
`208411Orig1s000SumR.pdf. Accessed 16 Apr 2019.
`11 . Kerr D, Kelly AM, Dietze P, Jolley D, Barger B. Randomized
`controlled trial comparing the effectiveness and safety of
`intranasal and intramuscular naloxone for the treatment of
`suspected heroin overdose. Addiction. 2009;104(12):2067-74.
`12. McDonald R, Danielsson Glende 0, Dale 0, Strang J.
`International patent applications for non-injectable naloxone
`for opioid overdose reversal: exploratory search and retrieve
`analysis of the PatentScope database. Drug Alcohol Rev.
`2018;37(2):205-15. https://doi.org/10.1111/dar.12571 .
`13. Adapt Pharma Inc. Narcan - naloxone hydrochloride spray In:
`Drugs@FDA: FDA approve drug products. Adapt Pharma Inc.
`Available from: https://www.accessdata.fda.gov/drugsatfda_docs/
`label/2015/2084lllbl.pdf. Accessed 16 Apr 2019.
`14. Keegan F, Bell RG, Crystal R, Weiss MB. Nasal drug products
`and methods of their use. US Patent. 2017;9,775,838.
`15. Crystal R, Weiss MB. Nasal drug products and methods of their
`use. US Patent. 2015;9,211,253.
`16. Sharma S, Kulkarni J, Pawar A. Permeation enhancers in the
`transmucosal delivery of macromolecules. Pharmazie.
`2006;61(6):495-504.
`17. Wyse J, DeHart MP. Intranasal naloxone compositions and
`methods of making and using same. US Patent. 2015;9,192,570.
`18. Tylleskar I, Skulberg AK, Nilsen T, Skarra S, Jansook P, Dale
`0. Pharmacokinetics of a new, nasal formulation of naloxone.
`Eur J Clin Pharmacol. 2017;73(5):555--62. https://doi.org/
`10.1007 /s00228-016-2191-1 .
`19. McDonald R, Lorch U, Woodward J, Bosse B, Dooner H,
`Mundin G, et al. Pharmacokinetics of concentrated naloxone
`nasal spray for opioid overdose reversal: phase I healthy
`volunteer study. Addiction. 2018;113(3):484-93. https://doi.org/
`10.1111/add.14033.
`20. Mundin G, McDonald R, Smith K, Harris S, Strang J.
`Pharmacokinetics of concentrated naloxone nasal spray over
`first 30 minutes post-dosing: analysis of suitability for opioid
`overdose reversal. Addiction. 2017;112(9):1647-52. https://
`doi.org/10.1111/add.13849.
`21. Wheatley M, Dent J, Wheeldon E, Smith P. Nasal drug delivery:
`an in vitro characterization of transepithelial electrical
`
`properties and fluxes in the presence or absence of enhancers.
`J Control Release. 1988;8(2):167-77.
`22. Bechgaard E, Gizurarson S, J11Srgensen L, Larsen R. The
`viability of isolated rabbit nasal mucosa in the Ussing chamber,
`and the permeability of insulin across the membrane. Int J
`Pharm. 1992;87(1-3):125-32.
`23. Chemuturi NV, Hayden P, Klausner M, Donovan MD. Com(cid:173)
`parison of human tracheal/bronchial epithelial cell culture and
`bovine nasal respiratory explants for nasal drug transport
`studies. J Pharm Sci. 2005;94(9):1976-85.
`24. Leonard AK, Sileno AP, MacEvilly C, Foerder CA, Quay SC,
`Costantino HR. Development of a novel high-concentration
`galantamine formulation suitable for intranasal delivery. J
`Pharm Sci. 2005;94(8):1736-46. https://doi.org/10.1002/jps.20389.
`25. Van Breemen RB, Li Y. Caco-2 cell permeability assays to
`measure drug absorption. Expert Opin Drug Metab Toxicol.
`2005;1(2):175-85.
`26. Hispira, Inc. Naloxone hydrochloride- naloxone hydrochloride
`injection, solution. In: FDA Label Search Available from:
`https://www.accessdata.fda.gov/spl/data/a91e6c30-fb00-4ae0-
`be36-ccdlf2363e15/a91e6c30-fb00-4ae0-be36-ccdlf2363e15.xml.
`Accessed 16 Apr 2019.
`27. Mylan Institutional LLC. Naloxone hydrochloride- naloxone
`hydrochloride injection, solution. In: FDA Label Search.
`Available from: https://www.accessdata.fda.gov/spl/data/
`69401599-0f6a-4055-9dde-0e57d66afe93/69401599-0f6a-4055-
`9dde-0e57d66afe93.xml. Accessed 16 Apr 2019.
`28. Marttin E, Verhoef J, Romeijn S, Zwart P, Merkus F. Acute
`histopathological effects of benzalkonium chloride and absorp(cid:173)
`tion enhancers on rat nasal epithelium in vivo. Int J Pharm.
`1996;141(1-2):151-60.
`29. Fabrizio B, Giulia BA, Fabio S, Paola R, Gaia C. In vitro
`permeation of desmopressin across rabbit nasal mucosa from
`liquid nasal sprays: the enhancing effect of potassium sorbate.
`Eur J Pharm Sci. 2009;37(1):36-42.
`30. Cho E, Gwak H, Chun I. Formulation and evaluation of
`ondansetron nasal delivery systems. Int J Pharm.
`2008;349(1):101-7.
`31. Tomita M, Hayashi M, Awazu S. Comparison of absorption(cid:173)
`enhancing effect between sodium caprate and disodium ethyl(cid:173)
`enediaminetetraacetate in Caco-2 cells. Biol Pharm Bull.
`1994;17(5):753-5.
`32. Tomita M, Hayashi M , Awazu S. Absorption-enhancing mech(cid:173)
`anism of EDTA, caprate, and decanoylcarnitine in Caco-2 cells.
`J Pharm Sci. 1996;85(6):608-11. https://doi.org/10.1021/
`js9504604.
`33. Grassin-Delyle S, Buenestado A, Naline E, Faisy C, Blouquit(cid:173)
`Laye S, Couderc L-J, et al. Intranasal drug delivery: an efficient
`and non-invasive route for systemic administration: focus on
`opioids. Pharmacol Ther. 2012;134(3):366-79.
`34. England RJ, Homer JJ, Knight LC, Ell SR. Nasal pH
`measurement: a reliable and repeatable parameter. Clin
`Otolaryngol Allied Sci. 1999;24(1):67-8.
`35. Cho DY, Hwang PH, Illek B, Fischer H. Acid and base
`secretion in freshly excised nasal tissue from cystic fibrosis
`patients with ~F508 mutation. Int Forum Allergy Rhino!.
`2011;1(2):123-7. https://doi.org/10.1002/alr.20028.
`36. Washington N, Steele R, Jackson S, Bush D, Mason J, Gill D,
`et al. Determination of baseline human nasal pH and the effect
`of intranasally administered buffers. Int J Pharm.
`2000;198(2):139-46.
`Inactive ingredient database U.S. Food and Drug Administra(cid:173)
`tion. 2018. Available from: https://www.accessdata.fda.gov/
`scripts/cder/iig/index.cfm. Accessed 16 Apr 2019.
`
`37.
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