Quantitative Estimation of the Effect of Nasal Mucociliary Function on

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Quantitative Estimation of the Effect of Nasal Mucociliary Function on In Vivo Absorption of Norfloxacin after Intranasal Administration to Rats Daisuke Inoue, Shunsuke Kimura, Akiko Kiriyama, Hidemasa Katsumi, Akira Yamamoto, Ken-ichi Ogawara, Kazutaka Higaki, Akiko Tanaka, Reiko Yutani, Toshiyasu Sakane, and Tomoyuki Furubayashi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00464 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 31, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Quantitative Estimation of the Effect of Nasal Mucociliary Function on In Vivo Absorption of Norfloxacin after Intranasal Administration to Rats Daisuke Inoue1, Shunsuke Kimura2, Akiko Kiriyama2, Hidemasa Katsumi3, Akira Yamamoto3, Ken-ichi Ogawara4, Kazutaka Higaki4, Akiko Tanaka5, Reiko Yutani5, Toshiyasu Sakane5, Tomoyuki Furubayashi1*. 1

Department of Pharmaceutics, School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara,

Naka-ku, Okayama 703-8516, Japan, 2Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo, Kyotanabe-shi, Kyoto 610-0395, Japan, 3Department of Biopharmaceutics, Kyoto Pharmaceutical University, 5 Misasagi-nakauchi-cho, Yamashina, Kyoto 607-8414, Japan, 4Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan, 5Laboratory of Pharmaceutical Technology, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada, Kobe 658-8558, Japan.

KEYWORDS. Nasal absorption, mucociliary clearance, prediction system, nasal formulation, norfloxacin.


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ABSTRACT. Nasal drug delivery has attracted significant attention as an alternative route to deliver drugs having poor bioavailability. Large-molecule drugs such as peptides, and central nervous system drugs, would benefit from intranasal delivery. Drug absorption after intranasal application depends on the nasal retention of the drug, which is determined by the nasal mucociliary clearance. Mucociliary clearance (MC) is an important determinant of the rate and extent of nasal drug absorption. The aim of the present study was to clarify the effect of changes in MC on in vivo drug absorption after nasal application, and to justify the pharmacokinetic model to which the MC parameter was introduced, to enable prediction of bioavailability after intranasal administration. The pharmacokinetics of norfloxacin (NFX) after intranasal administration were evaluated following the modification of nasal MC by pretreatment with the MC inhibitors propranolol and atropine and the MC enhancers terbutaline and acetylcholine chloride. From the relationship between nasal MC and bioavailability after nasal application, prediction of drug absorption was attempted based on our pharmacokinetic model. Propranolol and atropine enhanced the bioavailability of NFX by 90% and 40%, respectively, while the bioavailability decreased by 30% following terbutaline and 40% following acetylcholine chloride. As a result of changes in MC function, nasal drug absorption was changed depending on the nasal residence time of the drug. Based on our pharmacokinetic model, the nasal drug absorption can be precisely predicted, even when the MC is changed. This prediction system allows the quantitative evaluation of changes in drug absorption due to changes in nasal MC, and is expected to contribute greatly to the development of nasal formulations.


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1. Introduction Nasal drug delivery is an alternative administration route for poorly absorbed drugs. Examples include: high molecular weight drugs, peptides1-4, vaccines 5, 6 and drugs acting on the central nervous system (CNS) that can be directly transported from the nose to the brain via olfactory or trigeminal nerves7. Despite many studies reporting the predominance of nasal drug delivery, relatively few nasal drug preparations for systemic delivery are available on the market7. The absence of a system that allows for accurate estimation of nasal drug absorption may be one of the reasons why these formulations have not progressed. Species differences between animals and human, and inter-individual variability are frequently observed. These problems may make the development of nasal preparations difficult. Drug absorption after intranasal application depends on the nasal retention of the drug, which is determined by nasal mucociliary clearance (MC)8, 9. The primary function of MC is to protect against exogenous substances, such as bacteria and viruses, deposited by inhalation10, 11. Drugs applied to the nasal cavity are cleared toward the pharynx by MC and are transported to the stomach after swallowing. Given the large difference observed between nasal and intestinal absorption, MC is an important determinant of the rate and extent of drug absorption after nasal application. In our previous study, we determined the importance of MC in drug absorption after nasal administration, and established the first pharmacokinetic (PK) model to estimate nasal drug absorption12, 13. Since normal MC is assumed in this first PK model, the nasal bioavailability cannot be estimated when the normal MC is changed. Some studies have reported that the in vivo–in vitro correlation of MC is not constant14 and that the MC parameter can change


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significantly15. MC is impaired in respiratory diseases such as primary ciliary dyskinesia (PCD), chronic occlusive pulmonary disease (COPD), cystic fibrosis (CF) and asthma16. Many investigations on MC have measured the transport velocity of materials such as dyes and microparticles within nasal and tracheal tissues. Nasal or tracheal tissues from various animal species have been utilized to date, such as the nasal mucosa of human17-20 and rats21-24, tracheal mucosa of rats25, frog palates14, 19, 26-28 and bovine tracheal tissues29, 30. In other studies, the clearance time of dyes and particles from the nasal cavity to the pharynx was evaluated by collecting the marker from the pharynx11. In vitro and in situ measurements in which the transport of particles on excised mucosa have been performed previously. Movement velocity of particles was regarded as an index of MC14, 18, 29, 30. In our previous study, an in vitro MC evaluation system has been developed by measuring the translocation velocity of fluorescent microspheres in excised rat nasal mucosa as an index of MC31, 32. The system allows us to investigate MC and evaluate the effects of drugs, pharmaceutical additives, and formulations on nasal MC, and to facilitate a better understanding of the nasal MC function. Using the information derived from the in vitro system, the first estimation system of nasal drug absorption by Furubayashi et al.12, 13 was optimized for the development of nasal formulations. The aim of the present study was to clarify the effect of changes in MC on in vivo drug absorption after nasal application, and to evaluate the relationship between the nasal MC and in vivo nasal drug absorption. In addition, our PK model was amended to incorporate the MC parameter, the relationship was subsequently applied to our PK model for the prediction of bioavailability after intranasal administration, and the accuracy of the prediction was verified.


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2. Experimental Section 2.1. Materials. Hanks’ Balanced Salt Solution (HBSS) was obtained from Gibco (Life Technologies Japan Ltd., Tokyo, Japan). Ethyl carbamate (Urethane) was obtained from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Phosphate buffered saline (PBS, pH 7.4) and acetic acid were purchased from Nacalai Tesque (Kyoto, Japan). (±)-Propranolol hydrochloride, acetylcholine chloride, and atropine were obtained from Sigma-Aldrich (St. Louis, MO, USA). Terbutaline sulfate was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and methanol used for HPLC was purchased from Kanto Chemical Co., Inc (Tokyo, Japan). 2.2. Animal studies. Male Wistar rats weighing 250-300 g were used in all animal experiments. All animal studies were conducted under guidelines approved by the local committee of Animal Care of Shujitsu University in accordance with the Principles of Laboratory Animal Care (NIH publication #85-23). 2.3. Pretreatment of rat nasal mucosa with MC modulators. Four drugs with different modes of pharmacological action were used as MC modulators, as described previously31, 32. Propranolol (PPL, 0.1 mM) and atropine (ATRP, 0.1 mM) are MC inhibitors, while terbutaline (TBL, 1.0 mM) and acetylcholine chloride (ACH, 0.1 mM) are MC enhancers. According to the previous reports, the drug concentrations used in this study are sufficient to exhibit the pharmacological action33-36. Additionally, it has been confirmed in our previous study37 that in vivo nasal residence of substances in the nasal cavity in rats was changed by the pretreatment of these MC modulators.


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2.4. In vivo study on norfloxacin pharmacokinetics after oral and intravenous administration. Norfloxacin (NFX) was used as a model drug. In order to obtain the basic information on the absorption and disposition of NFX, the plasma clearance and the gastrointestinal (GI) absorption of NFX were evaluated. NFX was dissolved in PBS (pH 6.0) at a concentration of 1.0 mg/mL. For oral application, the right femoral artery was cannulated with polyethylene tube (SP-31, 0.50 mm I.D. and 0.80 mm O.D., Natsume Co., Ltd., Tokyo, Japan) for blood sampling. The surgery was performed under light inhalation anesthesia with diethylether. After complete recovery from anesthesia, 1.0 mg/mL NFX solution was administered orally into stomach (1 mg/kg body weight) with the syringe equipped with an animal feeding needle with a conical tip (15G, 80 mm length, KN348, Natsume Co., Ltd., Tokyo, Japan). The rat was kept in the cage (KN326 Type III, Natsume Co., Ltd.) and 200 µL of blood samples were collected at appropriate time interval up to 360 min. For intravenous dosing, rats were anesthetized with urethane, and 1.0 mg/mL NFX solution was injected into the left femoral vein (1.0 mg/kg body weight). Blood samples (200 µL) were obtained from a cannula inserted into the right femoral artery at the appropriate time interval up to 120 min. 2.5. Effect of MC modulators on in vivo drug absorption after intranasal administration. For blood sampling, the right femoral artery was cannulated with polyethylene tubing (SP-31) under light inhalation anesthesia with diethylether. MC modulators were dissolved in PBS (pH 7.4) and 40 µL of each solution was instilled into the right nasal cavity of the rat at 10 mm depth from the nostril, using the microsyringe equipped with 10 mm polyethylene tube (SP-10, 0.28 mm I.D. and 0.61 mm O.D., Natsume Co., Ltd.) 15 min prior to the application of NFX solution. NFX solution (5 µL of 1.0 mg/mL, pH 6.0) was instilled into the right nasal cavity under light diethylether anesthesia in the same manner as the application of MC modulators. After drug


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application, rats were kept in the cage (KN326 Type III) and blood samples (200 µL) was collected at appropriate time intervals up to 360 min. It was clarified using Calu-3 monolayer that no MC modulators used in the study affected the transepithelial transport of NFX (data not shown). 2.6. Analysis of the drug in blood samples. Blood samples (200 µL) were centrifuged for 5 min at 16,100 x g to obtain plasma, which was stored at −40°C until analysis. Plasma samples (100 µL) were deproteinated by the addition of 1.2 mL methanol, and the supernatant (1 mL) of the mixture was evaporated to dryness. The residue was reconstituted in 100 µL mobile phase (100 mM acetic acid:methanol = 90:10) for HPLC analysis. The concentration of NFX was determined by HPLC (LC-10AD, Shimadzu, Kyoto, Japan) equipped with an ODS column (YMC-Pack Pro C18, 4.6 mm × 150 mm, YMC CO., LTD, Kyoto, Japan). Flow rate and column temperature were 1.2 mL/min and 40°C. NFX was detected fluorometrically (fluorescent detector, RF-10, Shimadzu, Kyoto, Japan) at 278 nm excitation and 448 nm emission. The parameters on the accuracy and precision of HPLC analysis and on the analytical limitation of HPLC are listed in Table 1 and Table 2, respectively.

Table 1. Analytical parameters of HPLC for determination of norfloxacin concentration in the plasma Spiked concentration (ng/mL) 2 20 200

Measured concentration (ng/mL) 1.96 20.21 200.08

Precision

Accuracy

(%RSD) 3.42 3.15 2.58

(%) 101.79 98.97 99.96

(n= 4) (n= 4) (n= 4)


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Table 2. Analytical limitation of HPLC for plasma concentration of norfloxacin

Norfloxacin

LOD (ng/mL)

LOQ (ng/mL)

0.30

0.89

LOD; limitation of detection LOQ; limitation of quantification

2.7. PK modeling of drug absorption after intranasal administration. We have previously established the PK model to which the MC parameter was introduced12, 13. Since the estimation of nasal drug absorption with this system is limited under normal MC conditions, the system has been improved for the estimation of drug absorption changes due to changes in MC37. According to the previous model, total bioavailability (BA) after nasal administration (FnTotal) is the sum of the nasal BA (Fn) and the intestinal BA (Fgi), as shown in Eq. 1. FnTotal = Fn + Fgi

Eq. 1

Assuming that drug absorption and drug clearance by MC is the first-order process, Fn can be calculated according to Eq. 2 from the first-order rate constants of absorption (kn) and MC (kmc). The theory and kinetic model for these parameters were described in detail in the previous report13.

Fn =

kn kn + kmc

Eq. 2

Fgi can be calculated as the product of oral bioavailability (Fpo) and the fractional clearance to the GI tract (Eq. 3), assuming the negligible drug degradation in the nasal cavity.


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Fgi = Fpo ⋅ (1 − Fn)

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Eq. 3

The total bioavailability (FnTotal) can be expressed as Eq. 5 by substitution of Eq. 2 and Eq. 3 into Eq. 1.

FnTotal = Fn + Fpo ⋅ (1 − Fn) 　= (1 − Fpo) ⋅ Fn + Fpo

FnTotal = (1 − Fpo) ⋅

kn + Fpo kn + kmc

Eq. 4 Eq. 5

By introducing the MC parameter in place of kmc, Eq. 5 can be extended to allow the prediction of nasal drug absorption, when MC is changed. 2.8. Statistical analysis. Results are expressed as the mean ± S.E. Statistical significance was determined using JMP® software (SAS Institute Japan, Tokyo, Japan), based on Dunnett’s test. Pearson’s method was applied to the analysis of the statistical significance of the correlation.

3. Results 3.1. Absorption of NFX after nasal and oral application. The plasma concentration-time profiles of NFX following intravenous (IV), intranasal (IN) and oral (PO) administration are shown in Figure 1. Pharmacokinetic parameters are listed in Table 3. The bioavailability of NFX after IN and PO were 23.3% and 3.6%, respectively. Drug absorption after IN was faster (time to reach the maximum plasma concentration; Tmax = 5 min) and the bioavailability was significantly higher than that after PO (p

Quantitative Estimation of the Effect of Nasal Mucociliary Function on

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