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Title:
PLGA MICROPARTICLES LOADED WITH A FLUOROQUINOLONE FOR THE TREATMENT OF RESPIRATORY DISEASES
Document Type and Number:
WIPO Patent Application WO/2018/011040
Kind Code:
A1
Abstract:
This invention relates to PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability, the method of preparation thereof and applications thereof.

Inventors:
OLIVIER JEAN-CHRISTOPHE (FR)
DA COSTA GASPAR MARISA (FR)
Application Number:
PCT/EP2017/066831
Publication Date:
January 18, 2018
Filing Date:
July 05, 2017
Export Citation:
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Assignee:
INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECH MÉDICALE) (FR)
UNIV POITIERS (FR)
International Classes:
A61K9/00; A61K9/16; A61K31/00
Domestic Patent References:
WO2008157614A22008-12-24
WO2004089291A22004-10-21
WO2006125132A22006-11-23
WO2012054498A12012-04-26
Foreign References:
US20100209478A12010-08-19
US20080124400A12008-05-29
CN102885783B2014-04-16
CN102302458B2013-01-09
Other References:
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GOVENDER T; STOLNIK S; GARNETT MC; ILIUM L; DAVIS SS: "PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug", JOURNAL OF CONTROLLED RELEASE., vol. 57, no. 2, 1999, pages 171 - 85
SAELIM N; SUKSAWAENG K; CHUPAN J; TECHATANAWAT I: "Biopharmaceutics classification system (BCS)-based biowaiver for immediate release solid oral dosage forms of moxifloxacin hydrochloride (moxiflox GPO) manufactured by the government pharmaceutical organization (GPO", ASIAN JOURNAL OF PHARMACEUTICAL SCIENCES, 2015
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Attorney, Agent or Firm:
CABINET PLASSERAUD (FR)
Download PDF:
Claims:
CLAIMS

1. A PLGA microparticle loaded with a fluoroquinolone wherein:

the size of microparticle is comprised between 1 and 10 μιη, and

the fluoroquinolone content is comprised between 1 and 30 wt. % in relation to the total weight of the loaded microparticle.

2. The PLGA microparticle loaded with a fluoroquinolone according to claim 1, wherein the PLGA has ratio of lactide to glycolide comprised between 95:5 and 40:60, preferably between 85: 15 and 45:55, more preferably between 75:25 and 50:50, more preferably between 70:30 and 50:50, more preferably between 65:35 and 50:50, more preferably between 60:40 and 50:50, and even more preferably between 55:45 and 50:50.

3. The PLGA microparticle loaded with a fluoroquinolone according to claim 1 or 2, wherein the PLGA has a weight average molecular weight higher or equal to 500 Mw and inferior or equal to 240000, 116000, 70000, 40000, 28000, 18000, 15000, 7000 and 4000 Mw.

4. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 3 wherein the fluoroquinolone is selected from the group consisting of levofloxacin, ofloxacin, gatifloxacin, moxifloxacin and lomefloxacin, and isomers thereof.

5. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 4 wherein the fluoroquinolone is levofloxacin.

6. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 5 wherein the content of fluoroquinolone is comprised between 1 and 30 wt. , preferably between 5 and 20 wt. , and even more preferably between 5 and 15 wt. % in relation to the total weight of the loaded microparticle.

7. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 6 which has a size comprised between 1 and 10 μιη, preferably between 1 and 7 μιη and even more preferably between 1 and 5 μιη.

8. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 7 which is obtainable by a double emulsion method.

9. A population of the PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 8 which has a size of volume distribution (Dv) comprised between 1 and 10 μιη, preferably between 1 and 7 μιη and even more preferably between 1 and 5 μιη.

10. A composition comprising the population of the PLGA microparticle loaded with a fluoroquinolone according to claim 9.

11. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 8 or the composition according to claim 10 for use as a medicament.

12. The PLGA microparticle loaded with a fluoroquinolone according to anyone of claims 1 to 8 or the composition according to claim 10 for use in the treatment of pulmonary infections.

13. The PLGA microparticle loaded with a fluoroquinolone according to claim 11 or 12 or the composition according to claim 11 or 12 for aerosol administration

14. Method for producing the PLGA microparticles loaded with a fluoroquinolone according to anyone of claims 1 to 8 comprising the step of:

a) preparing a water-in-oil (W/O) emulsion wherein said emulsion comprises a fluoroquinolone solution, PLGA and a solvent, and

b) dispersing the water-in-oil (W/O) emulsion obtained in step (a) in an aqueous solution saturated with the fluoroquinolone.

Description:
PLGA MICROPARTICLES LOADED WITH A FLUOROQUINOLONE FOR THE TREATMENT OF RESPIRATORY DISEASES

INTRODUCTION

This invention relates to PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability, the method of preparation thereof and applications thereof.

BACKGROUND OF THE INVENTION

Fluoroquinolones are known to be used for the treatment of several bacterial infections. Typically, fluoroquinolones are used in the treatment of pulmonary infections.

Among fluoroquinolones, fluoroquinolones with high mucosal permeability are particularly interesting given that they exhibit specific properties and have particular distribution profiles. More particularly, levofloxacin is a highly water soluble fluoroquinolone characterized by a high permeability profile through the broncho-alveolar barrier.

It is well-known that fluoroquinolones are administered orally and intravenously. However, such administration routes require high doses of antibiotics and have undesirable side effects. The search for more efficient therapeutic approaches has driven to the development of inhaled fluoroquinolones. However, such products request relatively frequent administration to deliver the therapeutic dose, fastidious hygienic procedures and are not particularly adapted to fluoroquinolones with high mucosal permeability.

In order to solve the above-mentioned technical problems, the inventors of the present invention have developed a specific combination of PLGA and a fluoroquinolone with high mucosal permeability. The present inventors have discovered that the specificity of this combination, in term of nature of PLGA, PLGA particle size and fluoroquinolone content, highlights interesting properties in terms of lung concentrations, antibacterial efficacy, systemic exposures and toxicity. Thus, the present invention provides PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability which enable to improve treatment efficiency and ease of use, and to reduce frequency of administration while reducing systemic toxicity.

SUMMARY OF THE INVENTION

A first object of the present invention relates to a PLGA microparticle loaded with fluoroquinolone and applications thereof, wherein the size of the PLGA microparticle is comprised between 1 and 10 μιη, and the fluoroquinolone content is comprised between 1 and 30 wt. % in relation to the total weight of the loaded microparticle.

A second object of the present invention relates to a population of the PLGA microparticle loaded with a fluoroquinolone.

A third object of the present invention relates to a composition comprising the population of the PLGA microparticle loaded with a fluoroquinolone and applications thereof.

Another object of the present invention relates to a method for producing the PLGA microparticles loaded with a fluoroquinolone. Thus, the inventors of the present invention have demonstrated that PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability enable to solve the above mentioned technical problems, i.e. to provide a sustained-release PLGA microsphere dry powder in the form of aerosol, advantageous in terms of treatment efficiency, ease of use and frequency of administration while reducing systemic toxicity.

More particularly, the present inventors have found that the above-mentioned technical problems could be solved with specific PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability. Indeed, the microparticles of the present invention are specific in term of nature of PLGA, PLGA particle size and fluoroquinolone content. DEFINITIONS

The term "PLGA" of the present invention refers to the poly lactic-coglycolic acid, i.e. a copolymer of poly lactic acid (PLA) and poly glycolic acid (PGA).

The term "fluoroquinolones" of the present invention refers to fluoroquinolones with high mucosal permeability, i.e. fluoroquinolones characterized by a high permeability profile through the broncho-alveolar barrier.

The term "mucosal permeability" of the present invention refers to the permeability of the respiratory system mucosa.

The term "fluoroquinolone sustained-release" of the present invention refers to the release of the fluoroquinolone at a slow but steady rate over a specific period of time allowing a prolonged-action. The term "Fine Particle Fraction (FPF)" of the present invention refers to the fraction of particles having a diameter equal to or less than 5 μιη.

The term "pharmaceutically acceptable carrier or excipient" of the present invention refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the host at the concentration at which it is administered. Said carriers and excipients are selected from the usual excipients known by a person skilled in the art. The term "treatment" of the present invention refers to a method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease; (2) bringing about amelioration of the symptoms of the disease; or (3) curing the disease. A treatment may thus be administered after initiation of the disease, for a therapeutic action.

The term "effective amount" of the present invention refers to any amount of fluoroquinolone that is sufficient to fulfil its intended purpose(s), e.g. a desired biological or medicinal response in a cell, tissue, system or patient. The term "patient" of the present invention refers to a human or another mammal (e.g., primate, mouse, rat, rabbit, dog, cat, horse, cow, pig, camel, and the like). Preferably, the patient is a human.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One object of the present invention is a PLGA microparticle loaded with a fluoroquinolone wherein the size of microparticle is comprised between 1 and 10 μιη, and

the fluoroquinolone content is comprised between 1 and 30 wt. % in relation to the total weight of the loaded microparticle. PLGA is obtained by the co-polymerization of poly glycolic acid (PGA) and poly lactic acid (PLA). The release of the loaded fluoroquinolone depends on the PLGA properties, in particular on the PLGA ratio of lactide to glycolide (PLA/PGA), the PLGA molecular weight and the PLGA chain size.

In particular, a PLGA with higher molecular weight exhibits lower degradation rates than a PLGA with lower molecular weight.

In particular, a PLGA having longer polymer chains requires more time to degrade than a PLGA having smaller polymer chains.

In particular, a higher content of poly glycolic acid leads to quicker degradation rate of PLGA, excepted for 50:50 ratio of lactide to glycolide which exhibits the fastest PLGA degradation.

Thus, the person skilled in the art might choose the PLGA molecular weight, PLGA ratio of lactide to glycolide and PLGA chain size according to the desired sustained release. In a particular embodiment, the ratio of lactide to glycolide is comprised between 95:5 and 40:60, preferably between 85: 15 and 45:55, more preferably between 75:25 and 50:50, more preferably between 70:30 and 50:50, more preferably between 65:35 and 50:50, more preferably between 60:40 and 50:50, and even more preferably between 55:45 and 50:50. In a preferred embodiment, the ratio of lactide to glycolide is about 50:50.

In a particular embodiment, the PLGA weight average molecular weight is higher or equal to 500 Mw and inferior or equal to 240000, 116000, 70000, 40000, 28000, 18000, 15000, 7000 and 4000 Mw. In a particular embodiment, the PLGA weight average molecular weight is comprised between 500 and 240000, preferably between 500 and 116000, more preferably between 500 and 70000, more preferably between 500 and 40000, more preferably between 500 and 28000 and even more preferably between 500 and 15000 Mw. In a particular embodiment, the PLGA weight average molecular weight is comprised between 500 and 240000, preferably between 4000 and 240000, more preferably between 7000 and 116000, more preferably between 7000 and 70000, more preferably between 7000 and 40000, more preferably between 7000 and 28000 and even more preferably between 7000 and 15000 Mw. According to the present invention, PLGA microparticles are loaded with a fluoroquinolone with high mucosal permeability.

Fluoroquinolones constitute a family of antibiotics which exert antibacterial effect by acting on the bacterial DNA. In particular, fluoroquinolones inhibit the DNA gyrase and DNA topoisomerase IV (Karl Drlica. Mechanism of fluoroquinolone action. Current Opinion in Microbiology, Volume 2, Issue 5, 1 October 1999, Pages 504-508).

Examples of fluoroquinolones with high mucosal permeability are, but not limited to, levofloxacin, ofloxacin, gatifloxacin, moxifloxacin and lomefloxacin.

In a particular embodiment, the fluoroquinolone with high mucosal permeability according to the invention is selected from the group consisting of levofloxacin, ofloxacin, gatifloxacin, moxifloxacin and lomefloxacin, and isomers thereof.

Levofloxacin and ofloxacin are characterized by a high permeability profile through the broncho-alveolar barrier.

In a particular embodiment, the fluoroquinolone is levofloxacin or ofloxacin.

In a preferred embodiment, the fluoroquinolone is levofloxacin.

The size of the PLGA microparticle loaded with a fluoroquinolone is chosen so as to enable a pulmonary administration in aerosol form.

Furthermore, the size of the PLGA microparticle loaded with a fluoroquinolone and the fluoroquinolone content are chosen so as to obtain a fluoroquinolone sustained-release of at least 24 hours, preferably of at least 48 hours, and even more preferably of at least 72 hours in vivo and in vitro.

In a particular embodiment, at 24 hours, approximately 50 % of the initial content of the administered fluoroquinolone has been released, wherein the fluoroquinolone initial content represents approximately 10 wt. % in relation to the total weight of the loaded microparticle. In a particular embodiment, at 48 hours, approximately 55 % of the initial content of the administered fluoroquinolone has been released, wherein the fluoroquinolone initial content represents approximately 10 wt. % in relation to the total weight of the loaded microparticle. In a particular embodiment, at 72 hours, approximately 75 % of the initial content of the administered fluoroquinolone has been released, wherein the fluoroquinolone initial content represents approximately 10 wt. % in relation to the total weight of the loaded microparticle.

Typically the sustained-release is assessed by a release study in vitro in phosphate-buffered saline, pH 7.4, at 37°C and estimated in vivo through pharmacokinetic modelling of the blood plasma and ELF concentration-versus-time data. (John W. Skoug et al. Qualitative evaluation of the mechanism of release of matrix sustained release dosage forms by measurement of polymer release).

Typically, levofloxacin plasma concentrations versus time data are analyzed according to a non-linear mixed effects method with S-ADAPT software (v 1.52) using MC-PEM (Monte- Carlo Parametric Expectation Maximization) estimation algorithm and S-ADAPT TRAN translator (Bulitta JB, Bingolbali A, Shin BS, Landersdorfer CB. Development of a New Pre- and Post-Processing Tool (SADAPT-TRAN) for Nonlinear Mixed-Effects Modeling in S- ADAPT. The AAPS Journal. 2011;13(2):201-11).

In a particular embodiment, the PLGA microparticle loaded with a fluoroquinolone according to the invention has a content of fluoroquinolone comprised between 1 and 30 wt. , preferably between 5 and 20 wt. , and even more preferably between 5 and 15 wt. % in relation to the total weight of the loaded microparticle.

Typically, the content of fluoroquinolone is determined by spectrophotometry using an UV- Visible spectrophotometer after dissolution in DMSO using a fluoroquinolone calibration curve. In a particular embodiment, the PLGA microparticle loaded with a fluoroquinolone according to the invention has a size comprised between 1 and 10 μιη, preferably between 1 and 7 μιη and even more preferably between 1 and 5 μιη. The present invention also relates to a sustained-release dry powder formulation comprising PLGA microparticles loaded with a fluoroquinolone according to the invention.

In a particular embodiment, the PLGA microparticle loaded with a fluoroquinolone according to the invention is obtained by a double emulsion method.

Typically, an appropriate amount of fluoroquinolone is dissolved in an organic solvent, preferably dichloromethane or chloroform, and the resulting mixture is added to an oil phase consisting of PLGA (Wi/O).

Then, the Wi/O emulsion is added to a continuous phase of an organic solvent, preferably a polyvinylalcohol.

Another object of the present invention is a population of the PLGA microparticle loaded with a fluoroquinolone according to the invention which has a size of volume distribution (Dv) comprised between 1 and 10 μιη, preferably between 1 and 7 μιη and even more preferably between 1 and 5 μιη.

Typically, the size of volume distribution (Dv) is measured in purified water using laser light diffraction.

Another object of the present invention is a composition comprising the population of the PLGA microparticle loaded with a fluoroquinolone according to the invention.

The present invention also relates to a composition consisting of the population of the PLGA microparticle loaded with a fluoroquinolone according to the invention

Another object of the present invention is the use of the composition according to the invention for aerosol administration.

Thus, the present invention relates to aerosol administration comprising administering the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention in a subject. Therapeutic uses

Fluoroquinolones constitute a family of antibacterial agents. Fluoroquinolones are indicated for the treatment of several bacterial infections. Several bacterial infections include but are not limited to, respiratory infections such as bacterial bronchitis, bronchiolitis, pneumonia, tuberculosis, tonsillitis pharyngitis, otitis and sinusitis, septicaemia, typhoid fever, joint and bone infections, soft tissue and skin infections, gastrointestinal infections and urogenital infections. More particularly, fluoroquinolones are known to have an activity against a wide range of gram-positive and gram- negative organisms. The present inventors have found and demonstrated that the PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability of the present invention enable to improve the fluoroquinolone efficiency against bacterial agents in comparison to free fluoroquinolones. They have also demonstrated that a pulmonary administration of the PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability of the present invention, results in a prolonged release of the fluoroquinolone within the lung and in much higher fluoroquinolone concentrations in pulmonary system, in particular in lung epithelial lining fluid (ELF). They have also demonstrated that specific PLGA microparticles loaded with a fluoroquinolone, in term of nature of PLGA, PLGA particle size and fluoroquinolone content enable to reduce the frequency of administrations, to increase anti-infectious treatment efficiency while reducing systemic toxicity. Indeed, the PLGA microparticles loaded with a fluoroquinolone with high mucosal permeability of the present invention have fluoroquinolone sustained-release of at least 72 hours.

Thus, another object of the present invention relates to the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention for use as a medicament.

The present invention also provides a pharmaceutical composition comprising as active principle, the PLGA microparticles loaded with a fluoroquinolone according to the invention and a pharmaceutically acceptable excipient.

Another object of the present invention relates to the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention for use in the treatment of pulmonary infections.

In a particular embodiment, pulmonary infections are bacterial bronchitis, bronchiolitis and pneumonia. The present invention also relates to a method for treating a pulmonary infection, comprising administering to a patient an effective amount of the PLGA microparticle loaded with a fluoroquinolone according to the invention, or of the composition according to the invention. Typically, pulmonary infections are bacterial bronchitis, bronchiolitis and pneumonia.

The present invention also relates to a method for treating a patient having a pulmonary infection. Typically, pulmonary infections are bacterial bronchitis, bronchiolitis and pneumonia. The present invention also relates to the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention used in a method for the treatment of a human or an animal.

In a particular embodiment, the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention is used for aerosol administration

In a particular embodiment, the present invention relates to the PLGA microparticle loaded with a fluoroquinolone according to the invention or the composition according to the invention for aerosol administration destined to the treatment of pulmonary infections. Typically, pulmonary infections are bacterial bronchitis, bronchiolitis and pneumonia.

Suitable dosage ranges depend upon numerous factors such as the severity of the infection to be treated, the age and health of the subject. Furthermore, the dosage ranges depend on the sustained-release parameter and the fluoroquinolone content.

Typically, the dosage range of the PLGA microparticle loaded with a fluoroquinolone according to the invention is comprised between 1 and 10 mg/kg daily, preferably between 1 and 5 mg/kg daily, and more preferably between 1 and 2.5 mg/kg daily of body weight. In a particular embodiment, the dosage range of the PLGA microparticle loaded with a fluoroquinolone according to the invention is around 5 mg/kg of body weight every three days. Synthesis

Another object of the present invention relates to a method for producing the PLGA microparticles loaded with a fluoroquinolone according to the invention comprising the step of:

a) preparing a water-in-oil (W/O) emulsion wherein said emulsion comprises a fluoroquinolone solution, PLGA and a solvent, and

b) dispersing the water-in-oil (W/O) emulsion obtained in step (a) in an aqueous solution saturated with the fluoroquinolone. Typically, a levofloxacin solution is emulsified into a solution of PLGA and levofloxacin in dichloromethane or chloroform. The obtained Wi/0 emulsion is dispersed in a solution W 2 of polyvinylalcohol saturated with levofloxacin in PBS (phosphate buffered saline). The resulting W 1 /0/W 2 emulsion is then subjected to homogenization cycles, typically through a porous glass membrane. Then, it is poured into a solution of polyvinylalcohol saturated with levofloxacin in PBS. Dichloromethane is evaporated. Microspheres are washed through cycles of centrifugation, re-suspended in purified water and freeze-dried.

The present invention also relates to the PLGA microparticles loaded with a fluoroquinolone obtainable by the method according to the invention.

Although the exemplary embodiments of the present invention have been disclosed for illustrative purposes, a person skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

The present invention will now be illustrated using the following examples and figures, which are given by way of illustration, and are in no way limiting. BRIEF DESCRIPTION OF FIGURES

Figure 1. Structural pharmacokinetic model for intravenous or intratracheal administration of levofloxacin solutions and for intratracheal administration of chitosan loaded with levofloxacin or PLGA microsphere powder loaded with levofloxacin, with typical parameter estimates.

Vd, levofloxacin distribution volume; VELF, volume of ELF compartment; Vp, volume of peripheral lung compartment; CL, levofloxacin total clearance; Cldif, bidirectional transfer of levofloxacin clearance between plasma and ELF; Clout, unidirectional transfer of levofloxacin clearance from plasma to ELF; Cldist, levofloxacin distribution clearance; FELF, fraction of dose immediately released into the ELF compartment; FWeib, fraction of dose released according to a Weibull release model; a, time scale parameter; b, curve shape parameter, and CV (coefficient of variation), estimable inter-individual variabilities.

Figure 2. SEM (Scanning Electron Microscopy) images of PLGA microspheres after preparation (A and B) and after a one-week incubation at 37°C in PBS pH 7.4 under a 600- rpm magnetic stirring (C and D).

Figure 3. Experimental in vitro release data of PLGA microsphere loaded with levofloxacin in PBS, pH 7.4, at 37°C (mean + SD (square), n=3). Also represented is the in vivo release kinetic curve (dashed line) predicted by the pharmacokinetic model after intratracheal administration of PLGA microspheres and cumulating the immediate release (FELF = 19% of administered dose) and the slow release according to a Weibull model (FWeib = 73%, a = 27.7 h and b = 0.817). Figure 4. Observed levofloxacin plasma and ELF concentrations (mean + SEM, n = 3 to 5, log scale) versus time (symbols) and the respective pharmacokinetic model-predicted curves (solid lines, in grey for ELF and in black for plasma) after administration of a) an intravenous solution, b) an intratracheal solution (5.79 + 0.5 mg/kg dose), c) an intratracheal chitosan microsphere dry powder (4.73 + 2.0 mg/kg dose) and d) an intratracheal PLGA microsphere dry powder (3.06 + 1.5 mg/kg dose). Dotted lines correspond to the unbound plasma concentration curves. In the intratracheal PLGA microspheres panel, the abscissa (time) scale is different. EXAMPLES

A comparative pharmacokinetic study was conducted in rats after intratracheal aerosolization of levofloxacin:

- as a solution,

- as immediate-release chitosan microspheres, and

- as sustained-release PLGA microspheres.

A pharmacokinetic model was constructed to model levofloxacin concentrations both in plasma and in the lung epithelial lining fluid (ELF). Materials

Resomer® RG 502 H (PLGA 50:50, acid terminated) and dimethyl sulfoxide (DMSO) were obtained from Sigma- Aldrich® (France).

Rhodoviol 4/125 (polyvinylalcohol, degree of hydrolysis of 88%) was purchased from Prolabo (France).

Levofloxacin hemihydrate was provided by Tecnimede S.A. (Portugal).

Isoflurane (Forene®) was purchased from Abb Vie (France).

Dichloromethane BDH HipPerSolvTM for HPLC and formic acid 99-100% AnalaR (NormaPur) were obtained from VWR® (France).

Acetonitrile of HPLC grade was purchased from Carlo Erba reagents (France).

All other chemicals were of analytical grade or equivalent.

Purified water was produced using a MilliQ Gradient® Plus Millipore system.

The animal experiments were conducted in compliance with EC Directive 2010/63/EU after approval by the local ethic committee (COMETHEA) and were registered by the French Ministry of Higher Education and Research under the authorization number 2015042116017243.

Male Sprague Dawley® rats, RjHan:SD, (300 - 400 g, 8-9 weeks of age) were obtained from Janvier Laboratories (Le Genest-St-Isle, France). Male Sprague Dawley® rats were housed in ventilated and temperature-controlled wire cages under a 12-hour light-dark cycle for a minimum of 5 days before experiments, with ad libitum access to food (Product reference: 4RF21- PF1610; FLASH Aptitude, Gif-sur-Yvette, France) and water. The same conditions were maintained after the drug administration. Methods

Preparation of the levofloxacin solution for intravenous administration

On the day of the experiment, levofloxacin was dissolved in saline. The final concentration (1.5 - 2 mg/ml) was adapted to the rat weights in order to administer a maximum volume of 1 ml through the tail vein and to achieve 5 mg levofloxacin per kg body weight. This dose was calculated to be in the range of the levofloxacin inhalation solution (Aeroquin®) doses administered in the Cystic Fibrosis patients (Geller DE, Flume PA, Staab D, Fischer R, Loutit JS, Conrad DJ. Levofloxacin Inhalation Solution (MP- 376) in Patients with Cystic Fibrosis with Pseudomonas aeruginosa. American Journal of Respiratory and Critical Care Medicine, 2011;183(l l): 1510-6).

Preparation of the levofloxacin solution for intratracheal aerosolization

On the day of the intratracheal administration, levofloxacin was dissolved in saline at a 20 mg/ml final concentration in order to administer a fixed volume of 100 μΐ containing a targeted dose close to the intravenous dose (5mg/kg).

Preparation of chitosan microspheres for intratracheal aerosolization

Chitosan microspheres crosslinked with genipin and loaded with levofloxacin, were prepared by a spray-drying method and characterized by spectrophotometry using a UV-Visible spectrophotometer (Gaspar MC, Sousa JJS, Pais AACC, Cardoso O, Murtinho D, Serra MES et al. Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2015;96:65-75). The drug content was 48.4 + 5.8 wt. and mass median aerodynamic diameter (MMAD) was 5.4 + 0.2 μιη. In vitro release studies showed more than 90% release within 15 min in phosphate buffered saline (PBS), pH 7.4 at 37°C.

Preparation and characterization ofPLGA microspheres for intratracheal aerosolization PLGA microspheres loaded with Levofloxacin were prepared by a double emulsion - solvent evaporation method with premix membrane homogenization (Doan TV, Couet W, Olivier JC. Formulation and in vitro characterization of inhalable rifampicin-loaded PLGA microspheres or sustained lung delivery, International Journal of Pharmaceutics. 2011;414(l-2): 112-7). Briefly, 0.6 ml of a levofloxacin solution (250 mg/ml, adjusted to pH 6 with hydrochloric acid) was emulsified into 3 ml of a solution of PLGA (300 mg) and levofloxacin (100 mg) in dichloromethane using a Polytron® PT 3100D homogenizer equipped with a 7 mm homogenizing accessory (Kinematica AG, Switzerland) and set at 30000 rpm for 30 s. The obtained Wi/0 emulsion was dispersed in 7 ml of a solution W 2 of polyvinylalcohol (3% w/v) saturated with levofloxacin (35 mg/ml) in PBS at pH 7.4 under magnetic stirring (400 rpm). The resulting WyO/W 2 emulsion was subjected to three homogenization cycles through a Shirasu porous glass membrane (19.9 μιη porosity) under 25 kPa transmembrane pressure using an external pressure-type micro kit emulsification device (SPG Technology, Sadowara, Japan). It was immediately poured into 25 ml of a solution of 0.4 % (w/v) polyvinylalcohol saturated with levofloxacin (32 mg/ml) in PBS under magnetic stirring (400 rpm). Dichloromethane was evaporated off under vacuum at room temperature during 10 min using a rotary evaporator. Microspheres were washed through three cycles of centrifugation (3500 rpm, 5 min), resuspended in purified water (2 ml) and freeze-dried.

PLGA microspheres loaded with levofloxacin were then characterized according to their size, aerodynamic properties, drug content, morphology and release profile.

The mean size of the volume distribution (Dv) of microspheres was determined in purified water using laser light diffraction (Micro trac® XI 00 particle size analyzer) (Doan TV, Couet W, Olivier JC. Formulation and in vitro characterization of inhalable rifampicin-loaded PLGA microspheres for sustained lung delivery. International Journal of Pharmaceutics, 2011;414(l-2): 112-7 - Doan TVP, Olivier JC. Preparation of rifampicin-loaded PLGA microspheres for lung delivery as aerosol by premix membrane homogenization. International Journal of Pharmaceutics, 2009;382(l-2):61-6).

Yield (%) was calculated from the recovered mass of freeze-dried microspheres versus the initial weight of levofloxacin (in O and W phases) plus PLGA.

The microsphere drug content ( ), i.e. the amount of levofloxacin (mg) per 100 mg of microspheres (including entrapped levofloxacin), was determined by spectrophotometry at 300 nm using a Varian Cary 50 UV- Visible spectrophotometer after dissolution in DMSO using a levofloxacin calibration curve (0.625 - 10 μg/ml concentration range in DMSO). MMAD was determined with a Handihaler® Dry Powder Inhaler (DPI) using a Next Generation Impactor (NGI, Copley Ltd., Nottingham, UK), (Gaspar MC, Sousa JJS, Pais AACC, Cardoso O, Murtinho D, Serra MES et al. Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2015;96:65-75). The powder remaining in the capsule and deposited in the inhaler, induction port and all the stages was collected with DMSO (dimethyl sulfoxide) for levofloxacin spectrophotometric determination.

Microspheres were examined by scanning electron microscopy (SEM) using a Jeol JSM 6010 LV electron microscope (Tokyo, Japan) at 15 kV, after gold-sputtering the microspheres in an argon atmosphere.

For in vitro release studies under sink conditions, microspheres loaded with levofloxacin (5 mg) were dispersed in 10 ml of PBS, pH 7.4, and incubated at 37 °C under magnetic stirring (600 rpm) (Gaspar MC, Sousa JJS, Pais AACC, Cardoso O, Murtinho D, Serra MES et al. Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2015;96:65-75). One ml aliquots were collected at pre-determined time points over 7 days and centrifuged at 3500 rpm for 5 min (Hettich® Zentrifugen Universal 320R, Germany). Then, 100 μΐ of supernatant were collected and levofloxacin determined by HPLC, as previously described (Gaspar MC, Sousa JJS, Pais AACC, Cardoso O, Murtinho D, Serra MES et al. Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy. European Journal of Pharmaceutics and Biopharmaceutics, 2015;96:65-75). The remaining 900 μΐ were vortex-mixed and added back to the flasks.

Pharmacokinetic study

The targeted levofloxacin dose was 5 mg per kg body weight.

The rats (n=80), divided in four groups, were anaesthetized with inhaled isoflurane before dosing.

Group 1 (n=15) received an intravenous bolus injection of a levofloxacin solution in saline in a tail vein.

Group 2 (n=15) was treated intratracheally with aerosolized 20 mg/ml levofloxacin solution in saline (100 μΐ) using a IA-1C liquid Microsprayer® Aerosolizer (Penn-Century Inc., Philadelphia, USA), (Marchand S, Gobin P, Brillault J, Baptista S, Adier C, Olivier J-C et al. Aerosol Therapy with Colistin Methanesulfonate: a Biopharmaceutical Issue Illustrated in Rats. Antimicrobial Agents and Chemotherapy, 2010;54(9):3702-7).

The immediate-release chitosan microspheres (4 mg, corresponding to 2 mg of levofloxacin, Group 3, n=15) or the sustained release PLGA microspheres (20 mg, corresponding to 2 mg levofloxacin, Group 4, n=35) were aerosolized intratracheally using a Dry powder InsufflatorTM DP-4 (Penn-Century Inc., Philadelphia, USA) device that was weighed before and after the administration in order to measure the actual administered dose. To perform the intratracheal administrations, anaesthetized rats were placed on a rodent work stand inclined at an angle of 45° (Tern, Lormont, France) and the tip of the microsprayer or of the powder insufflator was introduced into the rat' s trachea with visualization of the vocal cords using an otoscope (Gagnadoux F, Pape AL, Lemarie E, Lerondel S, Valo I, Leblond V et al. Aerosol delivery of chemotherapy in an orthotopic model of lung cancer. Eur Respir J, 2005;26(4):657-61). After the intravenous or the intratracheal aerosol administrations, rats were returned to individual cages with free access to food and water.

At pre-determined time points, rats (3 to 5 per time point) were re-anesthetized with inhaled isoflurane for broncho-alveolar lavage (BAL) and blood sampling . After rat immobilization in a supine position with cervical hyperextension, the trachea was exposed and incised between two rings. A polyethylene catheter (0.58 mm i.d. and 0.96 mm o.d.; Harvard, Les Ulis, France) connected to a syringe filled with 1 ml of saline at 37 °C was inserted into the trachea (50 mm deep). After injection of saline, BAL samples (300 to 800 μΐ) were immediately collected by aspiration via the same catheter. A blood sample was then collected by cardiac puncture. BAL and blood samples were centrifuged (3500 rpm for 5 min and 3000 rpm for 10 min, respectively, at 4°C) and supernatants stored at -20 °C until levofloxacin and urea assays. For BAL sampling, conditions for centrifugation were optimized in a preliminary study in order to ensure that all the PLGA microspheres potentially withdrawn during the BAL procedure were sedimented. ELF levofloxacin concentrations (CELF) were estimated from the BAL sample levofloxacin concentrations (CBAL) using a dilution factor calculated from the urea concentrations in plasma (Urea plasma) and in BAL (Urea BAL) samples using the equation CELF = CBAL (Urea plasma/ Urea BAL) (Gontijo AVL, Brillault J, Gregoire N, Lamarche I, Gobin P, Couet W et al. Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grep ofloxacin. Antimicrobial Agents and Chemotherapy, 2014;58(7):3942-9.). Levofloxacin determination

Sample preparation

For plasma samples, 50 μΐ of plasma were mixed with 200 μΐ of the ciprofloxacin internal standard solution (0.1 μg/ml) in acetonitrile. Protein precipitate was separated by centrifugation at 14000 rpm for 15 min and 200 μΐ of supernatant were collected and vortex- mixed with 400 μΐ of 0.1 % (v/v) formic acid prior to analysis.

The same procedure was applied to levofloxacin calibration standards (2 to 400 ng/ml) prepared in blank rat plasma samples.

For BAL samples, 20 μΐ of supernatant were mixed with 80 μΐ of ciprofloxacin internal standard solution (0.05 μg/ml) in 0.1% (v/v) formic acid before analysis.

For BAL sample analysis, levofloxacin calibration standards (2 to 400 ng/ml) were prepared in saline.

Liquid chromatography tandem-mass spectrometry (LC-MS/MS)

Levofloxacin concentrations were determined in plasma and BAL samples using a validated LC-MS/MS method. The system consisted of a Waters Alliance 2695 separations module equipped with a binary pump and an autosampler thermostatically controlled at 4°C, and of a Waters Micromass® Quattro micro API triple quadrupole tandem mass spectrometer. Reversed-phase chromatography was performed on a Phenomenex JupiterTM C18 300 A column (5.0 μιη, 50 x 2.1 mm).

The mobile phase was composed of 0.1% (v/v) formic acid in acetonitrile and 0.1% (v/v) formic acid in water (25:75 (v:v)). The flow rate was 0.20 ml/min and the injection volume 20 μΐ.

The mass spectrometer was operated in the positive-ion mode. Ions were analyzed via multiple reaction monitoring (MRM) employing the transition of the [M + 2H] 2+ precursor to the product ions for the analyte and for the internal standard. Transition ions were 362.2 to 318.2 m/z for levofloxacin and 332.2 to 314.2 m/z for the internal standard. Optimal MS/MS set up parameters were: +3.25 kV ion spray voltage, 600 L/h and 350°C desolvation gas (N 2 ) flow and temperature respectively, 10 L/h cone gas (N 2 ) flow, 120°C source temperature, 25 V cone potential for the analyte and for the internal standard, 20 V collision energy for the analyte and the internal standard, 500 ms dwell time.

The lower limit of quantification (LLOQ) for levofloxacin determinations in plasma and BAL samples was 2 ng/ml, and no experimental measurements were outside the standard curves (2 to 400 ng/ml). Intra- and interday variabilities were characterized at four concentration levels (including LLOQ) with precision and accuracy lower than 15% for 400, 40, and 5 ng/ml concentrations and lower than 20% for the LLOQ. Urea determination

For urea determination in plasma, a photometric detection was applied using a modular automatic analyzer (Roche, France). The urea concentration in BAL samples was evaluated by LC-MS/MS (Gontijo AVL, Brillault J, Gregoire N, Lamarche I, Gobin P, Couet W et al. Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grepafloxacin. Antimicrobial Agents and Chemotherapy. 2014;58(7):3942-9).

Pharmacokinetic analysis and modeling strategy

Levofloxacin plasma concentrations versus time data were analyzed according to a non-linear mixed effects method with S-ADAPT software (v 1.52) using MC-PEM (Monte-Carlo Parametric Expectation Maximization) estimation algorithm and S-ADAPT TRAN translator (Bulitta JB, Bingolbali A, Shin BS, Landersdorfer CB. Development of a New Pre- and Post- Processing Tool (SADAPT-TRAN) for Nonlinear Mixed-Effects Modeling in S-ADAPT. The AAPS Journal. 2011;13(2):201-11).

Observed concentrations were log-transformed for the analysis. Various structural models were tested and compared based on the likelihood ratio tests (p < 0.05) of their objective functions and on visual inspection of diagnostic plots. The structural pharmacokinetic model (Fig. 1) was derived from an initial generic hybrid compartment model, with a mono- compartmental model for the systemic pharmacokinetics and a bi-compartment model for the ELF pharmacokinetics.

The model for levofloxacin systemic pharmacokinetics was monocompartmental with a distribution volume (Vd) and a total clearance (CL).

The distribution between plasma and ELF was described by two different processes, on the one hand by a bi-directional transfer characterized by a clearance (Cldif) and on the other hand by a unidirectional transfer from plasma to ELF characterized by a clearance (Clout). Only the unbound fraction of levofloxacin in plasma (55%) (Hurtado FK, Weber B, Derendorf H, Hochhaus G, Dalla Costa T. Population Pharmacokinetic Modeling of the Unbound Levofloxacin Concentrations in Rat Plasma and Prostate Tissue Measured by Microdialysis. Antimicrobial Agents and Chemotherapy. 2014;58(2):678-86) was assumed to distribute between plasma and ELF.

A bi-compartmental pharmacokinetics of levofloxacin in the ELF was necessary for a good fitting of the observed data, with the ELF compartment characterized with a volume VELF estimated by the modelling and directly linked to the plasma compartment, and a peripheral compartment of volume Vp characterized with a distribution clearance (Cldist). Both VELF and Vp were estimated through modelling.

The release process of levofloxacin from the intratracheally-aerosolized formulations was divided into two components: a fraction of the dose FELF that was immediately released into the ELF compartment (burst release), and a fraction of the dose FWeib that was released according to a Weibull release model, expressed as a differential equation for pharmacokinetic modeling,

where Q is the amount of levofloxacin not yet released, t is time, a is the time- scale parameter and b the shape parameter.

Inter-individual variabilities were expressed as coefficient of variation (CV) and modeled as log-normal.

The residual variability was estimated with an additive error model on the log scale, back- transformed into a proportional error model on normal scale for both plasma and ELF data. Plasma drug concentrations below the LLOQ were handled using the Beal M3 method (Beal SL. Ways to fit a PK model with some data below the quantification limit. J Pharmacokinet Pharmacodyn. 2001 ;28(5):481-504).

Results and discussion

PLGA microsphere characterization and in vitro levofloxacin release profile

The double emulsion - solvent evaporation method with premix membrane homogenization produced narrowly size-distributed PLGA microspheres with a mean size of volume distribution Dv = 5.0 ± 1.7 μηι and a yield of 50.0 + 4.9 wt.%. The drug content of 10.5 + 1.4 wt.% was considered satisfactory taking into account that highly water soluble drugs, such as levofloxacin, are generally poorly entrapped within the hydrophobic PLGA polymer matrix (Govender T, Stolnik S, Garnett MC, Ilium L, Davis SS, PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug, Journal of Controlled Release. 1999;57(2): 171-85). The SEM of the freeze-dried PLGA microsphere powder showed spherical particles with a smooth and poreless surface (Fig. 2A and 2B) though some small surface features were visible and attributed to residual polyvinylalcohol (Fig. 2B).

Using the Handihaler® DPI to aerosolize the powder, the MMAD was 7.1 + 0.2 μιη, and the fine particle fraction (FPF) was 30.2 + 2.3 %. The fact that MMAD was slightly higher than Dv was attributed to microspheres aggregation, as shown by SEM (Fig. 2A). In PBS at 37°C, under sink conditions, the levofloxacin release (Fig. 3) was characterized by a "burst" release of 40 % of the levofloxacin microspheres content within the first 30 min, followed by a gradual release up to at least 72 h. At 72h, approximately 75% of the drug content was released. After 1 week in PBS at 37°C (Fig. 2C and 2D), the microspheres appeared to be extensively degraded with obvious signs of surface alteration (Fig. 2D) (Diez S, Tros de Ilarduya C. Versatility of biodegradable poly(d,l-lactic-co-gly colic acid) microspheres for plasmid DNA delivery. European Journal of Pharmaceutics and Biopharmaceutics. 2006;63(2): 188-97), while 25% of levofloxacin initial content still remained in the microsphere polymer matrix (not shown). The low molecular weight PLGA 50:50 Resomer® 502H is therefore expected to minimize pulmonary accumulation of polymer after microsphere lung deposition, especially in the case of repeated administrations.

Pharmacokinetic experimental and model-predicted results

The administration of intravenous and intratracheal levofloxacin solutions as well as intratracheal chitosan or PLGA microsphere powder to the rats did not cause apparent signs of toxicity. The rats that were maintained up to 72 h after the intratracheal treatment with aerosolized PLGA microspheres showed normal weight gain. Through the intravenous route, rats were dosed accurately with 5 mg levofloxacin per kg body weight, but through the intratracheal route, due to the fixed volume of liquid administered with the microsprayer or the variable dosing efficiency of the powder insufflator, the targeted levofloxacin dose was not achieved and actual doses are presented in Table 1.

A population pharmacokinetic approach was used to characterize mainly the intra-pulmonary pharmacokinetics of levofloxacin after intratracheal aerosolization of the two dry microsphere powder formulations. The study design included the intravenous and intratracheal administrations of a levofloxacin solution in order to get comparators and to improve the modeling output since the population pharmacokinetic approach allows simultaneous analysis of various data sets. It is also the most appropriate modeling procedure when only one data set (i.e. simultaneous plasma and ELF concentrations) can be collected in each individual. The pharmacokinetic model is presented with the pharmacokinetic parameter estimates on Fig. 1. No inter-individual variability could be estimated for CL, Vd and Vp. Eventually, the selected pharmacokinetic model provided a reasonably good description of the experimental data over time, both in plasma and ELF, after intravenous administration or intratracheal aerosolization with the various formulations, as illustrated on Figure 4. Residual errors of the model were 13% in plasma and 18% and 21% in ELF depending on whether levofloxacin was administered intravenous or intratracheal (all formulations taken together), respectively. The much higher ELF exposure after aerosolization of PLGA microspheres loaded with levofloxacin with high concentrations sustained over time was adequately reported by the model. However, the analysis of the pharmacokinetic study needs to take into account some limitations. Rapid initial absorption or distribution phases are often difficult to characterize in pharmacokinetics, especially when sampling procedures are not instantaneous (which is the case of the BAL sampling method). Early ELF concentrations after intratracheal aerosolization of the dry microsphere powder may indeed depend on multiple uncontrolled parameters, including the depot characteristics and the onset of drug release from the microspheres or/and of levofloxacin solubilization within the small volume of the ELF (Fig. 1). In addition, the invasive intratracheal aerosolization procedure may induce by itself a transient alteration of the lung physiology which may affect levofloxacin disposition. All these factors would result in a high variability of the data at early sampling times, which would require extensive data, therefore a large number of animals, in order to interpret properly and to computerize the initial resorption and distribution phases. Therefore, it was decided not to collect plasma and BAL samples earlier than 30 min post administration. As a consequence, the pharmacokinetic model with an early burst release is probably a crude description of a more complex reality and the very high initial ELF concentrations of levofloxacin predicted by the model (between 20 mg/ml and 100 mg/ml, depending on the administered formulation), together with the ELF AUC between time 0 and 0.5h which contributed dramatically to the total AUC should be taken with caution. Accordingly, AUC from 30 min to the last time of measurable concentration (AUC0.5-t) were considered in order to compare levofloxacin exposures (Table 1). Table 1. Pharmacokinetic parameter estimates of expo

*dose estimated by weighing the powder insufflator before and after the dosing

**corresponding to free and protein-bound levofloxacin

Intratracheal or intravenous administration of the levofloxacin solutions resulted in similar experimental levofloxacin concentrations in plasma and ELF (Fig. 4a and 4b) with an elimination half-life of 0.96h. The bioavailability for the intratracheal solution was estimated to be 98%, with a direct release of levofloxacin into the ELF compartment. The distribution between the ELF and plasma compartments was very rapid, with an estimated half-life of transfer between the two compartments lower than 1 min. The estimated levofloxacin passive diffusion clearance Cldif (Fig. 1) was close to the value determined for moxifloxacin (Gontijo AVL, Brillault J, Gregoire N, Lamarche I, Gobin P, Couet W et al. Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grepafloxacin. Antimicrobial Agents and Chemotherapy. 2014;58(7):3942-9.), in consistency with their close log D values (Brillault J, De Castro WV, Couet W. Relative Contributions of Active Mediated Transport and Passive Diffusion of Fluoroquinolones with Various Lipophilicities in a Calu-3 Lung Epithelial Cell Model. Antimicrobial Agents and Chemotherapy. 2010;54(l):543-5) and their reported high permeability (Saelim N, Suksawaeng K, Chupan J, Techatanawat I. Biopharmaceutics classification system (BCS)- based biowaiver for immediate release solid oral dosage forms of moxifloxacin hydrochloride (moxiflox GPO) manufactured by the government pharmaceutical organization (GPO). Asian Journal of Pharmaceutical Sciences. 2015). For both routes of administration of the levofloxacin solutions, the ELF-to-plasma AUC0.5-72h ratios were slightly above 1. The Clout term which improved the modeling reflected the higher ELF levofloxacin concentrations than the levofloxacin unbound plasma concentrations, independently of the route of administration. It is of note that the ELF-to-plasma AUC0.5-t ratio is slightly above 2 when considering unbound concentrations in plasma, which reflected the ratio of the plasma- to-ELF clearances to the ELF-to-plasma clearance, i.e. (Clout+ Cldif)/Cldif. Considering that levofloxacin intracellular accumulation into macrophages was reported to be low and intermediate between those of ciprofloxacin and moxifloxacin (Serai C, Barcia-Macay M, Mingeot-Leclercq MP, Tulkens PM, Van Bambeke F. Comparative activity of quinolones (ciprofloxacin, levofloxacin, moxifloxacin and garenoxacin) against extracellular and intracellular infection by Listeria monocytogenes and Staphylococcus aureus in J774 macrophages. Journal of Antimicrobial Chemotherapy. 2005;55(4):511-7), a systematic overestimation of levofloxacin concentrations in ELF due to a potential lysis of alveolar macrophages (Gontijo AVL, Brillault J, Gregoire N, Lamarche I, Gobin P, Couet W et al. Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grepafloxacin. Antimicrobial Agents and Chemotherapy. 2014;58(7):3942-9) was excluded. The Clout term may therefore characterize a levofloxacin efflux transport mechanism as previously reported in vitro (Brillault J, De Castro WV, Couet W. Relative Contributions of Active Mediated Transport and Passive Diffusion of Fluoroquinolones with Various Lipophilicities in a Calu-3 Lung Epithelial Cell Model. Antimicrobial Agents and Chemotherapy. 2010;54(l):543-5) and in vivo (Zimmermann ES, Laureano JV, dos Santos CN, Schmidt S, Lagishetty CV, de Castro WV et al. A simultaneous semi-mechanistic population analysis of levofloxacin in plasma, lung and prostrate to describe the influence of efflux transporters on drug distribution following intravenous and intratracheal administration. Antimicrobial Agents and Chemotherapy. 2015). However, as predicted by in vitro permeability studies on a Calu-3 lung epithelial model (Brillault J, De Castro WV, Couet W. Relative Contributions of Active Mediated Transport and Passive Diffusion of Fluoroquinolones with Various Lipophilicities in a Calu-3 Lung Epithelial Cell Model. Antimicrobial Agents and Chemotherapy. 2010;54(l):543-5) the impact of efflux transport was modest compared to what was reported with moxifloxacin, and the Clout value estimated for levofloxacin (11.8 ml/h/kg) was much lower than the one estimated for moxifloxacin (57.1 ml/h/kg (Gontijo AVL, Brillault J, Gregoire N, Lamarche I, Gobin P, Couet W et al. Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grepafloxacin. Antimicrobial Agents and Chemotherapy. 2014;58(7):3942-9).

At the first sampling time point (30 min) after intratracheal aerosolization of the chitosan microsphere powder (Fig. 4c), plasma and ELF concentrations were similar. Considering that levofloxacin was shown in vitro to be almost immediately released from the chitosan microspheres (Gaspar MC, Sousa JJS, Pais AACC, Cardoso O, Murtinho D, Serra MES et al. Optimization of levofloxacin-loaded crosslinked chitosan microspheres for inhaled aerosol therapy. European Journal of Pharmaceutics and Biopharmaceutics. 2015;96:65-75) and was predicted by the pharmacokinetic model to be immediately released into the ELF compartment after intratracheal administration (Fig. 1), the low bioavailability of levofloxacin compared to the intratracheal solution or to the PLGA microsphere powder was attributed to the loss of chitosan microsphere powder during the intratracheal administration step. During the administration with the insufflator the aerosol was indeed observed to be partly dispersed back into the environment. The ELF-to-unbound plasma AUC0.5-t ratio was above 2 (Table 1), in consistency with intravenous or intratracheal administrations of the levofloxacin solution. Therefore, the immediate release microspheres did not substantially differ, in terms of ELF concentration or systemic exposure to levofloxacin, from intravenous or aerosolized administration of the levofloxacin solution. After intratracheal aerosolization of the PLGA microsphere powder loaded with levofloxacin (Fig. 4d), pharmacokinetic profiles dramatically differed from profiles obtained after intravenous or intratracheal administration of solutions or after the intratracheal administration of the chitosan microspheres. ELF concentrations were much higher than plasma concentrations, resulting in a high ELF-to-plasma AUC0.5h-t ratio (Table 1). Moreover, plasma concentrations declined much more slowly than after intravenous administration or intratracheal aerosolization of the levofloxacin solution and could be measured up to 24 h versus 4 h after dosing. They declined in parallel to ELF concentrations with an approximate half-life of 18 h (vs. 0.96 h after intravenous administration), thus illustrating a flip-flop phenomenon. This flip-flop observed in plasma corroborated the sustained release of levofloxacin in ELF that followed the intratracheal aerosolization of the PLGA microsphere powder loaded with levofloxacin. The bioavailability of levofloxacin released from the PLGA microspheres was 92%, with 19% of the dose released immediately (burst release) into the ELF and 73% released slowly over more than 72 h into the ELF from a depot compartment, according to a Weibull model (a = 27.7 h; b = 0.817). The in vivo release profile of levofloxacin from the PLGA microspheres that was estimated by the model is presented on Fig. 3 for comparison with the in vitro release profile. The "burst" release was lower in vivo than in vitro (19% (= FELF =) vs. 40 %). This difference may be explained by the fact that the in vitro release was performed in sink conditions under stirring and at constant pH (7.4), whereas in vivo levofloxacin saturation concentration may be reached rapidly due to the small volume of the ELF compartment (Fig. 1). Subsequently, the release became faster in vivo than in vitro. Beyond 18 hours, the cumulated in vivo released amount overpassed the in vitro results. Inhaled microspheres with diameter below 10 μιη are likely to be phagocytosed by lung macrophages (Hirota K, Kawamoto T, Nakajima T, Makino K, Terada H. Distribution and deposition of respirable PLGA microspheres in lung alveoli. Colloids and Surfaces B: Biointerfaces. 2013;105:92-7). The levofloxacin release from the PLGA microspheres may be impacted by the microsphere accumulation in intracellular compartments like phagolysosomes characterized by an acidic pH where levofloxacin is more soluble than at pH 7.4. In addition, in the case of the biodegradable polymeric PLGA microspheres, it is assumed that after the initial burst release the sustained release process results from the combination of drug diffusion within the polymer matrix and of the polymer matrix erosion (Shen J, Burgess DJ. Accelerated in vitro release testing methods for extended release parenteral dosage forms. The Journal of pharmacy and pharmacology. 2012;64(7):986-96). The polymer erosion in vitro was demonstrated by SEM analysis (Fig. 2C and 2D). Polymer erosion may be faster in vivo than in vitro, thus explaining the higher levofloxacin amount released between 18 and 72 hours. Conclusions

The intratracheal administration of the immediate release chitosan microsphere formulation provided pharmacokinetic profiles comparable to the intravenous or the intratracheal levofloxacin solutions, with the benefits inherent to dry powder formulations.

In particular, the plasma and ELF experimental concentration profiles versus time were similar for the intravenous and intratracheal levofloxacin solutions and for the intratracheal administration of chitosan microsphere dry powder loaded with levofloxacin, indicating that levofloxacin diffused almost instantaneously through the broncho-alveolar barrier and that the chitosan microspheres released levofloxacin very rapidly, as anticipated from in vitro release studies.

The bioavailability for the intratracheal levofloxacin solution and intratracheal chitosan microspheres was estimated to be 98% and 71%, respectively, both with a direct release into the ELF compartment. The ELF-to-unbound plasma AUC ratios were slightly above 2 and may result from an efflux transport. For the intratracheal PLGA microspheres, a high ELF-to- unbound plasma AUC concentration ratio (311) was observed and high levofloxacin concentrations were maintained in ELF for at least 72h in consistency with the in vitro release studies. The bioavailability was 92%, with 19% of the dose released immediately (burst release) into the ELF and 73% released slowly into the ELF from a depot compartment, i.e. the PLGA microspheres, according to a Weibull model.

Thus, the intratracheal administration of PLGA microspheres loaded with levofloxacin resulted in a prolonged release of levofloxacin within the lungs and in much higher levofloxacin concentrations in ELF than in plasma. Such a sustained-release formulation is expected to reduce the frequency of administrations compared to a levofloxacin solution for inhalation, and to increase anti-infectious treatment efficiency while reducing systemic toxicity. In particular, these results highlight the benefit of using sustained-release microspheres administered as aerosols to provide and to maintain high pulmonary concentrations of a highly water soluble antibiotic characterized by a high permeability profile through the broncho-alveolar barrier, i.e. levofloxacin. The sustained-release microsphere dry powder aerosol may therefore provide a promising alternative to the solutions or to pure drug dry powders for inhalation in terms of treatment efficiency, ease of use and frequency of administration, which should have a positive impact on the patients' compliance to their treatments. REFERENCES

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