Inhalation of Respirable Crystalline Rifapentine Particles Induces

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Brief Article

Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation Thaigarajan Parumasivam, Anneliese S Ashhurst, Gayathri Nagalingam, Warwick J. Britton, and Hak-Kim Chan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00905 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Molecular Pharmaceutics

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Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation

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AUTHORS

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Thaigarajan Parumasivam1,2, Anneliese S. Ashhurst3,4, Gayathri Nagalingam3,4, Warwick J. Britton3,4,

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Hak-Kim Chan1

7 8

1

9

Australia

Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, NSW, 2006,

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2

School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Pinang, 11800, Malaysia

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3

Tuberculosis Research Program, Centenary Institute, Sydney, NSW, 2042, Australia

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4

13

Sydney, NSW, 2006, Australia

Discipline of Infectious Diseases and Immunology, Sydney Medical School, The University of

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Corresponding author: [email protected]

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GRAPHICAL ABSTRACT

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ABSTRACT

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Rifapentine is an anti-tuberculosis (TB) drug with a prolonged half-life, but oral delivery results in

3

low concentrations in the lungs because of its high binding (98 %) to plasma proteins. We have shown

4

that inhalation of crystalline rifapentine overcomes the limitations of oral delivery by significantly

5

enhancing and prolonging the drug concentration in the lungs. The delivery of crystalline particles to

6

the lungs may promote inflammation. This in vivo study characterises the inflammatory response

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caused by pulmonary deposition of the rifapentine particles. The rifapentine powder was delivered to

8

BALB/c mice by intratracheal insufflation at a dose of 20 mg/kg. The inflammatory response in the

9

lungs and bronchoalveolar lavage (BAL) was examined at 12 h, 24 h and 7 days post-treatment by

10

flow cytometry and histopathology. At 12 h and 24 h post-treatment, there was a significant influx of

11

neutrophils into the lungs, and this returned to normal by day 7. A significant recruitment of

12

macrophages occurred in the BAL at 24 h. Consistent with these findings, histopathological analysis

13

demonstrated pulmonary vascular congestion and significant macrophage recruitment at 12 h and 24 h

14

post-treatment. In conclusion, the pulmonary delivery of crystalline rifapentine caused a transient

15

neutrophil-associated inflammatory response in the lungs that resolved over 7 days. This observation

16

may limit pulmonary delivery of rifapentine to once a week at a dose of 20 mg/kg or less. The

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effectiveness of weekly dosing with inhalable rifapentine will be assessed in murine Mycobacterium

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tuberculosis infection.

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KEYWORDS: Tuberculosis, inhalable rifapentine, aerosol delivery, pulmonary inflammation,

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toxicity

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INTRODUCTION

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Tuberculosis (TB) is a leading cause of death globally and is predominantly a respiratory

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infectious disease caused by Mycobacterium tuberculosis. It is estimated that one-third of the world’s

4

population has been exposed to M. tuberculosis with almost 9.6 million new cases and 1.5 million

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deaths annually.1 The current treatment of TB is based on oral administration of at least three first-line

6

drugs in combination (i.e. isoniazid, rifampicin, ethambutol, pyrazinamide) with direct observation of

7

patient compliance. This strategy has been widely promoted and implemented by the World Health

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Organization (WHO) as DOTS (directly observed treatment, short course).2 However, the treatment is

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challenging owing to the requirement of at least 6 months therapy with high dose antibiotics and the

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associated side effects that often reduce patient compliance and treatment success.

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Inhalation drug delivery, following the natural route of infection, is a rational approach to

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improve the current anti-TB regimen. The advantages of inhalation drug delivery are: (1) it delivers

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the drug directly to the lungs that results in a higher local drug concentration than can be achieved by

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oral or parenteral administration,3, 4 (2) it reduces the systemic exposure of the drug,5 and (3) it avoids

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degradation of the drug by the liver and the acidic gastric environment.6 These features of pulmonary

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delivery can reduce the drug dose and adverse effects, and therefore has the potential to shorten the

17

treatment duration.

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Rifapentine is an US Food and Drug Administration (FDA) approved drug administered

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intermittently for treatment of latent TB. Following oral administration, however, rifapentine fails to

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reach effective concentrations in the lungs owing to high plasma protein binding (98 %)7. In addition,

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the bioavailability of the drug is highly influenced by concomitant food intake and induction of its

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own metabolism.8, 9 Studies have shown that these barriers can be overcome if rifapentine is delivered

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directly to the lungs.10,

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inhalable crystalline rifapentine to mice at a dose of 20 mg/kg produced a lung Cmax 100-fold higher

25

than the intraperitoneal administration at same dose.10 Furthermore, the lung Tmax was extended to 2 h

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when the drug was given by the intratracheal route compared to 1 h after intraperitoneal delivery.10

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The blood plasma and lung drug concentrations were also maintained above the minimum inhibitory

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In a previous study, we demonstrated that the intratracheal delivery of


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concentration (MIC) against M. tuberculosis (0.03 – 0.06 µg/mL)12 for more than 24 h post-

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treatment.10 These findings suggest that the inhalable crystalline rifapentine significantly enhances

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and prolongs the drug concentration in the lungs, and this has the potential to reduce the therapeutic

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dose and the treatment duration whilst maintaining systemic concentrations.

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Inhalation therapy is now accepted treatment for a number of respiratory diseases, such as

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asthma and cystic fibrosis,13 but no inhaled medication has been approved for the treatment of TB.

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One limitation on the use of inhalable antibiotics for treatment of TB is the potential to exacerbate

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inflammation in the lungs. Therefore, in this report, we have examined the effects on pulmonary

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inflammation and cellular influx after intratracheal insufflation of respirable-size crystalline

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rifapentine particles at a dose of 20 mg/kg in mice. We report the changes in the leukocyte

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populations in the lungs and the bronchoalveolar lavage (BAL), and the histopathological changes in

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the lungs after drug exposure. Although the cytotoxicity of rifapentine on in vitro respiratory cells has

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been documented,14-17 this is the first study to characterise in vivo the effects of pulmonary delivery of

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respirable-size rifapentine crystals. This is a necessary foundation prior to proceeding using the

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rifapentine in future murine TB efficacy studies and human clinical trials. Furthermore, this study

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contributes to better understand the safety of inhaled therapies for TB.

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MATERIALS AND METHODS

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Rifapentine dry powder production and characterisation. The rifapentine powder was produced

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following a method detailed in Chan et al.17 Briefly, rifapentine (Hangzhou ICH Imp & Exp Co. Ltd.,

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Hangzhou, China) was dissolved in a co-solvent system consisting of acetone (Ajax Finechem Pty

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Ltd., NSW, Australia) and UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific,

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NSW, Australia) (1:1, v/v) at a concentration of 4 mg/mL. The system was then heated at 67°C to

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evaporate the acetone and formed a crystalline suspension. The suspension was homogenised at 10

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000 rpm for 2 minutes (Silverson L4RT High Shear Mixer, Silverson, Chesham, UK) and spray dried

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using a B-290 mini spray dryer (Buchi Labortechnik AG, Flawil, Switzerland) in an open-loop

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configuration using the following parameters: inlet temperature of 60 °C, atomiser of 60 mm,

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aspirator of 100 %, and feed rate of 2 mL/min. 5 ACS Paragon Plus Environment


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The morphology of the particles was examined using scanning electron microscopy (SEM)

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(FESEM, Hitachi S4500, Japan) at an acceleration voltage of 5 kV. The volume equivalent diameters

3

of the particles were measured using laser diffraction coupled with a Scirocco dry powder disperser

4

(Mastersizer 2000, Malvern Instruments, Worcestershire, UK) at a refractive index (RI) of 1.63 and

5

dispersion air pressure of 3.5 bars.

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The aerodynamic properties of the powder was tested at flow rates of 60 L/min and 100 L/min

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for 4.0 secs and 2.4 secs, respectively with both representing an air volume of 4.0 L. A capsule (size

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3, Capsugel, NSW, Australia) containing 10 ± 0.5 mg powder was loaded into an Osmohaler® and

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actuated through a multi-stage liquid impinger (MSLI) (Copley, Nottingham, UK) to simulate an

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inspiration. The dispersion was performed in triplicate in a climate controlled cabinet: temperature, 20

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± 5°C and relative humidity (RH), 50 ± 3 %. Methanol:water (3:1, v/v) with 2 % ascorbic acid was

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used as a rinsing solvent. The drug mass was quantified using high-performance liquid

13

chromatography (HPLC).16,

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3.0):acetonitrile:tetrahydrofuran at a ratio of 53:42:5 (%, v/v), and the stationary phase was a

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µBondapakTM C18 (3.9 × 300 mm2) column (Waters, Milford, Massachusetts). The system (Model

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LC-20, Shimadzu, Kyoto, Japan) was operated at an isocratic flow rate of 1 mL/min for 10 min. UV

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detection was performed at a wavelength of 250 nm (SPD-20A UV/VIS detector, Shimadzu) and the

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injection volume was 20 µL. A calibration curve with a R2 value of 1.0 was obtained with standard

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concentrations ranging from 0.001 to 0.1 mg/mL. The emitted dose was calculated as the amount of

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drug exited the device and capsule relative to the total drug recovery from HPLC. The recovered fine

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particle fraction (FPFrecovered) was represented as the percentage of particles with an aerodynamic

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diameter less than 5 µm relative to the total drug recovery from HPLC. Mass median aerodynamic

23

diameter (MMAD) and geometric standard deviation (GSD) were calculated from the log-probability

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plot of the MSLI data, following Appendix XII C of the British Pharmacopoeia.

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The mobile phase consisted of 50 mM phosphate buffer (pH

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Mice and ethics statement. Female 6 - 10 week old BALB/c mice were obtained from Animal

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BioResources (Moss Vale, NSW, Australia) and were housed in specific-pathogen-free facilities at

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the Centenary Institute, Camperdown, NSW, Australia. The experiment was conducted in accordance 6 ACS Paragon Plus Environment

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with the Australian code of practice for the care and use of animals for scientific purpose and

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approved by the Sydney Local Health District Animal Welfare Committee (protocol number

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2014/031A).

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Intratracheal delivery of rifapentine in a murine model. Rifapentine was administered to mice via

6

insufflation (DP-4M, Penn-Century, Philadelphia) at a well tolerated oral dose used in mice and

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humans (20 mg/kg).9,

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intraperitoneal injection and secured on an intubation platform. The insufflator was then inserted into

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the trachea until the tip was immediately adjacent to the first bifurcation and the drug directly

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aerosolised onto the lung surface. The insufflator was weighed before and after the aerosolisation to

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measure the delivered dose. The mouse was then injected intraperitoneally with atipamezole (0.0125

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mg) and rested on a heating pad and monitored until self-righting. Mice were euthanised at 12 h, 24 h

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and 7 days post-treatment by CO2 asphyxiation, and lungs (perfused with PBS + 20 U/mL Heparin)

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and BAL were collected. Three lobes of the lungs and BAL were processed for total cell counts and

15

immune cell characterisation via flow cytometry. One lobe of the lungs was used for histopathological

16

analysis. Another one lobe was used for drug quantification (data not reported). The experiments were

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performed independently twice.

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The mouse was anaesthesised with ketamine/xylazine (90/10 mg/kg) by

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The control mice were untreated (Nil) and did not undergo insufflations to determine basal

19

values. Morello et al. reported that insufflation (air only) in female BALB/c mice resulted only in a

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minor congestion and hemorrhage, and resolved over time.19 Similarly, in the present study, no

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macroscopic indication of significant trauma was observed in the lungs of the insufflated/treated mice.

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In addition, there was no significant increase in total leukocyte counts in the lungs or BAL of the

23

treated mice compared to the untreated group. Therefore, the inflammation caused by insufflation

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alone is likely to be negligible.

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Flow cytometry analysis. Lung tissue was digested in RPMI 1640 media (Bacto Laboratories, Mt.

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Pritchard, NSW, Australia) containing fetal calf serum (FCS) (10 %, v/v), collagenase IV (50 U/mL,

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Sigma-Aldrich, Castle Hill, Australia) and DNAse I (13 µG/mL, Sigma-Aldrich, Castle Hill, 7 ACS Paragon Plus Environment


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Australia) for 30 min at 37°C. After processing the lung tissue and BAL to single cell suspensions,

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erythrocytes were removed utilising ammonium-chloride-potassium (ACK) lysis buffer and the cells

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were then re-suspended in complete media. Fc receptors were blocked by incubation of cell

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suspensions with anti-CD16/CD32 (BD Pharmingen, San Jose, CA) in FACS buffer (PBS with 2 %

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FCS), followed by antibody staining for major leukocyte subsets. Antibody panel utilised: anti-CD11b

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AF700 (M1/70, BD Pharmingen, San Jose, CA), anti-CD11c PECy7 (HL3, BD Pharmingen, San

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Jose, CA), anti-F4/80 PE (T45-2342, BD Pharmingen, San Jose, CA), anti-MHCII APC

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(M5/114.15.2, eBioscience, San Diego, CA), anti-Ly6G PerCPCy5.5 (1A8, Biolegend, San Diego,

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CA), anti-B220 APCCy7 (RA3-6B2, BD Pharmingen, San Jose, CA), anti-CD45.2 PB (104,

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Biolegend, San Diego, CA), anti-CD3 FITC (145-2C11, BD Pharmingen, San Jose, CA) and live/dead

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fixable blue dead cell stain (Invitrogen, Carlsbad, CA). Data were acquired on the BD LSRFortessa

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and analysed with FlowJo (TreeStar, Ashland, OR).

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Histopathology examination. The lung lobes designated for histopathology were inflated and

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immersed in 10 % neutral buffered formalin, and stained with hematoxylin and eosin (H & E).

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Histopathological evaluation was measured by microscopy on 5 µm sections of the stained lung.

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Inflammation was scored by two independent histopathologists who were blinded to the treatment

18

conditions.

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Statistical analysis. Data were expressed as mean ± standard deviation (SD) or standard error of the

21

mean (SEM) as specified. Differences between treatment groups were evaluated by ANOVA with the

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Bonferroni post hoc comparison (GraphPad Prism 6 software), and p < 0.05 was considered

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statistically significant.

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RESULTS

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Rifapentine powder characterisation. The rifapentine particles were elongated crystals with

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physical width × length of at least 0.5 × 1.0 µm as shown in the scanning electron micrograph (Figure

4

1), similar to those reported in our previous study.17 The volume median diameter (D50) of the

5

particles measured by laser diffraction was 1.47 ± 0.12 µm with a span of 1.56 ± 0.09. When the

6

powder was dispersed at flow rates of 60 L/min and 100 L/min with an air volume of 4.0 L, the fine

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particle fraction (FPF < 5 µm) was 69.5 ± 1.2 % and 78.4 ± 0.2 %, respectively (Table 1). This was

8

corresponding to a MMAD of 2.21 ± 0.1 µm and 1.85 ± 0.1 µm, respectively. The GSD was

9

indifferent at both conditions with values between 1.75 and 1.78. Irrespective of the flow rates, the

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drug mostly deposited in stages 3 and 4, and filter stage (Figure 2). However, significantly higher

11

device retention was observed at 60 L/min (22.3 ± 1.3 %) than 100 L/min (12.4 ± 3.3 %).

12 13

Figure 1.

14 15

Figure 2.

16 17

Table 1.

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Flow cytometry analysis. A significant influx of neutrophils was observed in the lungs at 12 h and 24

20

h after treatment (Figure 3), an increase of approximately 20 % and 10 %, respectively compared to

21

the untreated control (p < 0.01). However, the neutrophil population returned to normal by day 7.

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There were no significant differences in the other leukocytes populations in the lungs, except there

23

was a trend for increase in macrophage/monocyte populations at 12 h and 24 h (p > 0.05), but this also

24

returned to baseline by day 7.

25

Similar cellular changes were also noted in the BAL after treatment (Figure 4). At 12 h and 24

26

h post-dosing, there was a significant recruitment of neutrophils so that they comprised 80 % and 60

27

%, respectively of all leukocytes in the BAL (p < 0.05). Again, neutrophil counts returned to baseline

28

by 7 days. There was a significant increase in the macrophage/monocyte and alveolar macrophage 9 ACS Paragon Plus Environment


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populations in the BAL at 24 h (p < 0.05), along with a small reduction in the T-cell, B-cell and

2

dendritic cell (DC) populations at 12 h and 24 h post-dosing (p > 0.05).

3

In summary, pulmonary delivery of rifapentine caused a transient neutrophil-associated

4

inflammation in the lungs that had resolved over 7 days. Please refer to the Supporting Information

5

for a detailed analysis of immune cell populations in the lungs and BAL (Figure S1 and S2).

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Figure 3.

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Figure 4.

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Histopathology examination. Selected photomicrographs from the lungs of mice are depicted in

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Figure 5 and the interpretations are summarised in Table 2. When compared to control mice (Figure

13

5A), lungs of mice taken at 12 h after treatment showed a moderate cytoplasmic vacuolation and

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blebbing of the respiratory epithelium that suggested an epithelial cell degeneration (Figure 5B).

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Furthermore, mild necrosis occurred in the large to small bronchioles on the epithelium lining with

16

mucin and karyorrhectic particles adhered to these small eroded surfaces. A mild perivascular

17

inflammation characterised by neutrophil influx was also observed (Figure 5C).

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The examination at 24 h post-treatment (Figure 5D) showed a severe congestion in the

19

parenchyma, compared to that of observed in the control mice or 12 h post-treatment. Extra- and

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intra-pulmonary airways including the terminal bronchiole displayed cytoplasmic vacuolation in at

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least 50 % of the epithelial cells and nearly one-third of the vacuole was mainly solitary. Moreover,

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the epithelial cells had shed and/or proliferated in small bronchioles with mononuclear cells in the

23

peribronchial zone. In some airways, particularly in small bronchioles, a focal increase of epithelial

24

cells with smaller nuclear to cytoplasmic (NC ratio) was present, suggesting early regeneration of

25

cells. A significant recruitment of alveolar macrophages and edema was also observed (Figure 5E).

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The day 7 post-treatment (Figure 5F) observations showed that at least 60 % of the

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parenchyma was conspicuously congested compared to the untreated lung. The vascular outline of the

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alveolar walls was enhanced by proteinaceous deposits that obscured capillary and its lumen. The 10 ACS Paragon Plus Environment

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clumps of granular proteinaceous material also occupied some alveolar sacs together with few

2

attached mononuclear and macrophage cells.

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In summary, a prominent vacuolar cytoplasmic degenerative change occurred at 12 h that had

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intensified by 24 h. In addition, at 24 h a minor focal loss of bronchial epithelium was accompanied

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by an early stage of regenerative hyperplasia. This vascular congestion continued up to day 7.

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Nonetheless, no significant pleural changes were observed.

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Figure 5.

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Table 2.

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DISCUSSION

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The rifapentine powder was found suitable for aerosolisation with a high FPF between 69.5 %

14

and 78.4 %. The elongated morphology of the particles reduced the cohesive van der Waals forces and

15

deposited via interception for a high respirable fraction.17 This is also reflected in a minimal

16

deposition in the throat and stage 1, and higher retention in stage 3 to filter stage of the MSLI.

17

However, the FPF produced at 100 L/min (78.4 ± 0.2 %) was significantly higher compared to when

18

dispersed at 60 L/min (69.5 ± 1.2 %). The inferior FPF was due to insufficient powder emptying from

19

the device at 60 L/min, suggesting the elongated particles required high energy input to disperse the

20

powder as revealed by the marked pressure drop at 100 L/min (~4.0 kPa) than that of 60 L/min (~1.4

21

kPa). Nonetheless, this high device retention in clinical application is likely to be optimised by

22

multiple breathing inspirations. Thus, the crystalline rifapentine demonstrated in vitro aerosol

23

properties suitable for inhaled administration.

24

In our previous studies, we have established that solubilised rifapentine was tolerated by

25

human airway epithelial (Calu-3), human alveolar basal epithelial (A549) and human THP-1

26

monocyte-derived macrophage cell lines up to a concentration of 60 µg/mL.14,

27

tobramycin, a currently FDA-approved drug for treatment of cystic fibrosis via the inhalation route, is

28

non-toxic to lung epithelial cells up to a concentration of 1.0 g/mL.21 However, the cytotoxicity of

16, 17, 20

In contrast,



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rifapentine could be overestimated in the in vitro assays as the drug was dissolved in an organic

2

solvent (i.e. DMSO) prior to use in the assays, while a gradual dissolution is expected after deposited

3

in the lungs. The in vitro assays also differ from the actual characteristics of in vivo delivery to the

4

lungs, such as mucociliary clearance of the pulmonary region that will have a significant impact on

5

the particle dissolution and retention in the lungs. Therefore, assessing the impact of pulmonary

6

delivery in vivo is essential for determining a rational dosage regimen for further preclinical and

7

clinical studies.

8

A single dose pulmonary insufflation of rifapentine caused a neutrophilic inflammation in the

9

lungs at 12 h and 24 h post-exposure that resolved by 7 days. Neutrophils are the first leukocytes

10

recruited to phagocytose and denature foreign materials deposited in the lungs through the release of

11

reactive oxygen species, defensins, lactoferrin, cathepsins and lysozymes.22, 23 Increased secretion of

12

reactive oxygen species, such as superoxide anion and proteolytic enzymes (e.g. neutrophil elastase)

13

can cause significant pulmonary inflammation and lung damage. The physiological effect of the

14

inflammation caused by the neutrophil influx in the present study is unknown. None of the mice were

15

distressed, suggesting the insufflation of rifapentine had not caused any acute lung damage for 7 days

16

after the treatment. The histology further revealed that the lung damage was reversible with evidence

17

of some regeneration of epithelial cells and no significant pleural changes after 24 h and 7 days post-

18

treatment. Similar results were reported when a single dose of a cationic lipid suspension was

19

administered to BALB/c mice by nasal instillation.24 This caused a significant influx of neutrophils

20

into the lungs and BAL at 1 to 3 days that returned to baseline by 14 days. There were also increased

21

levels of the pro-inflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and

22

interferon-γ (IFN- γ) that peaked at 1 to 2 day post-treatment and returned to baseline by 14 days.24

23

On the other hand, Patil-Gadhe et al. showed that repeated intratracheal insufflations (28 days daily)

24

of rifapentine proliposomes at doses of 1 and 5 mg/kg in Wistar rats did not elicit pulmonary

25

irritation, as revealed by minimal changes in neutrophil populations. However, when the multiple

26

doses were increased to 10 mg/kg which is only 50 % of the dose used in this study, there was a

27

significant mortality due to excessive inflammatory responses in the lungs.15 Therefore, careful


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attention is required if multiple doses of rifapentine at 20 mg/kg are to be delivered via the pulmonary

2

route.

3

Another important finding was the increased number of alveolar macrophages (~15-20 %)

4

and to lesser extent macrophage/monocyte populations in the BAL at 24 h post-exposure, both of

5

which resolved by day 7. This was consistent with the lung histology analysis where the alveolar

6

spaces were dominated by foamy macrophages at 24 h (Figure 5E). This may be due to the activation

7

of the alveolar macrophages to phagocytose the rifapentine particles, as well as recruitment of

8

inflammatory monocytes from the peripheral blood that differentiate into tissue macrophages.25

9

Similarly, Freimark et al. showed an influx of alveolar macrophages in the lungs of C57BL/6 mice

10

after intratracheal instillation of liposome suspensions containing an immunogenic plasmid. This

11

resulted in enhanced immune responses, possibly through the increased cellular uptake of the

12

particles.26 Therefore, both alveolar and monocyte-derived macrophages may have phagocytosed the

13

slowly dissolving rifapentine particles. This has the potential to directly target M. tuberculosis

14

residing within macrophages and possibly shorten the duration of treatment for TB.

15

The DC, B-cell and T-cell counts in the BAL were slightly reduced at 12 h and 24 h time

16

points and returned to normal by day 7, suggesting the rifapentine deposition likely affected the

17

antigen presenting cells (APC) distribution in the lungs. A previous study demonstrated that the DCs

18

were efficient at phagocytosing polystyrene nanoparticles (50 nm and 500 nm diameter in size) and

19

transporting them from lung to the draining lymph node.27 Furthermore, the inflammation in the lungs

20

prior to the particle insufflation was found to enhance the maturation and migration of DCs, and

21

increased the drainage of the particles via the lymphatic system.27, 28 A similar study also reported that

22

the footpad injection of polystyrene nanoparticles with a diameter of 20 nm in diameter were taken up

23

by DCs, macrophages and B-cells, and trafficked the particles to the lymph node.29 These findings

24

support the possibility that the rifapentine particles trafficked from lungs to the draining lymph nodes.

25

The pharmacokinetic data also supports this possibility as intratracheal insufflation of rifapentine at

26

the same dose resulted in systemic delivery of the drug with a Tmax of 12 h in the spleen.10

27

Presumably, the clearance of rifapentine particles from the lungs was driven by a combination of cell-



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Page 14 of 27

1

mediated transport and drainage via the lymphatic system, and also by macrophages through

2

mucociliary clearance.

3

One of the critical issues in inhalation toxicology is the dose for delivery. Unlike oral and

4

intravenous routes, it is difficult to determine the delivered dose following inhalation exposure as only

5

a fraction of the inhaled dose will deposit in the lungs. In the present study, an intratracheal device

6

(DP-4M® insufflator, Penn-Century) was used which has been proven to have high accuracy and

7

reproducibility for delivery of drugs directly to the lungs.30,

8

distributes the powder throughout the lungs of mice, and prevents pharyngeal and upper airway

9

losses.31 However, one limitation of this method is that the insufflator is only efficient for delivering a

10

minimum dose of 0.4 mg corresponding to 20 mg/kg of rifapentine. One of the approaches to

11

overcome this barrier is to reduce the proportion of the active ingredient with a carrier, such as lactose

12

or mannitol, in order to increase the dry powder mass loaded into the insufflator. The addition of such

13

a carrier, however, may alter the pharmacokinetics of the drug after deposition in the lungs.32

14

Furthermore, the risk of inflammation due to pulmonary delivery of rifapentine may be overestimated

15

in this model as the drug was delivered using a DP-4M® insufflators under positive pressure which is

16

different to aerosol deposition during tidal breathing. In the present study, 200 µL air pulses were

17

utilised as recommended by the manufacturer33 for delivery of the powder under positive pressure. It

18

is therefore possible that some particles may have been deposited in regions of the lungs that are not

19

readily accessible by inhalation under ambient pressure. Hoppentocht et al. reported that an air

20

volume of 200 µL was insufficient for aerosolisation, with 500 µL and 1000 µL air pulses providing

21

superior deagglomeration of powders.34 However, the maximum air volume that is safe to use for

22

insufflation in mice is 250 µL.19 Furthermore, during normal breathing, larger crystals would deposit

23

in the upper airways and be eliminated before reaching the lungs. The forcible deposition of these

24

larger crystals or agglomerates directly into the lungs by insufflations may have exacerbated the

25

neutrophil influx. It has also been reported that even spray instillation of saline, a solution considered

26

as “safe” and commonly used in nebulisation caused a transient neutrophil influx in rats.35 Therefore,

27

it would be valuable to assess the pulmonary inflammation when rifapentine is delivered via

28

inhalation during tidal breathing in the future.

31

This delivery technique also widely


Page 15 of 27

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1


CONCLUSION

2

We have shown that the intratracheal delivery of respirable crystalline rifapentine at a dose of

3

20 mg/kg caused a neutrophil-associated transient pulmonary inflammation that resolved over 7 days

4

period. This may limit the pulmonary delivery of rifapentine to once a week at the given dose or less.

5

The efficacy of weekly dosing with inhalable rifapentine in a murine model of M. tuberculosis

6

infection is ongoing. Furthermore, this finding suggests that the inhaled antibiotics may be more

7

suitable to be administered as an adjunct with an oral regimen where patients may require intermittent

8

dosing such as weekly, but this will depend on the powder formulation and pharmacokinetic profile of

9

the drug.

10 11

ACKNOWLEDGMENT

12

Thaigarajan Parumasivam and Anneliese Ashhurst are recipients of the Malaysian

13

Government Scholarship and Australian Postgraduate Award, respectively. The authors are thankful

14

to Tina Cardamone and Rolfe Howlett from Histopathology and Organ Pathology, The University of

15

Melbourne, Australia for the histopathological analysis. The authors acknowledge Australian

16

Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and

17

Microanalysis, The University of Sydney for their scientific and technical assistance. The work was

18

supported by the Australian Research Council’s Discovery Project (DP120102778), the National

19

Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control

20

(APP1043225) and project grant (APP104434), and the NSW Government Infrastructure Grant to the

21

Centenary Institute.

22 23

Supporting Information

24

Detailed analysis of immune cell populations in the lungs and BAL (Figure S1 and S2).

25 26 27 28 15 ACS Paragon Plus Environment


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Page 18 of 27

1 2

Figure 1. Elongated crystalline rifapentine particles viewed under scanning electron microscope.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20


Page 19 of 27

40 60 L/min 100 L/min Recovered mass (%)

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30

20

10

0 Capsule

Device

Adaptor

Throat

Stage 1 Stage 2 Stage 3 Stage 4 Filter (10.07 µm) (5.27 µm) (2.40 µm) (1.32 µm) (

Inhalation of Respirable Crystalline Rifapentine Particles Induces

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