Pharmacokinetics of Inhaled Rifampicin Porous Particles for

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Pharmacokinetics of Inhaled Rifampicin Porous Particles for Tuberculosis Treatment: Insight into Rifampicin Absorption from the Lungs of Guinea Pigs Lucila Garcia-Contreras, Jean Sung, Mariam Ibrahim, Katharina Elbert, David Edwards, and Tony Hickey Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00046 • Publication Date (Web): 05 May 2015 Downloaded from http://pubs.acs.org on May 11, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 36

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

Molecular Pharmaceutics

Pharmacokinetics of Inhaled Rifampicin Porous Particles for Tuberculosis Treatment: Insight into Rifampicin Absorption from the Lungs of Guinea Pigs

Lucila Garcia Contreras* 1, Jean Sung* 2,3, Mariam Ibrahim 1 , Katharina Elbert2, David Edwards2, Anthony Hickey 4 * These authors contributed equally to this work 1

The University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2 Harvard University, Cambridge, MA; 3 Pulmatrix, Lexington, MA; 4 RTI International, RTP, NC

Correspondence: Lucila Garcia-Contreras, Ph.D. The University of Oklahoma Health Sciences Center 1110 N. Stonewall Ave., Oklahoma City, OK 73126-0901 Telephone: 405-271-6593 Ext. 47205 Fax: 405-271-6593 Email: [email protected]

1 ACS Paragon Plus Environment


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

Page 2 of 36

ABSTRACT Tuberculosis (TB) is a life-threatening infection that requires a lengthy treatment process that is often associated with adverse effects. Pulmonary delivery of anti-TB drugs has the potential to increase efficacy of treatment by increasing drug concentrations at the lungs, the primary site of infection. The aim of the present study is to evaluate the disposition of rifampicin (RIF) after its pulmonary administration as porous particles (PPs) to guinea pigs and contrast it to that after oral administration. Methods: RIF microparticles were prepared by spray drying a solution of RIF and LLeucine (9:1) and the resulting particles were characterized for their physicochemical properties. Animals received RIF either as intravenous solution (IV), oral suspension of micronized RIF (ORS) and RIF-PPs (ORPP) or by insufflation of the PPs (IRPP). Plasma samples were collected at preselected time points and bronchoalveolar lavage (BAL) was performed at the end of the study. RIF concentrations in biological samples was analyzed by HPLC. Plasma concentration versus time data was analyzed by compartmental and non-compartmental methods. Results: RIF PPs were thin walled porous particles with mass median aerodynamic diameter (MMAD) of 4.8±0.1 µm, GSD = 1.29±0.03 and fine particle fraction below 5.8 µm of 52.9 ± 2.0%. RIF content in the resulting particles was 91.8±0.1%. Plasma concentration vs. time profiles revealed that the terminal slope of the IV group was different than that of the oral or pulmonary groups indicating the possibility of flip-flop kinetics. RIF from IRPP appeared to be absorbed faster than that of ORPP or ORS as evidenced by higher RIF plasma concentrations up until 2 hours. Notably, similar AUC (when corrected by dose), similar CL, λ and half-life were obtained after oral


Page 3 of 36

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


administration of RIF at 40 mg/kg and pulmonary administration of RIF at 20 mg/kg. However RIF in the IRPP group had a shorter Tmax and higher bioavailability than orally dosed groups. In addition, RIF concentrations in the BAL of animals in the IRPP group were 3-4 fold larger than that in the orally dosed groups. The disposition in ORS and ORPP were best described by a model with two sequential compartments, whereas the disposition of IRPP was best described by a two parallel compartment model. Conclusion: The advantages of delivering RIF by the pulmonary route are demonstrated in the present study. These include achieving higher RIF concentrations in the lungs and similar systemic levels after pulmonary delivery of one-half of the oral nominal dose. This is expected to result in a more effective treatment of pulmonary TB, as shown previously in published efficacy studies.

Keywords: Tuberculosis, rifampicin, porous particles, flip-flop kinetics and pulmonary absorption



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

Page 4 of 36

1. INTRODUCTION Despite being declared a global health emergency by the World Health Organization (WHO) twenty years ago, tuberculosis (TB) is still a serious, life threatening infection worldwide 1. TB infection occurs by inhalation of aerosolized droplets dispersed from a person infected with the Mycobacterium tuberculosis (Mtb) bacteria. Inhaled bacteria reach the alveolar region of the lungs where the pathogenesis of TB begins 2. It is estimated that 9 million new cases of TB infection occur each year and 1.5-2 million of these cases result in death 3. Furthermore, coinfection of HIV patients with TB increases the severity of such disease and decreases the life span of these patients. TB is considered the largest single cause of death among AIDS patients 4, 5. The CDC still recommends the classical combination of first line anti-TB drugs of rifampicin (RIF), isoniazid, pyrazinamide and ethambutol, which are given orally in high doses for an average time of 6-9months 6. This lengthy and arduous process elicits unwanted side effects, which may lead to lack of patient compliance and ultimately the emergence of new resistant bacterial strains. 7 In addition, this prolonged treatment time increases the financial health burden, especially on resource limited countries where the disease is endemic. Therefore, new drugs that have limited or no side effects and that could decrease treatment times are urgently needed to increase patient compliance and decrease the cost of treatment. There are many challenges to achieving effective treatment for the complete global eradication of TB. First, the process of finding innovative TB treatments is very slow 8. Many new drugs were reported to be effective against TB, however, they are still


Page 5 of 36

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


early in the development process and only a few are close to reaching the market. Only two new drugs, bedaquiline and delamanid have been approved recently 3. Thus, new approaches, such as increasing the dose of RIF to more than 10mg/kg, have been proposed in an attempt to shorten TB treatment times 8. This is a potentially easy solution since RIF is an inexpensive drug and well known among physicians worldwide9. However, this approach has the potential to increase unwanted side effects such as hepatotoxicity 10, 11. Furthermore, repeated oral administration of high RIF doses yields auto-induction of RIF’s own metabolism leading to a decrease in its bioavailability 12. An alternative, more effective approach to treat pulmonary TB is to deliver drugs directly to the lungs to achieve high local drug concentration for extended duration. Also, depending on the physicochemical characteristics of the particular drug and the formulation, pulmonary delivery could also result in systemic bioavailability of the drug, if treatment of extra-pulmonary TB is desired 13. Such approach has the possibility of accelerating the onset of Mtb killing, decreasing the dose to achieve the therapeutic effect, which in turn would reduce systemic side effects, decrease the frequency of dosing and eventually shorten the treatment duration14. RIF has been formulated in different ways for pulmonary administration including as pure micronized powder 15, poly(lactic-co-glycolic acid) PLGA microspheres 11, 16-19, and sustained release microparticles 20. The goal of the present study was to evaluate the disposition of RIF after pulmonary administration of porous particles (PPs) to guinea pigs. Powder formulations consisting of PPs deliver drug to the alveoli more efficiently than other powder formulations, because they can avoid natural clearance mechanisms in the respiratory



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

Page 6 of 36

tract 21. The disposition of RIF after delivery by the pulmonary route was also compared to the oral and IV routes of administration. An extensive pharmacokinetic (PK) analysis was performed to characterize RIF disposition after its administration by different routes to guinea pigs, a widely used animal model of TB.

2. MATERIALS AND METHODS L-leucine and RIF were obtained from Spectrum Chemicals & Laboratory Products (Gardena, CA). RIF is an orange red powder, slightly soluble in water (~1.3 g/l) and soluble in methyl and ethyl alcohol. Ethanol USP grade and acetonitrile were purchased from Pharmco Products Inc. (Brookfield, CT). Water from a Millipore Corp. (Billerica, MA) Milli-Q water purification system was used. All other chemicals and reagents used were of pharmaceutical or analytical grade.

2.1.

Formulation of RIF PPs

The spray drying solution was prepared by dissolving 1.2 g of L-leucine (10%) and 10.8 g of RIF (90%) in 0.20 L of water. Ethanol (0.30L) was added to this mixture, while stirring. The solution was agitated until dissolution of all materials. RIF PPs were manufactured by spray-drying (LabPlant, Model SD-06), with inlet temperature of 170 °C and solution feed flow rate of 45 ml/min. The solution was pumped into the two-fluid nozzle of the spray dryer with a gas flow rate of 25 g/min and nozzle pressure of 33 psi. Spray dried powders were collected in a container at the outlet of the cyclone. The resulting powders were stored in a desiccator for characterization and analysis of drug


Page 7 of 36

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


content. Physicochemical characterization of powder was completed within two weeks of powder manufacture and PK studies were completed within 3 months of this time.

2.2.

Characterization of Dry Powders

2.2.1. Particle shape and morphology The dry particles were viewed using scanning electron microscopy (SEM). A LEO 982 field emission scanning electron microscope (Carl Zeiss, Inc., Thornwood, NY) was operated at 2 kV with a filament current of about 0.5 mA. Powder samples were prepared by deposition on a double coated carbon conductive tape tab (Ted Pella Inc., Redding, CA) mounted on a pin mount and dusted. The sample was then coated with a Platinum/Palladium layer with a Cressington Scientific Instruments Inc. (Watford, UK) 208HR sputter coater, operated for 60 s at a sputtering current of 40 mA.

2.2.2. Particle properties The volume diameter of the spray-dried particles was measured by laser diffraction with a HELOS system with RODOS dry dispersing unit (Sympatec Inc., Lawrenceville, NJ). Each test was done in triplicate at an applied pressure of 1-2 bars. The geometric standard deviation (GSD) of dry powders was determined as GSD= (d84%/d16%) 0.5. Tapped density of powders was determined by loading RIF PPs into 0.3 ml sections of a 1-ml plastic pipette, capped with NMR tube caps, and tapped approximately 600– 1,200 times using a Varian tapper (Cary, NC) until the volume of the powder did not change. The tapped density was calculated from the difference between the weight of the pipette before and after loading, divided by the volume of powder after tapping.



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

Page 8 of 36

An eight-stage Andersen non-viable 1ACFM cascade impactor (ACI) (Copley Scientific Limited, Nottingham, UK) was used to determine the mass median aerodynamic diameter (MMAD) and the fine particle fraction (FPF) of the total dose of powder less than or equal to an effective cut-off aerodynamic diameter of 5.8 µm.

2.2.3. Drug load of powders The RIF content in the spray-dried particles was determined by reverse-phase high performance liquid chromatography (HPLC) using an Agilent 1100 series HPLC system with Zorbax columns and ChemiStation software (Agilent Technologies Inc., Palo Alto, CA). The mobile phase was run on a linear gradient from 45% 0.02 M phosphate buffer, 41% methanol and 14% acetonitrile to 25% 0.02 M phosphate buffer, 56% methanol and 19% acetonitrile over 20 minutes plus 10 minutes at the final composition and 5minutes post-run. Analysis was performed on a 10 µL injection at a flow rate of 1.0 ml/min through an Aglient Zorbax Eclipse XDB-C18 (4.6 x 150 mm) column equipped with a matching guard-column and absorbance recorded at 337 nm. The standard curve for RIF ranged from 0.05 to 5.0 µg/mL and the limit of quantitation (LOQ) was 0.05 µg/mL. The overall validation precision across all standards was 1.43 to 4.56%.

2.3.

Pharmacokinetic studies

Male Dunkin-Hartley Guinea pigs (395 ± 32g) were employed for the pharmacokinetic studies. Animals were housed in a 12 h light/12 h dark cycle and constant temperature environment of 22°C. A standard diet of food and water were supplied ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee 8 ACS Paragon Plus Environment

Page 9 of 36

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


(IACUC) of the University of North Carolina at Chapel Hill in accordance with “Principles of Laboratory Animal Care” (NIH publication #85–23, revised in 1985). Before the study, each animal underwent cannulation of the right external jugular vein for continuous blood sampling and allowed to recover overnight 22. Guinea pigs were divided into 4 groups receiving different RIF formulations (table 1): solution, suspension or dry powder, delivered by different routes (IV, oral, pulmonary). The RIF solution for IV administration was prepared by dissolving RIF first in a small volume of DMSO and completing the volume with water for injection (WFI) for a final proportion of 1:5 DMSO:WFI. Suspensions for oral administration were prepared by dispersing either micronized RIF alone (ORS) or RIF PPs (ORPP) in WFI. Animals receiving RIF PPs were anesthetized, endotracheally intubated and RIF PPs were administered by insufflation using a small animal dry powder insufflator (Penn-Century, Wyndmoor, PA). After dosing, blood samples (0.35 ml) were collected from each animal into heparinized tubes with ascorbic acid (added to prevent RIF oxidation) at 0, 0.08, 0.25, 0.5, 1.0, 1.5, 2, 3, 4, 6, 8 and 12 h. Sterile saline solution was used to replace the blood volume lost through sample collection. Plasma was separated and stored at −80°C until analysis. After collection of the last blood sample, animals were anesthetized and euthanized by exanguination, and bronchoalveolar lavage (BAL) was conducted using 5 ml sterile saline. BAL fluid samples were stored separately at −80°C until analysis.

2.4.

Determination of RIF concentration in biological samples

RIF concentrations in plasma and BAL were determined by an HPLC method after liquid-liquid extraction with acetonitrile. The HPLC system (Waters Corp., Milford, MA)



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

Page 10 of 36

consisted of a model 510 pump, model 717 plus autosampler and model 480 ultraviolet detector set at a wavelength of 337 nm. The system was equipped with a Nova-Pak C18 guard column and a Waters C18 analytical column (5 µm and 150 x 4.6 mm). The mobile phase consisted of 43% phosphate buffer pH = 5.2, 40% methanol and 17% acetonitrile at a flow rate of 1 ml/min. The standard curve for RIF in plasma ranged from 0.1 to 7.0 µg/mL and the LOQ was 0.1 µg/mL. The recovery of RIF from plasma was 91.32%. The overall validation precision across all standards was 3.81 to 5.16%.

2.5.

Pharmacokinetic analysis

RIF plasma concentration-time data was initially analyzed by non-compartmental methods (LaGran, program for area and moments in pharmacokinetic analysis)23, to obtain area under the curve (AUC), apparent total body clearance (CL), volume of distribution (V), mean residence time (MRT) and half-life (t1/2). Estimates of the terminal slope (λ) were obtained from the data points in the terminal phase of the concentration versus time plots. Maximum RIF concentrations (Cmax) and time to obtain the maximum RIF concentration (Tmax) were determined from the plasma versus time profiles for each individual animal. Bioavailability (BA) was calculated using the following equation:

BA = Dose normalized (AUC∞) lung, oral Dose normalized (AUC∞) IV The mean absorbance time (MAT) was calculated by the following equation:


Page 11 of 36

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


MAT=MRTlung, oral - MRTIV

RIF data was subsequently analyzed by compartmental analysis using WinNonlin (Pharsight Corporation, Mountain View, CA). The compartmental PK analysis method was established through simultaneous fitting of the plasma levels of RIF for each extravascular route with those obtained after IV administration. Different models were proposed and the choice of the best model was based on the Akaike goodness-of-fit indicator (AIC), coefficient of variation (CV%) and width of confidence interval (CI). The PK parameters corresponding to the selected model are reported.

2.5.1. Statistical analysis Data for the PK study were subjected to analysis of variance (ANOVA) and the leastsquares significant-differences multiple comparison method, Student–Newman–Keuls. A probability level of 5% (p

Pharmacokinetics of Inhaled Rifampicin Porous Particles for

Recommend Documents