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Research Article Slow Release Formulations of Inhaled Rifampin Intira Coowanitwong,1 Vikram Arya,1 Poj Kulvanich,2 and Günther Hochhaus1,3 Received 24 July 2007; accepted 27 May 2008; published online 27 June 2008 Abstract. Rifampin microspheres were prepared by spray drying using either polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA) polymers in different drug to polymer ratios (90:10 to 5:95, w/w). The in-vitro release characteristics, particle-size distribution, and cytotoxicity (in an alveolar macrophage cell line) and pharmacokinetics in rats after pulmonary instillation were evaluated. Increasing the polymer content from 10% to 95% slowed down the in vitro drug release with PLGA particles showing a steeper change with increasing polymer content (100% to 20% drug release over 6 h) than PLA particles (88% to 42% drug release over 6 h). PLA microsphere formulations revealed lack of cytotoxicity and a mass median aerodynamic diameter (MMDA) of 2.22–2.86 μm, while PLGA particles were larger (MMDA of 4.67–5.11 μm). Pharmacokinetics differed among the formulations with the 10% PLA formulation showing a distinct sustained release (tmax of 2 h vs 0.5 h of free drug) and a systemic bioavailability similar to that of free drug. Formulations with high polymer content showed a lower relative bioavailability (30%). This suggested that an optimal release rate existed for which a distinct amount of drug was delivered over an extended period of time. KEY WORDS: cytotoxicity; inhalation; microspheres; pharmacokinetics; rifampin; sustained release. INTRODUCTION Tuberculosis (TB) remains a major cause of mortality. Approximately one of three of people worldwide are believed to be infected with the bacteria Mycobacterium tuberculosis (MTB) (1). Both the high prevalence of TB in patients infected with the human immunodeficiency virus (HIV) and the alarming increase in drug resistance have made tubercu- losis (TB) an emerging threat. In 1993 the World Health Organization (WHO) declared TB a global public health emergency based on the increasing number of infected patients and the high mortality rate (2,3). According to WHO, the largest number of new TB cases in 2004 occurred in the Southeast Asia region, which accounted for 33% of incident cases around the world (4). A variety of drugs have been used in combination for the treatment of MTB in order to ensure successful treatment and prevent the development of resistance (5,6). The drugs that comprise the first-line of defense include isoniazid, rifampin, pyrazinamide, streptomycin, and ethambutol. Isoni- azid, rifampin, pyrazinamide, and streptomycin have bacteri- cidal action while the ethambutol is bacteriostatic (7). Isoniazid and streptomycin are pharmacologically active against MTB only in the extracellular environment, where the bacilli grow most rapidly along the cavity wall. Pyrazina- mide is most effective in the acidic pH (pH 5.5) of the intracellular environment of the macrophages. Rifampin is the only anti-TB drug which, in addition to the activity via the aforementioned mechanisms, shows bactericidal activity in the extracellular environment where the slower growing semi- dormant organisms may demonstrate intermittent activity (8,9). Because of its favorable pharmacokinetics and unique mechanism of action, rifampin appears to be a suitable candidate for TB therapy, however it has been shown that chronic oral and i.v. therapy of rifampin resulted in adverse systemic side effects (such as rash, fever, and gastrointestinal disturbances, various symptoms related to the nervous system, and development of jaundice (7)) leading to poor patient compliance (7). Hence, alternate therapies are required that can reduce these concomitant side effects and help to localize the drug at the site of infection i.e., the lung. The sustained release dosage form was selected over the uncoated, immediate release form for the following reasons: (a) Increase in the local effects due to application at the site of infection. (b) Requirement of relatively small doses for effective therapy. (c) Reduction in systemic exposure (due to local application) leading to a reduction of systemic adverse effects (10,11). All these factors are expected to result in a higher benefit-to-risk ratio and better patient compliance. In this study, we investigated the use of a sustained release form of inhaled rifampin for TB therapy using animal models. Although there are several techniques to prepare the sustained release formulations, we selected a spray-drying method since it is a rapid, one-step process that converts liquid droplets to dried particulate form that can be efficiently inhaled (12–19). Thus, this approach can achieve a sustained 1550-7416/08/0200-0342/0 # 2008 American Association of Pharmaceutical Scientists 342 The AAPS Journal, Vol. 10, No. 2, June 2008 (# 2008) DOI: 10.1208/s12248-008-9044-5 1 Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610, USA. 2Department of Industrial Pharmacy, College of Pharmacy, Chula- longkorn University, Bangkok, Thailand. 3 To whom correspondence should be addressed. (e-mail: hochhaus@ cop.ufl.edu) presence of rifampin in the lung (increased pulmonary residence time) with sufficient pharmacological drug concen- trations. The pulmonary fate of inhalation drugs is rather complex and it is affected by a number of pharmacokinetic/ biopharmaceutical factors. In the case of slow-release for- mulations, it will depend, among other factors, on the release rate of the pharmacologically active drug and the pulmonary clearance of the particles, mainly due to the mucociliary clearance and other factors such as deposition efficiency and location. Here, we report the design of slow release, biodegradable rifampin particles that could be inhaled and our investigation of the relationship between the in vitro release rate and the pulmonary/systemic exposure. MATERIALS Rifampin was purchased from PCCA Inc. (Houston, TX, USA). Polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA) MW=75,000–125,000 and 40,000–65,000 Da, respec- tively) were acquired from Birmingham Polymers, Inc. (Birmingham, AL, USA). Dichloromethane was purchased from Shell Chemical (Bangkok, Thailand). Acetonitrile and methanol (HPLC grade), sodium chloride, dibasic sodium phosphate (anhydrous), monobasic sodium phosphate (anhy- drous), and ascorbic acid were obtained from Fisher Scientific Inc. (Suwanee, GA, USA). Extra-fine lactose monohydrate (particle size of 10–30 μm) was donated from EM Industries (Hawthrone, NY, USA). Number 2 capsules were provided by Pfizer Inc. (Greenwood, SC, USA). J774A.1 murine macrophage (MTB strain H37Ra), Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate buffer saline solution (PBS), and fetal bovine serum (FBS) were obtained from the American Type Culture Cell Collection (ATCC, Rockville, MD, USA). Middlebrook 7H9 and Middlebrook ADC Enrichment were purchased from BD Diagnostic Systems (Sparks, MD). Sodium chloride, Tween 80, microscope slides, and a Bacto Fluorescent Stain Set M were acquired from Fisher Scientific Inc. (Suwanee, GA, USA). 3-(4, 5-dime- thylthaizole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Male Sprague Dawley rats were obtained from Harlan Inc. (Indianapolis, IN, USA). METHODS Microsphere Formulations Different ratios of PLA and PLGA polymers to rifampin (Table I) were dissolved in 4 L of methylene chloride. The solutions were fed through a high-speed rotating spray nozzle of a Mobile Minor spray dryer (Niro, Switzerland) to produce small droplets (10–15 μm). The flow rate of the solution was controlled at 15 mL/min and the rotating pressure of the nozzle was at 4 bars. The temperature in the chamber (50°C) was higher than the boiling point of methylene chloride (47°C) enabling microsphere dry powders to be obtained in the collector after the methylene chloride evaporated. In addition, drug free PLA and PLGA microspheres, as well as polymer-free rifampin microspheres were prepared accord- ingly, using methylene chloride solutions of either polymer or drug. Product Characterization The information on particle size, shape, and surface morphology of the particles was obtained by S-4000 Fe-SEM Hitachi scanning electron microscope (Hitachi, USA). The samples were coated with gold using ion sputtering for 45 min prior to the experiment. Characterization was performed at 1–2 kV in a vacuum using different magnifications (×35, 50, and 100). An Image Pro Optical Analysis (MediaCybernetics Inc., USA) at two magnifications (×35 and ×100) was employed. Densities (ρ) of particles at 25°C were determined with an Ultrapycnometer 100 (Quantochrome, Boynton Beach, FL, USA). Helium (purity 99.9%) was used as the test gas. Rifampin Analysis during Particle Characterization The drug concentration in samples derived from phar- macokinetic, drug content and drug release samples was determined by reverse-phase HPLC (RP-HPLC) consisting of a Perkin Elmer Series 3B pump (Perkin Elmer Inc., USA) with a flow rate of 1.0 mL/min and a Waters C-18 (15 cm×ID 4.6 mm) column (Waters Co., USA) connected to a Perkin Elmer ISS100 autoinjector (Perkin Elmer Inc., USA). The detector was a Milton Roy SM-4000 (Milton Roy Co., USA) attached to a Hewlett Packard HP-3394A integrator (Hewlett- Packard Co., USA). The eluent was monitored at λ=254 nm. The mobile phase consisted of 50% acetonitrile in water with an additional 0.03% (v/v) trifluoroacetic acid (TFA). Six standard concentrations of rifampin (1, 2.5, 5, 10, 25, and 50 μg/mL) were prepared in the phosphate buffer pH 7.4, with 0.02% (v/v) of ascorbic acid as an antioxidant. An aliquot of 100 μL of methylprednisolone acetate (MPA) (100 μg/mL) was added into each 1 mL of the standard solutions, resulting in a final concentration of 10 μg/mL. The calibration curve was obtained using peak heights of the standard solutions. Table I. Percent Undissolved Drug Remaining in Sustained-release Rifampin Formulations After 6 h Polymer Percent undissolved drug Microspheres (PLA) PLA10 12.3±3.2 PLA20 18.6±2.6 PLA30 19.6±3.4 PLA50 19.7±1.8 PLA90 30.6±1.1 PLA95 57.7±0.5 Microspheres (PLGA) PLGA10 0 PLGA20 0 PLGA30 0 PLGA50 50.1±6.3 PLGA90 76.7±3.0 PLGA95 79.8±1.6 343Slow Release Formulations of Inhaled Rifampin Release Characteristics In vitro dissolution of free drug and dry powder formulations (500 mg of 2% rifampin drug in lactose) was tested using the USP dissolution instrument (Vankel Tech- nology Group, USA). One liter of phosphate buffer solution (pH 7.4) with 0.02% ascorbic acid as antioxidant was used as a dissolution medium. The dissolution apparatus was operat- ed at 37°C using a rotating speed of 40 rpm. One milliliter of the sample was withdrawn at various time points for 24 h (0, 2, 5, 10, 15, 30 min and 1, 1.5, 2, 3, 6, 10, 20 and 24 h) and assayed by HPLC. The same quantity of medium was added immediately after each sample withdrawal to keep the volume of the medium constant. A graph of percent- undissolved drug versus time was plotted. Cytotoxicity in Healthy Lung Macrophages The fully adherent murine alveolar monocyte–macro- phage cells (J774A.1) were grown in a DMEM supplement with 10% FBS without antibiotics at 37°C and 5% CO2. Cytotoxicity tests were performed using the [3-(4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay (20). Macrophages were seeded in a 96 well plate (Becton- Deckinson Labware, USA) with a concentration of 1×105 cells per well and were allowed to recover for approximately 24 h. Different rifampin concentrations (1,000, 100, 10, 1, 0.1, and 0.01 μg/mL) of free and sustained-release formulations, suspended in culture media were added. After 24 h, 25 mL of MTT solution (prepared in the PBS with final concentration of 5 mg/mL) was added. After 3 h of incubation, the supernatant was removed and 200 mL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the purple formazan crystals were dissolved using a plate shaker (Biometra, Germany) for 30 min. The absorbance was measured using a microplate spectrophotometer model Spectramex250 (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 570 nm. Fine Particle Fraction An eight-stage cascade impactor (Graseby Anderson, USA) was used to determine the fine particle fraction of the rifampin formulations. Eight metal plates were installed on the impactor machine before use. A throat piece (non-USP type) that simulates the human throat was connected on the top of the device. The dose (containing 70 mg of free drug) was weighed, loaded directly into the chamber using a respirable device, Cyclohaler™ (Novartis Pharma AG, Swit- zerland), and dispersed at an inspiration rate of 28.3 L/min (confirmed by a flow meter, Bios International, USA) for an inhalation time of 1 min. After each determination, the powder on each plate and throat piece of the impactor was dissolved with 10 mL methanol, containing 0.02% (w/v) ascorbic acid solution and analyzed by reverse phase HPLC. The experiment was performed in triplicate. The percentage of the total dose collected from stages 2 to 5, representing particles with the aerodynamic diameter 1.1–5.8 μm, was considered to be the fine particle fraction (21). Graphs between the percent cumulative of rifampin were plotted against the effective cut off diameter for that stage (9.0, 5.8, 4.7, 3.3, 2.1, 1.1, 0.7 and 0.4 μm for stage 0 to 7, respectively). A 100% drug cumulative was calculated as the total amount of drug deposited on the entire cascade impactor. Calculation of the mass median aerodynamic diameter (MMAD) of the formulations was performed using R program (R Foundation, USA). Animal Procedures The project was approved by the Animal Care Commit- tee, University of Florida, an AAALAC approved facility. Male Sprague Dawley rats (Harlan, Inc., Indianapolis, IN, USA), weighing 250–330 g, were housed for 12 h in a light/ dark, constant temperature environment prior to the exper- iment. They were fed a standard pellet diet and water as required but were food-fasted overnight prior to each experiment. Each rat was weighed and placed in a rat holder for intraperitoneal administration of anesthetics (fresh prepara- tion of the combination of 1.5 mL of 10% (v/v) ketamine, 1.5 mL of 2% (v/v) xylazine, and 0.5 mL of 1% (v/v) acepromazine, at the dose of 1 mL/kg). The depth of the anesthesia was checked by tail pinch or pedal withdrawal reflex. After the rats were completely anesthetized, one inch of a special round-tipped cannula attached to a delivery device for administration of the dry powders (Penn-Century Inc., Pennsylvania, USA) was introduced into the trachea through the mouth. A mixture of 10±0.5 mg rifampin formulation in extra-fine lactose (to allow a similar insufflation volume for the different formulations), equivalent to 600 μg of rifampin free drug was placed in the chamber of the device and instilled into the lungs with insufflations of 3 mL of air. The rats were decapitated at 0.5, 1, 2, 4, 6, and 8 h after drug administration and 5 mL of blood was collected from each rat. The blood was centrifuged at 4°C for 15 min to obtain the plasma and 0.02% ascorbic acid (w/v) was added to the plasma. The plasma was snap frozen and stored at −70°C until further processing. Since rifampin has been shown to be very stable in plasma at −20°C (22,23), this procedure was judged to provide sufficient stability. Plasma samples were thawed at room temperature and 25 μL of MPA (10 μg/mL) was added to 0.5 mL aliquots as an internal standard, resulting in a final concentration of 0.5 μg/mL. Ethanol (0.5 mL of 30%) was added to the plasma samples for protein precipitation. The samples were refrigerated for 15 min then centrifuged for 15 min at 4°C at 3,000×g using a CR422 centrifuge (Jouan Inc., USA). Endcapped Supelco C-8 car- tridges (3 mL, Sigma-Aldrich Co., USA) were washed with 2 mL of water and 2 mL of 30% methanol and 1 mL of the supernatant was transferred onto the cartridges for solid phase extraction. The analytes were eluted from the column with 3 mL ethyl acetate. After 3 h evaporation, 100 μL of mobile phase was added to each tube for reconstitution. After centrifuging for 3 min at 6,000×g the drug concentration in the reconstituted samples was determined using the reverse phase high performance liquid chromatography (RP-HPLC) setup consisting of a Perkin Elmer Series 3B pump (Perkin- Elmer Inc., USA) with a flow rate of 1.0 mL/min and a C-18 (30 cm×4.6 mm) column (Waters, USA) connected to a Perkin Elmer ISS200 autoinjector. The detector was an SPD-10A 344 Coowanitwong, Arya, Kulvanich and Hochhaus (Shimadzu, USA) attached to a computer system. Turbochrom Navigator® software version 4.0 was used for the data analysis (Perkin-Elmer Inc., USA). The eluent was monitored at λ= 254 nm. The mobile phase consisted of a mixture of 35:65 acetonitrile to pH 3.5-phosphate buffer. The calibration curve for rifampin was linear in the range of 8 ng/mL to 0.5 μg/mL (r2>0.995), with linearity extending well beyond this range. Slopes from the calibration curves were used to calculate sample concentrations. This assay was validated to ensure both precision and accuracy in accordance with the FDA guidelines for bioanalytical method validation (24). The intra-day precision (percent coefficient of variation [CV]) and accuracy (percent bias) of the quality control (QC) samples were 8.49– 11.9% and 101.9–102.4%, respectively. Pharmacokinetic Analysis The plasma concentration vs time data, obtained after intratracheal instillation of free rifampin and rifampin microspheres were analyzed using the noncompartmental model (extravascular input) in WinNonlin® (Pharsight Corp, USA). The various pharmacokinetic parameters estimated were: maximum plasma concentration (Cmax), time to reach maximum concentration (Tmax), area under the curve from time zero to last time point of the experiment (AUC0− t). The relative bioavailability (Frel) for each formulation was calculated as: Frel ¼ AUCSusRIFAUCFreeRIF ð1Þ where AUCSusRIF is the area under the curve of the sustained-release formulations and the AUCFreeRIF is the area under the curve of the free drug. Statistics The difference in pharmacokinetic parameters and respirable fractions between each formulation was tested for significance using ANOVA (Design Expert version 6 Soft- ware for Experiment Design, Stat Ease Inc., USA) assuming α=0.05. RESULT AND DISCUSSION Morphology Scanning electron microscopy (SEM) of the microspheres showed morphological differences directly related to the type and content of the polymers. Free drug microspheres showed a number of fractures thereby indicating the lack of particle elasticity and flexibility (data not shown). Figure 1 shows the rifampin microsphere formulations containing 0% to 100% of the PLA and PLGA, respectively. The microspheres of PLA and PLGA bulk polymer were spherical with rounder edges, but of shriveled and raisin-like shape (Fig. 1). Microspheres with increasing polymer content had a more defined round shape indicating that polymers can help to increase the elasticity and flexibility of the particles. It should be noted however, that the presence of shriveled and raisin-like shape microsphere might be due to the lack of a plasticizer. We did not use a plasticizer in our experiments due to the possible toxic residue in the lung after inhalation of the formulations. Based on SEM measurements, the sizes of PLA and PLGA was comparable. In Vitro Release Characteristics The dissolution profiles of the microspheres prepared with various contents of PLA and PLGA are shown in Figs. 2 and 3. Fig. 1. SEM micrograph of rifampin microspheres containing PLGA and PLA 345Slow Release Formulations of Inhaled Rifampin The dissolution profiles of the microsphere for