Direct Lung Delivery of Para-Aminosalicylic Acid by Aerosol Particles

ARTICLE IN PRESS

Tuberculosis (2003) 83, 379–385

Tuberculosis

www.elsevierhealth.com/journals/tube Direct lung delivery of para-aminosalicylic acid by aerosol particles

N. Tsapisa,b, D. Bennettc, K. O’Driscollc, K. Sheac, M.M. Lippc,K.Fuc, R.W. Clarkec, D. Deaverc, D. Yaminsb, J. Wrightc, C.A. Peloquind, D.A. Weitza,b, D.A. Edwardsa,b,* aDivision of Engineering and Applied Sciences, Department of Physics, Harvard University, 29 Oxford St., Cambridge, MA 02138, USA bMedicine in Need (MEND), Harvard University, 29 Oxford St., Pierce Hall, Cambridge, MA 02138, USA cAlkermes Inc., 88 Sidney St., Cambridge, MA 02139, USA dInfectious Diseases Pharmacokinetics Laboratory, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206, USA Accepted 5 August 2003

KEYWORDS Summary Para-aminosalicylic acid (PAS), a tuberculostatic agent, was formulated into Tuberculosis treatment; large porous particles for direct delivery into the lungs via inhalation. These particles Lung delivery; possess optimized physical properties for deposition throughout the respiratory tract, a Aerosol drug loading of 95% by weight and physical stability over 4 weeks at elevated temperatures. Upon insufflation in rats, PAS concentrations were measured in plasma, lung lining fluid and homogenized whole lung tissue. Systemic drug concentrations peaked at 15 min, with a maximum plasma concentration of 1171 mg/ml. The concentration in the lung lining fluid was 148762 mg/ml at 15 min. Tissue concentrations were 65720 mg/ml at 15 min and 3.270.2 mg/ml at 3 h. PAS was cleared within 3 h from the lung lining fluid and plasma but was still present at therapeutic concentrations in the lung tissue. These results suggest that inhalation delivery of PAS can potentially allow for a reduction in total dose delivered while providing for higher local and similar peak systemic drug concentrations as compared to those obtained upon oral PAS dosing. Similar particles could potentially be used for the delivery of additional anti- tuberculosis agents such as rifampicin, aminoglucosides or fluoroquinolones. & 2003 Elsevier Ltd. All rights reserved.

Introduction local and systemic applications.1,2 A principal advantage of LPPs relative to conventional inhaled Large porous particles (LPPs), characterized by therapeutic aerosol particles is their aerosolization 3,4 geometric sizes greater than 5 mm and mass den- efﬁciency. This permits the delivery of large drug sities less than 0.4 g/cm3, have achieved recent masses (several tens of milligrams per puff) from a 1 popularity for drug delivery to the lungs for both simple inhalation device. In addition, these particles possess the potential for avoidance of alveolar macrophage clearance due to their large geometric *Corresponding author. Division of Engineering and Applied size,5–7 enabling sustained drug release in the Sciences, Department of Physics, Harvard University, 29 Oxford 8 St., Cambridge, MA 02138, USA. Tel.: þ 1-617-495-1328; fax: lungs. These traits suggest that inhalation of LPPs þ 1-617-495-9837. may provide an advantage in the treatment of E-mail address: [email protected] (D.A. Edwards). tuberculosis (TB) by targeting the drugs directly to

380 N. Tsapis et al. the primary site of infection in order to achieve light using aluminum foil. All solutions were used therapeutic local drug concentrations with less immediately after preparation. Solutions were systemic exposure than oral dosing. Inhalation spray dried utilizing a NIRO Atomizer Portable delivery in general has been demonstrated to spray-drier system (Columbus, MD) equipped with possess several advantages for TB treatment com- a two-fluid nozzle for atomization. The 800 ml pared to oral delivery in theory9 and in practice.10 ethanol solutions were each mixed with 200 ml of Since the lungs are directly targeted, total body distilled water immediately prior to atomization. doses are lower, leading to a decrease of potential Spray-dried LPP-PAS powders were collected via a drug resistance build-up. In addition, by avoiding the cyclone. gastro-intestinal tract, side effects often associated with high oral doses (e.g. up to 12 g daily for para- aminosalicylic acid (PAS)11 canbeavoided.These Characterization of the spray-dried powders arguments suggest that inhalation delivery of PAS by dry LPPs could, if proven equally efficacious as oral The volume median geometric diameters (d) of two dosing, help to increase patient compliance, while representative batches of the LPP-PAS powders increasing local concentrations of the drug with a were measured by light diffraction (Rodos, Sympa- practical and convenient inhalation system. tec, Lawrenceville, NJ) at an applied pressure of While previous researchers have conducted ani- 1 bar. mal and human studies to explore pulmonary drug A two-stage Anderson Cascade Impactor (ACI-2, delivery for TB treatment,12,10 these systems in- Thermo Andersen, Smyrna, GA) was used to volved liquid (nebulized) delivery. Nebulization is determine the fine particle fraction (FPF) of the time consuming and cumbersome relative to stan- LPP-PAS powders. In practice, a capsule containing dard therapies for asthma and COPD, which typically approximately 10 mg of powder was placed in a employ pressurized metered dose inhalers and dry hand-held, dry-powder, breath-activated inhaler powder inhalers. This article presents results for device (the AIRs inhaler4). The capsule was dry powder LPP delivery of a specific drug, PAS, punctured, a pump was actuated to simulate an commonly used to treat cases of multidrug resistant inspiration (utilizing an air flowrate of 60 l/min for TB.13 We have formulated LPPs containing 95% of a duration of 2 s) and the powder was deposited on PAS by weight (LPP-PAS), confirmed that these different stages covered with glass fiber filters formulations possess physical and aerosol properties depending on the aerodynamic diameter of the suitable for use for the treatment of TB via particles. The filters were weighed before and after inhalation and delivered them to rats via pulmonary the experiment; the fractions of particles with an insufflation to assess drug concentrations in plasma, aerodynamic diameter below 5.6 and 3.4 mm were lung lining fluid (assayed via bronchoalveolar determined from the deposited weights. The mass lavageFBAL), and lung tissue homogenate. mean aerodynamic diameter (MMAD), written daer; is related to the geometric diameter d (assumingpffiffiffi 14 particle sphericity) by the formula : daer ¼ d r; where r is the particle density normalized by the Materials and methods density of water (1 g/cm3). Scanning Electron Microscopy (SEM) was per- Materials formed using a LEO 982 (LEO Electron Microscopy, Inc., Thornwood, NY) operating between 1 and 5 kV 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine with a filament current of approximately 0.5 mA. (DPPC, MW ¼ 734.05, purity X99%) was purchased LPP-PAS powder samples were deposited onto SEM from Genzyme Corp. (Cambridge, MA). PAS in the stubs covered with adhesive tape and then coated free acid form was purchased from Spectrum with a gold–chromium layer using a Polaron SC7620 Chemical, Co. (New Brunswick, NJ). Distilled water sputter coater operated for 90 s at a sputtering USP grade was purchased from B. Braun Medical, current of 18 mA. Inc. (Irvine, CA) and ethanol USP grade was purchased from PharmCo (Brookfield, CT). Accelerated physical stability Particle production via spray drying LPP-PAS powder samples were placed into a 401C Solutions for spray drying were prepared by oven at 15% relative humidity to test accelerated dissolving 0.25 g of DPPC and 4.75 g of PAS in physical stability. FPF and d were measured at 0, 2 800 ml of ethanol. Solutions were protected from and 4 weeks. ARTICLE IN PRESS

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Animal experiments tized by intraperitoneal injection of ketamine and xylazine as described above. The LPP-PAS powders This research adheres to the Guide for the Care and were administered via an insufflation device Use of Laboratory Animals (National Academy (PennCentury, Philadelphia, PA). Once sedation Press, 1996). Male Sprague Dawley rats were was confirmed, animals were placed onto a obtained from Taconic Farms (Germantown, NY). modified slant board to optimize visual placement The animals weighed between 357 and 534 g (mean of the insufflation cannula into the trachea. The tip weight was 431.5677.29 g). The animals were in of the insufflation device was placed in the trachea good health upon arrival and remained so until use; just above the carina and 5 mg of LPP-PAS powder no clinical signs of illness were observed at any was delivered through the insufflation device by time. They were housed two per cage while on rapidly pushing a 3 cm3 bolus of air through the study in accordance to NIH guidelines prior to device a total of 3 times. Pharmacokinetic sampling surgery in standard plastic shoebox cages with time started immediately after the final 3 cm3 bolus heat-treated laboratory grade Bed-O’ Cobs (Ander- of air was injected. All blood samples were taken son, Maumee, OH) bedding. The light/dark cycle via the implanted jugular catheter. Blood was was 12/12 h. The temperature in the animal room collected pre-dose, and at 15, 30, 180, and was ambient room temperature of approximately 420 min following pulmonary insufflation. Approxi- 251C and the ambient humidity was in the range of mately 0.4 ml of blood was taken at each time approximately 35–60%. Animals were housed in point. All blood samples were collected in EDTA accordance with the Guide for the Care and Use of tubes. Plasma was separated in a tabletop centri- Laboratory Animals (ILAR). Animals were allowed fuge (Eppendorf 5415C, VWR, Boston, MA) at access to food and water ad libitum throughout the 14,000 rpm for 30 s. The retrieved plasma was duration of the study. The food was Lab Diet- subsequently snap frozen on dry ice and stored at Rodent Diet #5001 (PMI Nutrition International, 801C until analysis. Inc., Brentwood, MO). The water was from a clean For BAL to assess lung lining fluid PAS levels, tap source from the City of Cambridge, Massachu- animals were euthanized via intraperitoneal injec- setts Municipal Water Supply. tion of 216 mg of euthasol (Delmarva, Midlothian, Indwelling jugular catheters were inserted under VA) and exsanguinated per an approved IACUC general anesthesia using a ketamine (80–90 mg/kg) protocol. The trachea was cannulated using an 18- (Fort Dodge, Inc., Fort Dodge, IA) and xylazine (10– gauge needle adaptor for subsequent injection and 13 mg/kg) (Phoenix Pharmaceuticals, St. Joseph, retrieval of broncho-alveolar lavage (BAL) fluid. MO) cocktail by intra-peritoneal injection 24 h prior Prior to BAL, the left main bronchus was clamped to dosing. The catheterization method was per- and subsequently tied off to isolate the large left formed per an approved procedure.15 Following lobe for whole tissue analysis. BAL was performed surgery, the study subjects were numbered upon on the remainder of the lung (lavage buffer: pre-dose blood collection and placed into individu- 145 mM NaCl (8.5 g/l), 5 mM KCl (0.37 g/l), 1.9 mM ally numbered cages overnight. The animals were NaH2PO4 (0.26 g/l for monohydrate; 0.228 g/l for used as presented in Table 1 for the different types anhydrous), 0.5 mM glucose (1 g/l) pH ¼ 7.4). One of samples. Five-milligram samples of LPP-PAS ml of lavage buffer was slowly inserted via a 5 ml powders were weighed out into hydroxypropyl- syringe and as much as possible of the fluid was methyl cellulose capsules (Shionogi, Japan) and retrieved for subsequent analysis. Retrieved BAL stored at 41C prior to dosing. Following catheter- fluid was clarified by centrifugation at 250 RCF ization and prior to treatment, animals were (1060 rpm) for 2 min. Following centrifugation, the distributed equally based on body weight through- supernatant was reserved, snap-frozen on dry ice, out the treatment groups. Animals were anesthe- and stored at 801C until analysis.

Table 1 Utilization of the rats for the different types of samples. Group # rats 0 min 15 min 30 min 180 min 420 min 1 5 Plasma Plasma Plasma/BAL lung 2 5 Plasma Plasma Plasma Plasma/BAL lung 3 5 Plasma Plasma/BAL lung

A total of 15 rats were insufflated in this study. At each timepoint, plasma samples were taken from a minimum of five animals. At terminal sacrifice timepoints (15, 30, and 420 min), BAL and whole lung homogenization were completed on a minimum of 5 sets of lungs. ARTICLE IN PRESS

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Following BAL, the lungs were removed from the chest cavity for homogenization. The isolated left lobe was retrieved intact, weighed and placed into a 15 ml conical tube. The tissue was snap frozen on dry ice and stored at 801C until homogenization. For homogenization, lung tissues were thawed and 3.0 ml of lavage buffer was added to each 15 ml tube. Lungs were homogenized using a Polytron PT- 1200 Homogenizer (Kinematica AG, Luzerner- strasse, Switzerland). The subsequent tissue slurry was snap-frozen on dry ice and stored at 801C until analysis. Plasma, BAL fluid and homogenized lung tissue PAS concentrations were determined by an HPLC assay described in the literature.16 Briefly, all samples were analyzed using high-performance liquid chromatography (HPLC) with the following equipment: a Waters model 510 pump and model 680 gradient controller and solvent select valve (Milford, MA), a Spectra Physics model 8875 fixed- volume autosampler (San Jose, CA), a McPherson model FL-750A fluorescence detector (Acton, MA), a Macintosh IIci computer (Apple Computers, Inc., Cupertino, CA), and the Rainin Dynamax HPLC data management system (Woburn, MA). The standard curves for PAS were 1.0–100.0 mg/ml. The lower limit of detection was approximately 0.01 mg/ml, and the low standard was 1 mg/ml. The best fits of the standard curves were achieved using a weight of 1=Y 2: The recovery of PAS from serum was 94% (range, 91–97%). Testing for PAS within sample precision produced a percent coefficient of variation (CV) of 2.0%. Testing for PAS day-to-day assay Figure 1 SEM images of the PAS LPPs just after spray- precision produced a coefficient of variation of drying (top) or after 4 weeks at 401C and 15% relative 2.4% (low: 0.1% at 1 mg/ml; high: 5.0% at 10 mg/ml). humidity (bottom). Scale bars represent 5 mm. A total of 15 animals were insufflated in this study, five per treatment group. The time of terminal sacrifice was staggered to increase the aerodynamic properties of two representative LPP- pharmacokinetic and tissue sample size at each PAS batches are shown in Table 2; values of d were timepoint. For the PK studies according to Table 1, in the range of 7.0–7.5 mm whereas FPFs below 15 animals were pre-bled to establish background 5.6 mm ranged from 0.59 to 0.63 and below 3.4 mm levels. At 15 and 30 min post-dosing, 10 samples ranged from 0.31 to 0.39. These particle charac- were collected and at the 180 and 420 min post- teristics are typical of particles that deposit dosing timepoints, 5 samples were collected. To throughout the respiratory tract, with good deposi- generate the PK data, a total of 5 samples were tion in the central airways and the alveolar region, assayed from each timepoint. For BAL samples, at based on human data for similar powders.17 each terminal sacrifice, 5 samples were collected for each timepoint. Accelerated physical stability

Given the observation via SEM of the presence of Results and discussion crystalline phases in the particles (conﬁrmed via DSC, data not shown), the stability of aerosol Particle physical properties properties to physical changes was tested in an accelerated stability environment (401C and 15% SEM pictures reveal the particles to be porous and relative humidity). The geometric diameter at 1 bar partly crystalline (Fig. 1, top). The geometric and and the FPF were tested at 2 and 4 weeks; neither ARTICLE IN PRESS

Direct lung delivery of PAS by aerosol particles 383

SEM (Fig. 1, bottom). The PAS formulation thus Table 2 Physical properties of the PAS particles for two different batches. appears to be physically stable over 4 weeks under elevated temperature exposure, an important Batch Volume median FPF feature for practical therapy. geometric diameter (d in mm) o5.6 mm o3.4 mm Animal experiments 1 7.47 0.596 0.311 The pharmacokinetic data (Fig. 3) indicate that PAS 2 7.07 0.629 0.392 delivered as 5 mg of LPP-PAS powder via insufflation Variability from batch to batch is acceptable for animal reached the systemic circulation within 15 min of experiments since the insufflation method homoge- delivery to the lungs with a plasma peak concen- neously delivers air powders to the lung. tration of 1171 mg/ml (Table 3) and a time to peak concentration in the range of 0–30 min, with total clearance by 3 h post-exposure. For general com- parison, the peak concentration that we observed in rats is an order of magnitude larger than the minimum inhibitory concentration (MIC) of PAS16,18 and in the same range of what is observed for humans upon oral dosing. Normalizing the inhaled PAS results for rat body mass, the total body dose is approximately 11 mg/kg via the lungs compared to an estimated value of 57 mg/kg for oral dosing of PAS in humans (assuming a single dose of 4 g administered to a 70 kg person). Owing to limits of detection, we were not able to measure PAS concentration in the BAL fluid beyond 15 min. At 15 min, we found this concentration to be 5.772.4 mg/ml. To attempt to estimate from this value the corresponding ‘‘true’’ lung fluid concentration (i.e. to account for the fact that the BAL buffer acts as a diluent), an estimated dilution factor was calculated. An estimate of the total lung lining fluid volume for a rat has been reported to be 80.2 ml.19 Since only the right side of

Figure 2 Geometric diameter at 1 bar (top) and FPF (bottom, K represents o5.6 mm and mo3.4 mm) over 4 weeks at 401C and 15% relative humidity.

displayed statistically signiﬁcant changes (Fig. 2). Figure 3 PAS pharmacokinetics in the plasma after Additionally, no observable increases or changes in insufﬂation of 5 mg of powders to rats. Data shown is crystalline content were observed over time with mean7SEM of nX5 guinea pigs at each timepoint. ARTICLE IN PRESS

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promising to argue for further dose-ranging safety Table 3 Measured and calculated PAS concentrations in plasma, lung fluid and lung tissue after and efficacy testing. insufflation. 15 min (mg/ml) 180 min (mg/ml) Acknowledgements Blood 11710 Lung fluid 148762 0 Authors would like to thank Prof. J. Y. Kim for his Lung tissue 65720 3.270.2 support, and Prof. A. J. Hickey for very fruitful Data shown is mean7SEM of nX5 guinea pigs. discussions. the lung was lavaged, it was estimated that 40.1 ml References of lung lining fluid was diluted in 1 ml of BAL buffer. Accounting for this dilution, the PAS concentration 1. Edwards DA. The delivery of biological agents by aerosols. in the lung lining fluid at 15 min was calculated to Am Inst Chem Eng J 2002;48:2–6. be 148762 mg/ml (Table 3), which is approximately 2. Edwards DA, Hanes J, Caponetti G, Hrkach J, BenJebria A, thirteen times the measured PAS concentration in Eskew ML, Mintzes J, Deaver D, Lotan N, Langer R. Large porous biodegradable particles for pulmonary drug delivery. plasma at this time point. Science 1997;276:1868–71. The concentrations in the homogenized lung 3. Dunbar CA, Hickey AJ, Holzner P. Dispersion and charac- tissue were determined to be 3.371 mg/ml at terization of pharmaceutical dry powder aerosols. KONA 15 min and 0.1670.01 mg/ml at 3 h. To estimate 1998;16:7–45. the dilution due to the buffer, the left lobe was 4. Edwards DA, Dunbar CA. Therapeutic aerosol bioengineer- ing. Annu Rev Biomed Eng 2002;4:93–107. removed, placed in 3 ml of buffer and homoge- 5. Kawaguchi H, Koiwai N, Ohtsuka Y, Miyamato M, Sasakawa S. nized: the new volume recorded was 3.16 ml. Thus Phagocytosis of latex-particles by leukocytes 1. Dependence the lung contribution to the volume is 0.16 and the of phagocytosis on the size and surface-potential of dilution factor is 0.16/3.16 ¼ 0.0506. The concen- particles. Biomaterials 1986;7:61–6. tration in the lung tissue is finally estimated to be 6. Krenis LJ, Strauss B. Effect of size and concentration of latex 7 7 particles on respiration of human blood leukocytes. Proc Soc 65 20 mg/ml at 15 min and 3.2 0.2 mg/ml at 3 h Exp Biol Med 1961;107:748. (Table 3). This indicates that, although not detect- 7. Rudt S, Muller RH. In vitro phagocytosis assay of nanopar- able in the blood or the lung fluid, PAS remains at ticles and microparticles by chemiluminescence 1. Effect of therapeutic concentrations in the lung tissue for at analytical parameters, particle size and particle concentra- least 3 h after insufflation. tion. J Controlled Release 1992;22:263–71. 8. Vanbever R, Ben-Jebria A, Mintzes J, Langer R, Edwards DA. Sustained release of insulin from insoluble inhaled particles. Drug Dev Res 1999;48:178–85. 9. Flume P, Klepser ME. The rationale for aerosolized anti- biotics. Pharmacotherapy 2002;22(3 Pt 2):71S–9S. Conclusion 10. Sacks LV, Pendle S, Orlovic D, Andre M, Popara M, Moore G, Thonell L, Hurwitz S. Adjunctive salvage therapy with inhaled aminoglycosides for patients with persistent We have formulated PAS, a tuberculostatic agent, smear-positive pulmonary tuberculosis. Clin Infect Dis into LPPs containing 95% PAS by weight via 2001;32:44–9. spray drying. These LPP-PAS formulations are for 11. Peloquin CA, Berning SE, Huitt GA, Childs JM, Singleton MD, direct delivery to the lungs by inhalation. Their James GT. Once-daily and twice-daily dosing of p-aminosalicylic acid granules. Am J Respir Crit Care Med 1999; physical properties are optimal for deposition 159:932–4. throughout the respiratory tract, from the central 12. O’Hara P, Hickey AJ. Respirable PLGA microspheres contain- airways to the alveolar region, and these physical ing rifampicin for the treatment of tuberculosis: manufac- properties are stable at accelerated storage ture and characterization. Pharm Res 2000;17:955–61. ! conditions. 13. Mitnick C, Bayona J, Palacios E, Shin S, Furin J, Alcantara F, Sanchez! E, Sarria M, Becerra M, Smith Fawzi MC, Kapiga S, The systemic and local concentrations observed Neuberg D, Maguire JH, Kim JY, Farmer P. Community-based after insufflation of LPP-PAS in rats suggest that therapy for multiresistant tuberculosis in Lima, Peru. N Engl greater lung exposure to PAS can be achieved at J Med 2003;348:119–28. much less total body drug dose than those typically 14. Gonda I. In: Crommelin DJA, Midha KK, editors. Topics in used for oral delivery. It is unclear what these pharmaceutical sciences. Stuttgart: Medpharm Scientific; 1991. p. 95–115. pharmacokinetic data mean precisely in terms of 15. Waynforth HB, Flecknell PA. Catheterisation of the jugular the safety and efficacy of an inhaled PAS product, vein. In: Experimental and surgical technique in the rat, 2nd though we believe the results to be sufficiently ed. Boston: Academic Press; 1992. ARTICLE IN PRESS

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16. Peloquin CA, Zhu M, Adam RD, Singleton MD, Nix DE. 18. Yew WW, et al. Serum pharmacokinetics of antimi- Pharmacokinetics of para-aminosalicylic acid granules under crobial drugs in patients with multidrug-resistant tuber- four dosing conditions. Ann Pharmacother 2001;35:1332–8. culosis during therapy. Int J Clin Pharm Res 1999;XIX: 17. Dunbar CA, Scheuch G, Sommerer K, Delong M, Verma A, 65–71. Batycky R. In vitro and in vivo dose delivery characteristics 19. Hatch G. Comparative biochemistry of airway lining ﬂuid. In: of large porous particles for inhalation. Int J Pharm Parent RA, editor. Comparative biology of the normal lung. 2002;245:179–89. Boca Raton, FL: CRC press; 1992.