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Dry powder nitroimidazopyran antibiotic PA-824 aerosol for inhalation
by
Jean C. Sung,1 Lucila Garcia-Contreras,2 Jarod L. VerBerkmoes,1 Charles A.
Peloquin, 3 Katharina J. Elbert,1 Anthony J. Hickey,2* and David A. Edwards1*
1Harvard School of Engineering and Applied Sciences, Cambridge, MA 02138
2University of North Carolina, School of Pharmacy, Chapel Hill, NC 27599
3National Jewish Medical and Research Center, Denver, CO 80206
*Authors to whom correspondence should be addressed at:
David A. Edwards
Harvard School of Engineering and Applied Sciences,
322 Pierce Hall, 29 Oxford Street, Cambridge, MA 02138
ph 617 495 1328; fax 617 495 9837
email: [email protected]
Anthony J. Hickey
University of North Carolina at Chapel Hill, 1310 Kerr Hall, CB #7360, Chapel
Hill, NC 27599
Ph 919-962-0223; fax 919-966-0197
Email: [email protected]
Running title: Inhaled PA-824 for TB treatment
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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.01389-08 AAC Accepts, published online ahead of print on 12 January 2009
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Abstract 1
We formulated PA-824, a nitroimidazopyran with promise for the treatment of 2
tuberculosis, for efficient aerosol delivery to the lungs in a dry powder porous 3
particle form. The objectives of this study were to prepare and characterize a 4
particulate form of PA-824, assess the stability of this aerosol formulation at 5
different environmental conditions and determine the pharmacokinetic 6
parameters of the powder after pulmonary administration. The drug was spray 7
dried into porous particles containing a high drug load and possessing desirable 8
aerosol properties for efficient deposition in the lungs. Physical, aerodynamic 9
and chemical properties of the dry powder were stable at room temperature for 10
six months and refrigerated conditions for at least one year. Pharmacokinetic 11
parameters were determined in guinea pigs after pulmonary administration of the 12
PA-824 powder formulation at three doses (20, 40, 60 mg/kg) and compared to 13
intravenous (20 mg/kg) and oral (40 mg/kg) delivery of the drug. Oral and 14
inhaled delivery of PA-824 achieved equivalent systemic delivery at the same 15
body dose within the first twelve hours of dosing. However, animals dosed by 16
the pulmonary route showed drug loads remaining locally in the lungs 32 hours 17
post-exposure whereas those given oral drug cleared more rapidly. Therefore, 18
we expect from this pharmacokinetic data that pulmonary delivery may achieve 19
the same efficacy as oral at the same body dose, with a potential improvement in 20
efficacy related to pulmonary infection. This may translate into the ability to 21
deliver lower body doses of this drug for the treatment of tuberculosis by aerosol. 22
23
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Introduction 24
The global epidemic caused by the infectious disease tuberculosis (TB) has 25
garnered attention recently due to the growing concern over the emergence of 26
multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant 27
tuberculosis (XDR-TB), which are caused by Mycobacterium tuberculosis that 28
show resistance to multiple first-line drugs or that are virtually untreatable by 29
existing TB antibiotics (6). An investigational nitroimidazopyran, PA-824, is one 30
of the most promising TB drug candidates in thirty years. PA-824 appears to 31
have bactericidal activity against both active replicating and persisting or latent 32
M. tuberculosis with the potential of treating MDR-TB (21). It has been proposed 33
that this activity against static bacilli could help sterilize TB lesions faster (26). 34
Therefore, reduced treatment times may be possible with drug regimens that 35
include PA-824. Any advance in TB therapy that addresses drug resistant 36
tuberculosis and reduces frequency of dosing, dose required and duration of 37
treatment has the potential to improve patient treatment. 38
39
Preclinical evaluation in animal models of PA-824 alone and in combination with 40
moxifloxacin, rifampicin, isoniazid and/or pyrazinamide indicated potential for the 41
treatment of TB and MDR-TB (9, 13-15, 21, 22, 25). Drugs were delivered orally 42
in these studies, with likely only a fraction of the administered dose reaching the 43
lung tissue, the primary infection site of TB. Thus, direct delivery of PA-824 to 44
the lungs could improve the effectiveness of treatment by increasing the local 45
concentration of drug in the lungs thereby targeting pulmonary TB, while still 46
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delivering drug systemically for non-pulmonary TB. It is also possible that this 47
non-invasive route of administration may require smaller doses compared to 48
standard oral treatment, thus, potentially reducing side effects and shortening 49
time of treatment. Inhaled antibiotics have been demonstrated to improve 50
treatment of TB (18) and other diseases such as cystic fibrosis (19). However, 51
these treatments were given by nebulization, a process which requires lengthy 52
treatment times and sterile water to achieve relatively low delivery efficiencies 53
(8). 54
55
The dry powder inhaler is an alternative method for pulmonary drug delivery, with 56
the ability to deliver stable formulations easily from simple inhalers. The porous 57
particle system, a dry particle formulation, is well suited for efficient delivery to 58
the lungs (1). This novel particulate aerosol has also been explored for TB 59
treatment with the tuberculostatic agents para-aminosalicyclic acid (PAS) and 60
capreomycin (2, 3, 23). PAS, delivered as a dry powder, was shown to achieve 61
greater lung exposure in rats at a lower total body drug dose than the typical oral 62
dose (23). Pulmonary delivery of capreomycin gave promising efficacy results in 63
a guinea pig model of tuberculosis, with lower wet organ weights and bacterial 64
burden in the lungs than animals given intravenous or intramuscular treatment (2, 65
3). Thus, pulmonary administration of PA-824 in a porous particle form may 66
provide an additional therapeutic advantage for this promising new drug. In the 67
present study, PA-824 was formulated for aerosol delivery into a porous particle 68
form and characterized for physical and aerosol properties that would be 69
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desirable for deposition in the lungs. The powder was tested for physical, 70
aerodynamic and chemical stability at a variety of environmental conditions over 71
a period of one year. 72
73
The disposition of PA-824 after oral delivery was determined previously in mice 74
(10, 13), rats (20), dogs and monkeys (16), but not after pulmonary delivery to 75
guinea pigs. The TB infected guinea pig model is a clinically relevant animal 76
model with respect to human disease because the pathogenesis and lung 77
pathology in guinea pigs mimics that in humans with active TB (12, 17, 24). It is 78
likely that drug disposition will be different in TB infected animals than in healthy 79
animals. As the initial step to determine PA-824 disposition in guinea pigs, the 80
present studies were conducted in healthy animals to define baseline 81
pharmacokinetic parameters of PA-824 after intravenous administration of 82
solution and pulmonary administration of the dry powder formulation, and 83
compared to those obtained by the oral standard route of delivery. In addition, 84
local PA-824 concentrations were determined in the lungs after the drug 85
concentration became undetectable in systemic circulation to evaluate the 86
efficiency of delivery by both routes of administration. 87
88
89
90
91
92
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MATERIALS AND METHODS 93
Materials 94
L-leucine was obtained from Spectrum Chemicals & Laboratory Products 95
(Gardena, CA) and the phospholipid 1,2-Dipalmitoyl-sn-glycero-3-96
phosphocholine (DPPC) from Genzyme Pharmaceuticals (Cambridge, MA). PA-97
824 was received from the Global Alliance for TB Drug Development (New York, 98
NY). Acetonitrile, ethanol USP grade and methanol were purchased from 99
Pharmco Products Inc. (Brookfield, CT). Water from a Millipore Corp. (Billerica, 100
MA) Milli-Q water purification system was used. 101
102
Preparation of Porous Particles 103
The spray drying solution was prepared by dissolving L-leucine in pure water at 104
55 ºC. PA-824 and DPPC were then added to ethanol with heating and stirring. 105
The aqueous solution was added to the ethanol mixture immediately before 106
spray drying for a final solids concentration of 4 g/L and solvent ratio of 70% 107
ethanol and 30% water (v/v). The final formulation consisted of a ratio of 108
75:20:5, PA-824: L-leucine:DPPC (wt %). 109
110
Porous particles were prepared using a Niro, Inc. Mobile Minor spray dryer 111
(Columbia, MD). The inlet temperature was set at 107 ºC and the solution feed 112
rate at 60 ml/min. The solution was pumped into the two-fluid nozzle of the spray 113
dryer with a gas flow rate of 25 g/min. Spray dried powders were collected in a 114
container at the outlet of the cyclone. 115
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Particle Characterization 116
Characterization of Dry Powders 117
The volume diameter of the spray dried powder was measured by laser 118
diffraction using a HELOS system with RODOS dry dispersing unit (Sympatec 119
Inc., Lawrenceville, NJ). Each measurement was performed in triplicate at an 120
applied pressure of 2 bar. 121
122
The aerodynamic properties and particle distribution of the powder (fine particle 123
fraction, FPF; mass median aerodynamic diameter, MMAD; geometric standard 124
deviation, GSD) were determined with standard methodology using an eight-125
stage Andersen Cascade Impactor (ACI; Copley Scientific Limited, Nottingham, 126
UK). The fine particle fraction of the total dose of powder less than or equal to an 127
effective cut-off aerodynamic diameter of 5.8 µm (FPFTD) was calculated by 128
dividing the powder mass recovered from stages 1-7 of the impactor by the total 129
particle mass. 130
131
The morphology of the dry particles was evaluated using a 982 field emission 132
scanning electron microscope (SEM, LEO, Carl Zeiss, Inc., Thornwood, NY) after 133
coating powder samples with a Platinum/Palladium layer (208HR sputter coater, 134
Cressington Scientific Instruments Inc., Watford, UK). 135
136
137
138
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Drug Load of Powders 139
The PA-824 load of the spray dried powder was determined by a reverse-phase 140
high performance liquid chromatography (HPLC) method using an Agilent 1100 141
Series HPLC system with Zorbax columns and ChemStation software (Agilent 142
Technologies Inc., Palo Alto, CA). The mobile phase was run on a linear 143
gradient from 20% acetonitrile and 80% water to 60% acetonitrile and 40% water 144
over 30 minutes with 5 minutes of equilibration time. Analysis was performed on 145
a 50 µL injection at a flow rate of 1.5 ml/min through an Agilent Zorbax Eclipse 146
XDB-C18 (4.6 x 150 mm) column and absorbance recorded at 330 nm. An 147
Agilent Zorbax Eclipse XDB-C18 analytical guard column was also used. 148
149
Powder Stability Study 150
A spray dried PA-824 formulation of 75:20:5 // PA-824: L-leucine:DPPC (wt %) 151
was stored at different environmental conditions to assess physical, aerodynamic 152
and chemical stability over time. Powder aliquots were transferred to glass vials 153
in a controlled environment held at 10% relative humidity and then placed at 154
temperatures of 4°C, 25°C and 40°C, representing refrigerated, room 155
temperature and accelerated storage conditions. Sealed sample vials were 156
stored protected from light in polyethylene jars with anhydrous calcium sulfate 157
desiccant (W.A. Hammond Drierte Co., Xenia, OH). Samples were removed 158
from storage at 0, 0.5, 1, 3, 6 and 12 months and allowed to equilibrate to room 159
temperature before analysis. The powders were analyzed in triplicate for particle 160
size, fine particle fraction of the total dose and drug content. Statistical 161
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significance was determined by linear regression analysis, where p-values < 0.05 162
were considered to be statistically significant. 163
164
Pharmacokinetic Studies 165
Experimental Design 166
Male guinea pigs weighing 463.86 ± 65.11g were employed in the 167
pharmacokinetic (PK) studies. Animals were housed in a 12 h light/ 12 h dark 168
cycle and constant temperature environment of 22 °C. A standard diet and water 169
were supplied ad libitum. All animal procedures were approved by the 170
Institutional Animal Care and Use Committee of the University of North Carolina. 171
Twenty-four hours before the study, each animal underwent cannulation of the 172
right external jugular vein for continuous blood sampling (4, 5). 173
174
Animals were randomly divided into five groups (n = 6-7) receiving PA-824 as 175
follows: intravenously (IV) in solution at a dose of 20 mg/kg, oral gavage of the 176
drug in a mixed micelle suspension at 40 mg/kg, and as dry powder via 177
intratracheal insufflation by the pulmonary route as porous powders at nominal 178
doses of 20, 40 and 60 mg/kg. For the insufflation procedure, animals were 179
anesthetized and the trachea visualized with a laryngoscope. A small animal 180
insufflator (Penn Century, Philadelphia, PA) was inserted into the trachea and 181
placed at a distance of 1 cm from the carina. PA-824 powders were dispersed 182
with the help of 5 mL of air from an empty syringe. The insufflator chamber and 183
tube containing the dose of PA-824 powder were weighed carefully before and 184
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after delivery to the animal to estimate the actual delivered dose to be used for 185
the PK analysis for each animal. 186
187
PA-824 was formulated into a micelle suspension for oral dosing with 10% 188
hydroxypropyl-β-cyclodextrin and 10% lecithin as described previously (15). PA-189
824 doses were based on results from an efficacy study of oral administration of 190
PA-824 in a guinea pig model of tuberculosis where a dose of 40 mg/kg delivered 191
daily for 30 days showed a statistically significant reduction of bacilli in lungs and 192
spleen (21). After drug administration, blood samples (0.35 ml) were collected 193
from each animal into heparinized tubes at 0, 0.08, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 194
12 and 24 hours. Additional samples were collected at 28 and 32 hours in 195
animals dosed with powder at 60 mg/kg. Sterile saline solution was used to 196
replace the blood volume lost through sample collection. Plasma was separated 197
and stored at -80 °C until analysis. After collection of the last blood sample, 198
animals were euthanized by exanguination and bronchoalveolar lavage (BAL) 199
was conducted (5 ml sterile saline). The bronchoalveolar lavage sample was 200
centrifuged and the pellet and supernatant separated. 201
202
Sample Analysis 203
Plasma and BAL samples were analyzed by a validated HPLC assay for drug 204
content. Serum concentrations of PA-824 were determined using a system 205
consisting of a ThermoFinnegan P4000 HPLC pump (San Jose, CA) with model 206
AS1000 fixed-volume autosampler, a model UV2000 ultraviolet detector, a 207
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Gateway Series e computer (Poway, CA), and the Chromquest HPLC data 208
management system. The plasma standard curve for PA-824 ranged from 0.20 209
to 50 µg/ml. The absolute recovery of PA-824 from plasma was 88.2%. The 210
overall validation precision across all standards was 0.67 to 5.38%. Sample 211
preparation for the BAL supernatant was an adaptation of the plasma method, 212
with identical HPLC method. 213
214
Pharmacokinetic Analysis 215
The pharmacokinetic parameters area under the curve (AUC), elimination rate 216
constant (K), mean residence time (MRT), half-life (t1/2) and bioavailability (F) 217
were obtained by non-compartmental methods (WinNonlin, Pharsight 218
Corporation, Mountain View, CA). Maximum PA-824 concentrations (Cmax) and 219
time to obtain the maximum PA-824 concentration (Tmax) were determined from 220
the non-fitted plasma versus time profiles for each individual animal. 221
Bioavailability (F) was calculated by the following equation: 222
223
F = AUC lung, oral x D IV 224
AUC IV D lung, oral 225
226
227
where D is the dose. The subscripts indicate the routes of administration: 228
intravenous (IV), pulmonary (lung) and gastrointestinal (oral) delivery. 229
230
231
232
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Statistical Analysis 233
Data for the pharmacokinetic study was subjected to analysis of variance 234
(ANOVA) and least-squares significant-differences multiple comparison method. 235
A probability level of 5% (p < 0.05) was considered to be statistically significant. 236
237
RESULTS 238
Particle manufacture and characterization 239
Porous particles with a load of approximately 75% (w/w) PA-824 were prepared 240
by spray drying. The resulting dry powder formulation consisted of thin-walled 241
porous particle structures as shown in Figure 1. The median volume diameter of 242
the particles was 4.14 ± 0.04 µm, and had desirable aerosol properties for human 243
pulmonary delivery (7, 11) indicated by an MMAD of 4.74 ± 0.08 µm, GSD = 1.53 244
± 0.02 and FPFTD = 53.3 ± 2.7 %. The PA-824 content of the powder was 74.8 ± 245
0.5% of the total mass as estimated by HPLC with UV detection. 246
247
Powder stability study 248
The stability of a PA-824 powder formulation composed of 75:20:5, PA-824: L-249
leucine:DPPC (wt %) was studied for one year at refrigerated (4 °C), room 250
temperature (25 °C) and accelerated conditions (40 °C). The results for volume 251
median diameter, FPFTD and PA-824 content analysis over time are shown in 252
Figure 2. The powder showed no statistically significant differences (p > 0.05) in 253
physical or aerodynamic characteristics or chemical content for one year at 254
refrigerated conditions and for at least six months at room temperature. Powder 255
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at the accelerated condition (40 °C) maintained stability for at least three months 256
and only showed a significant decline in aerosol properties at six months (p = 257
0.000) and PA-824 content at one year (p = 0.039). 258
259
Pharmacokinetic studies 260
Average PA-824 plasma concentration versus time curves are shown in Figure 3. 261
The terminal phases of these curves appear to be linear for all treatments. A 262
dose dependency was observed among groups receiving particles by the 263
pulmonary route, with animals receiving the largest dose having the highest 264
plasma concentration and PA-824 remaining detectable for a longer period of 265
time. Interestingly, the plasma concentration versus time profiles for animals 266
dosed by the oral and pulmonary routes with 40 mg/kg of PA-824 appeared 267
almost superimposable for the first 8 hours, but plasma concentrations in animal 268
receiving an oral dose were detectable only 12 hours after dosing, whereas those 269
of animals dosed by the pulmonary route remained detectable 24 hrs after dose 270
administration. 271
272
The local PA-824 concentrations remaining in the lungs 32 hours after drug 273
administration to the different groups are shown in Figure 4. PA-824 was 274
detectable at levels well above the detection threshold of the analytical method 275
in the lungs of the three groups of animals dosed by the pulmonary route in a 276
dose dependent manner, whereas no drug was detected in the lungs of animals 277
receiving PA-824 by the intravenous or oral routes. 278
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PK parameters obtained by non-compartmental methods are presented in Table 279
1. AUC and Cmax after PA-824 administration by the pulmonary route increased 280
linearly with the dose (R2 = 0.999) with statistical differences between the three 281
values. AUC was statistically comparable in animals receiving 40 mg/kg by the 282
oral and pulmonary routes. However, PA-824 was eliminated (K) at a slower rate 283
when given at doses of 40 and 60 mg/kg by the pulmonary route, correlating with 284
significantly longer half-lives (t1/2) and mean residence time (MRT). Notably, 285
among animals receiving 40 mg/kg PA-824, t1/2 by the pulmonary route was 286
almost double that of those receiving the drug by the oral route. Cmax and Tmax 287
were statistically comparable in groups of animals receiving the 40 and 60 mg/kg 288
doses. Bioavailability was approximately 60% and statistically comparable in all 289
treatment groups. 290
291
DISCUSSION 292
The continuing TB epidemic, including a growing number of cases of MDR/XDR-293
TB and the increasing co-incidence of HIV and TB synergy, requires new and 294
improved treatment as part of the strategy for containment and reduction of the 295
infection. The first drug demonstrating promise in treating both active and latent 296
tuberculosis is the nitroimidazopyran PA-824, typically administered orally. Our 297
goal was to develop PA-824 in a form suitable for delivery as an aerosol directly 298
to the lungs, the primary site of TB infection. 299
300
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We have shown the potential for the antibiotic PA-824 to be formulated into a dry 301
powder porous particle form that can be delivered efficiently by the pulmonary 302
route. This high drug load formulation maintained physical, aerodynamic and 303
chemical stability at refrigerated conditions (4 ºC) for over a year and room 304
temperature (25 ºC) for more than six months. This room temperature stability 305
may reduce the need for a cold chain for storage and distribution. At accelerated 306
conditions (40 ºC), chemical stability was maintained throughout the 12 month 307
study duration, while aerodynamic properties showed significant decline at six 308
months at this elevated temperature. This heat sensitivity is typical for dry 309
powders to be delivered by inhalation (2). In summary, the stability of the 310
formulation is temperature dependent, with chemical and physical stability 311
maintained during the 12 month study duration at refrigerated conditions and for 312
more than six months at room temperature conditions, while likely allowing for 313
short-term storage excursions at higher temperatures. 314
315
Pharmacokinetic parameters after oral administration of PA-824 in guinea pigs 316
were determined in the present study, revealing that the disposition of PA-824 317
differed from literature reports for other different species. Half-life after oral 318
administration in guinea pigs (2.43 h) was comparable to that in monkeys and 319
rats (2-5 h) (16), but slightly longer than that in dogs (1-2 h), confirming the 320
observation that systemic absorption in dogs may be low due to poor absorption 321
and rapid metabolism (16). Tmax after oral administration to guinea pigs (4 h) was 322
also similar to that in monkeys (3.33 h), but significantly shorter than that in rats 323
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(6-8 h), which may indicate a shorter absorption time in guinea pigs. Cmax values 324
14- to 17-fold higher than MIC values of PA-824 against M. tuberculosis (0.015 -325
0.25 µg/ml) (21) were observed in guinea pigs after pulmonary and oral 326
administration of a 40 mg/kg dose, respectively. In contrast, Nuermberger et al. 327
(13) reported Cmax values of 80- to 110-fold higher than the MIC in mice after oral 328
administration of a 100 mg/kg dose. These observations may be the result of 329
administering different doses, but also variation in the disposition of this drug by 330
the two species. Therefore, caution should be exercised when selecting 331
pharmacokinetic parameters from one species to identify a dose or dosing 332
regimen including PA-824 for the treatment of TB in another species. 333
334
In the present study, Cmax and AUC showed a linear dose dependency for 335
powders administered to guinea pigs by the pulmonary route, as was previously 336
reported in mice after oral administration of PA-824 (10). 337
338
Differences in the disposition of PA-824 were observed in guinea pigs when 339
administered by different routes at the same dose. Systemic PA-824 levels 340
appeared similar following delivery by the pulmonary and oral routes within the 341
first 12 h after dosing of 40 mg/kg. However, the drug was not detectable in 342
plasma after 12 h in animals receiving oral doses, whereas systemic drug levels 343
remained detectable in animals dosed by the pulmonary route until 24 h. This 344
observation is consistent with the slower elimination rates and longer half-lives 345
and MRT obtained for animals dosed by the pulmonary route. Most notably, PA-346
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824 concentrations were sustained locally in the lung 32 h after dosing, even 347
after systemic concentrations were undetectable. Tissue concentrations 3 to 8-348
fold higher than in plasma have also been reported after oral administration of 349
PA-824 to rats (20). However, these observations were made at much earlier 350
time points (4-6 h) than in the present study (32 h). Thus, we show that 351
delivering drug directly to the lungs confers the advantage of increasing tissue 352
concentrations of drug at the primary site of TB infection for longer periods of 353
time than drug delivered orally. 354
355
Pharmacokinetic data reported for guinea pigs in the present study, indicate that 356
aerosol administration to the lungs achieves either comparable or lower systemic 357
exposure than the oral route, with elevated local lung concentrations. Therefore, 358
we propose that pulmonary delivery may achieve the same efficacy as oral at the 359
same body dose, with a potential improvement in effectiveness related to 360
pulmonary infection. This may translate into the ability to deliver lower body 361
doses with less frequency than standard dosing regimens. 362
363
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Acknowledgements 364
This work was supported by a grant from the National Institute of Health/NIAID 365
under Grant Number 5 U01 61336 and a National Science Foundation Graduate 366
Research Fellowship. 367
368
The authors acknowledge the Global Alliance for TB Drug Development for PA-369
824 material used in this study and Dr. Melvin Spigelman and Dr. Doris Rouse 370
for their assistance. We also thank Plastiape for their generous donation of 371
inhaler devices. 372
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472
473
474
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Table 1. Pharmacokinetic parameters obtained by non-compartmental 476
analysis after administration of PA-824 formulations by different routes and 477
at different doses (Mean ± standard deviation, n=6-7). 478
Parameter IV 20 mg/kg
Oral 40 mg/kg
Insufflation 20 mg/kg
Insufflation 40 mg/kg
Insufflation 60 mg/kg
AUC(0-t) (µgh/ml)
26.54±2.202 25.77±6.402 14.80±3.843 32.34±16.792 50.96±9.531
K (h-1) 0.37±0.041 0.30±0.052 0.24±0.012 0.17±0.053 0.13±0.053
t1/2 (h ) 1.91±0.243 2.43±0.563 2.83±0.102,3 4.38±1.062 5.91±2.511
MRT (h) 2.69±0.313 5.37±0.532 5.17±0.452 7.52±1.911 8.26±1.801
Cmax (µg/ml)
9.19±1.541 4.14±0.782 2.01±0.553 3.42±1.142,3 4.58±2.492
Tmax (h) 0.11±0.072 4.00±0.631 4.33±1.031 3.25±2.091 3.60±2.881
F(0-∞) - 0.56±0.121 0.59±0.161 0.63±0.321 0.62±0.101
F’ - - 2.11±0.561 1.12±0.572 0.74±0.122
479
AUC = Area under the curve; K = Elimination rate constant; t1/2 = half-life; MRT = 480
Mean residence time; Cmax = Maximum concentration; Tmax = Time at which Cmax 481
occurs; F = Absolute bioavailability; F’ = Relative bioavailability compared to oral 482
treatment. 483
Numeric superscripts show the relative ranks of values (starting from the highest 484
values). When the means are not significantly different, the same superscript is 485
used.486
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FIGURE LEGENDS 487
Figure 1. Scanning electron micrograph of spray dried PA-824 porous particles 488
(PP). [Scale bar represents 2 µm.] 489
490
Figure 2. PA-824 porous powder stability data for (a) volume median diameter, 491
(b) fine particle fraction of the total dose and (c) PA-824 content as a percentage 492
of the initial value at 4 °C (), 25 °C (∆) and 40 °C (■) over a period of 12 months. 493
494
Figure 3. Average PA-824 plasma concentration versus time curves (log scale) 495
after administration of PA-824 at the following treatments and doses: intravenous 496
solution (IV; 20 mg/kg), oral mixed micelle suspension (Oral; 40 mg/kg), and 497
insufflated PA-824 dry powder porous particles (Insuff; 20, 40, 60 mg/kg). (Mean 498
± standard deviation, n = 6-7). 499
500
Figure 4. PA-824 concentrations in bronchoalveolar lavage (BAL) fluid 32 hours 501
after dosing. While no PA-824 was found in the lung fluid for drug given as 502
intravenous solution or oral mixed micelle suspension, powders given by 503
insufflation showed sustained levels of PA-824 in the lungs after 32 hours. There 504
also appears to be a dose dependency on the drug levels remaining in the lungs 505
(Mean ± standard deviation, n = 6-7). 506
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507
Figure 1. Scanning electron micrograph of spray dried PA-824 porous particles 508
(PP). [Scale bar represents 2 µm.] 509
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3.5
4.0
4.5
Vo
lum
e m
ean
dia
mete
r (µ
m)
85
95
105
0 2 4 6 8 10 12
Time (months)
PA
-824 c
on
ten
t (%
of
init
ial)
20
30
40
50
60
70
FP
F TD (
%)
(a)
(b)
(c)
3.5
4.0
4.5
Vo
lum
e m
ean
dia
mete
r (µ
m)
85
95
105
0 2 4 6 8 10 12
Time (months)
PA
-824 c
on
ten
t (%
of
init
ial)
20
30
40
50
60
70
FP
F TD (
%)
(a)
(b)
(c)
510
Figure 2. PA-824 porous powder stability data for (a) volume median diameter, 511
(b) fine particle fraction of the total dose and (c) PA-824 content as a percentage 512
of the initial value at 4 °C (), 25 °C (∆) and 40 °C (■) over a period of 12 months.513
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514
Time (h)
0 10 20 30
PA
-824 p
lasm
a c
oncentr
ation
(µ
g/m
l)
0.1
1
10 IV (20 mg/kg)
Oral (40 mg/kg)
Insuff (20 mg/kg)
Insuff (40 mg/kg)
Insuff (60 mg/kg)
515
516
Figure 3. Average PA-824 plasma concentration versus time curves (log scale) 517
after administration of PA-824 at the following treatments and doses: intravenous 518
solution (IV; 20 mg/kg), oral mixed micelle suspension (Oral; 40 mg/kg), and 519
insufflated PA-824 dry powder porous particles (Insuff; 20, 40, 60 mg/kg). (Mean 520
± standard deviation, n = 6-7). 521
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0
2
4
6
8
10
12
14
16
18
IV solution
(20 mg/kg)
Oral micelle
suspension
(40 mg/kg)
Insuff PP
(20 mg/kg)
Insuff PP
(40 mg/kg)
Insuff PP
(60 mg/kg)
PA
-824 c
on
cen
trati
on
in
BA
L f
luid
( µµ µµ
g/m
L)
522
Figure 4. PA-824 concentrations in bronchoalveolar lavage (BAL) fluid 32 hours 523
after dosing. While no PA-824 was found in the lung fluid for drug given as 524
intravenous solution or oral mixed micelle suspension, powders given by 525
insufflation showed sustained levels of PA-824 in the lungs after 32 hours. There 526
also appears to be a dose dependency on the drug levels remaining in the lungs 527
(Mean ± standard deviation, n = 6-7). 528
529
530
531
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