AAC Accepts, published online ahead of print on 10 November 2014 Antimicrob. Agents Chemother. doi:10.1128/AAC.03951-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Pharmacokinetics, Microbial Response, and Pulmonary Outcomes of Multi-dose
2
Intravenous Azithromycin in Preterm Infants at Risk for Ureaplasma Respiratory
3
Colonization
4
Running Title: Azithromycin 20 mg/kg multi-dose in preterm infants
5
L. Marcela Merchan, MD1*; Hazem E. Hassan, PhD3*; Michael L. Terrin, MD2; Ken B.
6
Waites, MD4; David A. Kaufman, MD5; Namasivayam Ambalavanan, MD4; Pamela
7
Donohue, ScD6; Susan J. Dulkerian, MD1; Robert Schelonka, MD7, Laurence S.
8
Magder, PhD2, Sagar Shukla, PharmD3, Natalie D. Eddington, PhD3; and Rose M.
9
Viscardi, MD1#.
10 1
11
Departments of Pediatrics, and 2Epidemiology and Preventive Medicine, University of
12
Maryland, Baltimore School of Medicine, Baltimore, MD; 3University of Maryland,
13
Baltimore School of Pharmacy, Baltimore, MD; 4Departments of Pathology and
14
Pediatrics, University of Alabama at Birmingham School of Medicine, Birmingham, AL;
15
5
Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA; 6
16
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore,
17
MD; and 7Department of Pediatrics, Oregon Health and Science University, Portland,
18
OR.
19 20
*
21
#
22
e-mail: [email protected]
23
L.L.M. and H.E.H. contributed equally to the work. Corresponding Author: Rose M. Viscardi, M.D
24
ABSTRACT
25
The study objective was to refine the population pharmacokinetics model, determine
26
microbial clearance, and assess short-term pulmonary outcomes of multiple dose
27
azithromycin treatment in preterm infants at risk for Ureaplasma respiratory colon-
28
ization. Fifteen subjects (Ureaplasma-positive=7) received intravenous azithromycin 20
29
mg/kg every 24h for 3 doses. Azithromycin concentrations were determined in plasma
30
samples obtained up to 168h post-first dose, by a validated LC/MS/MS method.
31
Respiratory samples were obtained pre-dose and at three time points post-last dose for
32
Ureaplasma culture, PCR, antibiotic susceptibility testing, and cytokine concentrations.
33
Pharmacokinetic data from these 15 subjects as well as 25 additional subjects [single
34
10 mg/kg dose (N=12), single 20 mg/kg dose (N=13)] were analyzed using a non-linear
35
mixed-effect population modeling (NONMEM) approach. Pulmonary outcomes were
36
assessed at 36 wk post-menstrual age and 6 months adjusted age. A 2-compartment
37
model with all PK parameters allometrically scaled on body weight best described the
38
azithromycin pharmacokinetics in preterm neonates. The population pharmacokinetic
39
parameters’ estimates for clearance, central volume of distribution, inter-compartmental
40
clearance, and peripheral volume of distribution were 0.15 L/h x WT(kg)0.75, 1.88 L x
41
WT(kg), 1.79 L/h x WT(kg)0.75 and 13 L x WT(kg), respectively. The estimated
42
AUC24/MIC90 was ~4 hr. All post-treatment cultures were negative and there were no
43
drug-related adverse events. One Ureaplasma-positive infant died at 4 months of age,
44
but no survivors were hospitalized for respiratory etiologies during the first 6 months
45
adjusted age. A 3 day course of 20 mg/kg/day intravenous azithromycin shows
46
preliminary efficacy in eradicating Ureaplasma spp. from the preterm respiratory tract.
2
47
INTRODUCTION
48
Bronchopulmonary dysplasia (BPD) is the major pulmonary morbidity in infants born
49
preterm characterized by arrested alveolar development and chronic inflammation.
50
Studies of human infants and experimental animal models indicate that the central
51
event in BPD pathogenesis is the interruption of normal developmental signaling during
52
early stages of lung development by lung injury that may be initiated in utero by
53
intrauterine infection with a subsequent dysregulated inflammatory response (1-3). A
54
recent meta-analysis of 39 studies confirmed that respiratory tract colonization with the
55
genital mycoplasma species Ureaplasma parvum and Ureaplasma urealyticum
56
increases the risk for development of BPD in extremely low gestation infants (4). It has
57
not been established whether eradicating Ureaplasma spp. from the respiratory tract of
58
preterm infants prevents or attenuates Ureaplasma infection-mediated lung injury.
59 60
Azithromycin, an azalide antibiotic, has anti-inflammatory properties and antimicrobial
61
activity against Ureaplasma spp. in in vitro (5, 6) and in in vivo experimental models (7-
62
9). Although the efficacies of azithromycin and a related macrolide, clarithromycin, to
63
prevent BPD have been assessed in single-center studies of preterm infants (10-13),
64
the optimal dosing regimens for these antibiotics have not been determined in
65
pharmacokinetic and pharmacodynamic studies and the impact on long term pulmonary
66
and neurologic outcomes are unknown. Our first steps to address these questions has
67
been to conduct studies in the at-risk population to determine the optimal dose, safety,
68
and in vivo anti-infective efficacy of azithromycin in preparation for future phase III
69
randomized, placebo-controlled trials (14, 15). We previously characterized the
3
70
pharmacokinetics (PK) of a single dose of intravenous azithromycin (10 mg/kg and 20-
71
mg/kg) in preterm infants (14). We demonstrated both doses were safe in 24 to 28
72
weeks gestation mechanically ventilated infants, but the 20 mg/kg dose was more
73
effective in eradicating Ureaplasma from the respiratory tract (14,15). Neither dose
74
reduced the pulmonary inflammatory response. Simulation analysis using our previously
75
developed PK model suggested that a multiple-dose regimen of 20 mg/kg intravenous
76
azithromycin may be efficacious for microbial clearance as it could mostly maintain
77
azithromycin plasma levels above the MIC50 for at least 120 hr post I.V. infusion.
78 79
The current study of preterm infants treated with 3 doses of 20 mg/kg intravenous
80
azithromycin was designed to 1) determine the microbiological and short term
81
pulmonary outcomes of the multiple dose; 2) assess the safety of this regimen, and 3)
82
collect additional PK data to further refine the population PK model. We hypothesized
83
that intravenous azithromycin therapy will prevent BPD in Ureaplasma-colonized
84
preterm infants by accelerating pathogen clearance or down-regulating the pulmonary
85
inflammatory response.
86 87
MATERIALS AND METHODS
88
Subject Enrollment
89
We conducted a Phase IIa non-randomized, open-label, PK and safety study of
90
intravenous azithromycin 20 mg/kg every 24h x 3d in preterm infants who were at high
91
risk for Ureaplasma respiratory tract colonization and the development of BPD (FDA
92
IND78990). Study subjects were recruited from six clinical sites from December 2011 to
4
93
June 2012. The institutional review board of each institution approved the study, and
94
parental informed consent was obtained. Inclusion criteria were: 1) gestational age 240-
95
286 weeks; 2) appropriate size for gestational age; 3) 1.5 mg/dL);
103
8) corrected QT interval (QTc) ≥450 ms; 9) neonatal exposure to any other systemic
104
macrolide antibiotic; 10) clinically suspected Ureaplasma CNS infection or other
105
confirmed bacterial or viral infection; and 11) participation in other clinical trials involving
106
investigational products.
107 108
Drug administration and blood sampling
109
After baseline laboratory tests and respiratory specimens were obtained, study infants
110
received azithromycin 20 mg/kg administered at a concentration of 2 mg/ml by
111
intravenous infusion over 60 min within 24 h of enrollment. A total of 3 doses were
112
administered at 24 ± 0.5h intervals. Six blood samples (0.25 ml each) were obtained
113
from each subject during specified time periods post- first dose infusion and processed
114
as previously described (15). Optimum sampling periods were predicted using the PK
115
software ADAPT5 (Biomedical Simulations Resource (BMSR), U. Southern California,
5
116
Los Angeles, CA). Samples were collected anywhere between 1-2, 2-4, 6-8, 25-48, 49-
117
96 and 120-168 hr post first dose. Specifying windows for withdrawing plasma samples
118
helps to better characterize the disposition profile and is a more clinically practical
119
approach (14-17). Levels of azithromycin in plasma were measured using a validated
120
high-performance liquid chromatography-tandem mass spectroscopy (HPLC/MS/MS)
121
detection method as previously described (14, 15).
122 123
Pharmacokinetic data analysis
124
The plasma concentration vs. time data from the a) single 10 mg/kg (14), b) single 20
125
mg/kg (15) and c) multiple 20 mg/kg studies were compiled and analyzed
126
simultaneously using the nonlinear mixed-effects modeling software, NONMEM 7.2
127
(ICON, MD). The first-order conditional estimation procedure (FOCE) with interaction
128
was used for the analysis. Upon development of the base model (two compartment with
129
proportional residual error model), covariate-parameter relationships were explored.
130
The available covariates were weight (WT), gestational age (GA), sex, height, and body
131
surface area.
132 133
The likelihood ratio test was applied to discriminate the alternative nested models where
134
a drop of the objective function value by at least 3.84 points was necessary to declare
135
model improvement, data significance level of P = 0.05, and one degree of freedom.
136
Inter-occasion variability was also estimated where study one (single 10 mg/kg
137
azithromycin), study two (single 20 mg/kg azithromycin) and study three (multiple 20
138
mg/kg azithromycin) were considered as first-, second- and third-occasion, respectively. 6
139
The final model that best fit the data was a two-compartment structural model (ADVAN3
140
TRANS4 NONMEM subroutine) with all parameters allometrically scaled on body weight
141
with a fixed exponent of 0.75 for both Cl and Q and a fixed exponent of 1 for both V1
142
and V2. Post-hoc Bayesian estimates of the individual subjects’ PK parameters were
143
calculated.
144
A proportional error model was used to describe the residual error as follows:
145
Yij obs = Yij pred*(1 + εij)
146
where Yij obs is the observed azithromycin plasma concentration j in subject I, Yij pred is
147
the model-predicted azithromycin plasma concentration, and ε is a residual random
148
error for individual i and measurement j and is assumed to be normally distributed
149
ε~N(0,σ2).
150
An exponential variance model was used to describe the variability of PK parameters
151
across individuals as follows:
152
Pi = θκ exp (ηκi)
153
where Pi is the estimated parameter value for subject i, θκ is the typical population value
154
of parameter k, ηki is subject specific deviation from population mean for individual i and
155
parameter k and assumed to follow normal distribution η~N(0, ω2).
156 157
Ureaplasma culture, antibiotic susceptibility testing, and real-time PCR
158
Upon enrollment, 2 tracheal aspirate samples at least 2h apart and one nasopharyngeal
159
sample from intubated infants, or 2 nasopharyngeal samples at least 2h apart from non-
160
intubated infants were obtained pre-dose for Ureaplasma culture and PCR. Subsequent
161
samples were obtained at 2 and 4 or 5 days post last dose and 21 days postnatal age.
7
162
Each specimen was directly inoculated in 10B broth and frozen at – 80 o C for later
163
shipment to University of Alabama at Birmingham Diagnostic Mycoplasma laboratory for
164
10B broth and A8 agar quantitative culture, azithromycin susceptibility testing by the
165
10B broth microdilution method (18-20), and real-time PCR for detection and species
166
determination using species-specific primers(14, 15, 21). A culture was considered
167
negative if no growth was detected after 7 days incubation. A confirmed positive culture
168
was defined as a positive broth culture from either tracheal aspirate or nasopharyngeal
169
specimen confirmed by typical morphology on agar plates. All isolates were confirmed
170
and speciated by PCR. Since Ureaplasma is only transmitted vertically, neonates who
171
were positive by confirmed culture or PCR at any time point were considered a
172
colonized neonate. Ureaplasma eradication was defined as 3 negative cultures post-last
173
dose.
174 175
Tracheal aspirate cytokine analysis
176
Tracheal aspirates for cytokines were obtained at the same time points as the culture
177
specimens pre- and post-treatment and were separated by centrifugation into cell pellet
178
and supernatant fractions as previously described (14). The cytokines interleukin (IL)-
179
1ß, IL-6, IL-8, and IL-17 were measured in the supernatants using LuminexTM multi-
180
analyte immunoassay with reagents from Upstate Biotechnology. Cell pellet lysates
181
were assayed for myeloperoxidase (MPO) activity (indicator of neutrophil activation) as
182
previously described (22, 23).
183 184
Whole blood cytokines
8
185
Whole blood spots were collected on filter paper (50 µl) pre-dose, 4-5 d post-dose and
186
21d of age and immediately frozen at -80°C. The stored blood spots were eluted and
187
analyzed by the multiplex LuminexTM immunoassay for the same cytokines as the
188
tracheal aspirates, as previously described (24-26).
189 190
Clinical outcomes
191
All infants were monitored for safety including vital signs, frequency of apnea and
192
bradycardia, cardiac rhythm, pulse oximetry up to 8 h post dose, clinical laboratory
193
testing pre-dose and up to study day 28, and adverse events until hospital discharge.
194
Infants were assessed for morbidities associated with prematurity, concomitant
195
medications, hearing screen results, and adverse events until discharge or transfer.
196 197
For the BPD endpoint, infants who were receiving supplemental oxygen or positive
198
pressure support at 36 weeks postmenstrual age (PMA) were considered to have BPD.
199
For infants receiving supplemental oxygen by nasal cannula, the delivered fraction of
200
inspired oxygen concentration or “effective FiO2” (27) was calculated by the technique
201
described by Benaron et al. (28) that was used in the STOP-ROP trial (29) which is
202
based on weight, oxygen liter flow, and oxygen concentration. Study subjects who were
203
transferred or discharged at ≤35 weeks PMA on supplemental oxygen were considered
204
to have BPD.
205 206
Pulmonary Outcomes at Six Months Adjusted Age
9
207
Data were collected by structured parental interviews before discharge and follow-up
208
phone interview at 6 months adjusted age utilizing the validated Tucson Children’s
209
Respiratory Study questionnaires that were designed to elicit a complete history of
210
possible covariates such as family history of asthma or atopy, and important
211
environmental (e.g., smoking) and infectious exposures and detailed interval respiratory
212
health history (30, 31). Recurrent wheezing and chronic cough were defined as
213
episodes occurring more than twice per week. The primary clinical outcome was
214
occurrence of hospitalization for respiratory illnesses and secondary outcomes included
215
the need for home supplemental oxygen, doctor and emergency room visits, parental
216
report of chronic cough and wheezing, and use of respiratory medications.
217 218
RESULTS
219
Subject Characteristics
220
As shown in Figure 1, seventy-one of 192 infants (63%) with a gestational age of 240 to
221
286 weeks gestation who were less than 72h age upon NICU admission were eligible for
222
the study. Parents of 43 infants were approached for consent, and 15 consented to the
223
study. Maternal macrolide exposure [N=36 (30%)] was the most common reason for
224
non-eligibility. All enrolled subjects received the 3 doses of intravenous 20 mg/kg
225
azithromycin and survived to BPD assessment at 36 wk PMA.
226 227
Seven subjects (47%) were culture and PCR positive for Ureaplasma spp. pre-dose. As
228
previously observed, U. parvum [N=5, (71%)] was more commonly isolated than U.
229
urealyticum [N=2, (29%)]. In 6 subjects with paired tracheal aspirate and
10
230
nasopharyngeal samples, 4 were culture-positive in both specimens and 2 were positive
231
in nasopharyngeal, but not tracheal aspirate specimens. In one Ureaplasma-positive
232
subject who was not intubated at the time of study entry, the NP sample was culture
233
and PCR positive. There were no positive cultures with 20 mg/kg dosing at any follow-
234
up time point, but one subject was PCR positive 4-5d post-last dose. The baseline
235
characteristics and clinical outcomes of the study sample stratified by Ureaplasma
236
status pre-treatment are presented in Tables 1 and 2 respectively. The Ureaplasma-
237
positive and negative subjects were similar for birthweight, gestational age, and
238
respiratory support at study entry.
239 240
Four of 15 (26%) infants developed BPD at 36 weeks PMA (Table 2). Three of seven
241
(43%) Ureaplasma-positive subjects developed BPD compared to one of eight (12.5%)
242
Ureaplasma-negative infants (p NS by Fisher Exact test). There was no difference by
243
Ureaplasma culture status in duration of mechanical ventilation support or supplemental
244
oxygen. One Ureaplasma-positive and one Ureaplasma-negative infant were
245
discharged home on supplemental oxygen. Fourteen subjects survived to discharge and
246
one Ureaplasma-positive subject died at four months of age due to persistent
247
pulmonary hypertension as a complication of BPD.
248 249 250 251
Pharmacokinetic analysis Plasma samples (N=239) from 40 subjects (including the 2 single dose studies and
252
current multiple dose study) were included in the PK analysis to further refine our
253
previously reported population model. The disposition of azithromycin was biphasic
254
(Figure 3A) suggesting that the pharmacokinetics of azithromycin could follow a two
11
255
compartment model and since the magnitude of many body processes [e.g., Clearance
256
(CL) or Volume of distribution (V)] may change in a regular fashion as the magnitude of
257
a certain covariate changes (e.g., body weight), we allometrically scaled PK parameters
258
on body weight during model development. Indeed, the inclusion of body weight as a
259
covariate improved the model fit to the data and explained some of the inter-subject
260
variability as was the case in our previous reports (14, 15) and the final model was a 2-
261
compartment model with all PK parameters allometrically scaled on body weight (Table
262
3). Inclusion of other covariates and accounting for inter-occasion variability did not
263
further reduce the inter-subject variability for any of the parameters and resulted in no
264
significant drop in the objective function value, therefore they were not included in the
265
final model. Goodness-of-fit plots for the final population PK model indicated that the
266
model described the data well without systematic bias (Figure 2). Based on the final
267
model parameters, the elimination half-life (t1/2) was estimated to be 69 hours in a
268
typical neonate weighing 1 kg.
269 270
Azithromycin MICs were determined using Clinical and Laboratory Standards Institute
271
(CLSI) guidelines (18) for 65 non-duplicate ureaplasma isolates (22 isolates from the
272
three azithromycin study cohorts and 43 isolates from banked specimens from preterm
273
infants (20)). The MIC50 and MIC90 were 2 and 8 µg/mL, respectively. The MIC range for
274
isolates from the current cohort was 1-8 µg/mL. No azithromycin resistance (MIC ≥16
275
µg/mL) as designated by the CLSI (18) was detected among the isolates from study
276
participants or banked specimens. Following three doses of 20 mg/kg intravenous
277
administration, azithromycin observed plasma concentrations were mostly maintained
12
278
between MIC50 (2 µg/ml) and MIC90 (8 µg/ml) for ~120 hr post first dose (Figure 3A),
279
consistent with our previously reported simulation analysis (15). The estimated
280
AUC24/MIC90 value was 4 hr. Simulation analysis indicated that the model generally
281
captured the trend in azithromycin disposition in preterm neonates, but it under-
282
predicted some concentrations (Figure 3B).
283 284
Azithromycin Effects on Indices of Inflammation
285
We analyzed 4 cytokines previously associated with Ureaplasma respiratory tract
286
colonization and BPD (IL-1ß, IL-6, and IL-8) (32, 33) and BPD (IL-17A)(26). In the 7
287
subjects (4 Ureaplasma-negative and 3 Ureaplasma-positive) with paired pre- and post-
288
dose tracheal aspirates, there was a decrease in IL-1ß, IL-8, IL-6, and IL-17A two days
289
post-last dose, but no difference between baseline and post-4 days concentrations
290
except for IL-17A (Tables 4 and 5). These differences were only significant for IL-17A.
291
There were no significant differences for cytokines measured in blood samples (data not
292
shown). Myeloperoxidase activity in tracheal aspirate cell pellets was also not
293
influenced by azithromycin treatment.
294 295
Safety and Serious Adverse Events
296
All subjects were monitored for adverse events until discharge from the NICU. There
297
were no abnormal vital signs or arrhythmias noted during or within 4 hours of drug
298
administration nor episodes of feeding intolerance. There were no abnormal laboratory
299
values attributable to study drug. A 25 week gestation Ureaplasma-negative infant
300
experienced a spontaneous intestinal perforation three days after the third study drug
13
301
dose and subsequently developed necrotizing enterocolitis Bell Stage 2. There were
302
three subjects with intraventricular hemorrhage Grade ≥3, one of whom also had
303
periventricular leukomalacia noted on a head ultrasound obtained on day 3 of life and
304
subsequently developed post-hemorrhagic hydrocephalus requiring ventriculoperitoneal
305
shunt placement.
306 307
Six Months Adjusted Age Pulmonary Outcomes. None of the 14 survivors were
308
hospitalized for respiratory illnesses between NICU discharge and six months adjusted
309
age (Table 6). One Ureaplasma negative subject remained on supplemental oxygen at
310
6 months adjusted age. There were no differences in reports of doctor/ER visits for
311
cough or wheezing, or respiratory medication use.
312 313
DISCUSSION
314
The PK properties of azithromycin support short courses of therapy. The drug has a
315
relatively long elimination half-life, is concentrated in phagocytic cells with good tissue
316
penetration, especially in the lung, and persists in tissues for much longer than in
317
plasma (34). Therefore, in this study we tested a 3 day course of 20 mg/kg intravenous
318
dose of azithromycin in preterm neonates to evaluate its effectiveness in eradicating
319
Ureaplasma, to determine its safety in this population, and to further refine the
320
population PK model of azithromycin in extremely preterm infants at risk for Ureaplasma
321
respiratory tract colonization and BPD. This dosage regimen showed preliminary
322
efficacy to eradicate ureaplasma and safety, and the model PK parameters were
323
estimated with high precision. The developed model was based on data from our
14
324
previously reported 2 single dose studies (14, 15) and the current multiple dose.
325
Allometric scaling of all four PK parameters (CL, V1, Q and V2) on body weight resulted
326
in significant model improvement and the PK parameter estimates were comparable to
327
our previously reported model (15). Overall, the model fit the data well as indicated by
328
the goodness-of-fit plots (Figures 2 and 3A), significant drop of the objective function
329
value and precision of parameter estimates (Table 3). To evaluate the final model and
330
determine its predictive accuracy we performed simulation analysis of the current study
331
with 200 replicates (n=15 subjects/each). Visual Predictive Check (Figure 3B) indicated
332
that the model is generally capable of predicting azithromycin concentrations in preterm
333
neonates, however, it under-predicted some concentrations that could be due to
334
random, unaccounted for inter-study variability. The model will be further refined and
335
inter-study variability can be better explained as data from more subjects become
336
available from current and future clinical studies.
337 338
Pharmacodynamic studies suggest that the AUC24/MIC90 ratio is most predictive
339
parameter of azithromycin efficacy (35). Results of experimental infections in animal
340
models suggest that increasing the dose early in the infectious process will result in
341
clearance of even marginally susceptible organisms (36). Although optimal AUC24/MIC90
342
ratios for azithromycin to eradicate other common organisms were reported to range
343
from 0.5 to 14.8 h (37), the optimal AUC24/MIC90 for azithromycin to effectively eradicate
344
Ureaplasma is unknown. In the prior 10 mg/kg and 20 mg/kg single dose studies, the
345
MIC90 of 4 µg/mL based on previously published reference data (38) was used to
346
calculate the AUC24/MIC90. For the current study, we based the MIC90 of 8 µg/ml on
15
347
azithromycin susceptibility testing of Ureaplasma isolates from study participants and
348
banked specimens from preterm infants. Our results indicated that an AUC24/MIC90 of >
349
4 hr would be effective to eradicate Ureaplasma. Since the activity of macrolides in vitro
350
is affected by pH of the medium (39, 40), the higher azithromycin MICs observed in
351
some isolates from the study cohorts as well as banked isolates may be due, in part, to
352
the acidic pH (6.0) of the 10B broth that is necessary for growth of the organisms in vitro
353
and measurement of the MIC endpoint. Therefore, at physiologic pH, the MIC may be 2-
354
4 dilutions lower, resulting in a higher AUC/MIC ratio. Furthermore, experimental
355
evidence in animal models and adult humans suggest that azithromycin concentrations
356
in alveolar macrophages in the lung will far exceed the Ureaplasma isolates MICs.
357
Following oral administration of 50 mg/kg azithormycin in rats, the AUC in alveolar
358
macrophages to AUC in plasma ratio was 648 (41). In human adults who received
359
either oral (42) or intravenous azithromcyin (43) for 5 days, the drug concentration was
360
increased more than 1500 fold in alveolar macrophages compared to plasma levels.
361 362
Based on the current study in preterm neonates, a dosage regimen of 3 intravenous
363
doses of 20 mg/kg/d azithromycin appeared to be safe and effectively eradicated
364
Ureaplasma from the respiratory tract in all culture-positive subjects. We suggest that
365
this azithromycin dosage regimen is appropriate for future randomized clinical trials to
366
eradicate Ureaplasma in preterm neonates.
367 368
Infants with BPD are at risk for adverse pulmonary outcomes during childhood. Up to
369
50% of BPD infants require re-hospitalization in the first year of life (44-47). Infants who
16
370
required home oxygen therapy are at highest risk for re-hospitalizations (45, 48).
371
Respiratory symptoms, primarily chronic cough and wheezing, and use of respiratory
372
medications (bronchodilators, steroids, oxygen) are more common in BPD than non-
373
BPD infants in early childhood. Maternal smoking during pregnancy and smoking in the
374
home also contribute to adverse respiratory outcomes in preterm infants (48).
375 376
Ureaplasma respiratory tract colonization has been proposed as an etiologic factor in
377
reactive airway disease in young infants. Wheezing in infants and children less than 3
378
years of age has been associated with isolation of Ureaplasma from the upper
379
respiratory tract (49). In a large study of almost 3000 women and their offspring in
380
Sweden, maternal vaginal colonization with Ureaplasma during pregnancy was
381
associated with a 2-fold increased risk for hospitalizations for asthma in the offspring
382
during the first 3 years of life (50). In preliminary data, we observed that 50% of
383
Ureaplasma-respiratory tract colonized infants compared to 27% of non-colonized
384
infants were re-hospitalized primarily for respiratory illnesses in the first year of life
385
(unpublished data). In the current study, none of the 14 survivors who had been treated
386
with the 3 day course of azithromycin were hospitalized during the first 6 months
387
(adjusted age) for respiratory illnesses.
388 389
Assessments of pulmonary outcomes beyond the neonatal period are important to
390
determine the larger impact of neonatal interventions to prevent BPD on lung health.
391
Treatment with intratracheal recombinant human CuZn superoxide dismutase at birth
392
did not reduce the rate of BPD at 28d, but did reduce the number of episodes of
17
393
wheezing, bronchodilator use, frequency of emergency room visits, and hospitalization
394
in treated infants in the first year of life compared to the placebo group (51). Exposure to
395
inhaled nitric oxide (iNO) between 7 and 21d of age in infants
1
Pharmacokinetics, Microbial Response, and Pulmonary Outcomes of Multi-dose
2
Intravenous Azithromycin in Preterm Infants at Risk for Ureaplasma Respiratory
3
Colonization
4
Running Title: Azithromycin 20 mg/kg multi-dose in preterm infants
5
L. Marcela Merchan, MD1*; Hazem E. Hassan, PhD3*; Michael L. Terrin, MD2; Ken B.
6
Waites, MD4; David A. Kaufman, MD5; Namasivayam Ambalavanan, MD4; Pamela
7
Donohue, ScD6; Susan J. Dulkerian, MD1; Robert Schelonka, MD7, Laurence S.
8
Magder, PhD2, Sagar Shukla, PharmD3, Natalie D. Eddington, PhD3; and Rose M.
9
Viscardi, MD1#.
10 1
11
Departments of Pediatrics, and 2Epidemiology and Preventive Medicine, University of
12
Maryland, Baltimore School of Medicine, Baltimore, MD; 3University of Maryland,
13
Baltimore School of Pharmacy, Baltimore, MD; 4Departments of Pathology and
14
Pediatrics, University of Alabama at Birmingham School of Medicine, Birmingham, AL;
15
5
Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, VA; 6
16
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore,
17
MD; and 7Department of Pediatrics, Oregon Health and Science University, Portland,
18
OR.
19 20
*
21
#
22
e-mail: [email protected]
23
L.L.M. and H.E.H. contributed equally to the work. Corresponding Author: Rose M. Viscardi, M.D
24
ABSTRACT
25
The study objective was to refine the population pharmacokinetics model, determine
26
microbial clearance, and assess short-term pulmonary outcomes of multiple dose
27
azithromycin treatment in preterm infants at risk for Ureaplasma respiratory colon-
28
ization. Fifteen subjects (Ureaplasma-positive=7) received intravenous azithromycin 20
29
mg/kg every 24h for 3 doses. Azithromycin concentrations were determined in plasma
30
samples obtained up to 168h post-first dose, by a validated LC/MS/MS method.
31
Respiratory samples were obtained pre-dose and at three time points post-last dose for
32
Ureaplasma culture, PCR, antibiotic susceptibility testing, and cytokine concentrations.
33
Pharmacokinetic data from these 15 subjects as well as 25 additional subjects [single
34
10 mg/kg dose (N=12), single 20 mg/kg dose (N=13)] were analyzed using a non-linear
35
mixed-effect population modeling (NONMEM) approach. Pulmonary outcomes were
36
assessed at 36 wk post-menstrual age and 6 months adjusted age. A 2-compartment
37
model with all PK parameters allometrically scaled on body weight best described the
38
azithromycin pharmacokinetics in preterm neonates. The population pharmacokinetic
39
parameters’ estimates for clearance, central volume of distribution, inter-compartmental
40
clearance, and peripheral volume of distribution were 0.15 L/h x WT(kg)0.75, 1.88 L x
41
WT(kg), 1.79 L/h x WT(kg)0.75 and 13 L x WT(kg), respectively. The estimated
42
AUC24/MIC90 was ~4 hr. All post-treatment cultures were negative and there were no
43
drug-related adverse events. One Ureaplasma-positive infant died at 4 months of age,
44
but no survivors were hospitalized for respiratory etiologies during the first 6 months
45
adjusted age. A 3 day course of 20 mg/kg/day intravenous azithromycin shows
46
preliminary efficacy in eradicating Ureaplasma spp. from the preterm respiratory tract.
2
47
INTRODUCTION
48
Bronchopulmonary dysplasia (BPD) is the major pulmonary morbidity in infants born
49
preterm characterized by arrested alveolar development and chronic inflammation.
50
Studies of human infants and experimental animal models indicate that the central
51
event in BPD pathogenesis is the interruption of normal developmental signaling during
52
early stages of lung development by lung injury that may be initiated in utero by
53
intrauterine infection with a subsequent dysregulated inflammatory response (1-3). A
54
recent meta-analysis of 39 studies confirmed that respiratory tract colonization with the
55
genital mycoplasma species Ureaplasma parvum and Ureaplasma urealyticum
56
increases the risk for development of BPD in extremely low gestation infants (4). It has
57
not been established whether eradicating Ureaplasma spp. from the respiratory tract of
58
preterm infants prevents or attenuates Ureaplasma infection-mediated lung injury.
59 60
Azithromycin, an azalide antibiotic, has anti-inflammatory properties and antimicrobial
61
activity against Ureaplasma spp. in in vitro (5, 6) and in in vivo experimental models (7-
62
9). Although the efficacies of azithromycin and a related macrolide, clarithromycin, to
63
prevent BPD have been assessed in single-center studies of preterm infants (10-13),
64
the optimal dosing regimens for these antibiotics have not been determined in
65
pharmacokinetic and pharmacodynamic studies and the impact on long term pulmonary
66
and neurologic outcomes are unknown. Our first steps to address these questions has
67
been to conduct studies in the at-risk population to determine the optimal dose, safety,
68
and in vivo anti-infective efficacy of azithromycin in preparation for future phase III
69
randomized, placebo-controlled trials (14, 15). We previously characterized the
3
70
pharmacokinetics (PK) of a single dose of intravenous azithromycin (10 mg/kg and 20-
71
mg/kg) in preterm infants (14). We demonstrated both doses were safe in 24 to 28
72
weeks gestation mechanically ventilated infants, but the 20 mg/kg dose was more
73
effective in eradicating Ureaplasma from the respiratory tract (14,15). Neither dose
74
reduced the pulmonary inflammatory response. Simulation analysis using our previously
75
developed PK model suggested that a multiple-dose regimen of 20 mg/kg intravenous
76
azithromycin may be efficacious for microbial clearance as it could mostly maintain
77
azithromycin plasma levels above the MIC50 for at least 120 hr post I.V. infusion.
78 79
The current study of preterm infants treated with 3 doses of 20 mg/kg intravenous
80
azithromycin was designed to 1) determine the microbiological and short term
81
pulmonary outcomes of the multiple dose; 2) assess the safety of this regimen, and 3)
82
collect additional PK data to further refine the population PK model. We hypothesized
83
that intravenous azithromycin therapy will prevent BPD in Ureaplasma-colonized
84
preterm infants by accelerating pathogen clearance or down-regulating the pulmonary
85
inflammatory response.
86 87
MATERIALS AND METHODS
88
Subject Enrollment
89
We conducted a Phase IIa non-randomized, open-label, PK and safety study of
90
intravenous azithromycin 20 mg/kg every 24h x 3d in preterm infants who were at high
91
risk for Ureaplasma respiratory tract colonization and the development of BPD (FDA
92
IND78990). Study subjects were recruited from six clinical sites from December 2011 to
4
93
June 2012. The institutional review board of each institution approved the study, and
94
parental informed consent was obtained. Inclusion criteria were: 1) gestational age 240-
95
286 weeks; 2) appropriate size for gestational age; 3) 1.5 mg/dL);
103
8) corrected QT interval (QTc) ≥450 ms; 9) neonatal exposure to any other systemic
104
macrolide antibiotic; 10) clinically suspected Ureaplasma CNS infection or other
105
confirmed bacterial or viral infection; and 11) participation in other clinical trials involving
106
investigational products.
107 108
Drug administration and blood sampling
109
After baseline laboratory tests and respiratory specimens were obtained, study infants
110
received azithromycin 20 mg/kg administered at a concentration of 2 mg/ml by
111
intravenous infusion over 60 min within 24 h of enrollment. A total of 3 doses were
112
administered at 24 ± 0.5h intervals. Six blood samples (0.25 ml each) were obtained
113
from each subject during specified time periods post- first dose infusion and processed
114
as previously described (15). Optimum sampling periods were predicted using the PK
115
software ADAPT5 (Biomedical Simulations Resource (BMSR), U. Southern California,
5
116
Los Angeles, CA). Samples were collected anywhere between 1-2, 2-4, 6-8, 25-48, 49-
117
96 and 120-168 hr post first dose. Specifying windows for withdrawing plasma samples
118
helps to better characterize the disposition profile and is a more clinically practical
119
approach (14-17). Levels of azithromycin in plasma were measured using a validated
120
high-performance liquid chromatography-tandem mass spectroscopy (HPLC/MS/MS)
121
detection method as previously described (14, 15).
122 123
Pharmacokinetic data analysis
124
The plasma concentration vs. time data from the a) single 10 mg/kg (14), b) single 20
125
mg/kg (15) and c) multiple 20 mg/kg studies were compiled and analyzed
126
simultaneously using the nonlinear mixed-effects modeling software, NONMEM 7.2
127
(ICON, MD). The first-order conditional estimation procedure (FOCE) with interaction
128
was used for the analysis. Upon development of the base model (two compartment with
129
proportional residual error model), covariate-parameter relationships were explored.
130
The available covariates were weight (WT), gestational age (GA), sex, height, and body
131
surface area.
132 133
The likelihood ratio test was applied to discriminate the alternative nested models where
134
a drop of the objective function value by at least 3.84 points was necessary to declare
135
model improvement, data significance level of P = 0.05, and one degree of freedom.
136
Inter-occasion variability was also estimated where study one (single 10 mg/kg
137
azithromycin), study two (single 20 mg/kg azithromycin) and study three (multiple 20
138
mg/kg azithromycin) were considered as first-, second- and third-occasion, respectively. 6
139
The final model that best fit the data was a two-compartment structural model (ADVAN3
140
TRANS4 NONMEM subroutine) with all parameters allometrically scaled on body weight
141
with a fixed exponent of 0.75 for both Cl and Q and a fixed exponent of 1 for both V1
142
and V2. Post-hoc Bayesian estimates of the individual subjects’ PK parameters were
143
calculated.
144
A proportional error model was used to describe the residual error as follows:
145
Yij obs = Yij pred*(1 + εij)
146
where Yij obs is the observed azithromycin plasma concentration j in subject I, Yij pred is
147
the model-predicted azithromycin plasma concentration, and ε is a residual random
148
error for individual i and measurement j and is assumed to be normally distributed
149
ε~N(0,σ2).
150
An exponential variance model was used to describe the variability of PK parameters
151
across individuals as follows:
152
Pi = θκ exp (ηκi)
153
where Pi is the estimated parameter value for subject i, θκ is the typical population value
154
of parameter k, ηki is subject specific deviation from population mean for individual i and
155
parameter k and assumed to follow normal distribution η~N(0, ω2).
156 157
Ureaplasma culture, antibiotic susceptibility testing, and real-time PCR
158
Upon enrollment, 2 tracheal aspirate samples at least 2h apart and one nasopharyngeal
159
sample from intubated infants, or 2 nasopharyngeal samples at least 2h apart from non-
160
intubated infants were obtained pre-dose for Ureaplasma culture and PCR. Subsequent
161
samples were obtained at 2 and 4 or 5 days post last dose and 21 days postnatal age.
7
162
Each specimen was directly inoculated in 10B broth and frozen at – 80 o C for later
163
shipment to University of Alabama at Birmingham Diagnostic Mycoplasma laboratory for
164
10B broth and A8 agar quantitative culture, azithromycin susceptibility testing by the
165
10B broth microdilution method (18-20), and real-time PCR for detection and species
166
determination using species-specific primers(14, 15, 21). A culture was considered
167
negative if no growth was detected after 7 days incubation. A confirmed positive culture
168
was defined as a positive broth culture from either tracheal aspirate or nasopharyngeal
169
specimen confirmed by typical morphology on agar plates. All isolates were confirmed
170
and speciated by PCR. Since Ureaplasma is only transmitted vertically, neonates who
171
were positive by confirmed culture or PCR at any time point were considered a
172
colonized neonate. Ureaplasma eradication was defined as 3 negative cultures post-last
173
dose.
174 175
Tracheal aspirate cytokine analysis
176
Tracheal aspirates for cytokines were obtained at the same time points as the culture
177
specimens pre- and post-treatment and were separated by centrifugation into cell pellet
178
and supernatant fractions as previously described (14). The cytokines interleukin (IL)-
179
1ß, IL-6, IL-8, and IL-17 were measured in the supernatants using LuminexTM multi-
180
analyte immunoassay with reagents from Upstate Biotechnology. Cell pellet lysates
181
were assayed for myeloperoxidase (MPO) activity (indicator of neutrophil activation) as
182
previously described (22, 23).
183 184
Whole blood cytokines
8
185
Whole blood spots were collected on filter paper (50 µl) pre-dose, 4-5 d post-dose and
186
21d of age and immediately frozen at -80°C. The stored blood spots were eluted and
187
analyzed by the multiplex LuminexTM immunoassay for the same cytokines as the
188
tracheal aspirates, as previously described (24-26).
189 190
Clinical outcomes
191
All infants were monitored for safety including vital signs, frequency of apnea and
192
bradycardia, cardiac rhythm, pulse oximetry up to 8 h post dose, clinical laboratory
193
testing pre-dose and up to study day 28, and adverse events until hospital discharge.
194
Infants were assessed for morbidities associated with prematurity, concomitant
195
medications, hearing screen results, and adverse events until discharge or transfer.
196 197
For the BPD endpoint, infants who were receiving supplemental oxygen or positive
198
pressure support at 36 weeks postmenstrual age (PMA) were considered to have BPD.
199
For infants receiving supplemental oxygen by nasal cannula, the delivered fraction of
200
inspired oxygen concentration or “effective FiO2” (27) was calculated by the technique
201
described by Benaron et al. (28) that was used in the STOP-ROP trial (29) which is
202
based on weight, oxygen liter flow, and oxygen concentration. Study subjects who were
203
transferred or discharged at ≤35 weeks PMA on supplemental oxygen were considered
204
to have BPD.
205 206
Pulmonary Outcomes at Six Months Adjusted Age
9
207
Data were collected by structured parental interviews before discharge and follow-up
208
phone interview at 6 months adjusted age utilizing the validated Tucson Children’s
209
Respiratory Study questionnaires that were designed to elicit a complete history of
210
possible covariates such as family history of asthma or atopy, and important
211
environmental (e.g., smoking) and infectious exposures and detailed interval respiratory
212
health history (30, 31). Recurrent wheezing and chronic cough were defined as
213
episodes occurring more than twice per week. The primary clinical outcome was
214
occurrence of hospitalization for respiratory illnesses and secondary outcomes included
215
the need for home supplemental oxygen, doctor and emergency room visits, parental
216
report of chronic cough and wheezing, and use of respiratory medications.
217 218
RESULTS
219
Subject Characteristics
220
As shown in Figure 1, seventy-one of 192 infants (63%) with a gestational age of 240 to
221
286 weeks gestation who were less than 72h age upon NICU admission were eligible for
222
the study. Parents of 43 infants were approached for consent, and 15 consented to the
223
study. Maternal macrolide exposure [N=36 (30%)] was the most common reason for
224
non-eligibility. All enrolled subjects received the 3 doses of intravenous 20 mg/kg
225
azithromycin and survived to BPD assessment at 36 wk PMA.
226 227
Seven subjects (47%) were culture and PCR positive for Ureaplasma spp. pre-dose. As
228
previously observed, U. parvum [N=5, (71%)] was more commonly isolated than U.
229
urealyticum [N=2, (29%)]. In 6 subjects with paired tracheal aspirate and
10
230
nasopharyngeal samples, 4 were culture-positive in both specimens and 2 were positive
231
in nasopharyngeal, but not tracheal aspirate specimens. In one Ureaplasma-positive
232
subject who was not intubated at the time of study entry, the NP sample was culture
233
and PCR positive. There were no positive cultures with 20 mg/kg dosing at any follow-
234
up time point, but one subject was PCR positive 4-5d post-last dose. The baseline
235
characteristics and clinical outcomes of the study sample stratified by Ureaplasma
236
status pre-treatment are presented in Tables 1 and 2 respectively. The Ureaplasma-
237
positive and negative subjects were similar for birthweight, gestational age, and
238
respiratory support at study entry.
239 240
Four of 15 (26%) infants developed BPD at 36 weeks PMA (Table 2). Three of seven
241
(43%) Ureaplasma-positive subjects developed BPD compared to one of eight (12.5%)
242
Ureaplasma-negative infants (p NS by Fisher Exact test). There was no difference by
243
Ureaplasma culture status in duration of mechanical ventilation support or supplemental
244
oxygen. One Ureaplasma-positive and one Ureaplasma-negative infant were
245
discharged home on supplemental oxygen. Fourteen subjects survived to discharge and
246
one Ureaplasma-positive subject died at four months of age due to persistent
247
pulmonary hypertension as a complication of BPD.
248 249 250 251
Pharmacokinetic analysis Plasma samples (N=239) from 40 subjects (including the 2 single dose studies and
252
current multiple dose study) were included in the PK analysis to further refine our
253
previously reported population model. The disposition of azithromycin was biphasic
254
(Figure 3A) suggesting that the pharmacokinetics of azithromycin could follow a two
11
255
compartment model and since the magnitude of many body processes [e.g., Clearance
256
(CL) or Volume of distribution (V)] may change in a regular fashion as the magnitude of
257
a certain covariate changes (e.g., body weight), we allometrically scaled PK parameters
258
on body weight during model development. Indeed, the inclusion of body weight as a
259
covariate improved the model fit to the data and explained some of the inter-subject
260
variability as was the case in our previous reports (14, 15) and the final model was a 2-
261
compartment model with all PK parameters allometrically scaled on body weight (Table
262
3). Inclusion of other covariates and accounting for inter-occasion variability did not
263
further reduce the inter-subject variability for any of the parameters and resulted in no
264
significant drop in the objective function value, therefore they were not included in the
265
final model. Goodness-of-fit plots for the final population PK model indicated that the
266
model described the data well without systematic bias (Figure 2). Based on the final
267
model parameters, the elimination half-life (t1/2) was estimated to be 69 hours in a
268
typical neonate weighing 1 kg.
269 270
Azithromycin MICs were determined using Clinical and Laboratory Standards Institute
271
(CLSI) guidelines (18) for 65 non-duplicate ureaplasma isolates (22 isolates from the
272
three azithromycin study cohorts and 43 isolates from banked specimens from preterm
273
infants (20)). The MIC50 and MIC90 were 2 and 8 µg/mL, respectively. The MIC range for
274
isolates from the current cohort was 1-8 µg/mL. No azithromycin resistance (MIC ≥16
275
µg/mL) as designated by the CLSI (18) was detected among the isolates from study
276
participants or banked specimens. Following three doses of 20 mg/kg intravenous
277
administration, azithromycin observed plasma concentrations were mostly maintained
12
278
between MIC50 (2 µg/ml) and MIC90 (8 µg/ml) for ~120 hr post first dose (Figure 3A),
279
consistent with our previously reported simulation analysis (15). The estimated
280
AUC24/MIC90 value was 4 hr. Simulation analysis indicated that the model generally
281
captured the trend in azithromycin disposition in preterm neonates, but it under-
282
predicted some concentrations (Figure 3B).
283 284
Azithromycin Effects on Indices of Inflammation
285
We analyzed 4 cytokines previously associated with Ureaplasma respiratory tract
286
colonization and BPD (IL-1ß, IL-6, and IL-8) (32, 33) and BPD (IL-17A)(26). In the 7
287
subjects (4 Ureaplasma-negative and 3 Ureaplasma-positive) with paired pre- and post-
288
dose tracheal aspirates, there was a decrease in IL-1ß, IL-8, IL-6, and IL-17A two days
289
post-last dose, but no difference between baseline and post-4 days concentrations
290
except for IL-17A (Tables 4 and 5). These differences were only significant for IL-17A.
291
There were no significant differences for cytokines measured in blood samples (data not
292
shown). Myeloperoxidase activity in tracheal aspirate cell pellets was also not
293
influenced by azithromycin treatment.
294 295
Safety and Serious Adverse Events
296
All subjects were monitored for adverse events until discharge from the NICU. There
297
were no abnormal vital signs or arrhythmias noted during or within 4 hours of drug
298
administration nor episodes of feeding intolerance. There were no abnormal laboratory
299
values attributable to study drug. A 25 week gestation Ureaplasma-negative infant
300
experienced a spontaneous intestinal perforation three days after the third study drug
13
301
dose and subsequently developed necrotizing enterocolitis Bell Stage 2. There were
302
three subjects with intraventricular hemorrhage Grade ≥3, one of whom also had
303
periventricular leukomalacia noted on a head ultrasound obtained on day 3 of life and
304
subsequently developed post-hemorrhagic hydrocephalus requiring ventriculoperitoneal
305
shunt placement.
306 307
Six Months Adjusted Age Pulmonary Outcomes. None of the 14 survivors were
308
hospitalized for respiratory illnesses between NICU discharge and six months adjusted
309
age (Table 6). One Ureaplasma negative subject remained on supplemental oxygen at
310
6 months adjusted age. There were no differences in reports of doctor/ER visits for
311
cough or wheezing, or respiratory medication use.
312 313
DISCUSSION
314
The PK properties of azithromycin support short courses of therapy. The drug has a
315
relatively long elimination half-life, is concentrated in phagocytic cells with good tissue
316
penetration, especially in the lung, and persists in tissues for much longer than in
317
plasma (34). Therefore, in this study we tested a 3 day course of 20 mg/kg intravenous
318
dose of azithromycin in preterm neonates to evaluate its effectiveness in eradicating
319
Ureaplasma, to determine its safety in this population, and to further refine the
320
population PK model of azithromycin in extremely preterm infants at risk for Ureaplasma
321
respiratory tract colonization and BPD. This dosage regimen showed preliminary
322
efficacy to eradicate ureaplasma and safety, and the model PK parameters were
323
estimated with high precision. The developed model was based on data from our
14
324
previously reported 2 single dose studies (14, 15) and the current multiple dose.
325
Allometric scaling of all four PK parameters (CL, V1, Q and V2) on body weight resulted
326
in significant model improvement and the PK parameter estimates were comparable to
327
our previously reported model (15). Overall, the model fit the data well as indicated by
328
the goodness-of-fit plots (Figures 2 and 3A), significant drop of the objective function
329
value and precision of parameter estimates (Table 3). To evaluate the final model and
330
determine its predictive accuracy we performed simulation analysis of the current study
331
with 200 replicates (n=15 subjects/each). Visual Predictive Check (Figure 3B) indicated
332
that the model is generally capable of predicting azithromycin concentrations in preterm
333
neonates, however, it under-predicted some concentrations that could be due to
334
random, unaccounted for inter-study variability. The model will be further refined and
335
inter-study variability can be better explained as data from more subjects become
336
available from current and future clinical studies.
337 338
Pharmacodynamic studies suggest that the AUC24/MIC90 ratio is most predictive
339
parameter of azithromycin efficacy (35). Results of experimental infections in animal
340
models suggest that increasing the dose early in the infectious process will result in
341
clearance of even marginally susceptible organisms (36). Although optimal AUC24/MIC90
342
ratios for azithromycin to eradicate other common organisms were reported to range
343
from 0.5 to 14.8 h (37), the optimal AUC24/MIC90 for azithromycin to effectively eradicate
344
Ureaplasma is unknown. In the prior 10 mg/kg and 20 mg/kg single dose studies, the
345
MIC90 of 4 µg/mL based on previously published reference data (38) was used to
346
calculate the AUC24/MIC90. For the current study, we based the MIC90 of 8 µg/ml on
15
347
azithromycin susceptibility testing of Ureaplasma isolates from study participants and
348
banked specimens from preterm infants. Our results indicated that an AUC24/MIC90 of >
349
4 hr would be effective to eradicate Ureaplasma. Since the activity of macrolides in vitro
350
is affected by pH of the medium (39, 40), the higher azithromycin MICs observed in
351
some isolates from the study cohorts as well as banked isolates may be due, in part, to
352
the acidic pH (6.0) of the 10B broth that is necessary for growth of the organisms in vitro
353
and measurement of the MIC endpoint. Therefore, at physiologic pH, the MIC may be 2-
354
4 dilutions lower, resulting in a higher AUC/MIC ratio. Furthermore, experimental
355
evidence in animal models and adult humans suggest that azithromycin concentrations
356
in alveolar macrophages in the lung will far exceed the Ureaplasma isolates MICs.
357
Following oral administration of 50 mg/kg azithormycin in rats, the AUC in alveolar
358
macrophages to AUC in plasma ratio was 648 (41). In human adults who received
359
either oral (42) or intravenous azithromcyin (43) for 5 days, the drug concentration was
360
increased more than 1500 fold in alveolar macrophages compared to plasma levels.
361 362
Based on the current study in preterm neonates, a dosage regimen of 3 intravenous
363
doses of 20 mg/kg/d azithromycin appeared to be safe and effectively eradicated
364
Ureaplasma from the respiratory tract in all culture-positive subjects. We suggest that
365
this azithromycin dosage regimen is appropriate for future randomized clinical trials to
366
eradicate Ureaplasma in preterm neonates.
367 368
Infants with BPD are at risk for adverse pulmonary outcomes during childhood. Up to
369
50% of BPD infants require re-hospitalization in the first year of life (44-47). Infants who
16
370
required home oxygen therapy are at highest risk for re-hospitalizations (45, 48).
371
Respiratory symptoms, primarily chronic cough and wheezing, and use of respiratory
372
medications (bronchodilators, steroids, oxygen) are more common in BPD than non-
373
BPD infants in early childhood. Maternal smoking during pregnancy and smoking in the
374
home also contribute to adverse respiratory outcomes in preterm infants (48).
375 376
Ureaplasma respiratory tract colonization has been proposed as an etiologic factor in
377
reactive airway disease in young infants. Wheezing in infants and children less than 3
378
years of age has been associated with isolation of Ureaplasma from the upper
379
respiratory tract (49). In a large study of almost 3000 women and their offspring in
380
Sweden, maternal vaginal colonization with Ureaplasma during pregnancy was
381
associated with a 2-fold increased risk for hospitalizations for asthma in the offspring
382
during the first 3 years of life (50). In preliminary data, we observed that 50% of
383
Ureaplasma-respiratory tract colonized infants compared to 27% of non-colonized
384
infants were re-hospitalized primarily for respiratory illnesses in the first year of life
385
(unpublished data). In the current study, none of the 14 survivors who had been treated
386
with the 3 day course of azithromycin were hospitalized during the first 6 months
387
(adjusted age) for respiratory illnesses.
388 389
Assessments of pulmonary outcomes beyond the neonatal period are important to
390
determine the larger impact of neonatal interventions to prevent BPD on lung health.
391
Treatment with intratracheal recombinant human CuZn superoxide dismutase at birth
392
did not reduce the rate of BPD at 28d, but did reduce the number of episodes of
17
393
wheezing, bronchodilator use, frequency of emergency room visits, and hospitalization
394
in treated infants in the first year of life compared to the placebo group (51). Exposure to
395
inhaled nitric oxide (iNO) between 7 and 21d of age in infants
