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Antimicrobial Agents and Chemotherapy, July 2000, p. 1974-1976, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Direct Measurement of the Anti-Influenza Agent
Zanamivir in the Respiratory Tract following Inhalation
Amy W.
Peng,1,*
Stefano
Milleri,2 and
Daniel S.
Stein1
Clinical Pharmacology Department, Glaxo
Wellcome Inc., Research Triangle Park, North Carolina
27709,1 and Glaxo Wellcome
S.p.A., Medicine Research Centre, Verona, Italy2
Received 7 October 1999/Returned for modification 23 January
2000/Accepted 5 April 2000
 |
ABSTRACT |
In a single-center, randomized study, zanamivir (Relenza)
concentrations in induced sputum samples and nasal washings of healthy adults following oral inhalation were measured. Concentrations in
sputum exceeded the median viral neuraminidase 50% inhibitory concentration at 6, 12, and 24 h, and those in nasal washings did
so at 6 and 12 h. There were no zanamivir-related adverse events
or laboratory abnormalities.
| |
TEXT |
Influenza virus is highly infectious
and can cause worldwide pandemics with significant morbidity and
mortality (3). A new strategy in the management of influenza
is the use of agents that inhibit the action of neuraminidase, the
glycoprotein component of influenza virus that is responsible for
liberating new virus particles from infected airway cells
(4). The first of these neuraminidase inhibitors approved
for influenza treatment was zanamivir (Relenza) (14).
Zanamivir is not metabolized and has a short plasma half-life (~2 h),
low protein binding (<10% of systemically circulating zanamivir), and
limited bioavailability (<20% following oral inhalation) (6). Twice-daily oral administration of 10 mg of zanamivir is effective in the treatment of naturally occurring influenza A and B
virus infections (8, 10). Recent clinical studies with
healthy adults showed that once-daily administration of 10 mg of
zanamivir prevents symptomatic, laboratory-confirmed influenza (11).
In order to provide data that further support the treatment and
prophylaxis dosing regimens for zanamivir, in the present study we
directly measured zanamivir concentrations in induced sputum and nasal
wash samples at 6, 12, and 24 h following a single, inhaled 10-mg
dose in male and female healthy subjects.
Methods and results.
A single-center, parallel group,
uncontrolled, open-label, randomized sampling-time design was used
(protocol NAI10902). All subjects provided written informed consent
prior to entry. Of 30 volunteers screened, 18 were enrolled to receive
treatment. The majority (10 of 12) of screening failures resulted from
poor quality or absence of induced sputum, as determined by a sputum induction test. Medical histories were recorded, and physical examinations and clinical laboratory tests were performed during screening.
Descriptive statistics, including the mean, standard deviation, median,
and range, were determined relative to sampling time. Statistical
analysis was performed to estimate the population mean zanamivir
concentration and its 95% confidence interval (CI) at each sampling
time by using an analysis-of-variance technique [log(zanamivir) versus
sampling time] (SAS Procedure Guide; SAS Institute, Cary, N.C.; 1999).
Subjects were randomized (six to each sampling group) to one of the
three postdose sampling time groups: 6, 12, or 24 h following
the
single zanamivir dose. Treatment groups were similar with
respect to
gender, age, weight, height, and body mass index (Table
1). Baseline FEV
1
measurements ranged from 91 to 133% of predicted
values (Table
1). All
laboratory values at screening were within
normal ranges, and there
were no significant changes in FEV
1 during
or after the
sputum induction procedure.
Subjects spent 1 to 2 days at the research study site for dosing and
sample collection. Subjects received one dose of 10 mg
(two 5-mg
blisters) of zanamivir via the Relenza Diskhaler, and
samples were
collected one time only at either 6, 12, or 24 h
postdosing.
Characteristics of the inhaler device and zanamivir
formulation have
been described previously (
5,
12). Safety
and tolerability
were monitored by using vital signs, clinical
laboratory assays, and
recorded adverse events during treatment
and follow-up.
At the specified sampling times (6, 12, or 24 h postdosing),
subjects underwent nasal washings (5 ml of hypertonic saline/nasal
opening) and sputum inductions according to the standard study
site
protocol. For sputum inductions, each subject inhaled hypertonic
saline
(3.5% [wt/vol]) via an ultrasonic nebulizer until 10 ml
of sputum
was produced. The quality of all sputum samples was
assessed by
counting cells in cytospin preparations according
to standard
procedures. All sputum samples met standards for acceptability
(<80%
squamous cells and >200 nonsquamous cells). Samples were
frozen at
20°C or lower until analysis. Blood samples were collected
for
determination of urea concentrations immediately after sputum
samples
were
collected.
The primary end point was the measurement of dilution-adjusted
zanamivir concentrations in sputum and nasal wash samples at
6, 12, and
24 h postdose in at least six evaluable individuals
per sampling
time. Liquid chromatography-tandem mass spectrometry
was used to detect
total zanamivir levels in sputum and nasal
wash samples (
1).
The analytical linear range (determined with
spiked sputum and nasal
washings) was 0.5 to 1,000 ng/ml, with
coefficients of variation of 3.8 to 11.7% over the linear range.
Urea concentrations in sputum, nasal
washings, and blood were
assayed with a high-resolution blood urea
nitrogen test (Sigma
BUN Reagent and Urea Standard Diluent)
(
13). Since recovered
sputum and nasal wash volumes differ
among individuals, all final
zanamivir concentrations were adjusted by
using a urea dilution
method to compare urea concentrations in body
fluid with those
in blood so that comparisons could be made between
subjects: dilution-adjusted
zanamivir concentration in sputum or nasal
washing = unadjusted
zanamivir concentration × (urea level
in blood/urea level in sputum
or nasal
washing).
Median (range) dilution-adjusted zanamivir concentrations at 6, 12, and
24 h postdosing were 1,336 (888 to 3,563), 304 (53
to 1,434), and
47 (16 to 258) ng/ml in sputum samples and 137
ng/ml (21 to 305), 122 ng/ml (below quantifiable levels to 212),
and below quantifiable levels
in nasal wash samples,
respectively.
The median (range) fold zanamivir concentrations above the median
influenza A and B virus neuraminidase 50% inhibitory concentration
(IC
50) (0.9 ng/ml [
16]) at 6, 12, and
24 h postdosing were 1,483
(985 to 3,958), 337 (58 to 1,593), and
52 (17 to 286) for sputum
samples and 151 (23 to 338), 135 (<5 to
234), and <5 for nasal
wash samples, respectively. The neuraminidase
IC
50 assay is the
most reproducible assay for predicting
the susceptibility of clinical
isolates to neuraminidase inhibitors
(
2).
The concentrations in sputum and nasal washings as a function of
sampling time are shown in Fig.
1A and B,
respectively. Zanamivir
concentrations detected in sputum and nasal
washings decreased
over time. Using the median
urea-adjusted zanamivir concentrations
at 6 and 12 h
postdose and assuming an essentially first-order
elimination of inhaled
zanamivir from the lungs up to 12 h postdose,
we determined
that the elimination half-life would be 2.8 h (elimination
rate constant = 0.2467 h
1).
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FIG. 1.
(A) Dilution-adjusted zanamivir concentrations in sputum
6, 12, and 24 h after oral inhalation of 10 mg of zanamivir
powder. There were six subjects for each time point. Model means (95%
Cls) at 6, 12, and 24 h 1,441 (569, 3,650), 235 (93, 594), and 58 (23, 148)are indicated by horizontal lines. (B) Dilution-adjusted
zanamivir concentrations in nasal washings 6, 12, and 24 h after
oral inhalation of 10 mg of zanamivir powder. There were six subjects
for each time point. Model means (95% Cls) at 6, 12, and 24 h106
(26, 435), 34 (8, 139), and below the quantification limitare
indicated by horizontal lines.
|
|
Within 2 days of zanamivir administration, symptom-directed physical
examinations, laboratory tests, and pregnancy tests for
female subjects
were performed. No drug-related adverse events
were reported, and no
laboratory tests showed clinically significant
abnormalities. Mild to
moderate headache occurred in five subjects
(one in the 6-h group and
two each in the 12- and 24-h groups).
No subjects withdrew from the
study.
Commentary.
Zanamivir is delivered directly to the primary
site of influenza virus infection and replication (the epithelial
lining of the respiratory tract) by use of a dry-powder inhaler
(Relenza Diskhaler), thereby minimizing systemic exposure. In the
present study, we directly measured zanamivir concentrations in sputum and nasal wash samples following a single 10-mg orally inhaled dose in
order to further substantiate the dosing regimen in the clinical use of
zanamivir. Zanamivir concentrations exceeded the neuraminidase
IC50 at all time points examined, except for nasal wash
samples at 24 h postdose. Therefore, these data provide support for a twice-daily dosing regimen of zanamivir in order to maintain effective concentrations throughout the respiratory tract during the treatment of subjects with active infection.
The development of viral resistance has been a limitation to therapy
with other antiviral agents (
15,
17). Resistance
to
zanamivir has so far not been found in samples collected from
clinical
trials (
2) or in animal models under conditions that
readily
select for variants resistant to amantadine and rimantadine
(
9). Achieving supermaximal inhibitory concentrations (above
the IC
50) at the site of viral replication is an effective
strategy
for preventing the emergence of viral resistance during
therapy,
since this substantially limits the percentage of the viral
population
that can replicate (
7). The high local
concentrations of zanamivir
maintained throughout the respiratory tract
following twice-daily
dosing may be responsible for the low level of
viral resistance
observed to date with
zanamivir.
This study also provides support for once-daily prophylaxis with
zanamivir, as pulmonary concentrations at least 17-fold higher
than the viral IC
50 were maintained throughout the entire
24-h
measurement period, in addition to significant zanamivir
concentrations
being found in nasal washings at 6 and 12 h
postdose. Thus, exposure
to the influenza virus in the nasal mucosa
during times when the
drug concentrations at this site are below the
IC
50 would not
result in pulmonary infection, as
concentrations of the drug in
the bronchi are maintained well above the
IC
50 throughout the
entire 24-h measurement
period.
A recent clinical prophylaxis study randomized subjects to receive
once-daily placebo or 10 mg of zanamivir by oral inhalation
for a
4-week period (
11). Zanamivir was 84% effective in
preventing
symptomatic laboratory-confirmed influenza with fever
present.
Interestingly, 79% of those infected in the zanamivir group
were
asymptomatic, compared to 56% in the placebo group. This finding
suggests that zanamivir prevents symptomatic disease as well as
infection, possibly due to the sustained pulmonary concentrations
after
inhalation, which would inhibit the spread of virus from
the upper
respiratory tract. Importantly, antibodies produced
in
asymptomatic infected individuals may be protective against
subsequent
infection with the same strain of influenza
virus.
A previous pharmacokinetic study indirectly estimated local
concentrations of zanamivir in the lungs within minutes after
a single
10-mg orally inhaled dose (
5). The zanamivir concentration
in the peripheral lung region within minutes postdose was estimated
to
be approximately 6,900 ng/ml, or 7,676-fold above the median
influenza virus neuraminidase IC
50 (0.9 ng/ml
[
16]). Since in
the present study we used a
direct method for the assessment of
respiratory tract zanamivir levels,
a comparison of the results
with those from the previous study
was made. The zanamivir concentration
minutes after a 10-mg inhaled
dose was calculated in the present
study to be 5,870 ng/ml (using
C0 =
Ct ×
ekt). Therefore, the indirect estimation of peripheral
lung concentration
minutes postdose (6,900 ng/ml) determined in the
previous scintigraphy
study (
5) appears to be a reasonable
method for the clinical
estimation of lung exposure with inhaled
medication.
In conclusion, this study demonstrates that zanamivir concentrations
significantly higher than the median viral neuraminidase
IC
50 are retained in the respiratory tract following a
single
10-mg zanamivir dose. Therefore, the sustained zanamivir
concentrations
in the respiratory tract following the recommended
dosing regimen
are sufficient to achieve effective inhibition of
influenza virus
replication.
| |
ACKNOWLEDGMENTS |
We thank Lisa Squassante for statistical analyses, Christine M. Grosse for sample analyses, and Patrice Ferriola for writing and
editing assistance.
This study was supported by Glaxo Wellcome Research and Development.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Pharmacology Department, Glaxo Wellcome Inc., 5 Moore Dr., Research
Triangle Park, NC 27709. Phone: (919) 483-5445. Fax: (919) 483-6380. E-mail: ap4872{at}glaxowellcome.com.
| |
REFERENCES |
| 1.
|
Allen, G.,
S. Brookes,
A. Barrow,
J. Dunn, and C. Grosse.
1999.
Liquid chromatographic-tandem mass spectrometric method for the determination of the neuraminidase inhibitor zanamivir (GG167) in human serum.
J. Chromatogr.
732:383-393[Medline].
|
| 2.
|
Barnett, J. M.,
A. Cadman,
D. Gor,
M. Dempsey,
M. Walters,
A. Candlin,
M. Tisdale,
P. J. Morley,
I. J. Owens,
R. J. Fenton,
A. P. Lewis,
E. C. Claas,
G. F. Rimmelzwaan,
R. DeGroot, and A. D. Osterhaus.
2000.
Zanamivir susceptibility monitoring and characterization of influenza virus clinical isolates obtained during phase II clinical efficacy studies.
Antimicrob. Agents Chemother.
44:78-87[Abstract/Free Full Text].
|
| 3.
|
Betts, R.
1995.
Influenza virus, p. 1546-1567.
In
G. Mandell, J. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases. Churchill Livingstone, New York, N.Y.
|
| 4.
|
Calfee, D. P., and F. G. Hayden.
1998.
New approaches to influenza chemotherapy. Neuraminidase inhibitors.
Drugs
56:537-553[CrossRef][Medline].
|
| 5.
|
Cass, L.,
J. Brown,
M. Pickford,
S. Fayinka,
S. Newman,
C. Johansson, and A. Bye.
1999.
Pharmacoscintigraphic evaluation of lung deposition of inhaled zanamivir in healthy volunteers.
Clin. Pharmacokinet.
36(Suppl. 1):21-31.
|
| 6.
|
Cass, L.,
C. Efthymiopoulos, and A. Bye.
1999.
Review of the pharmacokinetics of zanamivir after intravenous, oral, inhaled or intranasal dosing in healthy volunteers.
Clin. Pharmacokinet.
36:1-11.
|
| 7.
|
Drusano, G.,
J. Bilello,
D. Stein,
M. Nessly,
A. Meibohm,
E. Emini,
P. Deutsch,
J. Condra,
J. Chodakewitz, and D. Holder.
1998.
Factors influencing the emergence of resistance to indinavir: role of virologic, immunologic, and pharmacologic variables.
J. Infect. Dis.
178:360-367[Medline].
|
| 8.
|
Hayden, F. G.,
A. D. Osterhaus,
J. J. Treanor,
D. M. Fleming,
F. Y. Aoki,
K. G. Nicholson,
A. M. Bohnen,
H. M. Hirst,
O. Keene, and K. Wightman.
1997.
Efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza virus infections. GG167 Influenza Study Group.
N. Engl. J. Med.
337:874-880[Abstract/Free Full Text].
|
| 9.
|
Mendel, D., and R. Sidwell.
1998.
Influenza virus resistance to neuraminidase inhibitors.
Drug Resist. Updates
1:184-189[CrossRef][Medline].
|
| 10.
|
MIST,
K. Campion,
C. Silagy,
C. Cooper,
P. Bolton,
R. Watts,
T. Liaw,
K. Narayan, and F. Deloze.
1998.
Randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza A and B virus infections. The MIST (Management of Influenza in the Southern Hemisphere Trialists) Study Group.
Lancet
352:1877-1881[CrossRef][Medline].
|
| 11.
|
Monto, A.,
D. Robinson,
M. Herlocher,
J. Hinston,
M. Elliott, and A. Crisp.
1999.
Zanamivir in the prevention of influenza among healthy adults.
JAMA
282:31-35[Abstract/Free Full Text].
|
| 12.
|
Nielsen, K.,
L. Okamoto, and T. Shah.
1997.
Importance of selected inhaler characteristics and acceptance of a new breath-actuated powder inhalation device.
J. Asthma
34:249-253[Medline].
|
| 13.
|
Rennard, S.,
G. Basset,
D. Lecossier,
K. O'Donnell,
P. Pinkston,
P. Martin, and R. Crystal.
1986.
Estimation of volume of epithelial lining fluid recovered by lavage using urea as a marker of dilution.
J. Appl. Physiol.
60:532-538[Abstract/Free Full Text].
|
| 14.
|
von Itzstein, M.,
W. Y. Wu,
G. B. Kok,
M. S. Pegg,
J. C. Dyason,
B. Jin,
T. Van Phan,
M. L. Smythe,
H. F. White,
S. W. Oliver,
P. Colman,
J. Varghese,
D. Ryan,
J. Woods,
R. Bethell,
V. Hotham,
J. Cameron, and C. Penn.
1993.
Rational design of potent sialidase-based inhibitors of influenza virus replication.
Nature
363:418-423[CrossRef][Medline].
|
| 15.
|
Wade, R. C.
1997.
`Flu' and structure-based drug design.
Structure
5:1139-1145[Medline].
|
| 16.
|
Woods, J. M.,
R. C. Bethell,
J. A. Coates,
N. Healy,
S. A. Hiscox,
B. A. Pearson,
D. M. Ryan,
J. Ticehurst,
J. Tilling,
S. M. Walcott, and C. Penn.
1993.
4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro.
Antimicrob. Agents Chemother.
37:1473-1479[Abstract/Free Full Text].
|
| 17.
|
Zimmerman, R. K.,
F. L. Ruben, and E. R. Ahwesh.
1997.
Influenza, influenza vaccine and amantadine/rimantadine.
J. Fam. Pract.
45:107-122[Medline].
|
Antimicrobial Agents and Chemotherapy, July 2000, p. 1974-1976, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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