Key words
bioavailability - pharmacokinetics - pulmonary & respiratory pharmacology - absorption
- bioanalytical method - nasal spray - Fluticasone Furoate
Introduction
Fluticasone furoate is an enhanced-affinity intranasal corticosteroids (INCS)
approved for the treatment of allergic rhinitis (AR) in adults and children 2 years
of age and older [1]. It exhibits its
anti-inflammatory effect as an entire molecule and is therefore not a prodrug or a
salt [2]. The exact mechanism of action of
fluticasone furoate in treating AR is not full known, and may affect the early and
late phase inflammatory response [3]. It is
suggested that being a corticosteroid, it exhibits its anti-inflammatory effect on
multiple inflammatory cells (such as, mast cells, eosinophils, neutrophils,
macrophages, and lymphocytes) and mediators (such as, histamine, eicosanoids,
leukotrienes, and cytokines) [1]
[3]. Other theories suggest that fluticasone
furoate can suppress inflammatory gene activation through multiple mechanisms, such
as inhibition of pro-inflammatory transcription factors like the NFκB [2]
[4]. Symptom relief can be achieved
approximately 8 h after starting treatment and can last for up to
24 h [3].
Intranasal corticosteroids are considered the mainstay therapy of AR, are highly
effective in treating AR-associated symptoms of nasopharyngeal itching, sneezing,
rhinorrhea, and nasal congestion, help improving ocular symptoms, and are
recommended to be used on a continuous basis for optimum efficacy [5]
[6]
[7].
To date, there is no evidence that one INCS is more clinically effective than another
despite differences in potency [5]
[8]; however, different INCS are characterized
by different pharmacological and pharmacokinetic properties [8]. It is reported that fluticasone furoate has
the highest relative glucocorticoid receptor affinity and lipophilicity, an
extensive hepatic first-pass metabolism, and with one of the lowest systemic
exposures and potential risks among INCS used for AR [3]
[8].
The systemic bioavailability of fluticasone furoate is very low [9]
[10], with reported average oral
bioavailability of 1.26% [9] and
absolute bioavailability of 0.5% [10].
Following multiple doses of 800 µg IN fluticasone furoate every
8 h for 10 days, mean AUCt was reported to be
74.92 pg/mL*h (95%
CI=43.64–128.63 pg/mL*h), mean
Cmax was 20.53 pg/mL (95%
CI=16.04–26.27 pg/mL), and median was
Tmax 0.75 h (range=0.08–8.00 h). The
half-life (t1/2) and AUCinf were not derived due to
lack of quantifiable concentrations at the terminal phase [10].
Fluticasone furoate was shown to be highly bound (>99%) to plasma
proteins in vitro studies, and to undergo extensive first-pass metabolism by the
cytochrome P450 isozyme CYP3A4 to form the 17β-carboxylic acid metabolite
via hydrolysis. Following oral and intravenous (IV) administration, fluticasone
furoate was shown to be more than 90% eliminated in the feces, with minimal
(1–2.6%) urinary excretion. Following a single 250 µg IV
dose of fluticasone furoate showed a t1/2 of 15.12 h
(95% CI=11.82–19.35 h), volume of distribution of
608.4 L (95% CI=375.4–985.8 L), and clearance of
57.45 L/h (95%
CI=45.51–72.52 L/h) [1]
[2]. One study reported a mean
AUCinf of 4259.39 pg/mL*h (95%
CI=3869.68–4688.34 pg/mL*h),
t1/2 of 10.584 h (95%
CI=7.713–14.525 h), mean AUCt of
3787.47 pg/mL*h (95%
CI=3478.52–4123.86 pg/mL*h), mean
Cmax 6652.10 pg/mL (95%
CI=5803.01–7625.43 pg/mL), volume of distribution at
steady state of 361.7 L (95% CI=264.8–494.0 L), clearance of
58.70 L/h (95%
CI=53.33–64.60 L/h), and median Tmax of
0.29 h (range=0.08–0.33 h), following a single
250 µg IV dose of fluticasone furoate [10].
Previous studies have shown that fluticasone furoate plasma levels measured using
a
lower limit of quantitation (LLOQ) of 10 pg/mL were mostly
undetectable (below the limit of quantitation [BLQ]), except when IV or
supra-therapeutic IN doses were used, and that the elimination phase could not be
characterized following IN administration due to unquantifiable concentrations
during the terminal phase [9]
[10]
[11]. This study was designed to evaluate the
pharmacokinetic profile of fluticasone furoate using a validated analytical method
with a lower LLOQ, after a single dose in healthy subjects.
Methods
Study design
This was an open-label, single-dose, one-treatment, crossover study conducted at
Pharma Medica Research Inc., Saint Charles, Missouri, USA. The crossover design
was chosen to determine the intra-subject coefficient of variation of
fluticasone furoate (results not shown). The results of the study are presented
summarizes the pharmacokinetics of fluticasone furoate. Due to the exploratory
nature of this study, a planned sample size of 18 subjects was deemed
appropriate.
Previous reports have shown that at lower doses, there were a very low number of
samples with quantifiable fluticasone furoate concentrations [11]. A dose of 880 µg was
chosen for this study as it was deemed to be a reliable and safe dose to
estimate fluticasone furoate bioavailability and pharmacokinetic profile [1]
[2]
[10]
[11].
One formulation of fluticasone furoate nasal spray (from GSK Consumer Healthcare,
USA) containing 27.5 µg per spray was used in both periods. Each
subject received one total dose of 880 µg fluticasone furoate as
2 sprays in alternating nostrils until 16 sprays had been delivered in each
nostril within approximately 2 min. Subjects were given approximately
5–8 seconds to sniff or deeply inhale through their nose after every two
sprays, when alternating between each nostril. The time of the first spray was
considered the time of drug administration. The 2 periods were separated by a
washout of 7 days between drug administrations.
Subjects remained at the clinic for approximately 10 h before and
24 h after drug administration and fasted for approximately 10 h
before and 4 h after drug administration. Water was restricted from
1 h before until 1 h after drug administration. From screening
to end-of-study (EOS), the planned duration of the study was up to 37 days.
Study participants
Non-smoking male and female subjects were eligible for this study if they were 18
years of age or older, had a body mass index (BMI) of 18.0–33.0
kg/m2, inclusive, were healthy with no clinically
significant findings from medical history, 12-lead electrocardiogram (ECG),
laboratory evaluation, physical examination, and vital signs measurements, and
were willing to use acceptable effective methods of contraception.
Subjects were excluded from participation if they mainly: had a known history or
presence of clinically significant diseases (including conditions compromising
nasal absorption such as chronic postnasal drip, epistaxis, nasal ulcer, sores,
surgery, or trauma, chronic sinusitis, or significantly abnormal nasal passage),
infection, or any hypersensitivity to fluticasone or related drug substances;
were pregnant or lactating females; had recently participated in other clinical
trial and/or donated or lost whole blood within the safe acceptable
timeframe; had known history or suspected presence of tuberculosis; showed any
positive serology test, urine screen test, or breath alcohol test results; used
inhibitors or inducers of hepatic drug metabolism or drugs that alter
gastrointestinal pH/movement within 30 days prior to drug
administration. Subjects received financial compensation for their
participation.
Study endpoints
The primary endpoint of this study was to characterize the pharmacokinetics of
fluticasone furoate using our validated analytical method with a low LLOQ
following the administration of a single intra-nasal dose in healthy subjects.
The secondary objective was to assess safety and tolerability of fluticasone
furoate.
Sample collection for pharmacokinetic evaluation
In each period, a blood sample for pharmacokinetic analysis was collected by
direct venipuncture in a 10-mL tube containing K2EDTA as the
anticoagulant, before drug administration and at 5, 10, 15, 20, 30, and
45 min, and at 1 h, 1 h 20 min, 1 h
40 min, 2 h, 2 h 20 min, 2 h
40 min, and 3, 4, 5, 6, 8, 10, 12, 16, 24, and 36 h after drug
administration.
Whole blood samples were centrifuged at approximately 4°C for
approximately 10 min at 3000 rpm within 60 min of collection.
The separated plasma was divided into 2 approximately equal aliquots (using the
second aliquot as backup) in labeled polypropylene tubes. The plasma aliquots
were stored at −80±15°C within 60 min of whole
blood collection, pending assay, and shipped on dry ice to the bioanalytical
laboratory of Pharma Medica Research Inc., Ontario, Canada.
Bioanalytical method and procedures
At the bioanalytical facility, the plasma samples were analyzed for fluticasone
furoate, using fluticasone furoate-d5 as the internal standard. The
standard calibration range was 0.100–100 pg/mL using a
plasma sample volume of 0.800 mL. The concentration of the internal standard was
300 pg/mL. Plasma samples, treated with K2EDTA as the
anticoagulant, were processed by liquid-liquid extraction with
Methyl-tert-Butyl-Ether (MtBE):Hexane (60:40), the organic phase was dried and
the reconstituted sample was transferred for analysis. Samples were analyzed by
LC-MS/MS (Shimadzu Prominence UFLC & SCIEX API 6500) and reverse
phase chromatography under gradient conditions with mobile phases composed of
0.01% Ammonium Hydroxide in Water and Methanol (100%).
Chromatographic separation was achieved using serial analytical columns (C18,
50×3mm, 2.6 µm and Biphenyl, 50×3mm,
2.6 µm). Fluticasone furoate was analyzed using positive ion
scan mode and a parent-daughter mass to charge ion transition of 539–293
and 544–293 for the internal standard. The retention time for
fluticasone furoate was approximately 3.3 min.
Correlation was obtained between peak area ratios and the corresponding
calibration standard concentrations over the entire calibration range. A linear
equation (y=ax + b) with 1/x
2 weighting was used. The coefficients of determination of the
single-point calibration curves were ≥ 0.999. The recoveries of
fluticasone furoate and the internal standard were 93.7–94.1%
and 92.1–95.2%, respectively. The accuracy and precision of the
method are presented in [Table 1] and the
stability of fluticasone furoate in human samples is presented in [Table 2].
Table 1 Accuracy and Precision of the Validated Analytical
Method.
|
Precision (%)
|
Accuracy (%)
|
|
LLOQ intra-day
|
≤14.4
|
84.7–111.0
|
|
LLOQ inter-day
|
14.4
|
101.0
|
|
QC L, M, H intra-day
|
≤6.4
|
94.3–112.0
|
|
QC L, M, H inter-day
|
≤7.2
|
101.0–102.3
|
QC quality control, H high (80 pg/mL), L low
(0.3 pg/mL), LLOQ lower limit of quantitation
(0.1 pg/mL), M medium (50 pg/mL).
Table 2 Stability of Fluticasone Furoate in Human
Samples.
|
Condition
|
Stability
|
|
In whole blood
|
3.00 h at room temperature
|
|
3.00 h in ice-water bath
|
|
In plasma
|
|
Freeze-thaw
|
Four cycles at −80±15°C
|
|
Bench top
|
19.00 h at room temperature
|
|
20.00 h in ice-water bath
|
|
Processed samples
|
|
Autosampler
|
145.25 h at approximately 5°C
|
|
Storage of reconstituted samples
|
45.00 h at approximately 5°C
|
|
Storage of evaporated samples
|
2.50 h at room temperature
|
Safety evaluation
Physical examination, vital signs (blood pressure, heart rate, respiratory rate,
and temperature) measurements, and clinical laboratory tests (biochemistry,
hematology, and urinalysis) were performed at screening and EOS. Serology blood
tests (human immunodeficiency virus, hepatitis C antibody, and hepatitis B
surface antigen), 12-lead ECG recording, serum human chorionic gonadotropin
(hCG) test for female participants, urine drug screen, urine cotinine test, and
tuberculosis questionnaire evaluation were also obtained at screening. In
addition, in each period, urine drug screen, urine cotinine test, urine hCG test
for female participants, and breath alcohol tests were performed at check-in,
blood pressure and heart rate were measured prior to drug administration and at
1, 3, and 6 h post-dose, and temperature was measured daily during
confinement. All clinical laboratory tests were performed by Quest Diagnostics
Lenexa, Kansas, USA.
The use of herbal products, nutritional supplements, vitamins, grapefruit and
grapefruit-containing products, alcohol and alcohol-containing products,
caffeine- and xanthine-containing products, and inhalers-, nasal sprays-, or
steam inhalation-based practices were restricted during the study. Concomitant
medications were not allowed during the study unless requested or approved by
the investigator. Nondrug therapies that did deviate from protocol procedures
were allowed. Medical and adverse events (AE) were monitored from screening to
EOS.
Pharmacokinetic and statistical analysis
The pharmacokinetic parameters were estimated for fluticasone furoate using a
non-compartmental approach in Phoenix WinNonlin version 6.4 (Certara USA, Inc.,
Princeton, NJ). The actual post-dose sample collection times were used in the
pharmacokinetic analysis. The peak concentration (Cmax) and the time
to reach Cmax (Tmax) were determined from individual
plasma concentration-time profiles for FF. The area under the plasma
concentration-time curve (AUCt) was calculated using the linear
up-log down trapezoidal method from 0 to 36 h. The area under the plasma
concentration-time curve from zero to time infinity (AUCinf) was
calculated as AUCt + Ct/kel,
where Ct is the last measurable concentration and kel is
the terminal rate constant. The terminal half-life (t1/2) was
calculated as 0.693/kel. All obtained samples were assayed;
however, subjects with sufficient data to allow pharmacokinetic characterization
were included in the pharmacokinetics and statistical analyses. Descriptive
statistics for the pharmacokinetic parameters of fluticasone furoate were
calculated. Safety and tolerability of FF were assessed using descriptive
statistics for all subjects who participated in the study and were primarily
based on the occurrence and severity of AE.
Results
Subjects’ disposition and demographics
Eighteen healthy subjects were enrolled in and 17 subjects completed the study
([Fig. 1]). Overall, 11 female and 7
male subjects participated in the study. The subjects had a mean age of 40.5
years and BMI of 26.5 kg/m². Eight (44.4%) subjects were
white and 10 (55.6%) were black or African American ([Table 3]).
Fig. 1 Subjects disposition.
Table 3 Summary of Demographic Characteristics.
|
Demographics
|
Study Population N=18
|
|
Gender, n (%)
|
|
|
Female
|
11 (61.1)
|
|
Male
|
7 (38.9)
|
|
Age, years, mean±SD (range)
|
40.5±12.4 (22–59)
|
|
BMI, kg/m², mean±SD (range)
|
26.5±3.6 (19.6–31.4)
|
|
Weight, kg, mean±SD (range)
|
76.9±12.3 (155.8–183.6)
|
|
Height, cm, mean±SD (range)
|
170.2±7.9 (56.1–101.9)
|
|
Race, n (%)
|
|
|
White
|
8 (44.4)
|
|
Black or African American
|
10 (55.6)
|
|
Ethnicity, n (%)
|
|
|
Hispanic or Latino
|
0 (0.0)
|
|
Not Hispanic or Latino
|
18 (100.0)
|
Arithmetic means are reported in this table. BMI body mass index, SD
standard deviation.
Pharmacokinetic analysis
A total of 23 samples were collected from each of the 18 subjects. One subject
withdrew from the study approximately 10 min after dosing due to
personal reasons (difficult phlebotomy), and was thus excluded from the
pharmacokinetic and statistical analyses. All the remaining samples collected
from the 17 subjects had measurable fluticasone furoate plasma concentrations,
including concentrations during the elimination phase. As such, all the 17
subjects showed concentration-time profiles with a clearly defined terminal
elimination phase and so the AUCinf and t1/2 could
be confidently estimated.
Bioanalytical analysis
The concentrations of all the samples analyzed were within the validated range. A
calibration standard and quality control samples of at least 6% of the
total study samples at three different concentrations were extracted and
analyzed within each batch. All seven extracted batches during the entire study
conduct passed the acceptance criteria. The inter-day precision (%CV)
was ≤ 5.6% and accuracy was 91.8% to
101.6%. A total of 782 samples were analyzed in this study (391 samples
in period 1), of which 79 samples were randomly selected around the
Cmax and the elimination phase of each profile for incurred
sample reanalysis (ISR). The results of the ISR showed 96.4%
confirmation of the original values within±20%.
Statistical results
Following a single 880 µg dose of IN fluticasone furoate, median
Tmax was 1.33 h
(range=0.75–6.00 h), mean Cmax was
13.05±7.59 pg/mL, mean AUCt was
148.48±77.76 pg/mL*h, mean AUCinf was
279.07±187.81 pg/mL*h, and mean
t1/2 was 31.67±29.23 h ([Table 4], [Fig. 2]). The intra-subject variability
was estimated to be 22% for AUCt and 24% for
Cmax.
Fig. 2 Mean plasma fluticasone furoate concentration-Time
profiles in linear (a) and log-linear scale (b) following administration
of a single intranasal dose to healthy subjects. Error bars represent
the standard deviation about the mean.
Table 4 Pharmacokinetic Parameters Based on Plasma
Fluticasone Furoate Following Administration of Single Intranasal
Dose of 880 µg to Healthy Subjects.
|
PK Parameter
|
N
|
Mean±SD
|
|
AUCinf , pg/mL*h
|
17
|
279.07±187.81
|
|
AUCt , pg/mL*h
|
17
|
148.48±77.76
|
|
Cmax, pg/mL
|
17
|
13.05±7.59
|
|
t1/2 , h
|
17
|
31.67±29.23
|
|
|
Median (Range)
|
|
Tmax, h
|
17
|
1.33 (0.75–6.00)
|
Arithmetic means are reported in this table. AUCinf area under
the concentration versus time curve from time zero to infinity,
AUCt area under the concentration versus time curve, from
time zero to the time of the last measurable concentration (t),
Cmax maximum measured concentration over the sampling
period, PK pharmacokinetic, SD standard deviation,
t1/2 apparent elimination half-life,
Tmax time of the maximum measured concentration over the
sampling period.
Safety and tolerability
The administration of 880 µg of IN FF under fasted conditions was
well tolerated by the healthy subjects who participated in the study. Four
subjects (22.2%) experienced 4 AEs in total: 3 subjects (16.7%)
experienced 3 AEs (one venipuncture site reaction and 2 headache events) in
period 1 and one subject (5.6%) experienced 1 AE (venipuncture site
reaction) in period 2. In total, the 2 headache AEs reported by 2 subjects
(11.1%) in period 1 were considered to be possibly related to FF. All
AEs were mild in severity and resolved.
There were no AE-related withdrawals or serious AEs reported in this study. There
were no clinically meaningful trends in laboratory safety measurements, vital
signs, physical examinations, or ECGs reported during this study. All
measurements were either within normal range or were deemed by the investigator
to be not clinically significant for all subjects.
Discussion
With an increase in the development of newer drugs and dosage forms, a full
understanding of their pharmacokinetics is essential in characterizing their
disposition. The development of newer analytical methods that can quantify
concentrations at lower thresholds (i. e. having a low LLOQ) plays an
important role. The lack of a sensitive enough LLOQ poses a barrier in being able
to
confidently evaluate the complete pharmacokinetic behaviour of these drugs.
Fluticasone furoate is one of the newest INCS, is available as prescription or
over-the-counter medicine, and may be more preferred by patients compared to other
INCS [12]. It is one of the first-line
treatments recommended in AR treatment [13]
[14]
[15]. Previous pharmacokinetic
characterization of IN fluticasone furoate was based on an LLOQ of
10 pg/mL [1]
[9]
[10]
[11]
[16], rendering plasma levels at the terminal
elimination phase undetectable resulting in a limited amount of pharmacokinetic
information that can be obtained.
A dose escalation study involving the administration of IN fluticasone furoate over
a
dose range of 55–440 µg once daily for 2 weeks, showed that
from 1476 plasma samples collected from 502 patients, 12 years of age or older with
seasonal AR, only 5.3% of total samples from 11.8% of patients had
detectable fluticasone furoate levels when an LLOQ of 10 pg/mL was
used. In addition, with higher doses, more samples had quantifiable concentrations,
but did not exceed 15.4% of the samples collected at the
440 µg dose nor three times the LLOQ [11]. Furthermore, following the administration
of a multiple-dose IN fluticasone furoate regimen of 2640 µg daily
for 3 days followed by 880 µg on the day of PK sampling to 16
healthy subjects, 50.00% of subjects had BLQ plasma concentrations at
8 h post-dose, 6.25% had BLQ plasma concentrations at all time
points, and 6.25% had only 1 measurable plasma concentration [10].
This study implemented a validated analytical method able to measure fluticasone
furoate plasma concentrations using an LLOQ of 0.1 pg/mL following
administration of a single 880 µg dose of IN fluticasone furoate. As
a result, all the samples (100.00%) collected from all the subjects who
completed the study had detectable and quantifiable plasma levels of FF at all
collection time points, including the terminal linear phase, and as such allowed for
a better characterization of the pharmacokinetic profile of fluticasone furoate than
before. The results showed that sampling for a longer time would likely have led to
more measurable concentrations, thus allowing for a more complete evaluation of the
terminal pharmacokinetic parameters.
The results showed a higher mean AUCt
(148.48 pg/mL*h) and median Tmax
(1.33 h), and lower Cmax (13.05 pg/mL) compared
to previously reported values for IN fluticasone furoate
(74.92 pg/mL*h, 0.75 h, and
20.53 pg/mL, respectively) [10]. The Tmax range (0.75 to 6.00 h) fell within
previously reported values for IN fluticasone furoate (0.08 to 8.00 h) [10]. Differences in these parameters values can
be explained by the number of measurable concentrations used to calculate them owing
to the LLOQ used in the analytical method. When an LLOQ of 10 pg/mL
was used, Cmax and Tmax were derived from 15 subjects with at
least 2 measurable plasma concentrations, including 1 subject with only 1 measurable
plasma concentration, and AUCt was derived from 14 subjects with at least
2 measurable plasma concentrations, including several subjects with undetectable
plasma concentrations at 8 h post-dose [10]. In this study, however, Cmax, Tmax, and
AUCt were calculated based on data from 17 subjects who had all their
plasma concentrations quantifiable at all pharmacokinetic sampling time points,
using a 100 times lower lower limit of quantitation.
This study provides a breakthrough in the bioanalysis and pharmacokinetics of
fluticasone furoate given intra-nasally . It allowed for a more accurate
characterization of the concentration-time profile of fluticasone furoate following
a single intra-nasal dose in healthy subjects, such that the terminal elimination
phase was clearly defined allowing for a more confident estimation of
AUCinf and t1/2. In addition, the new method
eliminates the need to expose healthy subjects to multiple doses and allows
calculation of the pharmacokinetic parameters following a single dose even as low
as
110 mcg, which corresponds to the standard dose that is given clinically.
Conclusion
Using a lower limit of quantitation of 0.1 pg/mL, the complete
characterization of fluticasone furoate pharmacokinetics, including a clearly
defined terminal elimination phase, was achieved following a single dose given
intranasally. The improved bioanalytical method enabled further insight into the
pharmacokinetics of fluticasone furoate that was not possible with other analytical
methods that used a higher lower limit of quantitation. With this new proven
sensitivity, it will allow for more optimal study designs investigating intra-nasal
or inhaled formulations of fluticasone furoate as the expected concentrations
following both routes of adminstration are expected to be low. This improved
bioanalytical method with allow for further investigations into the pharmacokinetics
of fluticasone furoate.
Authors Contributions
MB contributed to the study concept and the bioanalytical method development and
validation. ZT contributed to the study design, pharmacokinetic and statistical data
analyses, study results interpretation, and writing of the initial drafts of the
article. MB and ZT have reviewed and approved the final manuscript for submission,
and MB holds the final responsibility of the article submission.
Compliance with Ethical Standards
Compliance with Ethical Standards
This study was approved by Salus Institutional Review Board (IRB), Texas, USA. All
study procedures involving human participants were conducted in conformance with the
1964 Declaration of Helsinki and its later amendments, the International Council for
Harmonisation (ICH) Good Clinical Practice (GCP) regulations, IRB ethical standards,
the United States standards and requirements and other statutes or regulations
regarding the protection of the rights and welfare of human subjects participating
in biomedical research. All subjects provided written informed consent before trial
initiation.
Data Availability Statement
Data Availability Statement
The datasets generated during and/or analyzed during the current study are
available from the corresponding author on reasonable request.
