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Quantitative and In Vitro Deposition of Zanamivir Dry Powder
Inhalation by HPLC
Yang Yang, Guo Na, ZiWei Yang, XingGuo Mei*
Beijing Institute of Pharmacology and Toxicology, No. 27, Taiping Road, Beijing
10085, People’s Republic of China
*Corresponding
author.
Tel./fax:
+86-10-66932644,
E-mail
address:
[email protected]
Abstract
Dry powder formulations for inhalation are often composed of micron-sized drug
particles and inert carrier particles. Currently, nearly all DPI products (Relenza®,
Seretide®, Spiriva®, Symbicort®, Beclophar®, Flixotide®) already on the market rely
on lactose as a carrier material. However, the use of lactose has some disadvantages,
such as its sugar-associated reducing function that may interact with functional groups
of drugs such as budesonide or peptides and proteins. Mannitol is widely used in
pharmaceutical formulations and food products. It may be an attractive alternative
carrier to lactose because it does not have the reducing effect and is less hygroscopic.
Zanamivir is a new anti-flu drug, which is being trumpeted by Glaxo Wellcome.
Although the zanamivir dry powder (Relenza®) presented good aerosolization
properties, drawbacks are linked to lactose. In our previous study, a number of dry
powder formulations were produced by jet mill zanamivir with mannitol as the carrier.
In this study, a high performance liquid chromatographic method was developed for
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the estimation of the content of zanamivir dry powder inhalation (DPI) and its in vitro
deposition. The separation was achieved by Luna SCX column (250×4.6 mm, 5 μm,
make: Phenomenex), a mixture of acetonitrile / buffer (60/40 and pH=3.7) as mobile
phase, at flow rate of 1 ml/min. Detection was carried out at 233 nm. The calibrated
linear plot of zanamivir was within 0.05- 0.15 mg/ml with the mean recovery within
99.60%- 102.81% and within-day and among-days RSD of less than 2%. The relative
standard deviation of the repeatability test was 0.12%. Developed method was found
to be accurate, precise, selective and rapid for estimation of the content of zanamivir
DPI and its in vitro deposition. The in vitro deposition was also evaluated after the
aerosolization of powders at 60 l/min via Aerolizer® inhaler into a Twin-Stage
Impinger (TSI) and a Next Generation Impactor (NGI) (analysed using high
performance liquid chromatography). It was found that the trends in the FPF values
from the NGI data broadly mirror those of the TSI study. The FPF values for
tanamivir DPI were lower by NGI than TSI, an effect most probably due to the lower
aerodynamic cut-off for the NGI.
Key words: Zanamivir, dry powder inhalation, content, in vitro deposition, HPLC
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INTRODUCTION
Zanamivir (5- acetylamino- 4- guanidino- 2, 6- anhydro-3, 4, 5- trideoxy- D- glycerolD- galacto- non- 2- enoic acid), also known as Relenza®, is a new anti-flu drug, which
is being trumpeted by its manufactures- the pharmaceutical giant Glaxo Wellcome- as
the first breakthrough in flu treatment for 30 year. It is a potent and highly selective
inhibitor of the influenza A and B virus neuraminidase. Mechanism of action has on
viral neuraminidase catalyzes cleavages of terminal sialic acid residues attaches to
glycoprotein and glycolipids, a process necessary for release of virus from host cell
surfaces (1). When administered directly to the human respiratory tract, zanamivir
was shown to have potent antiviral effects.
Inhalation of small molecule drugs and biopharmaceuticals is an efficient and
convenient local drug delivery method (2). We have produced a zanamivir dry
powder inhalation (DPI) using mannitol as carriers. Zanamivir in rat and monkey
plasma by positive ion hydrophilic interaction chromatography (HILIC)/ tandem mass
spectrometry were developed (3). Till the date there is no HPLC method available for
analysis of the content of main component in zanamivir DPI and its in vitro deposition.
The aim of this study was to determine the content and in vitro deposition of
zanamivir DPI by HPLC.
MATERIALS AND METHODS
Reagents and materials
Zanamivir (purity: 99.11%) was purchased from Haoxin pharmaceutical Company
(Wuhan, China). Mannitol was obtained from Bodi Chemicals (Tianjin, China).
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Methanol and acetonitrile of chromatographic grade were purchased from J. T. Baker
Company (USA). All other materials or solvents were of analytical or
chromatographic grade and were used as obtained commercially.
Preparation of sample solution
Zanamivir dry powder inhalation was prepared in mobile phase, diluted the solution
and reached the final concentration of 0.1 mg/ml of zanamivir. This final solution
marked as sample solution.
Chromatographic conditions
HPLC chromatograms were obtained using Agilent HPLC system with G1311A Quat
Pump, G1329A Autosampler, Diode array detection (Agilent Technologies, USA).
The HPLC analysis was completed using a Luna SCX column (250×4.6 mm, 5 μm,
make: Phenomenex). The detector was connected to a computer and the data were
analyzed by chemistry station (B.04.02). Determination of zanamivir was done by
using acetonitrile / buffer (60/40 and pH=3.7) at flow rate of 1 ml/min. The detector
wavelength was 233nm. The column was maintained at 30 °C through out the
analysis. A 20 μl sample as described in the sample preparation was injected. Buffer
solution was prepared by dissolving 0.77 g sodium acetate in 500 ml water. The final
pH was adjusted to 3.7 ± 0.05 with dilute acetic acid and filtered through 0.45 μm
before use.
Standard curve preparation
Standard solutions were prepared by weighing a known amount of zanamivir by serial
dilution with mobile phase, resulting in final concentrations of 0.15, 0.12, 0.1, 0.08,
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0.05 mg/ml. A blank solution was also prepared with mobile phase. Each set of
standards and a blank was analyzed chromatographically 3 times. The peak area was
recorded and standard curve was constructed by linear regression of mean peak area
and concentration.
Accuracy and precision
In accordance with ICH guideline (4), the accuracy and precision of the method were
studied by using 3 known concentration levels of zanamivir (0.05, 0.08 and 0.1 mg/ml)
and 3 replicates each of the total analytical procedure, within day and between 3 days.
These
samples
were
considered
as
unknown
samples
and
analyzed
chromatographically using the proposed procedure.
Repeatability
In accordance with Q2B-ICH guideline (5), repeated analysis of a homogeneous
sample was performed by the same analytical procedure and the same analyst, with
the same equipment and in the same laboratory.
Robustness
The robustness of method as carried out by changing the chromatographic conditions
such as flow rate and organic solvent portion of the mobile phase.
Content of zanamivir DPI
The powders (10 mg eq zanamivir) was accurately weighed and quantitatively
transferred into a 50 ml volumetric flask. The resulting solution was diluted with
mobile phase and then filtered through 0.45μm filter. Samples were analyzed by the
HPLC as described above.
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In vitro deposition
In vitro powder deposition was determined separately with a twin-stage impinger (TSI)
(Copley Scientific, UK) and a next generation impactor (NGI) (Copley Scientific, UK)
operated under standard conditions (Appendix XII part C, British Pharmacopoeia
2010).
The Aerolizer® inhaler (Schering, Co.) was attached to the throat of the TSI with a
rubber mouthpiece adaptor and tested at 60 l/min for 5 s. Ten of size 3 gelatin
capsules, manually filled (25 mg powder in each), were discharged into the TSI
before the apparatus was disassembled. The contents of the device, capsules,
mouthpiece and throat and each impinger stage were washed with mobile phase into
volumetric flasks, made up to volume and passed through a 0.45 μm cellulose acetate
filter. Samples were analyzed by the HPLC as described above.
The Aerolizer® inhaler was attached to the throat of the NGI with a rubber mouthpiece
adaptor and tested at 60 l/min for 4 s. Ten capsules as described above were
discharged into the NGI. After the run, the capsule, inhaler, adaptor mouthpiece,
throat, and NGI stages were washed with mobile phase into volumetric flasks, made
up to volume and passed through a 0.45 μm cellulose acetate filter. Samples were
analyzed by the HPLC as described above.
RESULTS AND DISCUSSION
Quantitative analysis
The peak purity of zanamivir was assessed by comparing the retention time of
standard zanamivir sample good correlation was obtained between the retention time
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of standard and sample. Placebo and blank was injected and there were no peaks.
There are no interferences hence method is specific. Representative chromatograms
are shown in figure 1, 2 and 3.
The calibration function was determined by linear regression over the range 0.05-0.15
mg/ml. The regression equation was Y= 23.50 X – 237.0, where X is the
concentration of standard samples (mg/ml), and the correlation factor was 0.9999.
The results of studying the accuracy and precision are shown in table 1 and 2,
respectively. The recoveries are 99.60%- 102.81%with relative standard deviations
between 0.01 %and 1.58%, which are in acceptable ranges. Also, as shown in table 3,
the relative standard deviation of repeatability test is acceptable (0.12%). The results
of robustness studies are shown in table 4 and 5. With the change of flow rate of 0.8
ml/min, 1 ml/min and 1.2 ml/min, change of organic solvent portion of the mobile
phase with of 10% less, actual, 10% more and their tailing factor, plate count obtained
within the limit.
Content of zanamivir DPI
The proposed HPLC method has been used in the analysis the content of zanamivir in
DPI. As a result, the 3 Batches contents of zanamivir DPI were 99.73% ± 0.35,
99.86% ±0.22 and 100.12% ±0.12, respectively.
In vitro deposition of zanamivir DPI
Firstly, the in vitro deposition testing was performed using TSI and the data analyzed
to give the recovered dose (RD, the total amount of drug collected in the device,
capsule, mouthpiece and throat, upper and lower stages), emitted dose (ED, the total
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amount of drug collected in the mouthpiece and throat and upper and lower stages)
and fine particle fraction (FPF, the ratio of drug in the lower stage to the RD). The
results are shown in Table 6. The lower stage of the TSI has a higher aerodynamic
particle size cut-off (6.4μm) than the NGI (4.46μm); as such, TSI is a less rigorous
challenge for each formulation than NGI and this manifest in higher FPF figures.
Secondly, the in vitro deposition testing was performed using NGI and the data
analyzed to give the RD (drug collected in the device, capsule, mouthpiece and throat,
pre-separator, stages 1–7 and MOC) and FPF (ratio of drug collected in stages
3-MOC to the RD). The results are shown in Table 6. For the powders, the greatest
amount of deposition was achieved on stage 4 followed by stages 3 and 2,
respectively. Very small amounts were deposited on stages 7 and MOC rendering it
difficult to obtain a sufficient sample size for full analysis. The remainders in each
case constitute deposits in the pre-separator as well as powder losses which occurred
during the experiment.
The trends in the FPF values from the NGI data broadly mirror those of the TSI study.
In all cases, FPF values for these formulations were lower by NGI than TSI, an effect
most probably due to the lower aerodynamic cut-off for the NGI as noted earlier. In
all cases, the ED for these formulations had an ED of over 90% of the total capsule
contents, which is probably a reflection of the (presumably lower) force of cohesion
in the powders.
CONCLUSION
The proposed method is simple, specific, accurate and precise and hence can be used
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in routine for estimation of zanamivir in DPI dosage. Statistical analysis of the results
has been carried out revealing high accuracy and good precision. The percentage RSD
for all parameters was found to be less than two, which indicates the validity of the
method and assay results obtained by this method are in fair agreement. The
developed method can be used for routine quantitative simultaneous estimation of
zanamivir in DPI dosage form and its in vitro powder deposition.
Acknowledgments: We are grateful to the financial support from the Important
National
Science
&
Technology
Specific
Projects
(Grant
No.
2012ZX09301003-001-009) of China.
REFERENCES
1. Cox RJ, Mykkeltvedt E, Sjursen H, Haaheim LR, The effect of Relenza treatment
on the early immune response induced after influenza vaccination, Int. Congress
Series., 1219, 2001, 829-834.
2. Saint-Lorant G, Leterme P, Gayot A, Flament MP, Influence of carrier on the
performance of dry powder inhalers, Int. J. Pharm., 334, 2007, 85-91.
3. Todd M, Baughman, Wayne L, Wright. Determination of zanamivir in rat and
monkey plasma by positive ion hydrophilic interaction chromatography (HILIC)
/ tandem mass spectrometry, J. chromatography B, 852, 2007, 505-511.
4. Pharmaceutical application data. Tokyo. 77. ICH-Guideline Q2A, Validation of
Analytical
Procedures:
Definition
and
Trminology,
http://www.fda.gov/cder/guidance/ichq2a.pdf (access: July 2006).
5. Pharmaceutical application data. Tokyo. 77. ICH-Guideline Q2B, Validation of
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Analytical
Procedures:
Methodology,
http://www.fda.gov/cder/gdlns/ichq2bmeth.pdf (access: July 2006).
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Figure 1.Chromatogram for blank
Figure 2.Chromatogram of Standard
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Figure 3.Chromatogram of the sample
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Table 1. Within-day accuracy and precision of the proposed HPLC method
expected concentration
mean determination
SD
Recovery (%)
RSD (%)
n
0.08000
0.08115
0.05
101.44
0.25
3
0.10000
0.10267
0.44
102.67
0.54
3
0.12000
0.11953
0.04
99.61
0.01
3
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Table 2. Between-day accuracy and precision of the proposed HPLC method
expected concentration Mean determination
SD
Recovery (%)
RSD (%)
n
0.08000
0.08102
0.07
101.28
0.32
3
0.10000
0.1023
1.02
102.31
1.26
3
0.12000
0.11954
0.18
99.62
0.06
3
0.08000
0.08177
0.08
102.21
0.39
3
0.10000
0.10281
1.09
102.81
1.33
3
0.12000
0.11952
0.04
99.60
0.01
3
0.08000
0.08153
0.14
101.91
0.64
3
0.10000
0.10243
1.28
102.43
1.58
3
0.12000
0.11953
0.20
99.61
0.06
3
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Table 3. Repeatability data
Injection
Peak areas
Injection-1
288877
Injection-2
288463
Injection-3
288139
Injection-4
287841
Injection-5
288628
Average
288389.6
% RSD
% 0.12
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Table 4. Robustness (flow rate variation)
System suitability results
No.
Flow rate (ml/min)
USP plate count
USP tailing
1
0.8
2187
1.1
2
1
2223
1.1
3
1.2
2072
1.12
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Table 5. Robustness (Mobile phase variation)
Change in organic composition
System suitability results
No.
in the mobile phase
USP plate count
USP tailing
1
10% less
2012
1.1
2
Actual
2230
1.1
3
10% more
2039
1.2
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Table 6. In vitro powder deposition of the powders (n=3)
Determined using TSI
Determined using NGI
Fine particle fraction (FPF)%
24.04 ±1.6
21.04 ±1.2
Emitted dose (ED)%
97.7 ±2.5
98.1 ±1.9
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FIGURE LEGENDS
Figure 1: Chromatogram for blank
Figure 2: Chromatogram of Standard
Figure 3: Chromatogram of the sample
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TABLE LEGENDS
Table 1: Within-day accuracy and precision of the proposed HPLC method
Table 2: Between-day accuracy and precision of the proposed HPLC method
Table 3: Repeatability data
Table 4: Robustness (flow rate variation)
Table 5: Robustness (Mobile phase variation)
Table 6: In vitro powder deposition of the powders (n=3)
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