In vitro and in vivo evidence of the power of inhaled engineered particles to treat invasive pulmonary aspergillosis

1. Reformula*ng voriconazole for
inhaled therapy
Philippe Rogueda 12 January 2016
1 © Copyright Aedestra Ltd 2016. All rights reserved.
Inhala*on products made easy
www.aedestra.com

2. Disclaimer & Copyrights
The informa*on contained in this presenta*on is copyrighted.
Copyrights: All copyrights reserved. Aedestra Ltd, 2016.
Viewers may use the informa*on contained within these slides for internal purposes according to
copyrights laws of Hong Kong. They may not use nor distribute the informa*on to any third party
without Aedestra’s explicit concern. Reference to Aedestra’s authorship and copyright ownership
must be made in any private or public disclosure.
This presenta*on contains the personal opinions and thoughts of the presenters and may not be
assumed to represent the opinion of Aedestra Ltd nor of any employer or company or learned
society with whom the presenters might be or might have been associated.
Aedestra Ltd makes no representa*on to the accuracy of the informa*on contained therein.
Aedestra Ltd declines any responsibility on the use of the informa*on contained in these slides.
Aedestra Ltd does not recommend any course of ac*on resul*ng directly or indirectly from the
informa*on presented in these slides.
© Copyright Aedestra Ltd 2016. All rights reserved.
2

3. © Copyright Aedestra Ltd 2016. All rights reserved.
Reformula6ng voriconazole for inhaled therapy
On Thursday 10th December 2015, Sumit Arora presented his work on the formula*on
of voriconazole for inhaled therapy at DDL27 in Edinburgh. It was one of the most fun
and interes*ng lectures I have listened to in many years at a conference. His work was
well carried out, well presented, and useful.
I am delighted to be able to bring to you these slides and manuscript that Sumit kindly
agreed for me to make public via the Aedestra website and on a number of public
forum.
Sumit’s work shows the power of inhaled engineered par*cles to treat invasive
pulmonary aspergillosis. He demonstrates this with in vitro work, stability at 3 months
and in vitro deposi*on, but also with in vivo studies in mice. The evidence he provides
is compelling and shows yet again how the inhaled route is a viable alterna*ve to many
intravenous and oral therapies.
Thank you Sumit.
Philippe Rogueda @ Aedestra
3

4. © Copyright Aedestra Ltd 2016. All rights reserved.
SURFACE MODIFIED VORICONAZOLE DRY POWDER INHALABLE FORMULATION FOR
THE TREATMENT OF INVASIVE PULMONARY ASPERGILLOSIS
4
A lecture presented by Sumit Arora at DDL27, December 2015:
hXp://ddl-conference.com/?q=ddl25-programme-programme-day-2
Sumit Arora’s CV summary can be seen on LinkedIn:
hXps://in.linkedin.com/in/sumit-arora-68911342
Recent awards: Pat Burnel’s new inves*gator award 2015
First Prize at Ranbaxy Sun Pharma Science Scholar Award 2015
Abstract and presenta6on can be found in the slides that follow, courtesy of Sumit
Arora

5. © Copyright Aedestra Ltd 2016. All rights reserved.
SURFACE MODIFIED VORICONAZOLE DRY POWDER INHALABLE FORMULATION FOR
THE TREATMENT OF INVASIVE PULMONARY ASPERGILLOSIS
5
Abstract
Courtesy of Sumit Arora

6. Surface Modified Voriconazole Dry Powder Inhalable Formulation for the Treatment of Invasive Pulmonary
Aspergillosis
Sumit Arora1, 2, 3
, Mehra Haghi2, 4
, Paul M. Young2
, Michael Kappl3
, Daniela Traini2
& Sanyog Jain1
1
Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical
Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) Punjab- 160062 INDIA
2
Respiratory Technology, Woolcock Institute of Medical Research and Discipline of Pharmacology, Sydney Medical
School, The University of Sydney, NSW 2037, Australia
3
Max Planck Institute for Polymer Research, 55128 Mainz, Germany
4
School of Pharmacy, Graduate School of Health, University of Technology, Sydney, NSW 2007, Australia
Summary
Background: Invasive pulmonary aspergillosis (IPA) is a severe disease in immunocompromised patients with
extremely high mortality rate. Voriconazole (VRZ) is a first line treatment drug for IPA, conventionally administered
orally or intravenous, resulting in a plethora of drug-drug interactions and off-target toxic effects. In the present
research work, we developed and characterised a highly dispersible dry powder inhalable formulation of VRZ using L-
Leucine as a dispersibility enhancer. Methods: VRZ and L-Leucine in varying concentrations were dissolved in
ethanol-water (70:30% v/v) and spray dried to yield inhalable dry powders. Powders were characterised in terms of
particle size, morphology and aerosol performance using the low resistance RS01 dry powder device with next
generation cascade impactor. Storage stability (chemical stability and aerosol performance) of the optimized
formulation was evaluated for 3 months. Calu-3 sub bronchial epithelial cell line was used to study cell viability (MTS
test). Finally, in vivo pharmacokinetic studies in mice were carried out to determine the lung bioavailability of the
optimised formulation. Results: Dry powder comprising VRZ (8 mg/mL) and L-Leucine (2 mg/mL) was found to be
suitable for inhalation therapy. Powder exhibited a volume median diameter of 2.64 ± 0.05 µm and superior
aerosolisation with MMAD of 3.79 ± 0.02 µm and fine particle fraction (% aerosol < 5 µm) of 60.00 ± 0.94 %. Powder
exhibited irregular morphology and demonstrated physico-chemical stability of up to 3 months at room temperature.
Formulation was found to be non-cytotoxic to Calu-3 cells. Moreover, lung bioavailability in murine model showed the
ability of inhaled formulation to attain higher concentration of VRZ in lungs as compared to intravenous administration.
Conclusion: A highly respirable dry powder VRZ formulation was developed for the treatment of IPA.
Introduction
Aspergillus fumigatus, the opportunistic fungi, causes IPA particularly in immunocompromised patients such as those
suffering from hematologic malignancies, cancer, AIDS and those undergoing solid organ transplantation.[1]
This
results in substantial mortality (nearly 80%) and huge financial burden. VRZ is the drug of choice for the treatment of
IPA.[2]
Oral or intravenous administration of VRZ have been associated with high inter- and intra-patient
pharmacokinetic variability, poor lung distribution particularly in patients undergoing lung transplantation, alteration of
enzyme levels in liver leading to numerous, sometimes lethal drug-drug interactions as well as the off-target toxic
effects. [3]
Pulmonary delivery of high doses of VRZ represent a potential viable therapeutic option for the targeted treatment of
IPA, whilst minimising systemic exposure and related toxicity.
Methods and Materials
VRZ was supplied by Ranbaxy Laboratories (Gurgaon, India) and L-Leucine was purchased from Sigma-Aldrich
(Sydney, Australia). Calu-3 cell line (HTB-55) was purchased from the American Type Cell Culture Collection (ATTC,
Rockville, USA). Dulbecco’s modified Eagle’s medium and L-glutamine from Invitrogen (Sydney, Australia). All
solvents were of analytical grade and used as supplied (Biolab, Victoria, Australia)
Preparation of L-Leucine modified VRZ microparticles
For the preparation of respirable particles, VRZ (8 mg/mL) and L-Leucine (2 mg/mL) were dissolved in ethanol-water
(70:30% v/v) and spray dried using a Buchi Mini Spray Dryer B-290 at the following conditions: feed concentration of
10 mg/ml, inlet temperature 125°C, outlet temperature was 78°C, atomiser 700 L/h, aspirator 40 m3/h and feed rate
5%.

7. Morphological and Particle Size Analysis
Morphology of the spray dried products was studied using a scanning electron microscope (SEM, JMC, 6000 JEOL,
Japan). Samples were coated with 15 nm gold (Sputter coater S150B, Edwards High Vacuum, Sussex, UK) and
images were taken at random locations. Size distribution of the VRZ alone and VRZ-Leucine particles was analysed
using laser light diffraction (Mastersizer 3000, Malvern, United Kingdom) using the Scirocco dry dispersion unit with a
feed pressure of 4 bar and a refractive index of 1.62 for VRZ.
In vitro aerosol performance characterisation
Aerosol performance of the spray dried products (5mg in a size 3 gelatin capsule) was evaluated using an RS01 dry
powder inhaler device (Plastiape, Italy) with a next generation impactor (NGI) operated at a flow rate of 60 L/min for 4
sec. Under these operating conditions, the volume of air drawn through the inhaler corresponds to 4 L, which
represent the normal inspiratory capacity of an average sized-adult male of 70 kg. Samples were recovered from each
stage of the NGI and the VRZ content was determined by a validated HPLC method. Mass median aerodynamic
diameter (MMAD), geometric standard deviation (GSD) and fine particle fraction (FPF) (% aerosol < 5 µm) of the
emitted dose were calculated from the NGI results.
Short Term Storage Stability
Storage stability of optimised formulation was determined as per USFDA guidelines.[4]
Optimised formulation was
stored under two conditions: Condition 1: 25ºC and 60% RH and Condition 2: 40ºC and 75% RH in climate controlled
cabinet and assessed for their chemical stability and aerosol performance for up to a 3 months.
Calu-3 cell viability
Calu-3 cell viability for the spray dried VRZ only and L-Leucine modified VRZ was carried out according to the
previous published method.[5]
Briefly, cells were seeded at the density of 5×104
cells/well, incubated overnight and
treated with the increasing equivalent concentrations of VRZ (1.2 nM to 300 µM) for both the spray dried products for
72 h. 20 μL of the CellTiter 96®
Aqueous assay (MTS reagent) (Promega, Madison, USA) was added to each well to
assess the viability of the cells. The plates were incubated for 3 hours at 37°C in humidified 5% CO2 atmosphere. The
absorbance was measured at 490 nm using a Wallac 1420 VICTOR 2 Multilabel Counter (Wallac, Waltham, USA).
In vivo lung bioavailability
Animal experimentation were carried out after obtaining ethical clearances from the Institutional Animal Ethics
Committee of the National Institute of Pharmaceutical Education and Research (NIPER), S.A.S Nagar India. Balb/c
mice of either sex (20-25 g) were divided in two groups: Group 1 (40 animals) were dosed with optimised inhalable
formulation (target VRZ dose 10 mg/kg) using a custom built in house apparatus while Group 2 (40 animals) received
an intravenous VRZ dose (10mg/kg). At predetermined time points (10 min, 30 min, 1, 2, 4, 8, 12 and 24 h), five mice
were euthanised with pentobarbital injection. Whole blood was collected following cardiac puncture and lungs were
also excised and stored at -20ºC until further analysis. VRZ was quantified by validated HPLC method following
homogenisation of lung tissue according Beinborn et al
protocol with minor modifications.[6]
Results and Discussion
Dry powder formulation containing VRZ (8 mg/mL) and
L-Leucine (2 mg/mL) was found to have optimum
characteristics for inhalation therapy. Figure 1 shows
the representative scanning electron microscopy images
of spray dried VRZ alone and optimised L-Leucine
modified VRZ microparticles (VRZ_LEU_20). Spray
dried VRZ exhibited irregular plate like morphology with
crystalline structure. However, with the inclusion of L-
Leucine in the spray drying feed, the morphology of
composite particles were found to be more regular and
spherical. Particle size analysis by laser diffraction
indicated median volume diameters (dv0.5) of 4.52 ±
0.07 μm and 2.64 ± 0.05 (n=3) for VRZ alone and VRZ_LEU_20, respectively.
10 µm
B
10 µm
A
Figure 1 Representative scanning electron microscopy
images (A) Spray dried VRZ alone and (B) VRZ LEU 20

8. The in vitro aerosolisation performance of the
spray dried VRZ alone and VRZ_LEU_20 is
shown in Figure 2. The MMAD and FPF (%
aerosol < 5µm) of VRZ alone was found to be
6.12 ± 0.18 µm and 20.86 ± 1.98 %,
respectively, while for VRZ_LEU_20, it was
found to be 3.79 ± 0.02 µm and 60.00 ± 0.94
%, respectively. Incorporation of L-Leucine
clearly lead to an improvement (p<0.05) of the
aerosolisation performance of the spray dried
composite particles. L-Leucine probably
increased aerosol performance by reducing
particle agglomeration, thus promoting particle
deagglomeration and delivery.[7]
The optimised formulation (VRZ_LEU_20) was
found to be chemically stable in terms when
stored for 3 months at room temperature as
well as accelerated storage conditions. No
significant change (p>0.05) in the aerosol
performance of VRZ_LEU_20 was observed
when powders were stored at 25ºC and 60%
RH for three months. However, nearly 10%
decrease in FPF (% aerosol < 5µm) of
VRZ_LEU_20 was observed when it was stored at 40ºC and 75% RH. This clearly revealed that the optimised
formulation should be protected from high humidity and high temperature conditions for its optimal performance.
The dose response cytotoxicity profile of spray dried VRZ alone and VRZ_LEU_20 on Calu-3 cells is shown in Figure
3. Calu-3 cells could tolerate (nearly 90% cell viability) a wide range of VRZ concentrations, from 1.2 nM to 300 µM
indicating that it can be safely administered to the lungs in this range.
Figure 4 shows the plasma and lung VRZ concentration time profiles following intravenous administration of VRZ
solution and inhalation delivery of optimised formulation (VRZ_LEU_20). In vivo lung bioavailability studies in murine
model suggested that inhalable VRZ formulation (VRZ_LEU_20) was able to reach higher VRZ concentrations in the
lungs compared to intravenous administration, thereby, enhancing the therapeutic effect of the drug at the disease
site. Total lung VRZ exposure AUC 0-∞ was found to be 524.31 ± 170.05 mg/kg h wet lung weight and 32.89 ± 9.95
mg/kg h wet lung weight when administered through inhalation and intravenous delivery, respectively. Similarly, Cmax
in the lungs was found to be 1095.25 ± 277.92 µg/g and 13.48 ± 5.35 µg/g when VRZ was administered through
inhalation and intravenous route of administration, respectively.
Concentration of VRZ (mM)
10-1
100
101
102
103
104
105
106
Calu-3CellViability(%)
60
80
100
120
140
160
(A)
Concentration of VRZ_LEU_20 (mM)
10-1 100 101 102 103 104 105 106
Calu-3CellViability(%)
40
60
80
100
120
140
(B)
D
eviceThroat
Stage
1Stage
2Stage
3Stage
4Stage
5
Stage
6Stage
7Stage
8
0
10
20
30
40
50
VRZ
VRZ_LEU_20
%VRZDeposition
Figure 2 Aerodynamic particle size distribution profile of VRZ and
VRZ_LEU_20 with NGI at a flow rate of 60 L/min. For each stage,
VRZ is shown as a percentage of its total actual recovered
amount. (n=3; mean ± SD)
Figure 3 The effect of VRZ (A) and VRZ_LEU_20 (B) on Calu-3 Cell viability following 72 h VRZ
treatment. (n=3; mean ± SD)

9. Conclusions
IPA is a serious disease in immunocompromised patients with unmet medical needs. Pulmonary delivery of high dose
of VRZ could serve as attractive therapeutic alternative for the treatment of IPA. The present study confirmed the
suitability of L-Leucine modified VRZ formulation for the inhalation therapy. The formulation was found to be high
dispersible, stable for 3 months under room temperature conditions and non-toxic to the pulmonary epithelial cells. In
addition, murine pharmacokinetics studies revealed that inhalable VRZ formulation can achieve higher concentrations
of VRZ in the lungs as compared to conventional intravenous administration, thereby, may lead to better therapeutic
outcome.
Acknowledgments
Authors are thankful to Director, NIPER, Woolcock Institute of Medical Research and Max Planck Institute for Polymer
Research for providing necessary infrastructure facilities. SA is the recipient of an Endeavour Research Fellowship
and German Academic Exchange Service (DAAD) Scholarship from the Australian and German government,
respectively, in 2014 and the work was carried out as a part of these fellowships.
References
1. Patterson, T. F: Advances and challenges in management of invasive mycoses. Lancet 2005; 366: pp1013-1025.
2. T.J. Walsh, E.J. Anaissie, D.W. Denning, R. Herbrecht, D.P. Kontoyiannis, K.A. Marr, V.A. Morrison, B.H. Segal,
W.J. Steinbach, D.A. Stevens, J.A. van Burik, J.R. Wingard, T.F. Patterson, A: Infectious Diseases Society of,
Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America, Clin Infect Dis
2008; 46: pp 327-360.
3. Hilberg, O., Andersen, C. U., Henning, O., Lundby, T., Mortensen, J., Bendstrup, E: Remarkably efficient inhaled
antifungal monotherapy for invasive pulmonary aspergillosis. Eur Respir J 2012; 40: pp 271-273.
4. F. Draft, Guidance for industry—metered dose inhaler (MDI) and dry powder inhaler (DPI) drug products,
Chemistry, manufacturing, and controls documentation Oct. 1998.
5. Haghi, M., Young, P. M., Traini, D., Jaiswal, R., Gong, J., Bebawy, M: Time- and passage-dependent
characteristics of a Calu-3 respiratory epithelial cell model. Drug Dev. Ind. Pharm. 2010; 36: pp 1207-1214.
6. N.A. Beinborn, J. Du, N.P. Wiederhold, H.D. Smyth, R.O. Williams, 3rd
: Dry powder insufflation of crystalline and
amorphous voriconazole formulations produced by thin film freezing to mice. Eur J Pharm Biopharm 2012; 81: pp 600-
608
7. L. Cruz, E. Fattal, L. Tasso, G.C. Freitas, A.B. Carregaro, S.S. Guterres, A.R. Pohlmann, N. Tsapis: Formulation
and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery. J Control Release
2011; 152: pp 370-375.
Time (h)
0 5 10 15 20 25 30
VRZConcentration(µg/g)
0.01
0.1
1
10
100
1000
Lung (IV)
Lung (IL)
Time (h)
0 5 10 15 20 25 30
VRZConcentration(µg/ml)
0.01
0.1
1
10
100
Plasma (IV)
Plasma (IL)
(A) (B)
Figure 4 Voriconazole (VRZ) concentration–time plots following intravenous (IV) and inhalation (IL)
delivery (mean ± standard deviation) (n = 5) for (A) Plasma and (B) Lung.

10. © Copyright Aedestra Ltd 2016. All rights reserved.
SURFACE MODIFIED VORICONAZOLE DRY POWDER INHALABLE FORMULATION FOR
THE TREATMENT OF INVASIVE PULMONARY ASPERGILLOSIS
6
Presenta6on
Courtesy of Sumit Arora

11. Sumit Arora
National Institute of Pharmaceutical
Education and Research (NIPER), S.A.S
Nagar, INDIA
10.12.2015
7

12. Background of the Research -
Discontent
Rationale for Selection of the Drug
and Formulation
Experimental Results: Formulation Design and
Characterisation
Conclusion and
Acknowledgements
8

13. "The person who
takes medicine must
recover twice, once
from the disease and
o n c e f r o m t h e
medicine."
- William Osler, M.D.
9

14. 10
IPA is an increasingly common opportunistic fungal infection usually
occurring in patients with neutropenia and/or corticosteroid exposure. The
lungs are involved in about 85% of cases of IPA. Mortality rate exceeds 50%
in neutropenic patients and reaches 90% in haematopoietic stem-cell
transplantation recipients
Inhalation of
spores
Infected
Lungs
http://www.jpmoldcontrol.com/faq/health-hazards.shtml Sabins et al; Lung
India, 2012, 29(2); pg 185-186

15. N N
F CH3
N
N
N
F
F
OH
M o l e c u l a r
Formula
C16H14F3N5O
M o l e c u l a r
Weight
349.311 g/mol
pKa 1.76
Log P 1.8
Mel6ng Point 127-130 °C
Solubility Low water solubility (0.7
VORICONAZOLE
(VRZ)
11
Commericially available VRZ
Formulations administered as
tablet or an intravenous injection

16. 12
Poor lung distribu*on following
peroral administra*on
Drug toxicity and interac*ons
associated with systemic
exposure
Erra*c and variable
pharmacokine*cs of VRZ

17. 13
Investigate the potential of VRZ microparticles as dry
powder for Inhalation

18. 14
VRZ and L-
leucine (varying
concentration) in
Ethanol and
Water Mixture
(70:30 v/v)
Spray Drying
L-leucine
Modified VRZ
powder
Inlet Temp 125ºC
Aspirator 100%
Q Flow Maximum
Feed Rate 5%
Outlet Temperature
78ºC
Feed Concentration 10

19. Particle Diameter (µm)
0.1 1 10 100
Volume(%)
0
2
4
6
8
VRZ
VRZ_LEU_10
VRZ_LEU_20
VRZ_LEU_30
Sample Sample Code VRZ
(mg/ml)
L-leucine
(mg/ml)
VMD
(µm)
Span Drug Loading
(%)
Entrapment
Efficiency (%)
1 VRZ 10.0 0.0 4.52 ± 0.07 2.13 ± 0.15 99.02 ± 1.79 99.02 ± 1.79
2 VRZ_LEU_10 9.0 1.0 2.63 ± 0.06 2.49 ± 0.09 89.82 ± 0.97 99.80 ± 1.08
3 VRZ_LEU_20 8.0 2.0 2.64 ± 0.05 2.50 ± 0.07 79.18 ± 1.84 98.98 ± 2.18
4 VRZ_LEU_30 7.0 3.0 2.31 ± 0.09 2.69 ± 0.04 69.92 ± 0.19 99.89 ± 0.27
15
Hydrophi
lic
Lipoph
ilic

20. 5 µm
VRZ
5 µm
VRZ_LEU
_10
5 µm
VRZ_LEU_20
5 µm
VRZ_LEU_30
Scanning electron microscopic images of (A) VRZ (B)
VRZ_LEU_10 (90:10) (C) VRZ_LEU_20 (80:10) and (D) 16

21. 17
-18
-8
2
10 60 110 160
VRZ
VRZ_LEU_10
VRZ_LEU_20
VRZ_LEU_30
50 100 150
-20
-10
0
Temp (ºC)
HeatFlow(mW)
10 20 30 40
2θ Scale
Intensity
(ArbitraryUnits)
VRZ
VRZ_LEU_10
VRZ_LEU_20
VRZ_LEU_30
Leucine
10 20 30 40
2θ ScaleIntensity
(ArbitraryUnits)
A B
A) DSC Thermograms of spray dried VRZ formulations and B) Powder X-
Ray Diffractograms of spray dried VRZ formulations
L-leucine interferes with the crystallization of VRZ during the
spray drying
132.6
°C

22. Aerodynamic particle size distribution profile of L-leucine
modified VRZ microparticles with NGI at a flow rate of 60
L/min. For each stage, VRZ is shown as a percentage of
its total actual recovered amount. (n=3; mean ± SD)
Next Generation Impactor
(NGI)
18
DeviceThroat
Stage1(>
8.06µm
)
Stage2(8.06
-4.46µm
)
Stage3(4.46
-2.82µm
)
Stage4(2.82
-1.66µm
)
Stage5(1.66
-0.94µm
)
Stage6(0.94
-0.55µm
)
Stage7(0.55
-0.34µm
)
Stage8(<
0.34µm
)
0
10
20
30
40
50
VRZ
VRZ_LEU_10
VRZ_LEU_20
VRZ_LEU_30
%DrugDeposition
Operating
Conditions
Flow Rate: 60L/
min
Time of operation:
4s

23. Formulati
on
MMAD GSD
FPF (<
5µm)
EDF (%) FPD (µg)
VRZ
6.12 ±
0.18
1.60 ± 0.02 20.86 ± 1.98 57.77 ± 2.01 703.92 ± 96.56
VRZ_LEU_
10
4.54 ±
0.08
1.49 ± 0.02 50.97 ± 1.82 72.07 ± 2.31 1419.79 ± 50.49
VRZ_LEU_
20
3.79 ±
0.02
1.70 ± 0.01 60.00 ± 0.94 81.88 ± 0.56 1892.98 ± 156.67
VRZ_LEU_
30
3.97 ±
0.35
1.66 ± 0.04 58.73 ± 5.50 79.95 ± 1.75 1583.98 ± 139.10
Formulation with 20% L-leucine showed optimal aerodynamic
properties and was selected for further cell and in vivo studies.
MMAD – Mass Median Aerodynamic Diameter GSD – Geometric
Standard Deviation
FPF – Fine Particle Fraction EDF – Emitted Dose Fraction
FPD – Fine Particle Dose
19

24. 20
Storage
Condition
MMAD
(µm)
GSD
FPF (%) (<
5µm)
EDF (%) FPD (µg)
25ºC and 60%
RH
3.71 ± 0.16
1.73 ±
0.03
60.26 ± 2.03
77.32 ±
3.43
1942.79 ±
76.28
40ºC and 75%
RH
4.38 ± 0.13
1.62 ±
0.01
50.78 ± 4.02
77.98 ±
0.58
1371.40 ±
208.38
Aerosol property of optimised formulation (VRZ_LEU_20) after storage at room and
accelerated conditions for 3 months. Data are represented as mean ± S.D (n = 3)
DeviceThroatStage
1Stage
2Stage
3Stage
4Stage
5Stage
6Stage
7Stage
8
0
10
20
30
3 month 25ºC and 60%RH
3 month 40ºC and 75%RH
0 month
%VRZDeposition

25. 21
Haghi, M., et al. (2014) Pharm Res 31:1779–1787
Schematic Diagram of In vitro Calu-3 cell
integrated Impactor

26. 22
Statistical analysis revealed no significant difference between release
profile of VRZ and VRZ_LEU_20. Co-spraying L-leucine with VRZ
did not influence the dissolution of VRZ.

27. Even at the highest concentration of VRZ and VRZ_LEU, cell
viability was above the 80%.
96 well plate 5 X 104 cells per well
1.2 nM to 300 µM
72 hours incubation Absorbance 490 nm
10-6
10-5
10-4
10-3
10-2
10-1
100
60
80
100
120
140
160
Concentration of VRZ (mM)
Calu-3viability(%)
10-6
10-5
10-4
10-3
10-2
10-1
100
60
80
100
120
140
Concentration of VRZ_LEU (mM)
Calu-3viability(%)
23

28. Voriconazole (VRZ) concentration–time plots following intravenous (IV) and inhalation (IL) delivery
(mean ± standard deviation) (n = 5) for (A) Plasma, (B) Lung, (C) Liver, (D) Kidney and (E) Spleen at
the dose of 10 mg/kg in mice model. 24

29. 25
Route/
Parameter
Plasma Lung Liver Kidney Spleen
Inhalation
AUC 0-∞ (mg/L h) 26.22 ±
9.69
N/D N/D N/D N/D
AUC 0-∞ (mg/kg h) N/D 524.31 ±
170.05
59.09 ±
18.81
47.61 ± 8.65 23.19 ± 4.75
C0 (µg/ml or µg/g) 0 1095.25 ±
277.92
0 0 0
Cmax (µg/ml or µg/
g)
8.92 ±
2.25
N/D 13.58 ±
3.97
10.03 ± 3.44 3.64 ± 0.97
Tmax (h) 0.167 N/D 2 2 2
Intravenous
AUC 0-∞ (mg/L h) 47.12 ±
7.77
N/D N/D N/D N/D
AUC 0-∞ (mg/kg h) N/D 32.89 ± 9.95 92.74 ±
20.52
66.49 ±
15.54
36.44 ± 7.77
C0 (µg/ml or µg/g) 16.13 ±
8.31
0 0 0 0
Cmax (µg/ml or µg/
g)
N/D 13.48 ± 5.35 25.14 ±
8.20
16.46 ± 5.66 7.88 ± 1.29
Tmax (h) 0 1.1 ± 0.55 1 1 1
Pharmacokinetic parameters of VRZ following inhalation and
intravenous administration in BALB/c mice (mean ± standard
deviation; n=5)
AUC0–∞, area under the concentration–time curve from time 0 to infinity; C0, Concentration at
time = 0 h; Cmax, maximum observed VRZ concentration; Tmax, time to Cmax; N/D, not
determined. Values for plasm is presented as per mL or per L while for tissue homogenates,
values are presented in per g or per Kg.
This clearly demonstrates that an inhalable VRZ dry powder delivered directly to the lung
results in high VRZ concentrations whilst simultaneously reducing its systemic exposure to
other tissues such as liver, kidney and spleen and hence reducing associated toxicities.

30. Spray dried VRZ formulation as inhalation powder
could be used as a new potential therapeutic
approach for the targeted treatment of Invasive
Pulmonary Aspergillosis
26
Further in vivo efficacy studies in animal models are
needed to be performed

31. 27
Supervisor
s
Post Doc
D r . M e h r a
Haghi
Dr. Paul M
Young
Dr. Daienla Traini
R e s p i t e c h
Group
Dr. Sanyog Jain
C P N
Lab
Dr. Michael
Kappl
AKA Butt
“None of us is as smart as all of us.” - Japanese Proverb

32. 28

33. With compliments
© Copyright Aedestra Ltd 2016. All rights reserved.
29
www.aedestra.com