Enhanced Aerosolization of High Potency Nanoaggregates of

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Enhanced Aerosolization of High Potency Nanoaggregates of Voriconazole by Dry Powder Inhalation Chaeho Moon, Alan B. Watts, Xingyu Lu, Yongchao Su, and Robert O. Williams Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00907 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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Molecular Pharmaceutics

1

Enhanced Aerosolization of High Potency Nanoaggregates of Voriconazole by Dry

2

Powder Inhalation

3

Chaeho Moon,1 Alan B. Watts,1 Xingyu Lu,2 Yongchao Su,2 Robert O. Williams III1,*

4 5 6

1College 2Merck

of Pharmacy, The University of Texas at Austin, TX, 78712, USA

Research Laboratories, Merck & Co., Inc., Kenilworth, NJ, 07033, USA

7

Abstract

8

Invasive pulmonary aspergillosis is a deadly fungal infection with a high mortality rate,

9

particularly in patients having undergone transplant surgery. Voriconazole, a triazole

10

antifungal pharmaceutical product, is considered as a first-line therapy for invasive

11

pulmonary aspergillosis, and exhibits efficacy even for patients who have failed other

12

antifungal drug therapies. The objective of this study is to develop high potency

13

nanoaggregates of crystalline voriconazole composition for dry powder inhalation

14

using the particle engineering process, thin film freezing. In this study, mannitol at low

15

concentrations acted as a surface texture-modifying agent, and we evaluated the

16

physicochemical and aerodynamic properties of the voriconazole formulations

17

containing different amounts of mannitol. In vitro aerosol performance data

18

demonstrated that powder formulations consisting of 90 to 97 % (w/w) voriconazole

19

were the optimum for inhalation with a fine particle fraction (% of delivered dose) as

20

high as 73.6 ± 3.2 % and mass median aerodynamic diameter of 3.03 ± 0.17 µm when

21

delivered by a commercially available device. The thin film freezing process enabled

22

phase-separated submicron crystalline mannitol to be oriented such as to modify the

23

surface texture of the crystalline voriconazole nanoaggregates, thus enhancing their

24

aerosolization. Addition of as low as 3 % (w/w) mannitol significantly increased the fine

25

particle fraction (% of metered dose) of voriconazole nanoaggregates when compared to

26

compositions without mannitol (40.8 % vs. 24.6 %, respectively). The aerosol

27

performance of the voriconazole nanoaggregates with 5 % (w/w) mannitol was

28

maintained for 13 months at 25 °C / 60 % RH. Therefore, voriconazole nanoaggregates

29

having low amounts of surface texture-modifying mannitol made by thin film freezing

30

are a feasible local treatment option for invasive pulmonary aspergillosis with high

31

aerosolization efficiency and drug loading for dry powder inhalation. 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1. Introduction

6

Invasive pulmonary aspergillosis (IPA) is a life threatening fungal infection.

7

Aspergillosis is one of the most prevalent fungal infections caused by Aspergillus, a

8

common mold found both indoors and outdoors. While some types of aspergillosis, like

9

allergic forms, are not life threatening,1 invasive pulmonary forms of aspergillosis have

10

a mortality rate of over 80 %,2 and were reported as the most common type of

11

pulmonary fungal infection with 69.9 % among the other types of aspergillosis.3

12

Patients having undergone transplant surgery, including solid organ transplant and

13

bone marrow transplant (BMT), are highly susceptible to fungal and bacterial infections.

14

During the post-transplant period, the most common infections include invasive

15

pulmonary aspergillosis (IPA) and invasive candidiasis,3-5 and these two invasive

16

fungal infections are diagnosed in up to 90% of fungal infections for transplant

17

recipients.5 While the mortality rate for invasive candidiasis is not as high, the mortality

18

rate for IPA is reportedly high, up to 100 % in these patients. Among transplant

19

recipients, IPA is the most prevalent in lung transplant recipients,5, 6 because the mode

20

of fungal infection is primarily through inhalation of the spores.7 There were about

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20,000 lung transplant recipients in the United States during the last decade,8 and over

22

9 % among these patients can acquire IPA.4 Since the number of transplant surgeries

23

continues to increase, more frequent occurrence of IPA is anticipated, while the

24

mortality rate in patients with IPA remains high.

25

Voriconazole is the primary treatment for IPA9 since it was first approved by the Food

26

and Drug Administration (FDA) in 2002. Other drugs used to treat IPA include

27

itraconazole, posaconazole, and amphotericin B. Voriconazole is reportedly more

28

efficacious than these other drugs, and the efficacy of voriconazole has been

29

demonstrated in IPA patients who have failed other antifungal drug therapies.10-12

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Isavuconazium sulfate was recently approved as a prodrug of isavuconazole, a new 2 ACS Paragon Plus Environment

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triazole antifungal agent for treatment of IPA by the FDA and European Medicines

2

Agency (EMA). However, isavuconazole was approved in patients 18 years of age and

3

older based on the clinical trial results showing noninferior efficacy of isavuconazole to

4

that of voriconazole. Overall response at end-of-treatment using isavuconazole was not

5

superior compared to voriconazole (35.0% vs. 38.9%, respectively).13 Therefore,

6

voriconazole remains the most successful treatment for IPA with a broader range of

7

ages above 12 years old.14

8

Oral and injectable forms of voriconazole are commercially available. However, oral or

9

intravenous administration of voriconazole to treat IPA is problematic for several

10

reasons. Firstly, a high dose is required when administrated orally or intravenously,

11

which may increase the incidence of adverse effects. Lower doses equate to fewer side

12

effects in patients.15, 16 In addition, the efficacy of voriconazole by oral or intravenous

13

administration could vary by individual. In humans, voriconazole is primarily

14

metabolized to N-oxide by cytochrome P450 enzymes, and the major metabolite is not

15

active against invasive aspergillosis. CYP2C19, which is the enzyme significantly

16

involved in the metabolism of voriconazole, exhibits genetic polymorphism. As a result,

17

up to 4-fold higher voriconazole exposure (AUCt) is expected from patients who are

18

poor metabolizers as compared to those who are homozygous extensive metabolizer

19

counterparts.17 Therefore, to overcome these problems, we believe that administration

20

of voriconazole directly to the site of infection, the lungs, is more beneficial to patients.

21

Until recently, delivery of aerosolized antifungal drugs to the lungs was limited to

22

amphotericin B.18, 19 However, Hilberg et al.20 reported that inhaled voriconazole is more

23

efficacious for treatment of IPA over that of inhaled amphotericin B, confirming that

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nebulized voriconazole formulation, initially reported by Tolman et al.,21 successfully

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treated patients with IPA who had previously failed with oral or injectable dosage

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forms of voriconazole with or without inhaled amphotericin B. Therefore, an inhaled

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voriconazole formulation can be an enhanced treatment option for IPA over oral or

28

intravenous administration of voriconazole and pulmonary administration of

29

amphotericin B to treat IPA.

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Tolman et al. reported inhaled voriconazole delivered to the lungs by nebulization.21, 22

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The emitted aerosol was suitable for inhalation (MMAD 2.98 µm, FPF 71.7 %), and

32

clinically relevant lung tissue and plasma concentrations were achieved in mice. 3 ACS Paragon Plus Environment


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However, the concentration of voriconazole in lung tissue decreased after 6 hours to

2

levels below the minimum detectable range.21 In addition, the potency of the nebulized

3

formulation was also very low, only 5.9 % (w/w) with sulfobutylether-β-cyclodextrin

4

sodium (SBECD) as an excipient. The safety of SBECD delivered by pulmonary route

5

has not been confirmed yet, and this high amount of inactive ingredient can cause

6

serious side effects.16 Voriconazole formulations for dry powder inhalation (DPI) were

7

reported using poly-lactide-co-glycolide nanoparticles by Sinha et al.23 and poly-lactide

8

microparticles by Arora et al.,24 but the drug loading was low for these particles (31 %

9

and 20 % w/w, respectively). Arora et al. reported another voriconazole powder

10

formulation for DPI using leucine as an excipient.25 However, all of these DPI powder

11

formulations include non-GRAS excipients that have not been used for inhaled drugs

12

approved by FDA. Beinborn et al. also developed amorphous and crystalline

13

voriconazole formulations suitable for dry powder inhalation using the particle

14

engineering technology, thin film freezing (TFF).26,

15

formulation contained 75 % (w/w) excipient and therefore has low potency, and the

16

drug absorption efficiency was low with rapid clearance based on in vivo

17

pharmacokinetic data in a mouse model. The AUC0-24h of the crystalline formulation

18

was significantly higher than that of the amorphous formulation in both lung (452.6

19

µg•h/g and 232.1 µg•h/g, respectively) and plasma (38.4 µg•h/g and 18.6 µg•h/g,

20

respectively). However, aerosol performance of the crystalline formulation was inferior

21

(FPF 37.8 %).

22

TFF is a particle engineering technology that employs an ultra rapid freezing rate of up

23

to 10,000 K/sec.28 Due to the high degree of supercooling, TFF was successfully utilized

24

to produce nanostructured aggregates.29 Using computational modeling, Longest et al.

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have found that nanoaggregates, once deposited, can be absorbed 57-fold higher than

26

microparticles, and achieve more uniform distribution in lung.30 By incorporation into a

27

nanostructured aggregate, we theorize that similar in vivo attributes will be imparted

28

through TFF manufacturing. The enhanced absorption and microdosimetry by

29

nanostructured aggregates could provide explicit benefits for targeting drug substances

30

to the small airways.30 Also, TFF was able to produce a single polymorph of mannitol,

31

while other freezing techniques generated heterogeneous polymorphic forms of

32

mannitol.31 Spray drying is a common technique to produce micro- or nano-scale

33

particles for DPI. However, particle formation during the drying process of spray

27

However, the amorphous

4 ACS Paragon Plus Environment

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drying generally takes longer32 than the freezing process of TFF, allowing particles

2

more time to grow, generating larger size of particles. Accordingly, typical spray drying

3

methods will not have advantages of enhanced uptake and microdosimetry, which

4

nanoaggregates have as described by Longest et al.30

5

The objective of this study is to develop high potency nanoaggregates of crystalline

6

voriconazole composition for DPI using TFF technology, a bottom-up micronization

7

method. This paper is focused on further enhancing aerosol performance of crystalline

8

voriconazole powder formulation reported by Beinborn et al.,26, 27 and using excipients

9

that are used in approved inhalation aerosol products. The hypothesis is that addition

10

of low amounts of excipients can enhance the aerosol performance of the high potency

11

crystalline nanoaggregates voriconazole powder formulation for DPI by TFF. High

12

potency, crystalline voriconazole nanoaggregates for DPI consisting of nano-particles

13

with modified surface texture were developed, and the characterization of the

14

formulation is presented in this paper.

15 16

2. Materials and methods

17

Materials

18

The following materials were purchased: Voriconazole (Carbosynth, San Diego, CA);

19

Kollidon® 25 (D-Basf, Ludwigshafen, Germany); acetonitrile (HPLC grade, Fisher

20

Scientific, Pittsburgh, PA); trifluoroacetic acid (TFA) (HPLC grade, Fisher Scientific,

21

Pittsburgh, PA); Tuffryn Membrane Filter (25 mm, 0.45 µm, Pall Corporation, Port

22

Washington, NY). Filtered water (Evoqua, Warrendale, PA) was used, and pyrogen free

23

mannitol, Pearlitol® PF, was generously donated from Roquette America Inc. (Geneva,

24

IL).

25

Preparation of powder for dry powder inhalation using TFF

26

Mannitol and voriconazole (30 to 100 % w/w) powders were dissolved in a mixture of

27

acetonitrile and water (50:50 v/v), and the solid content in the solution was kept as 1 %

28

w/v. Approximately 15 µL of each solution was dropped from a height of 10 cm onto a

29

rotating cryogenically cooled (-60 °C) stainless steel drum. The frozen samples were

30

collected in a stainless steel container filled with liquid nitrogen, and transferred into a 5 ACS Paragon Plus Environment


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80 °C freezer until transferred to a lyophilizer. A VirTis Advantage Lyophilizer (VirTis

2

Company Inc., Gardiner, NY) was used to remove the solvent. The samples were kept

3

at -40 °C for 21 hours, and the temperature was slowly increased to 25 °C over 21 hours,

4

and then kept at 25 °C for another 21 hours to dry. The pressure was kept at 100 mTorr

5

during the drying process.

6

X-Ray powder diffraction (XRPD)

7

Crystallinity of the powder samples was determined by X-ray diffraction (MiniFlex 600,

8

Rigaku Co., Tokyo, Japan) measuring from 5 to 35 ˚2θ (0.02 ˚ step, 3 ˚/min, 40 kV, 15

9

mA).

10

Scanning electron microscopy (SEM)

11

SEM (Zeiss Supra 40V SEM, Carl Zeiss, Heidenheim an der Brenz, Germany) was used

12

to identify the surface morphology of the samples. An aliquot of powder was placed

13

onto carbon tape, and sputter coated with 60/40 Pd/Au for 20 min before capturing the

14

images.

15

Modulated Differential Scanning Calorimetry (mDSC)

16

Thermal analysis of the powder samples was studied by differential scanning

17

calorimetry model Q20 (TA Instruments, New Castle, DE) equipped with a refrigerated

18

cooling system (RCS40, TA Instruments, New Castle, DE). Modulated DSC was

19

performed with modulation period of 50 sec, modulated amplitude of 1 ˚C, and average

20

heating rate of 5 ˚C/min. Tzero pan and Tzero hermetic lid manufactured by TA

21

Instruments were used to hold samples during the test, and a hole was made on the lid

22

with 20G syringe needle before placing the pan in the sample holder.

23

Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM/EDX)

24

SEM/EDX (Hitachi S5500 SEM/STEM, Hitachi America, Tarrytown, NY) was used to

25

identify elements of the powders produced by TFF.

26

Atomic force microscopy (AFM)

27

Two different types of atomic force microscopy were used during this study.

28

dimensional (3D) surface topography images of particles generated by TFF were

3-


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1

obtained by Asylum MFP-3D AFM (Oxford Instruments, Oxfordshire, United

2

Kingdom), equipped with an aluminum coated MikroMasch HQ:NSC15 cantilever

3

(NanoWorld AG, Neuchâtel, Switzerland), which has a resonance frequency of 325 kHz,

4

force constant of 40 N/m, and typical tip radius of 8 nm. Powders were affixed to an

5

AFM disc with carbon tape, and compressed nitrogen gas was used to blow out

6

particles which did not adhere to the carbon tape firmly. Topography was carried out

7

with tapping mode at a scan rate of 1.00 Hz, set point of 1.08 V, and integral gain of 20.0.

8

Feedback filter, drive amplitude and drive frequency were optimized for each sample,

9

and all images were collected with 512 x 512 resolution. Gwyddion software (64 bit

10

Windows version 2.50)33 was used to generate 3D topography images.

11

To obtain the image of nanoaggregates, Park XE-100 AFM (Park systems, Suwon, Korea)

12

was used, equipped with an aluminum coated Nanosensors PPP-NCHR cantilever

13

(NanoWorld AG, Neuchâtel, Switzerland), which has resonance frequency of 330 kHz,

14

force constant of 42 N/m, and tip radius of less than 7 nm. 380 µm single side polished

15

P-type silicon wafer was coated with Tween® 20 (VWR, Radnor, PA) prior to load

16

powder samples for AFM. Tween® 20 (1.5 % w/v) was previously dissolved in HPLC

17

grade methanol (Fisher Scientific, Pittsburgh, PA). The solution was dropped on to the

18

silicon wafer using a transfer pipette, and solution was removed by compressed

19

nitrogen gas. Powder was put into a DP4 insufflator (Penn-Century Inc., Wyndmoor,

20

PA), and aerosolized on to the silicon wafer using a 3 mL syringe. After aerosolized

21

powder was loaded on the silicon wafer, compressed nitrogen gas was used to remove

22

powder solids that were not strongly adhered to the silicon wafer. Tapping mode was

23

applied to collect images of 512 x 512 resolution with a scan rate of 0.30 Hz. Other

24

values for AFM were optimized for each sample. The topography image was processed

25

by Gwyddion software (64 bit Windows version 2.50).33

26

Aerodynamic particle size distribution analysis

27

Aerodynamic particle size was determined by a Next Generation Pharmaceutical

28

Impactor (NGI) (MSP Co. Shoreview, MN), connected with High Capacity Pump

29

(model HCP5, Copley Scientific, Nottingham, UK) and Critical Flow Controller (model

30

TPK 2000, Copley Scientific, Nottingham, UK). A #3 HPMC capsule (VCaps plus,

31

Capsugel, Morristown, NJ), containing TFF powder (approximately 5 to 10 mg), was

32

placed into high resistant RS01 dry powder inhaler (Plastiape, Osnago, Italy), and 7 ACS Paragon Plus Environment


Page 8 of 42

1

dispersed into the NGI through the USP induction port at the flow rate of 60 L/min for 4

2

seconds per each actuation. The pre-separator was not used for entire test. NGI

3

collection plates were coated with 2 % w/v polysorbate 20 in methanol and allowed to

4

dry for 20 min before use. After aerosolization, the powder was extracted with the

5

mixture of water and acetonitrile (50:50 v/v), and analyzed voriconazole contents by

6

HPLC. Mass median aerodynamic diameter (MMAD), geometric standard deviation

7

(GSD), and fine particle fraction (FPF) were calculated based on the dose deposited on

8

device (capsule and device), induction port (adapter and induction port), stages 1

9

through 7, and micro-orifice collector (MOC) using Copley Inhaler Testing Data

10

Analysis Software (CITDAS) version 3.10 (Copley Scientific, Nottingham, UK).

11

High-performance liquid chromatography (HPLC)

12

A Dionex Ultimate 3000 HPLC system (Sunnyvale, CA) and Shimadzu DGU 14A

13

degasser (Shimadzu, Kyoto, Japan) were used to measure the quantity of voriconazole

14

contents. A Waters Xbridge C18 column (4.6 x 150 mm, 3.5 µm) (Milford, MA) was used.

15

The method details are as follows: an isocratic method for aerodynamic properties

16

using a mobile phase of 40/60 (v/v) water/acetonitrile containing 0.1 % (v/v) TFA and a

17

flow rate of 0.8 mL/min for 4 min; and a gradient method for chemical degradants

18

during stability study. For the gradient method, acetonitrile containing 0.1 % (v/v) TFA

19

was gradually increased from 25 to 95 % (v/v) for 14 min, mixed with water containing

20

0.1 % (v/v) TFA, and a flow rate was 0.8 mL/min. For both methods, the samples were

21

analyzed at a detection wavelength of 254 nm at 25 ˚C. Linearity was performed

22

between 50 ng/mL and 100 µg/mL with using an injection volume of 15 µL.

23

Solution Nuclear magnetic resonance (Solution NMR)

24

1H

25

mannitol of the TFF-VCZ-MAN powders. All 1H NMR spectra were recorded in

26

dimethyl sulfoxide-d6 (DMSO-d6) at 600 MHz on a VNMR 600 (Varian, Palo Alto, CA)

27

spectrometer at 25 ˚C. Chemical shifts were recorded relative to 2.47 ppm of DMSO-d6.

28

Solid-state Nuclear Magnetic Resonance (ssNMR)

29

ssNMR Experiments were carried out on a Bruker Avance III HD 400 MHz

30

spectrometer (Bruker, Billerica, MA) at 25 ˚C, with a magic angle spinning (MAS)

31

frequency of 12 kHz. Bruker 4 mm triple resonance HFX probe was utilized in the

NMR was performed to calculate the weight ratio between voriconazole and


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1

double-resonance modes tuned to 1H/13C or 1H/19F frequencies. All samples were

2

packed under ambient conditions in 4 mm ZrO2 rotors (Wilmad-LabGlass, PA). One-

3

dimensional (1D)

4

with a linearly ramped power level of 80-100 kHz during a 2 ms contact period on the

5

1H

6

used at a field strength of 80 kHz. Same power parameters, contact time, MAS

7

frequency were employed for 2-dimensional (2D)

8

(HETCOR) experiments. Adamantine was used as an external standard for calibrating

9

13C

13C

and

19F

cross-polarization (CP) MAS experiment was conducted

channel for enhancing CP efficiency. High power SPINAL64 proton decoupling was 13C-1H

CP heteronuclear correlation

chemical shift, with the ethyl 13C peak referenced at 38.48 ppm.

10

Fourier-transform Infrared Spectroscopy (FT-IR)

11

Nicolet™ iS™ 50 FT-IR equipped with Smart OMNI-Sampler™ (ThermoFisher

12

Scientific, Waltham, MA) was used to study intermolecular activity between

13

voriconazole and mannitol of TFF-VCZ-MAN powders. The measurement was

14

performed with the sample as a dry powder, and a spectral range of 4000 to 700 cm-1

15

was recorded at aperture of 150, resolution of 4, and scan numbers of 32.

16

Brunauer-Emmett-Teller (BET) specific surface area (SSA) analysis

17

Monosorb™ rapid surface area analyzer model MS-21 (Quantachrome Instruments,

18

Boynton Beach, FL) was used to measure SSA of TFF-VCZ-MAN powders by single-

19

point BET method. Samples were outgassed with nitrogen gas at 20 psi at ambient

20

temperature for 24 hours to remove surface impurities. A mixture of nitrogen/helium

21

(30:70 v/v) was used as the adsorbate gas.

22

Shear force resistance test

23

To test shear force resistance of TFF-VCZ-MAN 95:5 powders, the powders were placed

24

into a stainless steel container (inner diameter 2⅞ inch, height 4 ¼ inch), and pre-

25

sheared by rolling the container at 85 rpm. The powder sample was taken at 15, 30, and

26

60 min, and the aerodynamic property was compared with the initial condition.

27

Dissolution test

28

An in vitro dissolution method was used to quantite dissolution of voriconazole from

29

powders processed by TFF technology. Franz cell apparatus was used to enable 9 ACS Paragon Plus Environment


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differentiation of voriconazole release from powders produced by TFF. A Next

2

Generation Pharmaceutical Impactor (NGI) (MSP Co. Shoreview, MN), connected with

3

High Capacity Pump (model HCP5, Copley Scientific, Nottingham, UK) and Critical

4

Flow Controller (Model TPK 2000, Copley Scientific, Nottingham, UK) was used to load

5

aerosolized powders on a Tuffryn membrane filter (25 mm, 0.45 µm, Pall Corporation,

6

Port Washington, NY). Five nozzles at stage 2 on the lid of NGI were blocked with lab

7

tape, and only 1 nozzle was left opened. A Tuffryn membrane filter was placed and

8

fixed with lab tape on the collection cup under the opened nozzle at stage 2. A #3

9

HPMC capsule (VCaps plus, Capsugel, Morristown, NJ), containing TFF powder

10

(approximately 5 to 10 mg), was placed into high resistant RS01 dry powder inhaler

11

(Plastiape, Osnago, Italy), and dispersed into the NGI through the USP induction port

12

at the flow rate of 60 L/min for 4 seconds per each actuation. A pre-separator was not

13

used. After aerosolization, the powder-loaded (approximately 0.5 to 1 mg) membrane

14

filter was carefully removed from the collection cup, and placed on top of a receptor

15

chamber of Franz cell that was previously filed with degassed 10 mM phosphate

16

buffered saline (PBS), pH 7.4 (5 mL). To minimize generation of air bubbles in a

17

receptor chamber during the test, the 10 mM PBS was preheated at 37 ˚C for two hours

18

while degassing. A donor chamber was placed on the membrane filter, and the

19

membrane filter was fastened between receptor and donor chambers with a pinch

20

clamp. Parafilm was used to cover the top of donor chamber. Dissolution test was

21

conducted at sink conditions at 37 ˚C while magnetic bars were stirring in receptor

22

chambers. Dissolution media (150 µL) was withdrawn at timed intervals of 0, 20, 40, 60,

23

120, and 180 min for HPLC analysis without dilution. Fresh dissolution media was

24

replaced after each sampling.

25

Collection of aerosolized particles and preparation of SEM samples during

26

dissolution

27

The Fast Screening Impactor (FSI) (Copley Scientific, Nottingham, UK) connected with

28

High Capacity Pump (model HCP5, Copley Scientific, Nottingham, UK) and Critical

29

Flow Controller (model TPK 2000, Copley Scientific, Nottingham, UK) was used to

30

record SEM images of aerosolized TFF-VCZ-MAN powders before and during the

31

dissolution test,. A #3 HPMC capsule (VCaps plus, Capsugel, Morristown, NJ),

32

containing the TFF powder (approximately 5 to 10 mg), was placed into a high

33

resistance RS01 dry powder inhaler device (Plastiape, Osnago, Italy), and dispersed into 10 ACS Paragon Plus Environment

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1

a glass fiber filter (MSP Co. Shoreview, MN) set in the FSI to collect particles of

2

aerodynamic size 5 µm or less at the flow rate of 60 L/min for 4 seconds. Once the

3

particles were collected on the filter, they were transferred on to carbon tape, previously

4

attached on the SEM specimen, by tapping the carbon tape on the filter, and the SEM

5

image was recorded.

6

To record SEM images during the dissolution test, a glass fiber filter loaded with

7

powders from the FSI was cut round (25 mm diameter). The glass fiber filter was then

8

placed between a donor chamber and a receptor chamber of Franz cell, previously filled

9

with PBS, pH 7.4, at 37 ˚C. The filter was left on the Franz cell for 5 min, and placed in to

10

a -80 ˚C freezer for 1 hour. A VirTis Advantage Lyophilizer (VirTis Company Inc.,

11

Gardiner, NY) was used to remove the solvent at 25 °C for 5 hours. Carbon tape,

12

previously attached on SEM specimen, was tapped on the glass fiber filter to transfer

13

the TFF-VCZ-MAN powders, and SEM image was recorded as described previously.

14

Stability study

15

TFF-VCZ-MAN 95:5 dry powders were pre-sheared in a glass bottle, as described in

16

shear force resistance test. Between 7.6 mg and 8.4 mg of the pre-sheared powder was

17

filled into a size #3 HPMC capsule (Capsugel, Morristown, NJ). 14 capsules filled with

18

powders were transferred in a scintillation vial, and the vial was purged with nitrogen

19

gas for 20 sec before closing with a cap. The vial was sealed in an aluminum foil (13 x 15

20

cm), previously purged with nitrogen gas inside for 30 sec, and the aluminum foils

21

were kept at 25 ˚C/60 %RH. Purity and aerosol performance were performed at each

22

time point of 1, 3, 6, and 13 months.

23

Statistical analysis

24

Aerodynamic performance and cumulative drug release were compared for statistical

25

analysis by the student t-test. P-value < 0.05 was considered as significantly different.

26

JMP® 10.0.0 was used to compare the significance of the data.

27

3. Results

28

Physicochemical properties of voriconazole dry powder formulations

29

The TFF technology was used to produce crystalline voriconazole powder formulations 11 ACS Paragon Plus Environment


Page 12 of 42

1

containing mannitol. XRPD and mDSC were mainly employed to determine

2

crystallinity of the formulations. TFF-VCZ powder formulations including mannitol

3

were identified as crystalline as shown in Figs. 1 and 2. The TFF-VCZ-MAN powder

4

formulations exhibited characteristic voriconazole peaks of XRPD corresponding to

5

voriconazole bulk powder (e.g., 12.4 °2θ and 13.6 °2θ) and δ-mannitol (e.g., 9.5 °2θ and

6

20.2 °2θ) as shown in Fig. 1. These indicate that the powder formulations consist of

7

crystalline voriconazole and δ-mannitol. The intensity of δ-mannitol peaks decreased as

8

amounts of mannitol (% w/w) were reduced in the TFF-VCZ-MAN powder

9

formulations, and the peaks corresponding to δ-mannitol were not detectable when the

10

powder formulations contained 5 % (w/w) mannitol. TFF-MAN dry powder was

11

mainly δ-form, while trace amounts of α- and β-forms were detected by XRPD (13.5 °2θ

12

and 14.5°2θ respectively).

13

mDSC also confirmed crystallinity of the TFF-VCZ-MAN powder formulations. Fig. 2

14

shows no glass transition detected in the TFF-VCZ-MAN 95:5 and TFF-VCZ-MAN 50:50,

15

but only endotherm peaks corresponding to melting of voriconazole and mannitol. TFF-

16

VCZ had a melting endotherm peak at 130.86 °C with a heat of fusion of 105.3 J/g. When

17

expected heats of fusions for voriconazole in TFF-VCZ-MAN powders are calculated

18

by % fraction (w/w), the heats of fusions for voriconazole in TFF-VCZ-MAN 95:5 and

19

TFF-VCZ-MAN 50:50 were 100.0 J/g and 52.6 J/g respectively. The measured heats of

20

fusion for voriconazole were 95.1 J/g for TFF-VCZ-MAN 95:5, and 33.7 J/g for TFF-VCZ-

21

MAN 50:50, and these were 95.1 % and 64.0 % of the expected values. TFF-MAN had a

22

melting endotherm peak at 167.31 °C with a heat of fusion of 187.5 J/g. The expected

23

heats of fusions for mannitol in TFF-VCZ-MAN 95:5 and TFF-VCZ-MAN 50:50 were

24

9.38 J/g and 93.8 J/g, respectively. The measured heats of fusion for mannitol were 2.63

25

J/g and 63.2 J/g, respectively, and these were 28.0 % and 67.4 % of the expected values.

26

Table 2 presents composition ratios of voriconazole to mannitol (voriconazole:mannitol

27

w/w) in the two formulations tested by mDSC. The ratios were calculated by integration

28

of proton peaks using 1H-NMR. Theoretical ratio of one proton for TFF-VCZ-MAN 95:5

29

is 1:0.1009, and the experimental ratio was calculated as 1:0.0992 that represented 98.3 %

30

of expected mannitol was found in TFF-VCZ-MAN 95:5. In the case of TFF-VCZ-MAN

31

50:50, 100 % of expected mannitol was detected by 1H-NMR.

32

Particle morphology of TFF-VCZ-MAN powders is presented in Fig. 3. Agglomeration

33

of micron-size particles was observed in the TFF-VCZ powders, and those particles 12 ACS Paragon Plus Environment

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1

were also found in other TFF-VCZ-MAN powder formulations. More porous matrix

2

was observed with TFF-VCZ-MAN powders containing higher amounts of mannitol.

3

3D topography and illustration of TFF-VCZ and TFF-VCZ-MAN 95:5 powders shown

4

in Fig. 4 confirms that the surface texture of TFF-VCZ-MAN 95:5 powders is rough,

5

while that of TFF-VCZ powders is smooth. High resolution topography of TFF-VCZ-

6

MAN 95:5 powders in Fig. 5 indicates that TFF-VCZ-MAN 95:5 powders are

7

nanoaggregates consisting of about 150 – 500 nm nano-particles. SSAs of these TFF-

8

VCZ-MAN powders are shown in Table 1. The TFF-VCZ powders indicated the lowest

9

SSA (8.36 m2/g), and the porous matrix of TFF-MAN dry powder exhibited the highest

10

SSA (17.11 m2/g). The SSA increased as more mannitol was added to TFF-VCZ-MAN

11

powder formulations. By SEM/EDX shown in Fig. 6, the micron-size particles were

12

identified as being composed of voriconazole nanoaggregates with detection of

13

nitrogen, oxygen, and fluorine. The porous matrix was identified as mannitol by

14

detection of oxygen without nitrogen and fluorine.

15

The FT-IR peak pattern of TFF-VCZ powder was matched with that of voriconazole

16

bulk powder, and the same peak pattern was also found with TFF-VCZ-MAN powders

17

containing different amounts of mannitol. The peak pattern of TFF-MAN was also

18

found from TFF-VCZ-MAN powders. Therefore, the peaks only corresponding to TFF-

19

VCZ and TFF-MAN were observed in TFF-VCZ-MAN powders, and no new peak was

20

found on the FT-IR spectrum of TFF-VCZ-MAN powders, as shown in Fig. 7. 1D 13C

21

and

22

overlap of

23

shows identical spectra in TFF-VCZ and TFF-VCZ-MAN. Moreover, the sharp 13C and

24

19F

25

voriconazole and mannitol. 2D 1H-13C HETCOR spectrum of TFF-VCZ-MAN 90:10 was

26

compared with spectrum of TFF-VCZ in Fig. 9. Intermolecular cross-peaks between

27

voriconazole and mannitol from TFF-VCZ-MAN 90:10 were not observed.

28

In vitro aerosol performance and stability

29

Aerodynamic particle size distribution of TFF-VCZ-MAN powder formulations was

30

determined by a NGI, and the FPF (% of metered) is presented in Table 1. Based on the

31

FPF (% of metered dose) data, TFF-VCZ-MAN powder formulations consisting of 90 to

32

97 % (w/w) voriconazole exhibited the highest aerosolization. FPF (% of metered dose)

19F

CP-MAS spectra by ssNMR are shown in Fig. 8. Voriconazole has no spectral 13C

peaks with mannitol and possesses all resonances in the

19F

spectra. It

peaks in the spectra of TFF-VCZ-MAN 90:10 confirm the crystallinity of both



Page 14 of 42

1

of TFF-VCZ-MAN 97:3 was significantly higher (p < 0.05) than that of TFF-VCZ with 66%

2

improvement in FPF (% of metered dose). Aerosol performance of TFF-VCZ-MAN

3

powders containing 90 to 97 % (w/w) voriconazole were not significantly different (p >

4

0.05). Aerosol performance of TFF-VCZ-MAN powder formulations declined when

5

greater than 10 % (w/w) mannitol was included in the composition.

6

The influence of physical force on aerosol performance of TFF-VCZ-MAN 95:5 powder

7

formulation was also investigated by measuring FPF using the NGI. As shown in Figs.

8

10 and 11, particle size distribution and aerosol performance changes by different time

9

of shear force was monitored. At 15, 30, and 60 min, FPFs (% of metered) were 44.3, 47.5,

10

and 42.4 % respectively, and FPFs (% of delivered dose) were 68.7, 73.6, and 69.5 %

11

respectively. The initial value before applying shear force was 40.0 % for FPF (% of

12

metered dose) and 58.8 % for FPF (% of delivered dose). While a change in MMAD was

13

also observed from 3.7 µm at the initial time to 3.2, 3.0, and 3.1µm at 15, 30, and 60 min,

14

respectively, no significant change was found for the GSD.

15

A stability study was performed at 25 °C/60 %RH, and the purity and aerosol

16

performance changes of TFF-VCZ-MAN 95:5 powder formulation were monitored for

17

13 months as shown in Fig. 12. Purity of voriconazole in TFF-VCZ-MAN 95:5 was

18

maintained, and no degradant was detected during test period of time. To compare

19

aerosol performance over the stability study, FPF (% of metered), FPF (% of delivered),

20

MMAD, and GSD were compared at each time point. There was no statistically

21

significant difference on FPF (% of metered) for 13 months, as well as FPF (% of

22

delivered) (both p > 0.05). While GSD after 1 month decreased from the initial value (p
0.05).

24

Dissolution of voriconazole dry powder formulations

25

For dissolution testing of TFF-VCZ-MAN powder formulations, pH 7.4 PBS was used as

26

the receptor media, and the top of donor chamber of the Franz-cells was covered with

27

parafilm to prevent loss of dissolution media by evaporation. The dissolution rate of

28

crystalline TFF-VCZ-MAN 95:5 was compared with amorphous TFF-VCZ-PVPK25

29

25:75, and the crystalline dry powder showed significantly slower cumulative drug

30

release over the test time period (p < 0.05) as shown in Fig. 13. Cumulative voriconazole

31

release at 3 hours for amorphous TFF-VCZ-PVPK25 was 63.2 %, while that for

32

crystalline TFF-VCZ-MAN 95:5 was only 22.8 %. Cumulative voriconazole released at 3 14 ACS Paragon Plus Environment

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1

hours for TFF-VCZ-MAN 25:75 and TFF-VCZ-MAN 50:50 was 46.3 and 35.3 %,

2

respectively.

3

4. Discussion

4

Decision of mannitol as a single excipient

5

Geometric particle size of voriconazole powder formulations made by TFF was tested

6

with different excipients, including mannitol, lactose, glycine, arginine, leucine, lecithin,

7

magnesium stearate, calcium carbonate, and titanium oxide (data not shown). Up to 3

8

different excipients with different level of amounts were tested, and the geometric

9

particle size of voriconazole powder formulation was found the smallest with mannitol

10

used as a single excipient. While lactose is popularly used as an excipient for many

11

other dry powder formulations, voriconazole powder formulation including lactose

12

made by TFF was not suitable, due to high hygroscopicity.

13

Characterizations of voriconazole dry powder formulations

14

Voriconazole27,

15

transition temperatures below room temperature. Therefore, TFF-VCZ-MAN was

16

hypothesized to be crystalline unless there are strong intermolecular interactions

17

between voriconazole and mannitol to prevent crystallization. The TFF-VCZ-MAN

18

powder formulations were crystalline based on the XRPD data and the sharpness of 1D

19

CP-MAS spectra, indicating that there are not sufficiently strong interactions between

20

voriconazole and mannitol.

21

While XRPD is useful to characterize the crystallinity of powders, it may not detect low

22

amounts of amorphicity in the formulations. Therefore, mDSC was conducted on TFF-

23

VCZ-MAN powders. The TFF-VCZ-MAN dry powders were crystalline, since only two

24

endothermic melting peaks of voriconazole and mannitol were detected. However,

25

melting point depression was observed for mannitol especially in the TFF-VCZ-MAN

26

95:5. The low heat of fusion of mannitol in TFF-VCZ-MAN 95:5 could have occurred

27

because of a relatively low amount of mannitol dissolved in melted voriconazole before

28

a temperature reaches the melting point of mannitol. Also, mannitol particles in TFF-

29

VCZ-MAN 95:5 are typically 100-200 nm, and these nanoscale particles can lower the

30

heat of fusion. To confirm a potency of mannitol in TFF-VCZ-MAN powders that

31

showed melting point depression, the molecular ratio between voriconazole and

34

and mannitol35 have the strong tendency to crystallize, and glass



Page 16 of 42

1

mannitol was determined by 1H-NMR. While NMR is commonly used for qualitative

2

analysis, quantitative NMR analysis is also applicable.36, 37 The experimental molecular

3

ratios between voriconazole and mannitol matched well with the theoretical values in

4

both of TFF-VCZ-MAN 95:5 and TFF-VCZ-MAN 50:50. Moreover, 13C and 19F ssNMR

5

have often been used to confirm crystalline polymorphism and identify low levels of

6

amorphous drug substance in solid dosage forms.38, 39 The identical peak positions and

7

line widths of voriconazole resonances in 13C and 19F CP-MAS spectra of TFF-VCZ and

8

TFF-VCZ-MAN 90:10 confirm the crystallinity and suggest no quantifiable amorphous

9

content.

10

FTIR was used to study chemical interactions between voriconazole and mannitol. The

11

hydroxyl group of voriconazole is related to its degradation pathway,40 and it could be

12

the most active site if there are any intermolecular interactions. If this occurred, this

13

would shift the FT-IR peaks of voriconazole ranging between 3100 cm-1 and 3500 cm-1.41

14

There are two peaks corresponding to voriconazole in this range, and they are at 3118.9

15

cm-1 and 3198.4 cm-1. These two peaks are observed in all of the TFF-VCZ-MAN and

16

TFF-VCZ powder formulations, and no shift of these peaks was discovered. In case of

17

mannitol in this range, a peak at 3276.6 cm-1 was observed, and no shift was observed. If

18

there are interactions between mannitol and aromatic secondary amines of voriconazole,

19

peak shifts can be noticed between 1230cm-1 and 1300 cm-1.41 Four peaks from

20

voriconazole at 1241.5 cm-1, 1248.8 cm-1, 1268.5 cm-1, and 1277.6 cm-1 were detected in

21

this range, but no significant peak shift was found when mannitol was included in the

22

voriconazole powder formulations. Therefore, these FT-IR data support that there is no

23

or very weak interactions between voriconazole and mannitol in TFF-VCZ-MAN

24

powder formulations.

25

While FT-IR is typically utilized to identify conformation and intermolecular

26

interactions, ssNMR can provide more in-depth atomic-level information for structural

27

investigation.42 In this research, 1D

28

conformational changes. All voriconazole peaks showed no difference in chemical shifts

29

between TFF-VCZ and TFF-VCZ-MAN 90:10. Moreover, 2D

30

were acquired for investigating structural perturbations at a better resolution. This

31

result confirms no

32

resolution, chemical shifts of all aliphatic and aromatic protons in the indirect

33

dimension also exhibit no observable changes. Besides, 2D

13C

13C

and

19F

CP-MAS were utilized to investigate 13C-1H

HETCOR spectra

chemical shift change in the direct dimension. With the given 13C-1H

HETCOR has been 16

ACS Paragon Plus Environment

Page 17 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

utilized for detecting drug substance-excipient interactions.43 No inter-molecular cross

2

peaks, i.e. interactions, have been observed between voriconazole and mannitol at the

3

given spectral intensity.

4

Two different shapes of particles were observed in TFF-VCZ-MAN powder

5

formulations, and it was initially thought that the micron-size particles were

6

voriconazole, and the porous matrices were mannitol, based on the observed particle

7

morphologies of TFF-VCZ and TFF-MAN. To confirm this, chemical compositions of

8

these particles were confirmed by SEM/EDX. However, the locations that detected

9

oxygen, fluorine, and nitrogen overlapped with each other during the initial SEM/EDX

10

run, presenting particles that consisted of both voriconazole and mannitol. The cause

11

was later identified. Since the measuring depth of EDX is micron scale, the detection

12

beam passed through all of particle depth of TFF-VCZ-MAN 50:50 powders tested. To

13

overcome this problem, the powder was dispersed widely on carbon tape on a

14

specimen holder, and a spot analysis was performed to determine chemical

15

compositions of two different morphologies of particles. By spot analysis, the micron-

16

size particle was identified as voriconazole nanoaggregates based on the chemical

17

compositions of oxygen, nitrogen, and fluorine, while the porous matrix was identified

18

as mannitol, showing chemical composition of oxygen without nitrogen and fluorine.

19

Therefore, it was concluded that crystalline mannitol was phase-separated from

20

crystalline voriconazole during the TFF process.

21

While the AFM image in Fig. 5 shows that TFF-VCZ-MAN powders are nanoaggregates,

22

the BET data is also supportive for the formation of voriconazole nanoaggregates. When

23

SEM images present that TFF-VCZ particles are much greater than high porous matrix

24

of TFF-MAN, the specific surface area of TFF-MAN is only about twice greater than that

25

of TFF-VCZ. This can be because voriconazole particles are nanoaggregates having

26

more specific surface area than visually seen on the SEM image.

27

Level of mannitol affects aerosol performance and dissolution rate

28

The amount of mannitol in the TFF-VCZ-MAN powders affected their morphology.

29

When low amount of mannitol was included, submicron mannitol particles were

30

formed by prevention of particle growth as a result of high supercooling during the TFF

31

process.28 These particles existed on the surface of voriconazole nanoaggregates, and

32

modified their surface texture. These submicron mannitol particles were not taken out 17 ACS Paragon Plus Environment


Page 18 of 42

1

from the surface of voriconazole nanoaggregates during aerosolization. This may be

2

explained by the difficulty in removing nano-size particles from the surface. While

3

cohesive and adhesion forces are proportional to the diameter of particles, removal

4

forces are proportional to the cube of the diameter for gravitational, vibrational, and

5

centrifugal forces.44 Therefore, submicron mannitol particles were difficult to separate

6

from voriconazole nanoaggregates, and the rough surface texture of voriconazole

7

nanoaggregates

8

aerosolization. As the amount of mannitol in TFF-VCZ-MAN powders increased, large

9

porous mannitol matrices were produced. These did not only exist on the surface of

10

voriconazole nanoaggregate particles, but also were surrounding them. Multiple

11

voriconazole nanoaggregates were assembled as the large porous mannitol matrix

12

caused them to remain together. These aggregate structures remained during

13

aerosolization. As a result, these large aggregated particles decreased aerosol

14

performance of TFF-VCZ-MAN powder formulations that contained more than 10%

15

(w/w) of mannitol.

16

Aerosol performance of formulations for DPI significantly relies on cohesive and

17

adhesive forces of the particles. These forces include van der Waals, surface tension of

18

adsorbed liquid films, and electrostatic forces.45 All these are influenced by particle

19

shape and size, surface roughness/texture, relative humidity, temperature, duration and

20

velocity of particle contact.44, 46-48 Among these forces, van der Waals forces are the most

21

important.44 Since van der Waals forces are attractive forces induced by dipoles

22

between molecules, they decrease greatly when the distance between surfaces of

23

particles reaches the separation distance.44 Therefore, rougher surfaces reduce van der

24

Waals forces critically by keeping further average particle distances. Surface roughness

25

affects not only van der Waals forces, but also surface tension, which is induced by

26

surface moisture. A smooth surface of particles and high relative humidity lead to

27

stronger surface tension. Electrostatic force, however, relies on the particle size. Particles

28

bigger than 0.1 µm can generate electrostatic force.44 This attractive electrostatic force is

29

stronger with larger particles, and is also related with relative humidity; low humidity

30

retains the charges on the particles for longer time. Still, the electrostatic force is

31

typically considered smaller than van der Waals and surface tension forces.44 Hence,

32

surface roughness and texture of particles plays a significant role in aerosol

33

performance of formulations for DPI.

was

maintained

during

aerosolization,

resulting

in

greater


Page 19 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

The morphological changes of the powder formulations caused by different amounts of

2

mannitol notably affected the aerosol performance of TFF-VCZ-MAN powder

3

formulations. The aerosol performance was altered by the change of cohesive and

4

adhesive forces of particles, and lowering these forces are related with the reduced

5

contact areas between particles,46 in addition to further distance between particles.44 By

6

including low quantities of submicron mannitol particles, the contact areas of TFF-VCZ-

7

MAN nanoaggregates was significantly reduced, and the distance between

8

voriconazole particles were further apart as shown illustrations in Fig. 4. Compared to

9

the TFF-VCZ powder, TFF-VCZ-MAN 99:1 powder showed a significant improvement

10

in FPF (% of metered dose) (p < 0.05). This improvement by the addition of mannitol

11

continued up to 3% (w/w) of mannitol was added in the formulation. An increase of

12

about 5 % in FPF (% of metered dose) was achieved by the addition of 1 % (w/w)

13

mannitol to formulations containing 97 % to 100 % (w/w) of voriconazole. Without

14

excipients added, crystalline voriconazole powder formulations for DPI have relatively

15

high deposition in device, when made by spray drying24 or TFF, and tested with RS01

16

dry powder inhaler. This causes lower emitted dose. However, TFF-VCZ-MAN 95:5

17

powders exhibited higher emitted dose compared to TFF-VCZ powders (57 % vs. 37 %

18

respectively). This enhanced emitted dose was accomplished as a result of reduced

19

adhesion forces of particles to the device. Since TFF produces TFF-VCZ-MAN powders

20

that contain very small amounts of moisture (less than 0.1 % w/w, data not shown), and

21

voriconazole and mannitol are not hygroscopic, the surface tension forces are expected

22

to be relatively low on these particles. Storing powders in low humidity environment

23

can generate electrostatic forces, but these forces are considered much smaller than van

24

der Waals and surface tension forces.44 Accordingly, reducing contact areas of particles

25

and furthering particle distance by modifying surface textures were primarily involved

26

in lowering cohesive and adhesive forces of the TFF-VCZ-MAN powder formulations

27

that led to the aerosol performance improvement. Young et al. similarly described the

28

relationship between aerosol performance and separation energy between particles49

29

that corresponds well with our results.

30

Different amounts of mannitol in the TFF-VCZ-MAN powders not only affected aerosol

31

performance, but also dissolution rate. TFF-VCZ-MAN powders containing higher

32

amount of mannitol exhibited increased dissolution rates, and this could be explained

33

by faster wetting of the powders by mannitol. For TFF-VCZ-MAN powders including 19 ACS Paragon Plus Environment


Page 20 of 42

1

high amount of mannitol, the surrounding mannitol particles, that were enclosing

2

voriconazole, were wetted and dissolved very quickly. Therefore, voriconazole

3

nanoaggregates were surrounded by the dissolution media in a short time, and the

4

dissolution rate became faster. SEM picture of TFF-VCZ-MAN 25:75 powders presented

5

that most mannitol particles dissolved in less than 5 min on the Franz-cells, while

6

submicron mannitol particles were still observed on the surface of voriconazole

7

nanoaggregates from TFF-VCZ-MAN 95:5 powders. This represented that voriconazole

8

nanoaggregates did not get wet quickly when only a small amount of mannitol was

9

included to the powder formulations.

10

Benefits of TFF process

11

High potency nanoaggregates of voriconazole powder formulations were made by TFF.

12

While DPI formulations without carriers have been reported previously,50, 51 carriers are

13

commonly included in DPI formulations. However, carrier based DPI formulations are

14

generally low drug potency. Also, many factors, such as particle size,52 size

15

distribution,53 and surface morphology52,

16

aerosol performance during aerosolization, and such factors have negative effects on

17

deposited dose uniformity.55 By using TFF, the maximum aerosol performance of TFF-

18

VCZ-MAN nanoaggregates was attained with as low as 3 % (w/w) mannitol; therefore

19

the potency of optimized TFF-VCZ-MAN powder formulation can be up to 97 % (w/w).

20

This high drug potency with a very low level of excipient requires less powder to be

21

delivered, and the issues, such as low potency and deposited dose nonuniformity,

22

generally caused by carriers can be eliminated.

23

High potency DPI formulations can be also made by other techniques, such as milling,

24

for example. Even though the size of particles produced by milling and suitable for lung

25

delivery is a few microns, such particles are considered as single discrete micron-size

26

particles. As nanoaggregates, voriconazole DPI formulations made by TFF can have

27

significantly higher total lung absorption efficiency and uniformity of dose distribution

28

based on the study by Longest et al.30 These voriconazole nanoaggregates are expected

29

to allow for better epithelial coverage where fungal colonies are present. TFF was able

30

to produce nanoaggregates, because rapid nucleation with a freezing rate of up to

31

10,000 K/sec allowed for a narrower particle size distribution and lower Ostwald

32

ripening, producing a larger number of nuclei and preventing particle growth during

54

of carrier particles, influence the powder


Page 21 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

the freezing process.28, 56 The small size of unfrozen channels and the rapidly increased

2

viscosity of unfrozen solution28 made similar size of voriconazole nanoaggregates.

3

Surface modification of particles can be also accomplished by TFF. Begat et al.

4

previously reported surface modification of particles using hydrophobic materials, such

5

as lecithin, leucine, and magnesium stearate. While particles processed by dry

6

mechanical fusion processes, like mechanofusion, presented improved aerosol

7

performance with or without carriers by lowering surface free energy,57, 58 this process

8

was based on blending of drug substances with force controlling agents, like lecithin,

9

leucine, and magnesium stearate. Mechanofusion process requires mechanical energy

10

input to the formulation, and can cause chemical instability of the drug. In addition,

11

surface modification by blending may be applicable only to discrete micron-size

12

particles, not nanoaggregates due to possible deaggregation of aggregates by blending.

13

Kawashima et al. also reported surface modification of particles by various methods,

14

such as mechanical sheared mixing, freezing, or spray drying.59 With a hydrophilic

15

additive, such as light anhydrous silicic acid (AEROSIL), the surface of hydrophobic

16

particles converted to hydrophilic, and the surface modified particles presented

17

improved inhalation behaviors in vitro. However, this method uses discrete micron-

18

sized drug particles, and cannot be used for nanoaggregates. Therefore, these discrete

19

micron-sized particles processed by other methods cannot attain the enhanced uptake

20

and microdosimetry of those nanoaggregates described by Longest et al.30 By TFF,

21

however, energy input was not needed to modify surfaces of particles. Surface

22

modification of voriconazole nanoaggregates by phase-separated, submicron mannitol

23

particles, which individually existed on the surface of drug nanoaggregates, was carried

24

out due to rapid freezing rate that prevents particle growth.

25

5. Conclusion

26

High potency (up to 97 % w/w) nanoaggregates of crystalline voriconazole powder

27

formulations intended for dry powder inhalation were successfully developed using

28

TFF technology. A low amount of mannitol, used as a single excipient, favorably

29

enhanced the aerosol performance of voriconazole nanoaggregates by the phase-

30

separated submicron crystalline mannitol acting as a surface texture-modifying agent.

31

Voriconazole dry powder for inhalation made by TFF is a viable local treatment option

32

for invasive pulmonary aspergillosis with high aerosolization efficiency and drug 21 ACS Paragon Plus Environment


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1

loading while offering the potential benefits associated with deposition of

2

nanoaggregates in the airway.

3

Acknowledgements

4

The authors would like to thank Lung Therapeutics, Inc. for the financial support. The

5

authors are also thankful to Roquette America, Inc. for the kind donation of Pearlitol®

6

PF.

7 8 9 10 11 12

Table 1. Summary and aerodynamic property of voriconazole dry powder formulations

13

investigated using thin-film freezing (TFF) technology. Sample

Drug:Excipient ratio (w/w)

Dissolved solids

Solvent compositions

TFF-VCZ

No excipient

1.0% (w/v)

TFF-VCZ-MAN 99:1

99:1

1.0% (w/v)

TFF-VCZ-MAN 98:2

98:2

1.0% (w/v)

TFF-VCZ-MAN 97:3

97:3

1.0% (w/v)

TFF-VCZ-MAN 95:5

95:5

1.0% (w/v)

TFF-VCZ-MAN 93:7

93:7

1.0% (w/v)

TFF-VCZ-MAN 90:10

90:10

1.0% (w/v)

TFF-VCZ-MAN 85:15

85:15

1.0% (w/v)

TFF-VCZ-MAN 80:20

80:20

1.0% (w/v)

TFF-VCZ-MAN 70:30

70:30

1.0% (w/v)

TFF-VCZ-MAN 50:50

50:50

1.0% (w/v)

TFF-VCZ-MAN 25:75

25:75

1.0% (w/v)

Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v) Water:acetonitrile 50:50 (v/v)

Emitted dose (%) 36.5

FPF, of metered (%)

SSA (m2/g)

24.6

8.4

42.2

29.9

-

50.8

34.5

-

50.5

40.8

-

56.6

38.1

10.6

53.3

39.7

-

53.7

39.7

-

56.1

36.0

-

66.9

34.3

-

59.1

32.7

-

55.8

28.5

16.3

-

-

-


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TFF-MAN

No drug

1.0% (w/v)

TFF-VCZ-PVPK 25

25:75

1.0% (w/v)

Water:acetonitrile 50:50 (v/v) 1,4-dioxane

-

-

17.1

-

-

-

1 2 3 4 5 6 7 8

9 10

Figure 1. XRPD of (a) Voriconazole powder; (b) TFF-VCZ; (c) TFF-VCZ-MAN 95:5; (d)

11

TFF-VCZ-MAN 70:30; (e) TFF-VCZ-MAN 50:50; (f) TFF-MAN.

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1 2 3 4 5 6 7

8 9 10

Figure 2. Modulated DSC of (a) TFF-MAN; (b) TFF-VCZ; (c) TFF-VCZ-MAN 95:5; (d) TFF-VCZ-MAN 50:50.

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1 2 3 4 5 6 7 8 9 10

Table 2. Quantitative comparison of voriconazole and mannitol in TFF-VCZ-MAN 95:5

Voriconazole Chemical shift (ppm) TFF-VCZMAN (95:5) TFF-VCZMAN (50:50)

11

Mannitol Number of proton

Integratio n

3.91

1

10.13

3.91

1

1.02

Chemical shift (ppm)

Experimental 1H integration ratio (VCZ:MAN)

Number of proton

Integratio n

Theoretical 1H integration ratio (VCZ:MAN)

4.10

2

2.01

1:0.1009

1:0.0992

4.10

2

3.91

1:1.917

1:1.917

and TFF-VCZ-MAN 50:50 by 1H-NMR.

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1

2

3



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1 2

Figure 3. SEM images of TFF-VCZ-MAN: (a) TFF-VCZ; (b) TFF-VCZ-MAN 95:5

3

(×20,000); (c) TFF-VCZ-MAN 70:30; (d) TFF-VCZ-MAN 50:50; (e) TFF-VCZ-MAN 25:75;

4

(f) TFF-MAN; (g) aerosolized TFF-VCZ-MAN 95:5; (h) aerosolized TFF-VCZ-MAN

5

50:50; (i) TFF-VCZ-MAN 25:75, after 5 min in Franz cells ; (j) TFF-VCZ-MAN 95:5, after

6

5 min in Franz cell; (k) TFF-VCZ-MAN 95:5 (×1,000); (l) TFF-VCZ-MAN 95:5 (×5,000).

7

8


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1 2

Figure 4. SEM images of: (a) TFF-VCZ; (b) TFF-VCZ-MAN 95:5, 3D topography image

3

of: (c) TFF-VCZ; (d) TFF-VCZ-MAN 95:5, and illustration of contact area and distance

4

between particles of: (e) TFF-VCZ; (f) TFF-VCZ-MAN 95:5.

5

6 7

Figure 5. AFM topography image of aerosolized TFF-VCZ-MAN 95:5 by DP4 insufflator

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1 2

Figure 6. SEM/EDX data of TFF-VCZ-MAN 50:50: (a) SEM image; (b) elemental analysis

3

of spot A; (c) elemental analysis of spot B.

4 5

6 7

Figure 7. FT-IR of (a) voriconazole Powder; (b) TFF-VCZ; (c) TFF-VCZ-MAN 95:5; (d)

8

TFF-VCZ-MAN 70:30; (e) TFF-VCZ-MAN 50:50; (f) TFF-MAN.

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1 2 3 4 5 6

7 8

Figure 8. 1D CP-MAS spectra of (a) TFF-VCZ; and (b) TFF-VCZ-MAN 90:10. 13C and 19F

9

spectra are shown on the left and right column, respectively.

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1 2

3 4

Figure 9. 2D 1H-13C HETCOR spectra of (a) TFF-VCZ; and (b) TFF-VCZ-MAN 90:10.

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1 2

3 4

Figure 10. Aerodynamic particle size distribution profile of TFF-VCZ-MAN 95:5 by time

5

sheared: (blue) at 0 min; (red) at 15 min; (green) at 30 min; (purple) at 60min (n = 3;

6

mean ± SD).

7 8 9 10 11 12 13 14 15 16 17 18


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1

2 3

Figure 11. Aerodynamic properties of TFF-VCZ-MAN 95:5 by time sheared: (a) FPF, %

4

of delivered; (b) FPF, % of metered; (c) MMAD; (d) GSD (n = 3; mean ± SD). (FPF, % of

5

delivered = FPF dose / emitted dose; FPF, % of metered = FPF dose / recovered dose)

6 7 8 9 10



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1

2 3

Figure 12. Aerodynamic properties of TFF-VCZ-MAN 95:5 by time stored at 25°C/60%

4

RH: (a) FPF, % of delivered; (b) FPF, % of metered; (c) MMAD; (d) GSD (n = 3; mean ±

5

SD).

6 7 8 9 10 11


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1 2

Figure 13. Cumulative voriconazole release (%) of (a) TFF-VCZ-PVPK25 25:75

3

(amorphous); (b) TFF-VCZ-MAN 25:75; (c) TFF-VCZ-MAN 50:50; (d) TFF-VCZ-MAN

4

95:5 (n = 3; mean ± SD).

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1. Skowronski, E.; Fitzgerald, D. A. Life-threatening allergic bronchopulmonary aspergillosis in a well child with cystic fibrosis. The Medical journal of Australia 2005, 182, (9), 482-3. 2. Chamilos, G.; Kontoyiannis, D. P. Update on antifungal drug resistance mechanisms of Aspergillus fumigatus. Drug Resist Updat 2005, 8, (6), 344-58. 3. Lin, S. J.; Schranz, J.; Teutsch, S. M. Aspergillosis case-fatality rate: systematic review of the literature. Clin Infect Dis 2001, 32, (3), 358-66. 4. Pappas, P. G.; Alexander, B. D.; Andes, D. R.; Hadley, S.; Kauffman, C. A.; Freifeld, A.; Anaissie, E. J.; Brumble, L. M.; Herwaldt, L.; Ito, J.; Kontoyiannis, D. P.; Lyon, G. M.; Marr, K. A.; Morrison, V. A.; Park, B. J.; Patterson, T. F.; Perl, T. M.; Oster, R. A.; Schuster, M. G.; Walker, R.; Walsh, T. J.; Wannemuehler, K. A.; Chiller, T. M. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET). Clin Infect Dis 2010, 50, (8), 1101-11. 5. Vazquez, J. A.; Miceli, M. H.; Alangaden, G. Invasive fungal infections in transplant recipients. Ther Adv Infect Dis 2013, 1, (3), 85-105. 6. Patterson, J. E. Epidemiology of fungal infections in solid organ transplant patients. Transpl Infect Dis 1999, 1, (4), 229-36. 7. Munksgaard, B. Fungal infections. Am J. Transplant 2004, 4, (Suppl 10), 110-134. 8. Transplants in the U.S. by Recipient ABO. Jan.31, 2018 ed.; Health Resources and Services Administration, U.S. Department of Health & Human Services: Organ Procurement and Transplantation Network, 2018. 9. Walsh, T. J.; Anaissie, E. J.; Denning, D. W.; Herbrecht, R.; Kontoyiannis, D. P.; Marr, K. A.; Morrison, V. A.; Segal, B. H.; Steinbach, W. J.; Stevens, D. A.; van Burik, J.-A.; Wingard, J. R.; Patterson, T. F.; Infectious Diseases Society of, A. Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008, 46, (3), 327-60. 10. Herbrecht, R.; Denning, D. W.; Patterson, T. F.; Bennett, J. E.; Greene, R. E.; Oestmann, J.-W.; Kern, W. V.; Marr, K. A.; Ribaud, P.; Lortholary, O.; Sylvester, R.; Rubin, R. H.; Wingard, J. R.; Stark, P.; Durand, C.; Caillot, D.; Thiel, E.; Chandrasekar, P. H.; Hodges, M. R.; Schlamm, H. T.; Troke, P. F.; de Pauw, B.; Invasive Fungal Infections Group of the European Organisation for, R.; Treatment of, C.; the Global Aspergillus Study, G. Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis. N Engl J Med 2002, 347, (6), 408-15. 11. Chandrasekar, P. H.; Cutright, J.; Manavathu, E. Efficacy of voriconazole against invasive pulmonary aspergillosis in a guinea-pig model. The Journal of antimicrobial chemotherapy 2000, 45, (5), 673-6. 12. Garbino, J.; Rohner, P.; Kolarova, L.; Ondrusova, A.; Lew, D. Successful treatment of pulmonary invasive aspergillosis with voriconazole in patients who failed conventional therapy. Infection 2003, 31, (4), 241-3. 13. CRESEMBA®. Mar. 6, 2015 ed.; U.S. Food & Drug Administration: 2015. 14. VFEND®. Jul. 28, 2017 ed.; U.S. Food & Drug Administration: 2017. 38 ACS Paragon Plus Environment

Page 39 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43


15. Daughton, C. G.; Ruhoy, I. S. Lower-dose prescribing: minimizing "side effects" of pharmaceuticals on society and the environment. Sci Total Environ 2013, 443, 324-37. 16. Wong, Y. L. Adverse effects of pharmaceutical excipients in drug therapy. Ann Acad Med Singapore 1993, 22, (1), 99-102. 17. Li, T. Y.; Liu, W.; Chen, K.; Liang, S. Y.; Liu, F. The influence of combination use of CYP450 inducers on the pharmacokinetics of voriconazole: a systematic review. Journal of clinical pharmacy and therapeutics 2017, 42, (2), 135-146. 18. Le, J.; Schiller, D. S. Aerosolized Delivery of Antifungal Agents. Curr Fungal Infect Rep 2010, 4, (2), 96-102. 19. Borro, J. M.; Sole, A.; de la Torre, M.; Pastor, A.; Fernandez, R.; Saura, A.; Delgado, M.; Monte, E.; Gonzalez, D. Efficiency and safety of inhaled amphotericin B lipid complex (Abelcet) in the prophylaxis of invasive fungal infections following lung transplantation. Transplant Proc 2008, 40, (9), 3090-3. 20. 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, (1), 271-3. 21. Tolman, J. A.; Nelson, N. A.; Son, Y. J.; Bosselmann, S.; Wiederhold, N. P.; Peters, J. I.; McConville, J. T.; Williams, R. O., 3rd. Characterization and pharmacokinetic analysis of aerosolized aqueous voriconazole solution. Eur J Pharm Biopharm 2009, 72, (1), 199-205. 22. Tolman, J. A.; Wiederhold, N. P.; McConville, J. T.; Najvar, L. K.; Bocanegra, R.; Peters, J. I.; Coalson, J. J.; Graybill, J. R.; Patterson, T. F.; Williams, R. O., 3rd. Inhaled voriconazole for prevention of invasive pulmonary aspergillosis. Antimicrobial agents and chemotherapy 2009, 53, (6), 2613-5. 23. Sinha, B.; Mukherjee, B.; Pattnaik, G. Poly-lactide-co-glycolide nanoparticles containing voriconazole for pulmonary delivery: in vitro and in vivo study. Nanomedicine 2013, 9, (1), 94-104. 24. Arora, S.; Haghi, M.; Loo, C.-Y.; Traini, D.; Young, P. M.; Jain, S. Development of an inhaled controlled release voriconazole dry powder formulation for the treatment of respiratory fungal infection. Mol Pharm 2015, 12, (6), 2001-9. 25. Arora, S.; Haghi, M.; Young, P. M.; Kappl, M.; Traini, D.; Jain, S. Highly respirable dry powder inhalable formulation of voriconazole with enhanced pulmonary bioavailability. Expert opinion on drug delivery 2016, 13, (2), 183-93. 26. Beinborn, N. A.; Du, J.; Wiederhold, N. P.; Smyth, H. D. C.; Williams, R. O., 3rd. Dry powder insufflation of crystalline and amorphous voriconazole formulations produced by thin film freezing to mice. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e V 2012, 81, (3), 600-8. 27. Beinborn, N. A.; Lirola, H. L.; Williams, R. O., 3rd. Effect of process variables on morphology and aerodynamic properties of voriconazole formulations produced by thin film freezing. Int J Pharmaceut 2012, 429, (1-2), 46-57. 28. Engstrom, J. D.; Lai, E. S.; Ludher, B. S.; Chen, B.; Milner, T. E.; Williams, R. O., 3rd; Kitto, G. B.; Johnston, K. P. Formation of stable submicron protein particles by thin film freezing. Pharm Res 2008, 25, (6), 1334-46. 29. Sinswat, P.; Overhoff, K. A.; McConville, J. T.; Johnston, K. P.; Williams, R. O., 3rd. 39 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Page 40 of 42

Nebulization of nanoparticulate amorphous or crystalline tacrolimus--single-dose pharmacokinetics study in mice. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e V 2008, 69, (3), 1057-66. 30. Longest, P. W.; Hindle, M. Small Airway Absorption and Microdosimetry of Inhaled Corticosteroid Particles after Deposition. Pharm Res 2017, 34, (10), 2049-2065. 31. Moon, C.; Watts, A. B.; Williams III, R. O. Thin film freezing for production of an amorphous mannitol/lysozyme formulation for inhalation: A comparison of freezing techniques. Respir Drug Deliv 2016, 3, 611-616. 32. Wisniewski, R., Spray Drying Technology Review. In 45th International Conference on Environmental Systems, Bellevue, Washington, 2015. 33. Necas, D.; Klapetek, P. Gwyddion: an open-source software for SPM data analysis. Dent. Eur. J. Phys. 2012, 10, (1), 181-188. 34. Ramos, J. J. M.; Diogo, H. P. The slow relaxation dynamics in active pharmaceutical ingredients studied by DSC and TSDC: Voriconazole, miconazole and itraconazole. Int J Pharmaceut 2016, 501, (1-2), 39-48. 35. Yu, L.; Mishra, D. S.; Rigsbee, D. R. Determination of the glass properties of D-mannitol using sorbitol as an impurity. J Pharm Sci 1998, 87, (6), 774-7. 36. Espina, R.; Yu, L.; Wang, J.; Tong, Z.; Vashishtha, S.; Talaat, R.; Scatina, J.; Mutlib, A. Nuclear magnetic resonance spectroscopy as a quantitative tool to determine the concentrations of biologically produced metabolites: implications in metabolites in safety testing. Chemical research in toxicology 2009, 22, (2), 299-310. 37. Pauli, G. F.; Godecke, T.; Jaki, B. U.; Lankin, D. C. Quantitative 1H NMR. Development and potential of an analytical method: an update. J Nat Prod 2012, 75, (4), 834-51. 38. Correa-Soto, C.; Trasi, N. S.; Schmitt, P. D.; Su, Y.; Liu, Z.; Miller, E.; Variankaval, N.; Marsac, P. J.; Simpson, G. J.; Taylor, L. S. Second harmonic generation microscopy as a tool for the early detection of crystallization in spray dried dispersions. Journal of pharmaceutical and biomedical analysis 2017, 146, 86-95. 39. Offerdahl, T. J.; Salsbury, J. S.; Dong, Z.; Grant, D. J. W.; Schroeder, S. A.; Prakash, I.; Gorman, E. M.; Barich, D. H.; Munson, E. J. Quantitation of crystalline and amorphous forms of anhydrous neotame using 13C CPMAS NMR spectroscopy. J Pharm Sci 2005, 94, (12), 2591-605. 40. Shaikh, K. A.; Patil, A. T. A validated stability-indicating liquid chromatographic method for determination of degradation impurities and diastereomers in voriconazole tablets. Sci Pharm 2012, 80, (4), 879-88. 41. Silverstein, R. M.; Webster, F. X.; Kiemle, D. J., Spectrometric identification of organic compounds. 7th ed.; John Wiley & Sons: Hoboken, NJ, 2005; p x, 502 p. 42. Tian, D.; Li, T.; Zhang, R.; Wu, Q.; Chen, T.; Sun, P.; Ramamoorthy, A. Conformations and Intermolecular Interactions in Cellulose/Silk Fibroin Blend Films: A Solid-State NMR Perspective. The journal of physical chemistry B 2017, 121, (25), 6108-6116. 43. Pham, T. N.; Watson, S. A.; Edwards, A. J.; Chavda, M.; Clawson, J. S.; Strohmeier, M.; Vogt, F. G. Analysis of amorphous solid dispersions using 2D solid-state NMR and (1)H T(1) relaxation measurements. Mol Pharm 2010, 7, (5), 1667-91. 44. Hinds, W. C., Aerosol technology : properties, behavior, and measurement of airborne 40 ACS Paragon Plus Environment

Page 41 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38


particles. 2nd ed.; Wiley: New York, 1999; p xx, 483 p.

45. Hickey, J.; Concessio, N.; Platz, R. Factors influencing the dispersion of dry powders as aerosols. Pharm Tech 1994, 18, 58-64. 46. Beach, E. R.; Tormoen, G. W.; Drelich, J.; Han, R. Pull-off force measurements between rough surfaces by atomic force microscopy. J Colloid Interface Sci 2002, 247, (1), 84-99. 47. Tan, B. M. J.; Chan, L. W.; Heng, P. W. S. Improving Dry Powder Inhaler Performance by Surface Roughening of Lactose Carrier Particles. Pharm Res 2016, 33, (8), 1923-35. 48. Price, R.; Young, P. M.; Edge, S.; Staniforth, J. N. The influence of relative humidity on particulate interactions in carrier-based dry powder inhaler formulations. Int J Pharmaceut 2002, 246, (1-2), 47-59. 49. Young, P. M.; Cocconi, D.; Colombo, P.; Bettini, R.; Price, R.; Steele, D. F.; Tobyn, M. J. Characterization of a surface modified dry powder inhalation carrier prepared by "particle smoothing". J Pharm Pharmacol 2002, 54, (10), 1339-44. 50. Yazdi, A. K.; Smyth, H. D. Carrier-free high-dose dry powder inhaler formulation of ibuprofen: Physicochemical characterization and in vitro aerodynamic performance. Int J Pharm 2016, 511, (1), 403-14. 51. Yazdi, A. K.; Smyth, H. D. Hollow crystalline straws of diclofenac for high-dose and carrier-free dry powder inhaler formulations. Int J Pharm 2016, 502, (1-2), 170-80. 52. Du, P.; Du, J.; Smyth, D. C. Evaluation of Granulated Lactose as a Carrier for DPI Formulations: Effect of Drug Loading. 2014, 743-746. 53. Steckel, H.; Muller, B. W. In vitro evaluation of dry powder inhalers II: Influence of carrier particle size and concentration on in vitro deposition. Int J Pharm 1997, 154, (1), 31-37. 54. Flament, M.-P.; Leterme, P.; Gayot, A. The influence of carrier roughness on adhesion, content uniformity and the in vitro deposition of terbutaline sulphate from dry powder inhalers. Int J Pharmaceut 2004, 275, (1-2), 201-9. 55. Du, P.; Du, J.; Smyth, H. D. Evaluation of Granulated Lactose as a Carrier for Dry Powder Inhaler Formulations 2: Effect of Drugs and Drug Loading. J Pharm Sci 2017, 106, (1), 366-376. 56. Overhoff, K. A.; Johnston, K. P.; Tam, J.; Engstrom, J.; Williams III, R. O. Use of thin film freezing to enable drug delivery: a review. J. Drug Del. Sci. Tech. 2009, 19, (2), 89-98. 57. Begat, P.; Price, R.; Harris, H.; Morton, D. A. V.; Staniforth, J. N. The Influence of Force Control Agents on the Cohesive-Adhesive Balance in Dry Powder Inhaler Formulations. KONA Powder Part J 2005, 23, 109-121. 58. Begat, P.; Morton, D. A. V.; Shur, J.; Kippax, P.; Staniforth, J. N.; Price, R. The role of force control agents in high-dose dry powder inhaler formulations. J Pharm Sci 2009, 98, (8), 2770-83. 59. Kawashima, Y.; Serigano, T.; Hino, T.; Yamamoto, H.; Takeuchi, H. A new powder design method to improve inhalation efficiency of pranlukast hydrate dry powder aerosols by surface modification with hydroxypropylmethylcellulose phthalate nanospheres. Pharm Res 1998, 15, (11), 1748-52.

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