In Vitro Evaluation of Inhalable Verapamil-Rifapentine Particles for

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In Vitro Evaluation of Inhalable VerapamilRifapentine Particles for Tuberculosis Therapy Thaigarajan Parumasivam, John Gan Yar Chan, Angel Pang, Diana Huynh Quan, Jamie A. Triccas, Warwick J. Britton, and Hak-Kim Chan Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00833 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Molecular Pharmaceutics

In Vitro Evaluation of Inhalable Verapamil-Rifapentine Particles for Tuberculosis Therapy

AUTHORS

T. Parumasivam1, J. G. Y. Chan1, 2, A. Pang4, D. H. Quan3, J. A. Triccas3, W. J. Britton3,4 and H. K. Chan1*

1

Advanced Drug Delivery Group, Faculty of Pharmacy, The University of Sydney, 2006 Australia

2

JHL Biotech, Inc., Hsinchu County, Taiwan

3

Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, 2006 Australia

4

Tuberculosis Research Program, Centenary Institute, The University of Sydney, Sydney, 2006 Australia

*

Corresponding author. E-mail: [email protected]

1

ACS Paragon Plus Environment


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Abstract graphic

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1

ABSTRACT

2

Recent studies have demonstrated that drug efflux pumps of Mycobacterium tuberculosis (M. tb) provide

3

a crucial mechanism in the development of drug resistant to anti-mycobacterial drugs. Drugs that inhibit

4

these efflux pumps, such as verapamil have shown the potential in enhancing the treatment success. We

5

therefore hypothesized that the combined inhaled administration of verapamil and a first-line rifamycin

6

antibiotic will further improve the treatment efficacy. An inhalable dry powder consisting of amorphous

7

verapamil and crystalline rifapentine with L-leucine as an excipient was produced by spray drying. The in

8

vitro aerosol characteristic of the powder, its microbiological activity and stability were assessed. When

9

the powder was dispersed by an Osmohaler®, the total fine particle fraction (FPFtotal, wt. % of particles in

10

aerosol < 5 µm) of verapamil and rifapentine was 77.4 ± 1.1 % and 71.5 ± 2.0 %, respectively. The

11

combination drug formulation showed a minimum inhibitory concentration (MIC90) similar to that of

12

rifapentine alone when tested against both M. tb H37Ra and M. tb H37Rv strains. Importantly, the

13

combination resulted in increased killing of M. tb H37Ra within the infected macrophage cells compared

14

to either verapamil or rifapentine alone. In assessing cellular toxicity, the combination exhibited an

15

acceptable half maximal inhibitory concentration (IC50) values (62.5 µg/mL) on both human monocytic

16

(THP-1) and lung alveolar basal epithelial (A549) cell lines. Finally, the powder was stable after 3 months

17

storage in 0 % relative humidity at 20 ± 3°C.

18 19

KEYWORDS: Rifapentine, verapamil, efflux pump inhibitor, aerosols, tuberculosis

20 21 22 23 24 25

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1

INTRODUCTION

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Tuberculosis (TB) caused by the intracellular pathogen Mycobacterium tuberculosis (M. tb) is an ancient

3

lung infectious disease that remains a major cause of global mortality. In 2013, the World Health

4

Organization (WHO) reported that there were 1.5 million deaths attributed to the disease and 6.1 million

5

people with active infection1 with a further estimated 2 billion people have latent TB.2 In addition, there

6

are alarming rates of multi-drug resistant (MDR) and extensively drug resistant (XDR) M. tb and HIV-TB

7

co-infection that contributed to the increased rates of TB.3 Despite the limitations of long-term oral

8

chemotherapy and inadequate compliance to the current treatment regimen, only two new anti-TB drugs,

9

bedaquiline and delamanid have been successfully approved in the past 50 years.4,

5

Therefore, the

10

repurposing of existing drugs into advanced drug delivery systems could be an effective approach to

11

improve the existing arsenal of TB treatments.

12

In recent years, the role of bacterial and macrophage efflux pumps in promoting M. tb drug

13

resistance has gained significant attention. First, M. tb utilise these pumps to extrude ‘toxic’ compounds

14

including anti-TB agents from the interior of the bacterial cell into their extracellular environment.

15

Overexpression of these pumps leads to reduced drug concentration in the bacterial cytoplasm thus

16

enabling intrinsic and acquired drug resistance in M. tb.6-8 For instance, Rodrigues et al. demonstrated that

17

the overexpression of bacterial efflux pumps alone can result in isoniazid-resistance in M. tb without

18

requirement of genetic mutations.8 Second, M. tb within the infected macrophages are protected from

19

high antibiotic concentrations because the macrophage efflux pumps also extrude anti-TB agents to

20

provide a low intramacrophage drug concentration.7, 9 For example, Adams et al. revealed that exposure to

21

non-lethal concentration of rifampicin within the macrophage environment transcriptionally induced M. tb

22

efflux pump Rv1258 expression which allowed the bacteria to become resistant.10 These mechanisms

23

were also further confirmed in macrophages infected with several drug sensitive and resistant M. tb

24

strains in which the rapid development of tolerance to various anti-TB agents, including isoniazid,

25

moxifloxacin,

linezolid,

PA-824,

and

bedaquliline

was

established.11

4


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1

These bacterial and macrophage efflux pump-mediated resistant mechanisms may prolong the treatment

2

duration for TB and requires a multi-drug regime for cure.12

3

Adjunctive therapy with a drug that inhibits these efflux pumps could be a promising treatment

4

approach against TB. The efflux pump inhibitor could: (1) directly block the efflux system of M. tb and

5

reduce the development of bacterial phenotypic resistance;7,

6

macrophages thereby reducing intracellular drug level;7,

7

efflux pump of the phagolysosome, that causes acidification of the phagolysosome to activate hydrolytic

8

enzymes to degrade the intracellular M. tb.14 Hence, these three mechanisms of action have the potential

9

to shorten the current TB oral drug regimens and reduce the emergence of new MDR and XDR-TB

10

13

13

(2) block the efflux pump of infected

and (3) inhibit the calcium and potassium

strains.

11

Verapamil, a calcium-channel blocker is a most potent mycobacterial efflux pump inhibitors both

12

in vitro and in vivo TB models.10, 15-18 Verapamil has been approved by Food and Drug Administration

13

(FDA) since 1982 for oral treatment of cardiovascular disorders.19 In vitro studies have highlighted the

14

capacity of verapamil to significantly improve the anti-mycobacterial activity of many drugs including

15

rifampicin, streptomycin, isoniazid, ofloxacin, bedaquiline, and clofazimine.11, 13, 17, 18, 20 Verapamil also

16

reduces drug tolerance in the macrophage environment and amplifies the intracellular activity of isoniazid

17

and rifampicin against M. tb by enhancing the accumulation of these drugs inside the macrophage.10, 11

18

Furthermore, an in vivo study by Louw et al. demonstrated that verapamil can restore M. tb susceptibility

19

to first-line drugs and prevent the development of further drug resistance of mice infected with MDR-TB

20

isolate.21 Their study also showed that the incorporation of verapamil in the treatment of first-line anti-TB

21

drugs significantly reduced the bacillary load in the lungs compared to the anti-TB drugs alone. Similarly,

22

Gupta et al. demonstrated accelerated bacillary clearance and reduced relapse rates of standard anti-TB

23

therapy in M. tb infected mice when verapamil was co-administered.16 These findings are promising, but

24

clinical studies investigating verapamil as an adjunctive anti-TB agent are limited.

25

The under-utilisation of verapamil may be due to its limitations when administered orally with

26

rifampicin; a key first-line oral anti-TB drug that induces verapamil-metabolising enzyme CYP3A4.16, 22-24 5



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Thus, concomitant oral administration of these two drugs leads to the poor oral bioavailability of

2

verapamil

3

bioavailability of verapamil even in rifampicin naïve subjects after oral administration due to high plasma

4

protein binding of verapamil nearly 90 %.26, 27 These metabolic and protein binding limitations can be

5

overcome by inhaled delivery of verapamil in combination with rifamycin. Apart from avoiding first-pass

6

metabolism by the liver, pulmonary delivery offers additional advantages. First, targeted delivery to the

7

primary site of infection may increase local therapeutic concentration and anti-TB activity. Second, high

8

doses of efflux inhibitors at the site of infection could also enhance the activity of other orally-

9

administered anti-TB drugs, including the accumulation into infected macrophages. This dual mechanism

10

has the potential to shorten the treatment duration and reduce the side effects associated with prolonged

11

drug usage.

16, 22, 23

and severely reduces its clinical efficacy.25 There is also a significant variation in

12

The current study describes the manufacture of a combination inhalable dry powder consisting of

13

verapamil and rifapentine. Rifapentine was selected as it is a first-line anti-TB drug similar in action to

14

rifampicin, but has a longer pulmonary half-life and stronger anti-mycobacterial activity.28,

15

inhalable formulation of verapamil and rifapentine was produced at a drug mass ratio of 1:3, respectively

16

based upon current recommended oral dosages.30, 31 The physiochemical, in vitro aerosol performance and

17

storage stability, and in vitro microbiological activities including pulmonary cytotoxicity are reported.

29

The

18 19

METHODS AND MATERIALS

20

Materials. Verapamil hydrochloride, L-leucine, potassium dihydrogen orthophosphate, dimethyl

21

sulfoxide (DMSO), and phosphate-buffered saline (PBS) were obtained from Sigma-Aldrich, Castle Hill,

22

Australia. Rifapentine was from Hangzhou ICH Imp & Exp Company Ltd., Hangzhou, China.

23

Acetonitrile, methanol, tetrahydrofuran, and orthophosphoric acid were purchased from Ajax Finechem

24

Pty Ltd., Taren Point, Australia.

25

6


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1

Bacterial strain and cell culture. M. tb H37Ra (ATCC 25177), M. tb H37Rv (ATCC 27294), human

2

monocytic cell line (THP-1), and human alveolar basal adenocarcinoma epithelial cell line (A549) were

3

purchased from American Type Culture Collection (ATCC) (Manassas, VA). The M. tb strains

4

maintained in Middlebrook 7H9 media (enriched with 10 % albumin, dextrose and catalase (ADC)

5

supplement) with 20 % glycerol stock at -80°C. The THP-1 and A549 cells were grown in complete

6

RPMI-1640 media (RPMI-1640 medium, 0.05 mM 2-mercaptoethanol, 10 % fetal bovine serum) at 37°C

7

in 5 % CO2.

8 9

Formulation design. The single drug formulation of verapamil (Vrp_Leu) was produced by spray drying

10

a solution containing 2.7 mg/mL verapamil hydrochloride and 0.7 mg/mL L-leucine (equivalent to 20 %,

11

w/w) in distilled water. For the rifapentine only (Rpt) powder, a rifapentine crystalline suspension was

12

prepared as previously described.32 Briefly, a rifapentine solution at concentration of 4 mg/ml was

13

prepared in a co-solvent consisting of acetone:water (1:1, v/v). The solution was then heated to 67°C for

14

evaporation of acetone to form a crystalline suspension. This suspension was homogenized at 10 000 rpm

15

for 2 minutes (Silverson L4RT High Shear Mixer, Silverson, Chesham, UK) and then spray dried. For the

16

combination formulation (Vrp_Rpt_Leu), verapamil hydrochloride (at 1:3; verapamil:rifapentine, w/w)

17

and L-leucine (20 %, w/w) were solubilised in the homogenised rifapentine suspension and the mixture

18

was spray dried.

19

The spray drying process was carried out using a B-290 Mini Spray Dryer (Buchi Labortechnik

20

AG, Flawil, Switzerland) in an open-loop configuration using the following settings: inlet temperature of

21

60°C, atomizer of 60 mm (approximate 742 L/h), aspirator of 100 % (40 m3/h), and feed rate of 5 %

22

(equivalent to 2 mL/min). A spray dried L-leucine powder was also produced through spray drying a L-

23

leucine aqueous solution at a concentration of 0.7 mg/mL using the same parameters.

24 25

Physico-chemical characterization

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Particle morphology. Particle morphology was imaged using scanning electron microscopy (SEM) (Zeiss

2

Ultra Plus scanning microscope, Carl Zeiss, Oberkochen, Germany) at 5 kV acceleration voltage.

3

Powders were deposited onto carbon tape and sputter coated with 15 nm of gold using a K550X sputter

4

coater (Quorum Emitech, Kent, UK).

5 6

Particle sizing. The volumetric diameter was determined using laser diffraction coupled with a Scirocco

7

dry powder module (Mastersizer 2000, Malvern Instruments, Worcestershire, UK). The dry powder was

8

dispersed at a pressure of 3.5 bar. All the measurements were performed in triplicate. The refractive index

9

(RI) was set at 1.54, 1.63 and 1.60 (average RI of verapamil and rifapentine at ratio 1:3) for Vrp_Leu, Rpt

10

and the Vrp_Rpt_Leu, respectively.

11 12

X-ray power diffractometry. The crystalline property of the dry powders was analysed using x-ray

13

powder diffractometry (XRPD) (D500; Siemens, Karlsruhe, Germany). The powder was spread into the

14

cavity of the XRPD glass slide, and analysis was performed using Cu-Kα radiation at a generator voltage

15

of 40 kV and a current of 330 mA. The data were collected by the 2θ scan method with a scan rate of

16

0.04°/sec from 5-40°.

17 18

Thermal analysis. Differential scanning electron calorimetry (DSC) and thermogravimetric analysis

19

(TGA) (Mettler Toledo, Melbourne, Australia) were used to study the thermal profile of the dry powders.

20

For DSC, 5 mg of each powder was evenly spread into an aluminium crucible which was then crimped

21

with a perforated lid and heated from 30 to 300°C at 10°C/min under a continuous flow of nitrogen gas.

22

For TGA, 5 mg of each powder was weighed into an alumina crucible and then gradually heated at

23

10°C/min from 30 to 300°C under a dynamic nitrogen gas flow. Both sets of thermal data were recorded

24

and analysed through STARe software V.9.0x (Mettler Toledo, Greifensee, Switzerland).

25

8


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1

Dynamic vapour sorption. The moisture sorption behaviour of the powders was assessed using a dynamic

2

vapor sorption (DVS) instrument (DVS-1, Surface Measurement Systems, London, UK) operated at 25°C

3

with continuous nitrogen gas flow. The powders were exposed to a dual moisture ramping cycle of 0 to

4

90 % with 10 % increments of RH. The mass changes were recorded over time and equilibrium moisture

5

content determined by dm/dt of 0.02 % per minute.

6 7

Dissolution Profile. The dissolution profile of the dry powders was studied using a Franz-cell dissolution

8

system coupled with a heating stirrer station (Perm Gear Inc., Bethlehem, USA). The method was adapted

9

from Chan et al.33 Each cell reservoir was filled with 23 mL of PBS (pH 7.4) with 2 % ascorbic acid as an

10

antioxidant, to form a positive liquid meniscus. Magnetic stirrers were used to provide solute uniformity

11

within the reservoirs. The cells were connected to a water bath to maintain the internal liquid temperature

12

at 37°C.

13

Prior to dissolution, a capsule was filled with 20 mg of powder and actuated (Osmohaler®

14

(Plastiape, Italy) dry powder inhaler at an airflow of 100 L/min for 4 secs) into a next generation impactor

15

(NGI) containing cellulose filter papers (Whatman® qualitative filter paper Grade 5) in stages 3 and 4 that

16

has cut-off diameter of 2.2 and 1.3 µm, respectively. The filters holding the deposited powder were then

17

placed in between the buffer meniscus and the top compartment of the Franz-cell reservoir. Sampling

18

(500 µL) was performed at predetermined time points (15, 30, 45, 60, 90, 120, 180, 240, and 300 min)

19

and replaced with an equivalent amount of fresh buffer. At the final time point (5 h), the filter was

20

removed and washed thoroughly with 3 mL of fresh buffer solution.

21

Drug quantification was performed using a validated high-performance liquid chromatography

22

(HPLC) method.32 A µBondapakTM C18 (3.9 × 300 mm2) column (Waters, Milford, Massachusetts) was

23

coupled with a HPLC (Model LC-20, Shimadzu, Kyoto, Japan) operated at a detection wavelength of 250

24

nm (SPD-20A UV/VIS detector, Shimadzu) and an injection volume of 20 µL. The mobile phase was a

25

50 mM phosphate buffer (pH 3.0):acetonitrile:tetrahydrofuran at a ratio of 53:42:5 (%, v/v). The mobile

26

phase was run at an isocratic flow rate of 1.0 mL/min. Calibration curves with R2 values of 1.0 for 9



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1

verapamil and rifapentine were obtained using standard solutions with concentrations ranging from

2

0.0005 to 0.1 mg/mL. The data were expressed as a percentage of drug mass in the buffer solution relative

3

to the total mass recovery. Each formulation was tested in triplicate.

4 5

In Vitro Aerosol Performance. In vitro aerosol performance of the spray dried powders was analysed

6

using the Pharmacopeia method of the multi-stage liquid impinger (MSLI) (Apparatus C, British

7

Pharmacopeia 2012, Copley, Nottingham, UK) connected to a USP throat with a silicone mouthpiece

8

adapter.32 The rinsing solvent consisted of ascorbic acid (0.2 mg/mL) in methanol:water at 3:1. The MSLI

9

was filled with 20 mL of rinsing solvent in stages 1 to 4 and fitted with a glass fibre filter in stage 5. Size

10

3 hydroxypropyl methylcellulose capsules (Capsugel, Greenwood) were filled with 10 ± 0.5 mg of

11

powder. A capsule was punctured in an Osmohaler® (Plastiape, Italy), and actuated for 4 secs at 100

12

L/min (calibrated using airflow and flow controller (TSI 3063 airflow meter, TSI Instruments Ltd.,

13

Buckinghamshire, UK and Flutus Air, Novi Systems Ltd., Dulverton, UK, respectively)). Following the

14

dispersion, the components were washed with rinsing solvent as follows: 5 mL for the adaptor, and 10 mL

15

each for the capsule, device, throat and filter. Dispersions were performed at 20 ± 3°C and a relative

16

humidity (RH) of 50 ± 5 %. Each formulation was tested in triplicate and the drug mass was quantified

17

using HPLC as mentioned previously. The lower cut-off diameters for stages 1-4 of the MSLI at 100

18

L/min are 10.4, 4.9, 2.4, and 1.2 µm, respectively.

19

The total fine particle fraction (FPFtotal) was expressed as a mass percentage of particles with an

20

aerodynamic diameter less than 5 µm relative to the total recovered drug mass. The emitted fine particle

21

fraction (FPFemitted) was calculated as a mass percentage of particles with an aerodynamic diameter less

22

than 5 µm relative to the total recovered drug mass excluding the capsule and device. Mass median

23

aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the log-

24

probability plot of the MSLI data.

25

10


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1

Storage Stability Studies. Storage stability of Vrp_Leu and Vrp_Rpt_Leu was studied at 0 % and 60 %

2

RH at 20 ± 3°C for 1 and 3 month intervals. These study conditions were chosen because the powders

3

absorb significant amount of water at a RH above 60 % at 25°C as shown by the DVS analysis. The

4

stability of Rpt formulation has been reported in a previous study.32 The spray dried powders were stored

5

in loosely capped scintillation vials in a desiccator at 20 ± 3°C and protected from light. To maintain RH

6

close to zero, the desiccator contained gel desiccant beads and was under vacuum. For a 60 % RH and 20

7

± 3°C environment, the powders were stored in a climate controlled cabinet. At predetermined time points

8

a known quantity of powder was dissolved in MSLI rinsing solvent and quantified using HPLC to study

9

the chemical stability between verapamil and rifapentine. The drug content was expressed as a mass

10

percentage of each drug. Aerosol performance was also assessed on samples at these time points using the

11

aforementioned methods.

12 13

In vitro Microbiological and Cytotoxicity Studies

14

Resazurin Assay. The resazurin assay was performed in 96-well plates. The antibiotic powders were

15

dissolved in DMSO and serially diluted to the desired concentration in Middlebrook 7H9 media

16

supplemented with 10 % ADC supplement, 0.05 % glycerol, 0.05 % Tween-80 and 1 % tryptone.

17

Subsequently, mid-exponential phase (OD600 0.4-0.8) M. tb H37Ra or M. tb H37Rv cultures were added

18

and incubated for 5 days in a humidified incubator at 37°C. After the incubation, a mixture of 0.02 %

19

resazurin solution (30 µL) and 20 % Tween-80 (12.5 µL) was added to each well, then further incubated

20

for 48 h. Fluorescence was measured using a BMG Labtech Polarstar Omega (BMG Labtech, Ortenburg,

21

Germany) at an excitation wavelength of 530 nm and emission wavelength of 590 nm. Growth percentage

22

was determined in comparison to the growth of the untreated negative control after subtracting the

23

background fluorescence. Minimum inhibitory concentration (MIC90) was measured as the lowest

24

concentration that showed 90 % growth inhibition compared to the untreated cells.

25

11



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Page 12 of 41

1

Intracellular Inhibition Assay. THP-1 cells were grown in complete RPMI-1640 media (RPMI-1640

2

medium, 0.05 mM 2-mercaptoethanol, 10 % fetal bovine serum) at 37°C in 5 % CO2. The media

3

containing 50 ng/mL of phorbol 12-myristate 13-acetate and 1 × 105 cells/well were added to 96-well

4

plates and incubated for 48 h for the cells to adhere and differentiate. The cells were then infected with M.

5

tb H37Ra at a multiplicity of infection (MOI) of 5. After 4 h of infection, cells were washed 3 times with

6

PBS, replaced with fresh media and further incubated overnight for the cells to recover. The verapamil

7

and rifapentine in combination with various concentrations were dissolved in DMSO and then further

8

diluted in complete RPMI-1640 media to achieve the desired concentration. Diluted solutions were added

9

and incubated for 48 h. To determine the intracellular bacterial load, cells were lysed and bacterial

10

number (colony forming units, cfu) were determined after plating onto Middlebrook 7H11 (Bacto

11

Laboratories, Mt. Pritchard, NSW, Australia) supplemented with 10 % oleic acid, albumin, dextrose and

12

catalase (OADC) enrichment, and 0.5 % glycerol using standard procedures.34

13 14

Cytotoxicity Assay. The toxicity of the combination formulation and its individual components were

15

tested on THP-1 and A549 cell lines. A concentration of 5 × 105 cells/well was seeded to a 96-wells plate

16

and incubated for 48 h to allow cell adherence and differentiation. The drug powders were dissolved in

17

DMSO and diluted in complete RPMI-1640 media. Drugs (concentrations ranging from 0.00196 - 1.0

18

mg/mL) were added into the wells in two-fold dilutions and incubated for 4 days. On day 4, 10 µL of 0.05

19

% (w/v) resazurin was added and incubated for 4 h. Fluorescence readings were then taken at a

20

wavelength of 590 nm following excitation at 544 nm using the FLUOstar Omega Microplate Reader

21

(BMG Labtech, Ortenburg/Germany). The half maximal inhibitory concentration (IC50) was determined

22

through comparison to the untreated cells.

23 24

Statistics. All data were expressed as mean ± standard deviation with n=3. The statistical differences

25

were analysed using Microsoft Excel, t-test analysis of variance (Microsoft Office Excell 2007) and p

26

values < 0.05 were considered statistically significant. 12


Page 13 of 41

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1 2

RESULTS

3

Physico-chemical Characteristics

4

Particle Morphology. SEM analysis on the physical morphology of various powders is presented in

5

Figure 1. The raw verapamil was irregularly shaped, while processed Vrp_Leu was relatively spherical

6

with a highly corrugated surface. The rifapentine (Rpt) particles consisted of needle-like structures. A

7

mixture of nearly spherical and elongated particles was observed in the combination formulation. The

8

geometric size of verapamil particles were in the range of 1-3 µm while the rifapentine crystals were

9

about 10 × 2 × 2 µm of length × width × height.

10 11

Figure 1.

12 13

Particle Sizing. Volumetric median diameter (D50) of the raw verapamil powder was about 7.39 ± 1.70

14

µm, which was reduced to 1.42 ± 0.02 µm after being solubilised and spray dried with L-leucine in

15

Vrp_Leu (Table 1). The D50 of the Rpt (1.41 ± 0.06 µm) was similar to Vrp_Leu. A slight increase in size

16

was observed for the combined Vrp_Rpt_Leu particles (1.61 ± 0.03 µm) and the particles were within the

17

inhalable range of less than 5 µm.

18 19

Table 1.

20 21

X-ray Powder Diffractometry. Raw verapamil was shown to be crystalline as numerous diffraction peaks

22

were observed, notably at 10.59°, 14.45°, 17.07°, 18.1°, 18.84°, 20.29°, 21.32°, 23.06°, 23.75°, and

23

26.29° (Figure 2), similar to those reported in the literature.35 The Vrp_Leu showed a typical “halo”

24

pattern indicating the powder is largely amorphous. The Rpt powder demonstrated crystallinity with

25

peaks at 5.68°, 6.52°, 7.56°, 8.48°, 9.08°, 10.04°, 13.16°, 15.68°, 17.8°, 20.76°, and 23.52°. The

13



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Page 14 of 41

1

Vrp_Rpt_Leu powder also demonstrated crystalline peaks in similar regions as in Rpt but at lower

2

intensities. Crystalline peaks were also observed in the spray dried L-leucine.

3 4

Figure 2.

5 6

Thermal Analysis. Thermal properties of the Vrp_Rpt_Leu were compared to their individual

7

components as shown in Figure 3. The raw verapamil exhibited a broad endotherm from 25°C to 130°C

8

followed by a sharp endotherm peak at 146.65°C. In contrast, Vrp_Leu showed a broad exotherm from

9

approximately 80°C to 225°C and an endotherm peak at 246.93°C. Slight enthalpy changes were

10

observed in the DSC curves of Rpt and Vrp_Rpt_Leu from 25°C to 160°C, followed by an endothermic

11

peak at 174.90°C and 174.84°C, respectively. The spray dried L-leucine showed an extended endotherm

12

between 25°C and 275°C with a melting point of 293°C.

13

Figure 4 shows the TGA dynamic curves of the powders. Almost complete water evaporation

14

during the spray drying was observed because the measured weight loss was very low (< 0.5 %) for all

15

samples after heating from 25°C to 120°C. In addition, raw verapamil and Vrp_Leu begin to degrade at

16

around 225°C, while Rpt and Vrp_Rpt_Leu at 204°C and 162°C, respectively. Decomposition of spray

17

dried L-leucine began after 240°C.

18 19

Figure 3.

20 21

Figure 4.

22 23

Dynamic Vapour Sorption. The dynamic water sorption behavior of Vrp_Leu, Rpt and Vrp_Rpt_Leu

24

showed the formulations experienced a maximum mass change of 18.05 %, 13.70 % and 10.30 % (w/w),

25

respectively at 90 % RH (Figure 5). All formulations exhibited a similar reversible moisture sorption

26

trend at both the first and second moisture sorptions with no moisture-induced recrystallization. A double 14


Page 15 of 41

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1

sigmoid profile was observed during both moisture sorption and desorption cycles of Rpt; whereas only a

2

slight sigmoid profile was noticed in desorption cycles Vrp_Rpt_Leu which appeared after 60 % RH.

3 4

Figure 5.

5 6

Dissolution Profile. Figure 6 shows the dissolution properties of the Vrp_Leu, Rpt and Vrp_Rpt_Leu.

7

Verapamil, a hydrophilic drug, reached its maximal rate of dissolution within an hour in both Vrp_Leu

8

and Vrp_Rpt_Leu. However, the release rate of rifapentine was reduced by 20 % in Vrp_Rpt_Leu

9

compared to Rpt.

10 11

Figure 6.

12 13

In Vitro Aerosol Performance. The FPFtotal, FPFemitted, MMAD, and GSD of each formulation are

14

displayed in Table 2. Figure 7 presents the drug deposition of Vrp_Leu, Rpt and Vrp_Rpt_Leu powders

15

after MSLI dispersion.

16

The MMAD and GSD of Vrp_Leu were about 2.9 µm and 1.6, respectively, which resulted in an

17

FPFtotal of 67.3 ± 1.2 %. The FPFtotal of verapamil was increased to 77.4 ± 1.1 % in Vrp_Rpt_Leu, which

18

corresponds to MMAD and GSD of 2.10-2.16 µm and 1.65-1.66, respectively. In both formulations, a

19

higher percentage of verapamil was deposited in stage 3 and 4 compared to the other stages of the

20

impinger.

21

The FPFtotal and FPFemitted of Rpt were 83.9 ± 0.6 % and 93.9 ± 0.3 %, respectively. Notably, Rpt

22

also consists of approximately 24.5 ± 1.0 % of particles aerodynamically less than 1.32 µm, demonstrated

23

by the drug deposited in the filter stage. Rifapentine in Vrp_Rpt_Leu showed FPFtotal and FPFemitted that

24

was significantly reduced to 71.5 ± 2.0 % and 76.8 ± 2.2 %, respectively (p < 0.05).

15



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1

More importantly, for the combination formulation no significant differences were observed in

2

the deposition percentage between the two drugs in various stages of the MSLI (p > 0.05), indicating

3

uniform distribution of verapamil and rifapentine.

4 5

Table 2.

6 7

Figure 7.

8 9

Storage Stability Studies. Figure 8 shows the SEM images of Vrp_Leu and Vrp_Rpt_Leu exposed to 60

10

% RH for a month. The particles in both formulations were observed to be fused into a non-dipersible

11

solid aggregate. On the other hand, intact morphology (as in Figure 1) was observed in the powders stored

12

at 0 % RH. Therefore, further characterization was only carried out using powders stored at 0 % RH.

13

Table 3 shows the theoretical and actual drug content of verapamil and rifapentine in

14

Vrp_Rpt_Leu stored at 0 % RH over 3 months. The composition of the individual drugs remained

15

unchanged after 3 months of storage (p > 0.05). In regard to their aerosol performance, no significant

16

changes were seen in their FPFtotal, FPFemitted, MMAD and GSD as shown in Table 2 (p > 0.05). The

17

aerosol deposition profiles of Vrp_Leu and Vrp_Rpt_Leu stored at 0 % RH up to 3 months (Figure 9) did

18

not demonstrate any significant difference from their initial MSLI profile (p > 0.05).

19 20

Figure 8.

21 22

Table 3.

23 24

Figure 9.

25 26

In Vitro Microbiological and Cytotoxicity Studies 16


Page 17 of 41

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1

Resazurin Assay. The solubilised Rpt showed high degree of activity against M. tb H37Ra (MIC90 0.625

2

ng/mL) and M. tb H37Rv (MIC90 5.0 ng/mL). By contrast, the solubilised Vrp_Leu has no anti-

3

mycobacterial activity against both strains. The solubilised Vrp_Rpt_Leu demonstrated equivalent

4

activity against M. tb H37Ra (MIC90 0.625 ng/mL) and M. tb H37Rv (MIC90 5.0 ng/mL) as Rpt.

5

Furthermore, when the ratio of verapamil to rifapentine was increased, a decrease in the anti-

6

mycobacterial activity was observed. The excipient, L-leucine did not inhibit mycobacterial growth at the

7

concentrations used in the combinations of Vrp_Leu and Vrp_Rpt_Leu.

8 9

Intracellular Inhibition of M.tb H37Ra. In light of the efficacy of the solubilised Vrp_Rpt_Leu against

10

M. tb, we investigated whether verapamil and rifapentine at a ratio of 1:3 could restrict the mycobacterial

11

growth within the host THP-1 cell. This assay was only conducted with M. tb H37Ra due to biosafety

12

constraints on using virulent M. tb H37Rv strain.

13

As shown in Figure 10, the combined verapamil and rifapentine showed a dose-dependent

14

bactericidal activity on M. tb H37Ra growth over 2 days of infection. Furthermore, a statistically

15

significant (p < 0.05) difference was observed between the individual drug treatments compared to the

16

combination. For example on day 1, treatment with the combination (verapamil 0.05 µg/ml + rifapentine

17

0.15 µg/ml) resulted in 35 % and 20 % increased bacterial killing compared to verapamil (0.05 µg/ml) or

18

rifapentine (0.15 µg/ml) alone, respectively. A similar trend was also observed at the other two

19

concentrations tested. These findings indicated that the intracellular activity of rifapentine was enhanced

20

by the presence of verapamil.

21 22

Figure 10.

23 24

Cytotoxicity Assay. The safety of the formulations was investigated by determining the cytotoxic half

25

maximal inhibitory concentration (IC50) against THP-1 and A549 cell lines (Table 5). The IC50 of all

17



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Page 18 of 41

1

solubilised antibiotic powders were in the range of 62.5 µg/mL and 250 µg/mL. L-leucine did not have

2

any cytotoxic effect on either cell line.

3 4

Table 5.

5 6

DISCUSSION

7

To the best of our knowledge, this is the first study on the development of inhalable formulation of efflux

8

pump inhibitor, verapamil and rifapentine. The inhaled combination formulation is uniquely composed of

9

corrugated amorphous verapamil attached to elongated crystalline rifapentine particles. This distinct

10

combination showed a promising aerodynamic performance for both verapamil and rifapentine. When

11

verapamil alone was spray dried with L-leucine (Vrp_Leu), it had an FPFtotal above 65 % and the MMAD

12

was about 2.9 µm. The corrugated surface morphology of these amorphous particles reduces the

13

interparticle cohesive forces which concomitantly lead to lower particle adhesion forces and superior FPF

14

compared to smoother particles.36,

15

between the drugs for enhanced dispersion performance.38 When verapamil and L-leucine were further

16

combined with elongated rifapentine (Rpt) and spray dried (Vrp_Rpt_Leu), the FPFtotal of verapamil

17

increased to more than 75 % and the MMAD was reduced to about 2.1 µm (p < 0.05). This was due to a

18

larger deposition of the particles on stage 4 compared to stage 3 in Vrp_Leu, indicating that the elongated

19

Rpt particles act as a carrier for the amorphous verapamil to enhance the powder dispersibility. A similar

20

observation was also reported in an inhaled formulation that contained colistin and rifapentine.39 On the

21

other hand, lower deposition of rifapentine on the final filter stage (aerodynamically < 1.32 µm) reduced

22

the respirable fraction of rifapentine in the combination powder reduced to 70 % (p < 0.05) compared to

23

83 % in the rifapentine alone formulation. However, this reduction may not be clinically significant as the

24

particles likely to be submicron, hence exhaled due to negligible impaction and gravitational

25

sedimentation in the lung airways.40 More importantly, Vrp_Rpt_Leu also showed a uniform dose

26

distribution of both drugs at their intended dose (p < 0.05) in stages 1 - 4 of the MSLI, suggesting a

37

The addition of L-leucine also reduces intermolecular interaction

18


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1

potential homogeneous pulmonary deposition of both drugs. Therefore, the Vrp_Rpt_Leu combination

2

demonstrates suitability for pulmonary delivery at the intended dose with high lung deposition profile.

3

Another advantage of the Vrp_Rpt_Leu formulation was a slower in vitro dissolution rate of Rpt

4

particles. Raula et al. demonstrated a similarly slow dissolution rate for L-leucine coated salbutamol

5

sulphate (produced via spray drying) compared to pure drug particles.41 This phenomenon was explained

6

by the relatively low water solubility of L-leucine (approximately 24 g/L).41 On the basis of their finding,

7

it could be deduced that during the spray drying process L-leucine coated onto the surface of the Rpt and

8

slows the dissolution of the particles. In the context of TB treatment, the slow release of anti-TB drugs

9

may allow less frequent dosing as well as longer retention of physical drug particles to enhance

10

internalization by macrophages.42

11

The stability of the formulations was also addressed. A previous study has demonstrated that

12

amorphous rifapentine was chemically unstable after a month of storage at ambient (60 % RH) and dry (0

13

% RH) conditions.32 A unique crystalline form of rifapentine was therefore developed and shown to be

14

chemically stable for up to 3 months of storage under the aforementioned conditions.32 In contrast, spray

15

dried verapamil (without L-leucine) was very hygroscopic leading to immediate caking after being spray

16

dried at ambient RH (45 ± 5 %). In a recent phase 1, single-dose study by Dharmadhikari et al. used

17

inhalable capreomycin spray dried with 20 % L-leucine (w/w) with no severe adverse effects up to

18

administration of 300 mg.43 Hence, a similar proportion of L-leucine was used to stabilize verapamil

19

alone formulation.

20

The DVS moisture sorption profile demonstrates the Vrp_Leu still absorbs a significant amount

21

of water at elevated RH above 60 %. Nonetheless, the reversible water absorption and desorption pattern

22

as indicated by the identical reversible moisture sorption trend at both first and second moisture sorption

23

cycles suggesting absence of recrystallization. This could be due to the presence of L-leucine, which has

24

been reported to prevent water-induced recrystallization of amorphous pyrazinamide in an inhalable

25

antibiotic formulation consisting of rifapentine, moxifloxacin and pyrazinamide.44 Furthermore, a similar

26

reversible sorption profile was observed in Vrp_Rpt_Leu with an additional sigmoid profile after 60 % 19



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Page 20 of 41

1

RH. The presence of a sigmoid profile is attributable to the crystalline rifapentine, which forms a

2

monohydrate followed by a dihydrate.32

3

Both Vrp_Leu and Vrp_Rpt_Leu can be stored at validated conditions for long-term stability.

4

Vrp_Leu and Vrp_Rpt_Leu powders stored for a month at 60 % RH absorbed a significant amount of

5

water to become a non-dispersible cake. In contrast, Zhou et al. have shown that co-solvent spray drying

6

hygroscopic colistin with hydrophobic rifampicin at 1:1 protected the powder from storage moisture.

7

They proposed that this was due to the coating of the rifampicin onto the surface of the colistin.45

8

Although, Vrp_Rpt_Leu consists of 75 % of hydrophobic rifapentine, it was crystallised first before the

9

addition of verapamil prior to spray drying which resulted the drugs present as two distinctive forms. The

10

water absorption of Vrp_Rpt_Leu during storage therefore, could be mainly attributed to the relatively

11

hygroscopic verapamil particles. In contrast, the formulations stored for up to 3 months in a moisture-free

12

(vacuum) condition retained their physical morphology when observed under SEM. No statistical

13

difference was observed in the dispersion profile of these powders up to 3 months. Drug quantification

14

analysis also indicated the individual components of the combination remained at the intended mass ratio

15

after storage. The HPLC data revealed drug recovery of 95 – 105 % and no extra peaks that would

16

indicate oxidation or other unwanted chemical reactions. Thus, both Vrp_Leu and Vrp_Rpt_Leu should

17

be in moisture protective packaging for long term storage.

18

Verapamil and rifapentine in combination did not show synergistic inhibitory activity against M.

19

tb in vitro. The solubilised Vrp_Rpt_Leu formulation was anticipated to show a lower MIC90 than the

20

rifapentine alone formulation owing to efflux pump inhibition by verapamil. Unexpectedly, the MIC90

21

value did not decrease even with an increase in the proportion of verapamil. In contrast, Gupta et al.

22

reported that the MIC of bedaquiline and clofazimine against the laboratory H37Rv strain and clinical

23

isolates was reduced by 8 to 16 times in the presence of verapamil.17 Another study demonstrated

24

verapamil resulted at least a 4-fold reduction in the in vitro MIC of isoniazid against M. tb H37Rv.8

25

Rodrigues et al., who studied the contribution of efflux mechanism on isoniazid resistance in M.

20


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1

tuberculosis complex highlighted that activation of efflux pumps depends on the genotype of the strain

2

tested rather than the drug alone.8

3

Nevertheless, the killing of M. tb H37Ra within the macrophage host environment was enhanced

4

by the combination of verapamil with rifapentine when compared to treatment with the individual drugs.

5

This result could be due to verapamil that appears to inhibit the macrophage-induced M. tb efflux pumps,

6

leading to the increased killing of intracellular mycobacteria.10, 17

7

The cytotoxicity profiles were also studied to examine the tolerance of pulmonary cells to these

8

formulations. As the drugs are shown to accumulate in macrophages, tolerability was assessed in THP-1

9

derived macrophages. The tolerability was also assessed on a lung epithelial cell line due to the non-

10

specific nature of pulmonary delivery. For both cell lines, solubilised Vrp_Rpt_Leu demonstrated greater

11

toxicity than the Vrp_Leu solution, but this was similar to Rpt alone. This could be because the majority

12

(75 %) of the formulation was rifapentine. However, the cytotoxicity of solubilised Vrp_Rpt_Leu may

13

underestimate the potential pulmonary toxicity of the formulation as the Rpt particles may not undergo

14

rapid dissolution following lung deposition. In a previous study, Chan et al. reported the solubilised Rpt

15

alone was well tolerated in vitro by the macrophage and epithelial cell line models up to 50 µM,

16

compared 71.3 µM in the present study.32 Additionally pharmacokinetics analysis following a single

17

pulmonary dose of Rpt at 20 mg/kg showed that intratracheal delivery can achieve Cmax of 321 µg/ml in

18

the lungs with no adverse event in the animals during the treatment. This suggests that the pulmonary

19

tolerability of rifapentine may be even higher. Further pulmonary safety studies of Vrp_Rpt_Leu are

20

warranted.

21 22

CONCLUSION

23

In this study, we formulated a novel inhalable verapamil and rifapentine combination dry powder that

24

demonstrates excellent aerosol performance, enhanced activity against intracellular growth of M. tb in

25

macrophages relative to the single drugs, and a promising in vitro cytotoxicity profile. Murine studies are

26

underway to ascertain in vivo efficacy of this formulation. 21



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Page 22 of 41

1 2

ACKNOWLEDGEMENT

3

Thaigarajan Parumasivam is a recipient of Malaysian Government Scholarship. The authors acknowledge

4

Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy and

5

Microanalysis, The University of Sydney, for their scientific and technical assistance. The work was

6

supported by the Australian Research Council’s Discovery Project (DP120102778), the National Health

7

and Medical Research Council Centre of Research Excellence in Tuberculosis Control (APP1043225) and

8

Project grant (APP104434), and the NSW Government Infrastructure Grant to the Centenary Institute.

9 10

22


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REFERENCE 1. World Health Organization Global tuberculosis report 2014. (15/03/15), 2. Dye, C.; Scheele, S.; Dolin, P.; Pathania, V.; Raviglione, M. C.; for the, W. H. O. G. S.; Monitoring, P. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. JAMA 1999, 282, (7), 677-686. 3. Monedero, I.; Caminero, J. A. MDR-/XDR-TB management: what it was, current standards and what is ahead. Expert Rev Respir Med 2009, 3, (2), 133-145. 4. Zumla, A. I.; Gillespie, S. H.; Hoelscher, M.; Philips, P. P. J.; Cole, S. T.; Abubakar, I.; McHugh, T. D.; Schito, M.; Maeurer, M.; Nunn, A. J. New antituberculosis drugs, regimens, and adjunct therapies: needs, advances, and future prospects. Lancet Infect Dis 2014, 14, (4), 327-340. 5. Xavier, A. S.; Lakshmanan, M. Delamanid: A new armor in combating drug-resistant tuberculosis. J Pharmacol Pharmacother 2014, 5, (3), 222-224. 6. Ramón-García, S.; Martín, C.; Thompson, C. J.; Aínsa, J. A. Role of the Mycobacterium tuberculosis P55 efflux pump in intrinsic drug resistance, oxidative stress responses, and growth. Antimicrob Agents Chemother 2009, 53, (9), 3675-3682. 7. Szumowski, J.; Adams, K.; Edelstein, P.; Ramakrishnan, L., Antimicrobial efflux pumps and Mycobacterium tuberculosis drug tolerance: evolutionary considerations. In Pathogenesis of Mycobacterium tuberculosis and its interaction with the host organism, Pieters, J.; McKinney, J. D., Eds. Springer Berlin Heidelberg: 2013; Vol. 374, pp 81-108. 8. Rodrigues, L.; Machado, D.; Couto, I.; Amaral, L.; Viveiros, M. Contribution of efflux activity to isoniazid resistance in the Mycobacterium tuberculosis complex. Infect Genet Evol 2012, 12, (4), 695-700. 9. Cao, C. X.; Silverstein, S. C.; Neu, H. C.; Steinberg, T. H. J774 macrophages secrete antibiotics via organic anion transporters. J Infect Dis 1992, 165, (2), 322-328. 10. Adams, Kristin N.; Takaki, K.; Connolly, Lynn E.; Wiedenhoft, H.; Winglee, K.; Humbert, O.; Edelstein, Paul H.; Cosma, Christine L.; Ramakrishnan, L. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 2011, 145, (1), 39-53. 11. Adams, K. N.; Szumowski, J. D.; Ramakrishnan, L. Verapamil, and its metabolite norverapamil, inhibit macrophage-induced, bacterial efflux pump-mediated tolerance to multiple anti-tubercular drugs. J Infect Dis 2014, 210, (3), 456-466. 12. Berg, R. D.; Ramakrishnan, L. Insights into tuberculosis from the zebrafish model. Trends Mol Med 2012, 18, (12), 689-690. 13. Louw, G. E.; Warren, R. M.; Gey van Pittius, N. C.; McEvoy, C. R. E.; Van Helden, P. D.; Victor, T. C. A balancing act: efflux/influx in mycobacterial drug resistance. Antimicrob Agents Chemother 2009, 53, (8), 3181-3189. 14. Amaral, L.; Molnar, J. Why and how the old neuroleptic thioridazine cures the XDR-TB patient. Pharmaceuticals (Basel) 2012, 5, (9), 1021-1031. 15. Singh, K.; Kumar, M.; Pavadai, E.; Naran, K.; Warner, D. F.; Ruminski, P. G.; Chibale, K. Synthesis of new verapamil analogues and their evaluation in combination with rifampicin against Mycobacterium tuberculosis and molecular docking studies in the binding site of efflux protein Rv1258c. Bioorg Med Chem Lett 2014, 24, (14), 2985-2990. 16. Gupta, S.; Tyagi, S.; Almeida, D. V.; Maiga, M. C.; Ammerman, N. C.; Bishai, W. R. Acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an efflux inhibitor. Am J Respir Crit Care Med 2013, 188, (5), 600-607. 17. Gupta, S.; Cohen, K. A.; Winglee, K.; Maiga, M.; Diarra, B.; Bishai, W. R. Efflux inhibition with verapamil potentiates bedaquiline in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2014, 58, (1), 574-576.

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18. Machado, D.; Couto, I.; Perdigão, J.; Rodrigues, L.; Portugal, I.; Baptista, P.; Veigas, B.; Amaral, L.; Viveiros, M. Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis. PLoS ONE 2012, 7, (4), e34538. 19. Ogbru, O. Verapamil, Calan, Verelan, Verelan PM, Isoptin, Isoptin SR, Covera-HS. (14th Sep), 20. Spies, F. S.; Almeida da Silva, P. E.; Ribeiro, M. O.; Rossetti, M. L.; Zaha, A. Identification of mutations related to streptomycin resistance in clinical isolates of Mycobacterium tuberculosis and possible involvement of efflux mechanism. Antimicrob Agents Chemother 2008, 52, (8), 2947-2949. 21. Louw, G. E.; Warren, R. M.; Gey van Pittius, N. C.; Leon, R.; Jimenez, A.; Hernandez-Pando, R.; McEvoy, C. R. E.; Grobbelaar, M.; Murray, M.; van Helden, P. D.; Victor, T. C. Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux. Am J Respir Crit Care Med 2011, 184, (2), 269-276. 22. Fromm, M. F.; Busse, D.; Kroemer, H. K.; Eichelbaum, M. Differential induction of prehepatic and hepatic metabolism of verapamil by rifampin. Hepatology 1996, 24, (4), 796-801. 23. Mooy, J.; Böhm, R.; van Baak, M.; van Kemenade, J.; Vet, A. v. d.; Rahn, K. H. The influence of antituberculosis drugs on the plasma level of verapamil. Eur J Clin Pharmacol 1987, 32, (1), 107-109. 24. Fromm, M. F.; Dilger, K.; Busse, D.; Kroemer, H. K.; Eichelbaum, M.; Klotz, U. Gut wall metabolism of verapamil in older people: effects of rifampicin-mediated enzyme induction. Br J Clin Pharmacol 1998, 45, (3), 247-255. 25. Barbarash, R. A.; Bauman, J. L.; Fischer, J. H.; Kondos, G. T.; Batenhorst, R. L. Near-total reduction in verapamil bioavailability by rifampin. electrocardiographic correlates. Anglais 1988, 94, (5), 954-959. 26. Keefe, D. L.; Yee, Y.-G.; Kates, R. E. Verapamil protein binding in patients and in normal subjects. Clin Pharmacol Ther 1981, 29, (1), 21-6. 27. Kates, R. E. Calcium antagonists. Pharmacokinetic properties. Drugs 1983, 25, (2), 113-24. 28. Munsiff, S. S.; Kambili, C.; Ahuja, S. D. Rifapentine for the treatment of pulmonary tuberculosis. Clin Infect Dis 2006, 43, (11), 1468-1475. 29. Chan, J. G. Y.; Tyne, A. S.; Pang, A.; McLachlan, A. J.; Perera, V.; Chan, J. C. Y.; Britton, W. J.; Chan, H. K.; Duke, C. C.; Young, P. M.; Traini, D. Murine pharmacokinetics of rifapentine delivered as an inhalable dry powder. Int J Antimicrob Agents 2014, 43, (3), 319-323. 30. Rosenthal, I. M.; Zhang, M.; Williams, K. N.; Peloquin, C. A.; Tyagi, S.; Vernon, A. A.; Bishai, W. R.; Chaisson, R. E.; Grosset, J. H.; Nuermberger, E. L. Daily dosing of rifapentine cures tuberculosis in three months or less in the murine model. PLoS Med 2007, 4, (12), e344. 31. Micromedex Verapamil (Oral Route). (16th Sep 2014), 32. Chan, J. G. Y.; Duke, C. C.; Ong, H. X.; Chan, J. C. Y.; Tyne, A. S.; Chan, H.-K.; Britton, W. J.; Young, P. M.; Traini, D. A novel inhalable form of rifapentine. J Pharm Sci 2014, 103, (5), 1411-1421. 33. Chan, J. G. Y.; Chan, H.-K.; Prestidge, C. A.; Denman, J. A.; Young, P. M.; Traini, D. A novel dry powder inhalable formulation incorporating three first-line anti-tubercular antibiotics. Eur J Pharm Biopharm 2013, 83, (2), 285-292. 34. Pinto, R.; Saunders, B. M.; Camacho, L. R.; Britton, W. J.; Gicquel, B.; Triccas, J. A. Mycobacterium tuberculosis defective in phthiocerol dimycocerosate translocation provides greater protective immunity against tuberculosis than the existing bacille Calmette-Guérin vaccine. J Infect Dis 2004, 189, (1), 105-112. 35. Sahoo, J.; Murthy, P. N.; Biswal, S.; Manik. Formulation of sustained-release dosage form of verapamil hydrochloride by solid dispersion technique using Eudragit RLPO or Kollidon SR. AAPS PharmSciTech 2009, 10, (1), 27-33. 36. Adi, S.; Adi, H.; Tang, P.; Traini, D.; Chan, H.-k.; Young, P. M. Micro-particle corrugation, adhesion and inhalation aerosol efficiency. Eur J Pharm Sci 2008, 35, (1–2), 12-18.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24


37. Chew, N.; Tang, P.; Chan, H.-K.; Raper, J. How much particle surface corrugation is sufficient to improve aerosol performance of powders? Pharmaceutical Research 2005. 38. Chew, N. Y. K.; Shekunov, B. Y.; Tong, H. H. Y.; Chow, A. H. L.; Savage, C.; Wu, J.; Chan, H.-K. Effect of amino acids on the dispersion of disodium cromoglycate powders. J Pharm Sci 2005, 94, (10), 2289-2300. 39. Zhou, Q.; Sun, S.-P.; Chan, J. G. Y.; Wang, P.; Barraud, N.; Rice, S. A.; Wang, J.; Li, J.; Chan, H.-K. Novel inhaled combination powder containing amorphous colistin and crystalline rifapentine with enhanced antimicrobial activities against planktonic cells and biofilm of Pseudomonas aeruginosa for respiratory infections. Mol Pharm 2015, 12, (8), 2594-603. 40. Zhang, J.; Wu, L.; Chan, H.-K.; Watanabe, W. Formation, characterization, and fate of inhaled drug nanoparticles. Adv Drug Deliv Rev 2011, 63, (6), 441-455. 41. Raula, J.; Seppälä, J.; Malm, J.; Karppinen, M.; Kauppinen, E. Structure and dissolution of LLeucine-coated salbutamol sulphate aerosol particles. AAPS PharmSciTech 2012, 13, (2), 707-712. 42. Muttil, P.; Wang, C.; Hickey, A. Inhaled drug delivery for tuberculosis therapy. Pharm Res 2009, 26, (11), 2401-2416. 43. Dharmadhikari, A. S.; Kabadi, M.; Gerety, B.; Hickey, A. J.; Fourie, P. B.; Nardell, E. Phase I, single-dose, dose-escalating study of inhaled dry powder capreomycin: a new approach to therapy of drug-resistant tuberculosis. Antimicrob Agents Chemother 2013, 57, (6), 2613-2619. 44. Chan, J.; Tyne, A.; Pang, A.; Chan, H.-K.; Young, P.; Britton, W.; Duke, C.; Traini, D. A rifapentinecontaining inhaled triple antibiotic formulation for rapid treatment of tubercular infection. Pharm Res 2013, 31, (5), 1239-1253. 45. Zhou, Q.; Gegenbach, T.; Denman, J. A.; Yu, H.; Li, J.; Chan, H.-K. Synergistic antibiotic combination powders of colistin and rifampicin provide high aerosolization efficiency and moisture protection. AAPS J 2014, 16, (1), 37-47.

25 26 27 28 29 30 31 32 33 34 35 36 37 25



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

a

b

c

d

Page 26 of 41

1

Figure 1. Scanning electron micrographs of powder samples: (a) raw verapamil, (b) Rpt, (c) Vrp_Leu,

2

and (d) Vrp_Rpt_Leu.

3

26


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


Intensity

Page 27 of 41

Vrp_Rpt_Leu Vrp_Leu Rpt Raw verapamil Spray dried L-leucine 2

1

7

12

17

22 2θ (Degrees)

27

32

37

42

2

Figure 2. X-ray diffractograms of various powder formulations. Crystalline peaks were demonstrated by

3

Vrp_Rpt_Leu, Rpt, raw verapamil and spray dried L-leucine. Vrp_Leu was largely amorphous.

4 5 6 7 8 9 10 11 12 13 14 15

27



Spray dried L-leucine Vrp_Rpt_Leu

Exothermic →

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

Page 28 of 41

30 1

Rpt

Vrp_Leu Raw verapamil

60

90

120

150

180

210

240

270

300

Temperature (°C)

2

Figure 3. DSC thermograms of the spray dried combination formulation and its individual components.

3

The samples were heated from 30°C to 300°C under a dynamic flow of nitrogen gas.

4 5 6 7 8 9 10 11 12 13 14 15

28


Page 29 of 41

100

80

Raw verapamil Rpt

Weight (%)

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


Vrp_Leu

60

Vrp_Rpt_Leu Spray dried L-leucine 40

20

0 30

60

90

120

150 180 Temperature (°C)

210

240

270

300

1 2

Figure 4. TGA profiles of the spray dried formulation and its individual components. The samples were

3

heated from 30°C to 300°C under a dynamic flow of nitrogen gas.

4 5 6 7 8 9 10 11 12 13 14 15 29



Cycle 1 Sorp Cycle 2 Sorp

Change In Mass (%)

a

Cycle 1 Desorp Cycle 2 Desorp

20 18 16 14 12 10 8 6 4 2 0 0

20

40

60

80

100

RH (%)

b

Cycle 1 Sorp Cycle 2 Sorp

Cycle 1 Desorp Cycle 2 Desorp

14 Change In Mass (%)

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

Page 30 of 41

12 10 8 6 4 2 0 0

20

40

60

80

100

RH (%)

30


Page 31 of 41

c

Cycle 1 Sorp

Cycle 1 Desorp

Cycle 2 Sorp

Cycle 2 Desorp

12 Change In Mass (%)

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


10 8 6 4 2 0 0

20

40

60

80

100

RH (%)

1

Figure 5. Moisture sorption isotherms for (a) Vrp_Leu, (b) Rpt and (c) Vrp_Rpt_Leu. Humidity ramped

2

from 0 % to 90 % RH for 2-cycles.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

31


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

Cumulative amount (% of drug recovery)


Page 32 of 41

100

80

60

40 Verapamil in Vrp_Rpt_Leu Rifapentine in Vrp_Rpt_Leu Vrp_Leu Rpt

20

0 0

40

80

120

160 Time (min)

200

240

280

1 2

Figure 6. Dissolution profile of Vrp_Leu, Rpt and Vrp_Rpt_Leu using Franz-cell dissolution method

3

over a period of 5 h (mean ± SD, n=3).

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

32


Page 33 of 41

Recovery mass (%)

a

50 40

Vrp_Leu Rpt

30 20 10 0

b Recovered mass (%)

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


50 40

Verapamil in Vrp_Rpt_Leu Rifapentine in Vrp_Rpt_Leu

30 20 10 0

1

Figure 7. In vitro aerosol deposition profile for (a) Vrp_Leu and Rpt, and (b) Vrp_Rpt_Leu following

2

MSLI dispersion at a flow rate of 100 L/min at 0 months using Osmohaler® (mean ± SD, n=3).

3 4 5 6 7 8

33



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

a

Page 34 of 41

b

1

Figure 8. Scanning electron microscopy observation of (a) Vrp_Leu and (b) Vrp_Rpt_Leu exposed to 60

2

% RH for a month.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

34


a

50 40

Vrp_Leu Verapamil in Vrp_Rpt_Leu Rifapentine in Vrp_Rpt_Leu

30 20 10 0

b

50

Recovered mass (%)

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


Recovered mass (%)

Page 35 of 41

40

Vrp_Leu Verapamil in Vrp_Rpt_Leu Rifapentine in Vrp_Rpt_Leu 30 20 10 0

1

Figure 9. In vitro aerosol deposition profile for powders stored at 20±3ᵒC in 0 % RH for (a) 1 month and

2

(b) 3 months dispersed at a flow rate of 100 L/min via the Osmohaler® (mean ± SD, n=3).

3 4 5 6 7 8 35


Mycobacterial growth (CFU/mL)

a

Page 36 of 41

** 10

**

8

6

* *

*

4

** 2

0 No drug VRP (0.05) RPT (0.15) VRP/RPT VRP (0.5) (0.05/0.15)

RPT (1.5)

VRP/RPT (0.5/1.5)

VRP (5)

RPT (15)

VRP/RPT (5/15)

VRP (5)

RPT (15)

VRP/RPT (5/15)

Concentration (µg/mL)

1 Millions

b Mycobacterial growth (CFU/mL)

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

Millions


**

10

** 8

**

**

6

4

** 2

0 No drug VRP (0.05) RPT (0.15) VRP/RPT VRP (0.5) (0.05/0.15)

RPT (1.5)

VRP/RPT (0.5/1.5)

Concentration (µg/mL)

2 3

Figure 10. Bactericidal effects of verapamil (VRP) and rifapentine (RPT) at ratio of 1:3 on intracellular

4

infection of M. tb H37Ra in THP-1 cell treated for (a) 24 h and (b) 48 h (mean ± SD, n=3). Differences

5

between treatments were evaluated by analysis of variance (*p < 0.05, **p < 0.01).

6

36


Page 37 of 41

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


Table 1. Particle size distribution of the formulations (mean ± SD, n=3) D10 (µm)

D50 (µm)

D90 (µm)

Raw verapamil

2.27 ± 0.04

7.39 ± 1.70

24.37 ± 0.04

Rpt

0.74 ± 0.04

1.42 ± 0.02

2.72 ± 0.08

Vrp_Leu

0.80 ± 0.03

1.41 ± 0.06

2.66 ± 0.09

Vrp_Rpt_Leu

0.91 ± 0.03

1.61 ± 0.03

2.89 ± 0.05

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 37



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

Page 38 of 41

1

Table 2. FPFtotal, FPFemitted, MMAD, and GSD for Vrp_Leu, Rpt and Vrp_Rpt_Leu over 3 months in 0 %

2

RH (mean ± SD, n=3) Vrp_Leu

0 months

1 month

3 months

FPFtotal (%)

67.3 ± 1.2

68.6 ± 0.7

65.2 ± 2.9

FPFemitted (%)

73.6 ± 2.0

74.2 ± 0.6

71.1 ± 2.9

MMAD (µm)

2.87, 2.86, 2.80

2.93, 2.94, 2.58

2.86, 2.71, 2.94

GSD

1.60, 1.58, 1.59

1.61, 1.61, 1.60

1.58, 1.56, 1.58

Rpt

0 months

FPFtotal (%)

83.9 ± 0.6

-

-

FPFemitted (%)

93.9 ± 0.3

-

-

MMAD (µm)

1.76, 1.81, 1.79

-

-

GSD

1.69, 1.70, 1.69

-

-

0 months

1 month

3 months

Vrp_Rpt_Leu

verapamil

rifapentine

verapamil

rifapentine

verapamil

rifapentine

FPFtotal (%)

77.4 ± 1.1

71.5 ± 2.0

77.4 ± 1.1

71.8 ± 4.2

76.7 ± 1.5

69.6 ± 1.8

FPFemitted (%)

85.7 ± 1.1

76.8 ± 2.2

85.7 ± 1.1

78.2 ± 4.0

84.8 ± 1.2

77.8 ± 1.8

MMAD (µm)

2.11, 2.16,

2.29, 2.29,

2.21, 2.19,

2.39, 2.35,

2.10, 2.17,

2.35, 2.33,

2.10

2.19

2.16

2.34

2.19

2.32

1.66, 1.65,

1.66,1.64,

1.57, 1.61,

1.64, 1.63,

1.64, 1.62,

1.65, 1.63,

1.65

1.66

1.56

1.62

1.60

1.62

GSD

3

*MMAD and GSD from each replicate

4 5 6 7

38


Page 39 of 41

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

Table 3 Theoretical versus actual drug content of Vrp_Rpt_Leu over 3 months of storage in 0 % RH at

2

20°C (mean ± SD, n=3) Duration of storage

Drug content (%) Verapamil

Rifapentine

25

75

0 months

27.7 ± 1.5

72.3 ± 2.0

1 month

28.5 ± 3.2

71.5 ± 3.0

3 months

26.0 ± 2.5

73.0 ± 2.5

Theoretical

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

39



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

Page 40 of 41

1

Table 4. Minimum inhibitory concentration (MIC90) against M. tb H37Ra and M. tb H37RV of the three

2

drug formulations and the individual compounds using the resazurin assay Compound

MIC90 (µg/mL)* M. tb H37Ra

M. tb H37Rv

250

500

0.000625

0.005

> 62.5

> 1000

Vrp_Rpt_Leu (verapamil : rifapentine, 1:3)

0.000625

0.005

Verapamil : rifapentine, 1:1

0.00125

0.01


0.005

0.01


0.01

> 0.02

Vrp_Leu Rpt Spray dried L-leucine

3

*Mean MIC90 of three independent experiments performed in triplicate.

4 5 6 7 8 9 10 11 12 13 14 15

40


Page 41 of 41

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

Table 5. Half Maximal Inhibitory Concentration (IC50) of the inhalable dry powder formulations on

2

human monocytic (THP-1) and human alveolar basal carcinoma epithelial (A549) cell lines Compound

IC50 (µg/mL) THP-1

A549

Vrp_Leu

125

250

Rpt

62.5

62.5

Vrp_Rpt_Leu

62.5

62.5

>1000

>1000

Spray dried L-leucine 3

41


In Vitro Evaluation of Inhalable Verapamil-Rifapentine Particles for

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