SHetA2 DRY POWDER AEROSOLS FOR TUBERCULOSIS

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SHetA2 DRY POWDER AEROSOLS FOR TUBERCULOSIS: FORMULATION DESIGN AND OPTIMIZATION USING QUALITY BY DESIGN Mariam Ibrahim, Manolya Kukut Hatipoglu, and Lucila Garcia-Contreras Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01062 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Molecular Pharmaceutics

SHetA2 DRY POWDER AEROSOLS FOR TUBERCULOSIS: FORMULATION DESIGN AND OPTIMIZATION USING QUALITY BY DESIGN

Mariam Ibrahim, Manolya Kukut Hatipoglu, and Lucila Garcia-Contreras

Department of Pharmaceutical Sciences at University of Oklahoma Health Science Center, Oklahoma City, OK

Corresponding author: Lucila Garcia-Contreras, Ph.D. Assistant Professor Department of Pharmaceutical Sciences College of Pharmacy The University of Oklahoma Health Sciences Center 1110 N. Stonewall Ave. Suite 321 Oklahoma City, OK 73126-0901 Phone: (405)271-6593 Ext. 47205 Fax: (405) 271-7505 #Email: [email protected]

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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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Graphic for table of Contents use only


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ABSTRACT Tuberculosis (TB) is a life threatening pulmonary infection caused by Mycobacterium

tuberculosis (MTB). Current treatments are complex, lengthy and associated with severe side effects that decrease patient compliance and increase the probability of the emergence of drug resistant strains. Thus, more effective drugs with little to no side effects are needed to diversify the armamentarium against the global TB epidemic. SHetA2, an anticancer compound with null toxicity at doses much higher than the effective dose, was recently discovered to be active against MTB. In the present study, a dry powder formulation of SHetA2 for pulmonary delivery was developed to overcome its poor aqueous solubility and to maximize its concentration in the lungs, the main site of TB infection. Using quality by design methodology, three different formulations of SHetA2 microparticles (MPs) were designed, manufactured and optimized: SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs, to maximize the drug dose, target alveolar macrophages and increase drug solubility, respectively. The resulting three SHetA2 MP formulations had spherical shape with particle size ranging from 1 to 3 µm and a narrow size distribution, suitable for uniform delivery to the alveolar region of the lungs. Upon dispersion with the Aerolizer® dry powder inhaler (DPI), all three SHetA2 MP formulations had aerodynamic diameters smaller than 3.3 µm and fine particle fractions (FPF4.46) greater than 77%. SHetA2 remained chemically stable after MP manufacture by spray drying, but the drug transformed from the crystalline to the amorphous form, which significantly enhanced the solubility of SHetA2. Using a custommade dissolution apparatus, the FPF4.46 of SHetA2 MP dissolved much faster and to a greater extent (21.19 ± 4.40%) than the unprocessed drug (3.51 ± 0.9%). Thus, the physicochemical characteristics, in vitro aerosol performance and dissolution rate of the optimized SHetA2 MPs appear to be suitable to achieve therapeutic concentrations in the lungs.



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KEWORDS: Tuberculosis, SHetA2, pulmonary delivery, dry powder for inhalation, aerosol performance, spray drying, quality by design.


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1. INTRODUCTION Tuberculosis (TB) remains one of the major health problems of the world. In 2015, more than 10 million TB infections were reported worldwide and 1.8 million deaths, with most of these deaths occurring in developing countries1, whereas in developed countries, TB fatalities are mostly due to HIV TB co infection 2. Current TB treatments consist of oral administration of high doses of three to four drug combinations for 6-9 months 3-5. This long and complicated treatment causes severe side effects and decreases the quality of life of these patients to such extent that they stop the treatment before completion, which has led to the emergence of multi-drug resistant strains of MTB (MDR-TB). Therefore, new, safer and more effective treatments are urgently needed to reduce the incidence of side effects and treatment duration, thus improving treatment compliance and the quality of life of TB patients 3, 6. Within the last 40 years, only two new antitubercular drug, bedaquiline and delaminid, have been approved for TB treatment. Only bedaquiline has been approved by the FDA for treatment of MDR-TB in the United States 7 but information on this new drug is limited and its use is associated with severe side effects. In 2013, the World Health Organization issued an “interim policy guidance” in part due to an increase in the incidence of death among TB patients treated with bedaquiline 8. Delaminid has been approved for treatment of both susceptible and drug resistant TB in Europe and in Japan, but its use is also associated with severe side effects that can cause a life threatening abnormality of heart rhythm. Thus patients using delaminid require frequent heart monitoring increasing the burden of TB treatment 9, 10. Recently, our laboratory discovered that SHetA2, a flexible heteroarotinoid compound with anticancer activity, has antimicrobial activity against drug susceptible and drug resistant MTB 11. Formal studies to evaluate the disposition and toxicity of SHetA2 as an anticancer drug demonstrated that SHetA2 does not elicit adverse effects when given 5 ACS Paragon Plus Environment


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orally to rats and dogs at doses that were more than 30 times larger than the effective dose 12. However, SHetA2 has poor aqueous solubility and low oral bioavailability if administered without 30% Kolliphor, a solubilizing agent. Administration of SHetA2 by inhalation would not require solubilizing agents because by virtue of the small particle sizes employed for pulmonary delivery and the presence of surfactants in the lungs, its solubility may be enhanced and by consequence its bioavailability. In addition to the enhancement of dissolution of poorly water soluble drugs

3, 13

, the

pulmonary route of administration has several advantages for TB treatment, the most important being the delivery of antitubercular drug(s) directly to the primary site of infection: the lungs. This leads to other therapeutic advantages such as achieving higher local concentrations at the site of infection using smaller inhaled drug doses and decreasing the likelihood of side effects 3, 13-16. In addition, pulmonary drug delivery offers other advantages such as macrophage targeting

17-20

. However, the inhalable formulation must be carefully

designed and optimized to achieve these therapeutic and pharmaceutical advantages. The present work describes the design and manufacture of three formulations of SHetA2 MPs employing quality by design (QbD) methodology to optimize their physicochemical properties for efficient pulmonary delivery. The in vitro aerosol performance of SHetA2 MPs was determined when actuated from a commercial dry powder inhaler (DPI) into a cascade impactor and their dissolution was evaluated in a custom-made dissolution apparatus designed to mimic the conditions in the alveolar region of the lung.


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2. MATERIALS AND METHODS 2.1.

Materials SHetA2 was synthesized by Cayman Chemical company, Inc. under a contract from

Rapid Access to preventive Intervention Development (RAPID) National Cancer Institute (NCI) program. Poly (DL-lactide-co-glycolide) (50:50) (PLGA) was supplied by Birmingham polymers, Inc. United States. D-mannitol, were purchased from Sigma-Aldrich, United States. All solvents were of HPLC grade and were purchased from Sigma-Aldrich.

2.2.

Formulation and optimization of SHetA2 MPs QbD methodology was employed to design the formulation, manufacture and

optimization of SHetA2 MPs. Figure 1 shows the Ishikawa diagram illustrating the input (material attributes and process parameters) and output variables (quality and performance) considered in the optimization procedure. For the first step of QbD we defined the desired product performance and identified product critical quality attributes (CQAs) as: (1) particle size of 1-5 µm in diameter; (2) geometric standard deviation (GSD) of less than 2. For step 2 of QbD, we designed three SHetA2 MPs formulations: SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs, to maximize the drug dose, target alveolar macrophages and increase drug solubility, respectively. We selected spray drying as the process of manufacture due to its versatility to manipulate manufacturing conditions and use materials with different physicochemical properties. For step 3 of QbD, we employed the Design of Experiments (DoE) software (Design-Expert, version 8.0.1, Stat-ease®) to understand impact of material and process parameters on product CQAs. SHetA2 MPs were manufactured using Buchi-290 minispray dryer (Buchi, Switzerland). For the first two formulations, SHetA2 alone and SHetA2 PLGA MPs, SHetA2 alone or both SHetA2 and PLGA were dissolved in dichloromethane and spray dried at an 7 ACS Paragon Plus Environment


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atomization pressure of 5 bars and feed rate of 3 ml/min. For the third formulation, SHetA2 mannitol MPs, SHetA2 was dissolved in dichloromethane and mannitol in water and both solutions were concurrently spray dried using a 3 way fluid nozzle to accommodate the use of 2 different solvents, at the same feed rate and atomization pressure. The physicochemical characteristics of SHetA2 MPs were optimized by entering into the DoE software the four spray drying parameters of feed concentration, the nitrogen spray gas flow, inlet temperature and aspiration rate at two levels each. The DoE software randomized these parameters in a 24 factorial design for the manufacture of 16 batches each of SHetA2 alone and SHetA2 PLGA MPs whereas a 23 factorial design was used to randomize the spray gas flow, aspiration rate and inlet temperature for the manufacture of 8 batches of SHetA2 mannitol MPs. Table 1 lists the upper and lower limits for each of these spray drying parameters used in the factorial designs of SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs. Statistical analysis of the size and distribution for each MP batch performed by the software was used to determine the most influential manufacturing parameters of the spray drying process on the physical properties of the three SHetA2 MPs formulations.

2.3.

Physicochemical characterization of SHetA2 MPs

2.3.1. Particle morphology and size determination The morphologies of SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs were examined using scanning electron microscopy (HITACHI TM3000 SEM) at an acceleration voltage 5 Kv. Samples were prepared by deposition on a double-coated carbon conductive tape (Ted Pella Inc., Redding, CA), mounted on aluminum stubs then sputter coated with gold and palladium at the ratio of 6:4. The geometric diameter (Dg = X50) of the three MP formulations was measured directly from the SEM images (2-3 fields each having 25-30 MPs


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on average) with the aid of ImageJ 1.49n software (NIH, USA). The volume diameter (Dv = X50) of the three MP formulations was measured by laser diffraction using a HELOS system with RODOS dry dispersing unit (Sympatec Inc., Lawrenceville, NJ). Measurements were performed in triplicate at a dispersion pressure of 0.5 bars. GSD is calculated from the following equation 21: GSD = [D84% / D16%] 1/2 where D84% and D16% represent the diameters at the cumulative percentile of 84% and 16% of the particle size distribution after it has been “normalized”.

2.3.2. X-ray Diffractometry (XRD) The crystallinity of the drug in SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs was analyzed by powder X-ray diffraction (XRD) using a Rigaku Ultima IV X-ray diffractometer (Woodlands, TX). Cu-K-alpha radiation was used with a scintillation detector at a generation voltage of 40 kV and a current of 44 mA. Data were collected by the 2θ method at a step size of 0.04 for 2 seconds at the range of 5−40°.

2.3.3. Thermal analysis The thermal stability of unprocessed SHetA2 and SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs were assessed using differential scanning calorimetry (DSC) (DSC60, Shimadzu) (Kyoto, Japan). Approximately 5 mg of each formulation were loaded in aluminum pans, cribbed and heated from 30 to 210°C at the rate of 10°C/min. Differences in heat flow rate were measured against an empty reference pan. Exothermic and endothermic peaks were analyzed using ta60 software (version 2.21, Shimadzu).



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2.3.4. Fourier-transform infrared spectroscopy (FTIR) analysis The chemical stability of SHetA2 after spray drying and the likelihood of chemical interaction between SHetA2 and PLGA or mannitol were evaluated using FTIR (Bruker, MA, USA). Samples were prepared by mixing unprocessed or spray dried SHetA2 with potassium bromide (KBr), compressed into a pellet followed by FTIR analysis using Opus spectroscopy software.

2.3.5. Drug content uniformity In order to assess the uniformity of SHetA2 content in the different batches of MPs, three pre-weighed samples of SHetA2 PLGA and SHetA2 mannitol MPs were dissolved in 5 ml of acetonitrile: water mixture (85:15) followed by centrifugation at 12000 rpm for 10 minutes to separate any undissolved solids. After centrifugation, 15µl of the supernatants were collected, diluted with 85 µl of (85:15) acetonitrile water mixture and the SHetA2 content was determined using HPLC analysis.

2.3.6. SHetA2 quantification HPLC analysis was performed using a Waters Alliance HPLC System with a Vydac 201 TP C18 5 µm (250mm x 2.1 mm) column equipped with matching guard column and UV detection at 341 nm. The mobile phase consisted of 80% acetonitrile and 20% water at a flow rate of 0.3 ml/min. The injection volume was 10 µl and the retention time of SHetA2 peak was at 2.9 minutes.


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2.3.7. In vitro aerosol performance The aerosol performance of SHetA2 MP formulations upon dispersion by the Aerolizer® DPI (Novartis, Basel, Switzerland) was evaluated using next generation impactor (NGI) (Westech, NJ). To determine the fill mass that would allow the best powder dispersion by the Aerolizer, hydroxypropyl methyl cellulose (HPMC) capsules (Vcaps, Capsugel, size 3) were filled with masses of the different SHetA2 MP formulations ranging from 3 to 10 mg. Capsules were inserted in the Aerolizer® DPI and the device attached to the induction port of the NGI. After actuation of the device, the NGI was operated for 5 seconds at a flow rate of 60 L/min. The fill mass of 3.5 mg was determined as the most suitable to maximize the amount of powder in a #3 capsule, while leaving space inside the capsule for powder dispersion and fluidization by the Aerolizer® DPI. The induction port, collection cups and the filter were rinsed with an acetonitrile: water mixture (85:15) and the SHetA2 content was analyzed by HPLC. SHetA2 formulations were characterized according to their mass median aerodynamic diameter (MMAD), GSD, emitted dose (%) and fine particle fraction of the emitted dose at a cutoff diameter of 4.46 µm (FPF4.46). The MMAD was calculated as described in the USP chapter 22. The GSD for MMAD was calculated similarly to the GSD of Dv. The emitted dose (%) was calculated according to the following equation:

% =

2 × 100 2

The FPF % was calculated as the percentage of SHetA2 mass recovered from stages 3-7 in the NGI to the total drug mass recovered from the NGI.



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2.3.8. Apparent maximum solubility in phosphate buffer saline (PBS) The apparent solubility of the drug in SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs was determined in PBS containing 0.05% sodium dodecyl sulfate (SDS) using the unprocessed SHetA2 as a control. Excess amount of unprocessed SHetA2 and SHetA2 alone, SHetA2 PLGA and SHetA2 mannitol MPs were added to screw cap vials containing 5ml of PBS and 0.05% SDS and the vials and shaken for 24 hours at 37°C inside an incubator shaker (NewBranswick Scientific Co., Inc. Edison, NJ). At predetermined time points, 500µl samples were withdrawn, filtered using 0.2 µm polytetrafluoroethylene (PTFE) syringe filter and replaced with equal volumes of fresh medium. Filtered samples were diluted with equal volume of acetonitrile then injected into HPLC for SHetA2 content analysis.

2.3.9.Dissolution profile of the size range 2.82-4.46 µm of SHetA2 MPs in PBS containing 0.05% SDS Dissolution studies were performed using SHetA2 MPs of size range 2.82-4.46 µm to mimic the dissolution process of SHetA2 MPs in the alveolar region of the human lungs. The size range 2.82-4.46 µm of SHetA2 MPs was separated as previously described 23. A glass fiber filter paper (Westech Scientific Inc, GA, USA) was placed on the collection cup of stage 3 of the NGI, then SHetA2 MPs (3.5 mg) were weighed in a capsule and loaded into the Aerolizer® (Novartis, USA) and the device attached to the induction port of the NGI. After actuation of the device, the NGI was operated for 5 seconds at a flow rate of 60 L/min. After deposition of approximately 1 mg of SHetA2 MPs, the glass fiber paper containing SHetA2 MPs of sizes between 2.82 and 4.46 µm was transferred to a custom-made dissolution apparatus. The design of this custom-made dissolution apparatus was based on a few


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characteristics from those of Davies et al.24 and Wang et al. 23, but modified in a manner that simulated the limited volume of fluid available for dissolution in the alveolar region. A schematic diagram of the custom-made dissolution apparatus is shown in figure 2. It consisted of a jacketed, 20 ml volume, Franz diffusion cell (PermeGear, Inc., Hellertown, PA), and a dual channel pump (Cole-Parmer, USA). The dual pump was used to allow the continuous flow of dissolution medium of into the donor compartment and to circulate water into the jacket of the diffusion cell in a manner that the solution at the receiving compartment was maintained at 37 °C. The glass fiber paper holding the FPF of SHetA2 MPs was placed in the Franz cell between the donor and recipient compartments. The dissolution medium of PBS containing 0.05 % SDS was pumped unto the glass fiber paper at a flow rate of 2 ml/min and samples were collected from the receiving compartment every 15 minutes for 5 h. Throughout the study, SHetA2 MPs retained in the glass fiber paper were in contact with limited volume of dissolution medium (2 ml), which mimics the limited volume of fluid available in the lungs for dissolution. Dissolution studies were performed in triplicate.

2.3.10. Statistical analysis The values of the responses measured for each experiment dictated by each DoE (size and distribution) that were employed to optimize the three MP formulations were analyzed by the software as follows: First, the absolute values of the effect of each analyzed variable were displayed in a half normal probability plot and the magnitude of each effect was confirmed in a Pareto chart as a “t” value. After the effects with the largest magnitude (above the “t” value) were selected, the chosen model effects (variables) were analyzed with ANOVA based on Ftest; if (prob>F) was

SHetA2 DRY POWDER AEROSOLS FOR TUBERCULOSIS

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