The role of particle physico-chemical properties in pulmonary drug delivery for tuberculosis therapy.

http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–11 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.932029

REVIEW ARTICLE

The role of particle physico-chemical properties in pulmonary drug delivery for tuberculosis therapy

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Ninell P. Mortensen, Phillip Durham, and Anthony J. Hickey Technology for Industry and the Environment, Discovery Sciences Technologies Group, RTI International, Research Triangle Park, NC, USA

Abstract

Keywords

There is increasing interest in the use of inhaled aerosol drug therapy for the treatment of tuberculosis (TB). A number of methods of preparation of particles have been employed including spray drying, solvent evaporation, emulsion and phospholipid methods to create microparticles, macroaggregated nanoparticles, solid lipid nanoparticles and liposomes. Each of these methods involves the use of different proportions of additives to aid in the particle formation or to achieve important physico-chemical properties such as ease of dispersion. While these approaches all have merit their practical value is limited by constraints on dose and means of delivery as an aerosol in order to achieve a therapeutic effect. A review of a number of approaches is presented and placed in the context of the need for effective aerosol delivery systems for the treatment of TB as a guide to selection of appropriate excipients, processes and delivery strategies to support product development activities.

Bioavailability, dissolution rate, inhaled therapy, microparticles, spray drying

Introduction–pulmonary drug delivery using microparticles The lungs have a large surface area and a thin alveolar epithelium (Sakagami, 2006). Aerosol delivery of drugs for local action in the lungs has been a standard of care for asthma for decades (Ulrik and Lange, 2011, Barnes et al., 2012, Lipworth, 2013), and more recently pulmonary drug therapy has been implemented in the treatment of cystic fibrosis and diabetes (Patton and Byron, 2007). Inhaled therapy is capable of achieving a high local drug concentration in the lungs while minimizing systemic exposure. This is a desirable situation when treating a pulmonary infection and avoids possible systemic toxic side effects. Alveolar macrophages phagocytose material entering the lungs. Inhaled drug containing microparticles (MPs) are subject to macrophage uptake. This innate biological mechanism ensures high intracellular drug concentrations, a desirable situation when treating tuberculosis (TB) since Mycobacterium tuberculosis bacillus can survive and multiply inside the macrophages. Delivering the drug to the intended area of the lungs in a fashion that promotes its release, uptake and optimal bioavailability is challenging. This review focus on the use of microparticles (MP) in inhaled therapy of tuberculosis. The physiological conditions of the human airways that influence the MP deposition and disposition in the lungs, the physico-chemical properties of the MPs and an overview of the current literature in inhaled therapy to treat TB are discussed. In order to effectively deliver drugs to the lungs, it is important to understand the barriers to drug delivery. The following sections

Address for correspondence: Anthony J. Hickey, Technology for Industry and the Environment, Discovery Sciences Technologies Group, RTI International, 3040 East Cornwallis Road, Research Triangle Park, NC 27709, USA. Tel: +919 541 6771. E-mail: [email protected]

History Received 7 January 2014 Accepted 3 June 2014 Published online 29 July 2014

summarize the physiology and anatomy of the lungs important in deposition, clearance, targeting, and residence time of delivered therapeutic agents.

The human airway Estimates of human airway branching range from 23 to 32 generations and are divided into multiple, highly specialised compartments (Patton and Byron, 2007; Eixarch et al., 2010; Hofmann, 2011; Figure 1). The trachea, bronchi and bronchioles are collectively called the airways, and the branches of the organ system culminate in the alveoli. In an adult the surface area of the central airways is only a few m2 in contrast to the peripheral surface area of more than 100 m2. The airways are lined with many different cell types (epithelial, ciliated, goblet, secretory and basal cells) which are adapted to serve the specialized functions of each compartment. The major cell type of the airways is epithelial cells which in the bronchi of the upper airways has a columnar morphology with a height of 58 mm and gradually are thinning through to a height of 0.1–0.2 mm forming a cellular monolayer in the alveoli. Two important cell types in the lungs are mucus producing goblet cells and ciliated cells that are major components of the mucociliary escalator. Mucociliary transport removes any matter filtered from the conducted air and maintains the patency of the upper airways. The protective viscous mucus layer lining the epithelial cells is 8 mm thick in the bronchi and transitions through the airways into a 0.07 mm thick surfactant layer in the alveoli. The mucus layer is comprised of inorganic salts, proteins, glycoproteins (mucins), lipids and water, whereas the surfactant layer contains phospholipids, cholesterol and proteins (Eixarch et al., 2010). The presence and composition of this protective coating of the lungs is likely to affect both particle dissolution, diffusion towards the epithelium and the interaction between drugs and cell surfaces and/or receptors. Inhaled particles deposit


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N. P. Mortensen et al.

J Microencapsul, Early Online: 1–11

Figure 1. Schematic of the biology of the lungs (based on: Patton and Byron, 2007, Eixarch et al., 2010; Hofmann, 2011).

in the lungs and become imbedded in the mucus layer where they are transported up the airways and through the trachea by the ciliated cells and are delivered to the pharynx, where the material is expectorated and/or swallowed. In the unciliated respiratory bronchioles and alveolar space of the lungs macrophages are responsible for removing inhaled particles. The air interface of the alveoli is patrolled by alveolar macrophages, which phagocytise any deposited insoluble particles. Delivery of drugs to the lungs can be designed to avoid phagocytic cells or to take advantage of the phagocytic nature of the macrophages. The fate of inhaled drug particles in the lungs is also potentially influenced by the high relative humidity in the lungs, local metabolism, the presence of efflux transports (such as P-glycoprotein prominent in Type I cells), protein binding and cellular permeability (Eixarch et al., 2010). All metabolic enzymes found in the liver are also present in the lung, but generally at a lower expression level. For example cytochrome P450 (CYP450) enzymes are at 5–20 times lower levels than in the lung than the liver (Dahl and Lewis, 1993; Krishna and Klotz, 1994; Upton and Doolette, 1999; Labiris and Dolovich, 2003). Other protein transporters like breast cancer resistance protein, multidrug resistance associated protein and lung resistance protein found in the lungs are responsible for the efflux transport of many drugs, which leads to lower bioavailability than would otherwise be expected (Eixarch et al., 2010). All the biochemical and biophysical mechanisms listed are likely to differ between the various compartments of the lungs and may require consideration in the disposition of inhaled drugs and design of delivery systems. Also, in combination all the above biological functions will influence the rate and extent of drug absorption and retention in the lungs, which dictates the bioavailability of the drug. Rifampicin, a candidate for aerosol delivery to the lungs to treat TB, is subject to the action and an inducer of CYP P450 (sub-type 3A4) (Goodwin et al., 1999) and P-glycoprotein (Greiner et al., 1999).

The biology of the lungs and its influence on drug bioavailability is only part of the challenge when designing inhalation therapy. The physico-chemical properties of the aerosolised drug particles also play a large role in the disposition of the drug. These properties of particles, such as primary particle size, shape, density, electrostatic charge and aerodynamic particle size distribution, will dictate the fate of drug particles deposited in the lungs, and the prospects of reaching the intended target. Drug dose and proportion likely to enter the lungs, often referred to as fine particle fraction (FPF) are also important considerations in addressing the therapeutic effect. Finally, the pharmacokinetic properties of drug release, influenced by intrinsic solubility and dissolution rate, from particles and the absorption and elimination rate are important factors controlling the ability to achieve therapeutic concentrations.

Particle deposition in the human airways Deposition of drug particles in the lungs is primarily based on particle size and may be divided into five major mechanisms: Inertial impaction, sedimentation, diffusion by Brownian motion, interception and electrostatic precipitation (Patton and Byron, 2007; Carvalho et al., 2011; Hofmann, 2011). Inertial impaction occurs when the particle has sufficient momentum to follow its initial trajectory when the direction of the airstream changes. Consequently, it collides with the wall of the airways, usually at or near a bifurcation. Sedimentation occurs in response to the influence gravity and is a time-dependent process. Breathing cycle plays an important role in this kind of deposition, the longer the period of sedimentation (for example breath holding) the greater is the probability of particle to deposit. Particle diffusion happens when the particle is small enough to be subjected to random motion due to individual molecular bombardment (Carvalho et al., 2011). Impaction and sedimentation are the dominant mechanisms of deposition of therapeutic aerosols.

DOI: 10.3109/02652048.2014.932029

Particle physico-chemical properties in pulmonary TB drug delivery


Physico-chemical properties of particles and deposition Nominal particle diameters are descriptors rather than intrinsic dimensions and are usually referenced to standard spherical geometries according to the method used for measurement. Direct measurement of geometric particle diameter employs microscopes and pharmaceutically relevant indirect measurements of aerodynamic (da) and volume diameters employ inertial impactors and optical methods (laser light scattering). The da is the most appropriate descriptor of aerosolized drug particles in connection to inhalation therapy, since it relates to particle aerodynamic behaviour and has been linked to lung deposition. The optimal da of a particle intended to reach the lung is 1–5 mm (Goodman et al., 1994). Particles with a larger da than 5 mm are usually deposit in the oral cavity or pharynx, and particles smaller than 0.5 mm move by Brownian motion and settle very slowly. Small particles are not likely to deposit at all because they are exhaled before deposition occurs. The FPF is considered to consist of particles 55 mm in da. A simplified expression for the da is as follows: s ffiffiffiffiffiffiffiffiffiffiffiffiffi p ð1Þ da ¼ dg 0 where dp41 mm, o ¼ 1 g/cm3, dg is the particle geometric diameter, o is the particle density in the same unit as p, and is the dynamic shape factor of the particle. The shape factor is defined as the ratio of the drag force of the particle to that of a sphere of equivalent volume. The particle shape factor is influenced by shape, surface roughness and surface area which are all closely related to each other. The drag force on a nonspherical particle with high surface roughness will be greater than that for a spherical particle and the da, will as a result, be smaller than anticipated. Creating porous particles that have a similar mass of drug in a larger volume also increases the drag with respect to a solid particle of the same da (Edwards et al., 1997). Therefore, particle shape and density can be used to manipulate the aerodynamic properties of aerosols. The effect of particle shape on flowability, aerosolization and deposition properties of spherical, needle, cube, plate, and unique geometries of pollen has been studied. Pollen (approximately spherically with high surface roughness) exhibited the best flowability, highest emitted dose, and highest fine particle fraction (FPF; Hassan and Lau, 2009). Since drug dose is very important in therapy, the aerodynamic particle size distribution (APSD) is important. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) are descriptors of the APSD. The MMAD refers to the da above and below which 50% of the aerosol mass resides. Where the APSD is log-normal, meaning linear on a plot of cumulative percentage undersize on a probability scale against the logarithm of aerodynamic particle size (Martonen, 1983b) the GSD can be obtained, in the simplest manner by dividing the particle size at the 84th percentile by the MMAD (Martonen, 1983a). Particle da can also be affected by the humidity in the lung due to mostly to acquisition and occasionally removal (e.g. for hypotonic solutions) of water from the hygroscopic particle. Thereby deposition in the lung may be altered (Labiris and Dolovich, 2003). The increase of particle size is a function of the initial diameter of the particle and the time to equilibrate before deposition. For example, a particle 51 mm may increase five-fold in contrast to the two to three fold increase for a particle 42 mm (Swift, 1980). Specific hygroscopic growth functions can be calculated from first principles or measured for particles or droplets of known composition. The physico-chemical properties of the drug itself are also important. Molecular weight, lipophilicity (log P), solubility, pKa, protein binding, polar surface area, and charge, influence the

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permeability of a compound across the epithelial barrier (Eixarch et al., 2010). In general, lipophilic molecules cross the airway epithelium by passive transport, and hydrophilic molecules cross via extracellular pathways. However, particles, especially those that do not dissolve readily and are not deposited on the mucociliary escalator, are phagocytised by alveolar macrophages or absorbed into the pulmonary circulation (Labiris and Dolovich, 2003). The fate of the drug particle and the influence of its overall surface properties prior to dissolution are of great interest when considering disposition. Particle-particle and particle-cell surface interactions have to be taken into consideration when designing inhaled therapies. Van der Waal’s forces, hydrostatic interaction, mechanical interlocking, electrostatic and capillary forces are important contributors to particle interactions in the lungs. Ease of deaggregation to achieve the intended da dictates the dose delivered and the success of the intended therapeutic strategy. This subject that has been covered in a number of excellent reviews (Telko and Hickey, 2005; Patton and Byron, 2007; Hofmann, 2011; Misra et al., 2011; Hickey et al., 2013). The particle surface chemistry will also affect the composition of the protein corona formed on the particle in a biological, protein-rich environment (Cedervall et al., 2007; Lundqvist et al., 2008; Aggarwal et al., 2009; Mortensen et al., 2013). The protein corona forms immediately after introduction to a biological fluid and is dynamic over a period of several days following the Vroman effect. The Vroman effect dictates that the highly abundant proteins form the initial protein corona and are gradually replaced with the highest affinity proteins thereby transitioning from a loosely associated corona to a more tightly bound corona (Vroman, 1962; Vroman et al., 1980). This protein corona plays a role in both macrophage uptake and particle penetration through the various fluid and cellular barriers into the pulmonary circulation.

Tuberculosis TB is an insidious disease caused by M. tuberculosis. It is estimated that one-third of the worlds’ population is infected with M. tuberculosis (Suarez et al., 2001b; Elvang et al., 2009; Wang et al., 2009; Brennan et al., 2012; O’Garra et al., 2013), and 9 million new cases of TB occurred in 2011 with 1.4 million deaths (Hofmann, 2011; WHO, 2012). To complicate matters further, the occurrence of multi-drug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis strains are increasing (3.7% of new and 20% of previous cases (WHO, 2012)). The ideal scenario would be to immunize the population effectively against M. tuberculosis infections. However, the only available vaccine is the attenuated strain of Mycobacterium bovis, Bacille Calmette-Guerin (BCG), which has been used in humans for almost a century. However, the vaccine protection diminishes after 10–15 years following administration (Brewer, 2000) and fails to protect against TB in adults (ICMR, 1999). In addition to offering limited protection, BCG vaccines have proven unsafe for infants infected with HIV (Brennan et al., 2012). Several vaccines are in development and a few are in clinical trials, but the delay in improving TB vaccine strategy increases the need for more efficacious drugs for TB therapy. Most cases of TB are treatable but the current antibiotics require lengthy regimens at doses that result in considerable side effects (Table 1). The current standard of care for TB in most high-burden regions is a six month regimen, in which the initial two months include isoniazid, rifampin, pyrazinamide and ethambutol, followed by a four month continuation phase of rifampin and isoniazid. The introduction of rifampin and

Broad spectra antibiotic Broad spectra antibiotic

Ciprofloxacin

1.0 (Rastogi et al., 1996)

5–6 h in adults with normal renal function

^Minimal inhibitory concentration (MIC), mg/mL for H37Rv in broth. #(Drugbank.ca). *(mg/mL at 25 C) (Drugbank.ca).

Streptomycin

9

10 days following a single dose, 70 days after longterm, high-dose therapy. 4


0.5 (Rastogi et al., 1996, Shandil et al., 2007) 0.5 (Shandil et al., 2007)

Half-life in patients with normal renal function is 10 hr



Not available

7.58

Normal renal function, 3–4 h, impaired function, up to 8 h 9–10

28.3

30

Binds bacterial 30S ribosomal subunit, causing misreading of tRNA.

Course double stranded DNA breakages Course double stranded DNA breakages

Binds to bacterial DNA leading to disruption of cell cycle

2.25 104

Soluble

Is believed to inhibit protein synthesis, but mechanisms of action is unknown Competitively inhibits bacterial cell wall synthesis

Damage bacterial cell membrane and inhibits transtranscription

Inhibits DNA-dependent RNA polymerase in prokaryotic but not in eukaryotic cells. Is taken up by bacteria, but mechanism of action is unknown

Halts growth of resting organism, and kill dividing bacteria. Mechanism of action is unknown

Mechanism of action

Soluble in water as disulfate salt.

15

1.4

3.35 (+/0.66)

0.016–0.4 (Rastogi et al., 1996, Wanger and Mills, 1996, Hirata et al., 1995) 0.06–0.5 (Rastogi et al., 1996, Wanger and Mills, 1996)

0.25–1.0 (Wanger and Mills, 1996)

140

Water solubility*

Fast acetylators: 0.5–1.6 h. Slow acetylators: 2–5 h.

Half-life (hr)#

0.02–0.25 (Rastogi et al., 1996, Wanger and Mills, 1996)

MIC^

Intramuscular injection

Orally

Orally

Orally

Orally

Intramuscular injection

Orally

Orally

Orally

Orally

Route of administration

65–80% of an administered oral dose is excreted unchanged via the kidneys within 48 hr

Is distributed throughout the tissues and body fluids

Well absorbed and is widely distributed, penetrating well in the meninges

Readily absorbed from the GI, is widely distributed in the tissues and body fluids. Passes freely into mammalian cells Widely distributed in the tissues and body fluids. Enter phagocytoic cells. Is well absorbed, and can reach therapeutic concentration in CSF

Biodistribution

Includes nephrotoxicity and ototoxicity.

LD50 ¼ 5450 mg/kg (orally in mice)

Mainly on the central nerve system, ranging from headache and irritability to depression, convulsions and psychotic states. Includes abdominal symptoms, less frequently splenic infarction, bowel obstruction, and GI bleeding.

Included kidney damage, injury to the auditory nerve.

Includes, gout, GI upset, malaise, fever and can cause hepatic damage

About 5% of patients – allergic skin eruptions. Also, fever, hepatotoxicity, heamatological changes, arthritic symptoms and vasculitis. Relative infrequent, but includes skin eruption, fever and GI disturbance. Can cause liver damage Side effects are uncommon, but include optic neuritis.

Side effect


Olfoxacin

Antibacterial activity limited to mycobacteria

Antibiotic against enteric bacteria and other eubacteria Broad-spectrum antibiotic

Is inactive at neutral pH, but anti-TB at acid pH.

Broad spectra antibiotic and the most active anti-TB drugs. Only affects mycrobacteria.

Antibacterial activity limited to mycobacteria.

Clofazimine

Cycloserine

Second-line Capreomycin

Pyrazinamide

Ethambutol

Rifampicin

First-line Isoniazid

Description

Table 1. Antibiotics used in treatment of TB.


4 J Microencapsul, Early Online: 1–11


DOI: 10.3109/02652048.2014.932029


pyrazinamide into TB therapy allowed the duration of treatment to be reduced from at least 12 months to six months with 55% relapses. Further reductions in the duration of treatment, however, resulted in increasing rates of relapse after completion of treatment (Fox, 1981). Improving the efficacy of treatment of pulmonary TB by addressing the performance of the existing drugs is a way forward while we wait for the advent of affordable new drugs and improved vaccine strategies (Mitchison, 2000). One approach to improve the performance of existing drugs is to adopt alternative routes and means of delivery. Natural infection with TB occurs by inhaling bacteria-containing aerosols. Mycobacterium tuberculosis deposits in the lung periphery where they are phagocytised by alveolar macrophages initiating pulmonary infection. It has been proposed that targeted delivery of TB therapies directly to infected lungs will result in immediate contact with resident bacteria, leading to high local drug concentrations and rapid and effective killing. A number of TB drugs have been formulated in dry powder, nano- and microparticles for pulmonary delivery (Tsapis et al., 2003; GarciaContreras et al., 2007; Fiegel et al., 2008). Results of in vivo assessment of these drugs indicated that direct delivery to the lungs achieved high local concentrations and reduced bacterial burden compared to the same treatments delivered by other routes, offering the possibility of both reduced efficacious doses and reduced systemic side effects. However, despite these encouraging findings clinical inhalation therapy for TB has not been adopted.

Inhaled drug therapy in Tuberculosis Inhaled TB therapy of known drugs has not been proposed as an independent treatment but is usually considered as a complement to orally administered drugs or to potentially replace injected drugs. TB is a systemic disease and the most prominent sites of infection beside the lungs include liver, kidneys, spleen, uterus and bones. Therefore, treatment of TB is a complicated task because the bacteria are located in different organs and cell types and are present in different metabolic states. In the lungs M. tuberculosis are present in lesions which are poorly vascularised, and fortified with thick fibrous tissues. Dormant or resting M. tuberculosis with low metabolic activity are present as intracellular pathogens in antigen presenting cells, dendritic cells and alveolar macrophages within phagosomes where they survive, distribute by the cell mediated immune system, multiplying until the cells are ‘‘activated’’ by the T-helper lymphocytes. Successful treatment of TB means, targeting M. tuberculosis in intra- and extra-cellular pulmonary space, in both high and low metabolic states, and in other organs of the body. One of the potential advantages with inhalation drug therapy is that the local drug concentration in the lungs can be elevated without requiring high circulating drug concentrations, which reduces the incidence of systemic toxicity. For potent therapeutic agents it may be possible to achieve sufficient circulating concentrations to be therapeutic but not toxic. The drugs employed to treat TB may be divided into first-line antibiotics including isoniazid, rifampicin, ethambutol and pyrazinamide and second-line drugs, such as capreomycin, cycloserine, clofazimine, ciprofloxacin, ofloxacin and streptomycin (Table 1). Conventional drug therapy is lengthy (46 months) and several of the antibiotics are not well tolerated or readily absorbed from the gastrointestinal tract, which makes pulmonary delivery an attractive alternative (Table 1). The strategy of using the pulmonary route for drug delivery in TB therapy is not novel, but has received attention since the 1950s (Berishvili, 1954). Several excellent reviews have focused on inhaled drug delivery in TB therapy (Pandey and Khuller, 2005a;

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Muttil et al., 2009; Mitchison and Fourie, 2010; GonzalezJuarrero and O’Sullivan, 2011; Misra et al., 2011; Verma et al., 2011; Hanif and Garcia-Contreras, 2012; Mitchison and Davies, 2012), and additional studies have reported promising results of inhalation therapies (Tables 2 and 3). However, the fact remains that inhalation therapy for TB does not occur more than sixty years since first reported. Not all traditional TB antibiotics are suitable for inhaled therapy. If a drug already has high oral bioavailability and lowtoxicity this is the most desirable route. However, many TB drugs have low water solubility, biodistribution and exhibit significant side effects that limit its potential when delivered orally. In these cases the pulmonary route of administration is an attractive alternative route. First-line antibiotics that have been attracting attention as candidates for inhaled therapy are isoniazid (Muttil et al., 2007; Rojanarat et al., 2011), rifampicin (O’Hara and Hickey, 2000; Suarez et al., 2001a, 2001b; Hirota et al., 2010) and pyrazinamide (Rojanarat et al., 2012). Isoniazid is highly water soluble, but side effects occur in 5% of patients, and the half-life is low compared to many of the other antibiotics (Table 2). The introduction of rifampicin and pyrazinamide into TB therapy allowed the duration of treatment to be reduced from at least 12 months to 6 months with 55% relapses. However, unlike the poorly soluble rifampicin, the dose of pyrazinamide cannot be increased because of hepatic toxicity. Of the second-line antibiotics, capreomycin (Garcia-Contreras et al., 2007; GarciaContreras et al., 2012; Schoubben et al., 2013) and ofloxacin (Hwang et al., 2008; Palazzo et al., 2013; Park et al., 2013) have received most attention. More recently, clofazimine (Verma et al., 2013) has been studied as a candidate for pulmonary delivery. Capreomycin is administered by injection and is only water soluble as the disulfate salt. It’s side effects include both kidney damage and injury to the auditory nerve. The drug nitroimidazooxazin (PA-824) has also been investigated as a possible candidate for inhalation therapy of TB (Sung et al., 2009; Garcia-Contreras et al., 2010). Combination of several first-line antibiotics for simultaneous inhaled therapy is also an area of interest (Sharma et al., 2001; Pandey and Khuller, 2005b; Chan et al., 2012). Finding the most suitable antibiotic candidate based on its water solubility, half-life, bioavailability and distribution and side effects, is the first step in designing an inhaled therapy. The next step is to targeting the periphery of the lungs to ensure a requisite dose to achieve a local concentration exceeding the MIC for sufficiently long periods of time to ensure a bactericidal effect and while adding to the already increasing problem of resistant strains. Pulmonary drug delivery may be performed using a nebulizer, pressurized metered-dose inhaler (pMDI) or dry powder inhaler (DPI). Air jet nebulizers create a mist of droplets, using compressed air to disperse solution or suspension of drug. The pMDI generates a spray of aerosols using a formulation of propellant, active ingredient(s) and excipients. The device employs a metering valve to deliver an exact dose of drug particles to the patient. DPIs deliver a plume of aerosol by a variety of mechanisms, including shear, turbulent flow and impaction, occurring by exiting the inhaler on the patient’s breath. The drug is often micronised and blended with larger carrier particles to prevent aggregation (Telko and Hickey, 2005). However, the requirement for high doses frequently makes lactose blends undesirable and alternative strategies to allow powder dispersion are employed including the adoption of spray drying to achieve highly dispersible drug particles. All the devices have mechanisms to avoid particle aggregation to maintain the optimal da. Table 2 lists the reported da, MMAD, VMD, GSD and FPF for the different studies, and formulation and excipients used for aerosol generation. The most commonly used excipients are

Spray drying

Spray drying

Spray drying

Spray drying

Spray drying


Capreomycin Capreomycin

Clofazimine

Clofloxacin

Ofloxacin

Rifampicin (R), Isoniazid (H), Pyrazinamide (P)

6.214 1.7

3.5 ± 0.1

Nebulization

Spray drying

4.74 ± 0.08

4.45 ± 01

1.74

4.99

4.26–4.39

3.57

3.57

2.99–4.92

MMAD (mm)

Emulsion

Spray drying

PA-824

1–6

2–5

2.66–4.95

1–5

3.7 See table

2 (?)

da (mm)

4.14 ± 0.04

3.95 ± 0.01

2.76

2.66–2.75

VMD (mm)

1.9

1.288

1.53 ± 0.02

1.43 ± 0.07

1.41

1.57 2

1.41 1.38–1.57

GSD

45 ± 3

53.3 ± 2.7

57.6 ± 3.6

20–30

78.91 ± 8.4

78.91 ± 8.4

15–35

FPF (%)

6.25% H, 18.75% R, 75% PLA 10 mg each drug to 30 mg stearic acid (1:1 total drug to lipid) 400:150:75 (w/w) – P:R:H

70% ethanol solution, 75% (wt/wt) PA-824, 20% (wt/wt) L-leucine, 5% (wt/wt) DPPC

Hyaluronic acid (hyaluronan)

100%, 90%, 80% (app. 240, 480 or 720 mg)

80% Capreomycin, 20% Leucine in 50% EtOH

One part drug and two part L-PLA Encapsulation of PZA was 26–45%

1:2 drug:L-PLA 20–30% drug loading

Encapsulation of PZA was 18–30%

Formulation

(Chan et al., 2012)

(Pandey and Khuller, 2005b)

Stearic acid

Excipient free

(Sharma et al., 2001)

(Sung et al., 2009)

(Roy et al., 2012) (Garcia-Contreras et al., 2010)

(Park et al., 2013)

(Hwang et al., 2008)

(Palazzo et al., 2013)

(Verma et al., 2013)

(Garcia-Contreras et al., 2012) (Schoubben et al., 2013)

(Garcia-Contreras et al., 2007)

(Rojanarat et al., 2012)

(Muttil et al., 2007)

(Muttil et al., 2007) (O’Hara and Hickey, 2000) (Hirota et al., 2010) (Suarez et al., 2001a) (Suarez et al., 2001b)


Ref.

PLA

L-leucine, DPPC, ethanol

PLA, ofloxacin-palladium comples Hyaluronic acid (hyaluronan)

Oleate, linoleate or linolenate Leucine

Leucine

Pyrazinamide, soybean phosphatidylcholine, cholesterol and porous mannitol

L-PLA MP

Mannitol, soybean phosphatidylcholne and coloesterol from lanolin L-PLA MP PLGA MP PLGA MP PLGA MP

Excipients


Combination treatments Rifampicin (R), Isoniazid (H) Rifampicin (R), Isoniazid (H), Pyrazinamide (P)

Spray drying

Ofloxacin Other drugs Gentamicin PA-824

Emulsion

Spray drying

Pyrazinamide

Rifabutin

Spray drying Spray drying Spray drying Spray drying Solvent evaporation Spray drying

Spray drying


Isoniazid Rifampicin Rifampicin Rifampicin Rifampicin

Manufacturing method

Antibiotic

Table 2. Manufacturing and characterization of microparticles (MP) for TB treatment.


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DOI: 10.3109/02652048.2014.932029

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Table 3. Drug release, loading and biological endpoint. Antibiotic



Drug load (%)

Drug release

18–39

Isoniazid

50

Rifampicin

5.00 107 mg/MP

Rifampicin

30 (w/w)

Dissolution rate after 24 h: 75%, at pH 7.4, 52% pH 5.3.

Rifampicin

4.3 (w/w)

Dissolution rate up to 20–40% and 5–25% at pH 7.4 and pH 5.2.

Rifabutin

50

490% release in 12 h

Pyrazinamide


73.8

Capreomycin

Clofazimine

Ofloxacin

50.0 ± 2.5

Ofloxacin

27 (w/w)

Other drugs Gentamicin

Total release in 6 h

Biologic endpoint

References

MP showed higher bactericidal efficacy on M. bovis in MR8383 cells than free drug. MP targeted macrophages and not epithelial cells in a mouse inhalation study. Drug concentration in macrophages was 20 times higher with inhaled MP compared to drug solutions. Intracellular Rif concentration in NR8383 were higher for Rif-PLGA containing 0.25 and 2.5 mg Rif/ml than 5 mg/ml Rif solution, and had higher bactericidal effect on intracellular BCG. Guinea pigs treated with single and double doses of Rif-PLGA MP exhibited significantly reduced numbers of viable bacteria (H37Rv), inflammation and lung damage compared with lactose- PLGA, or Rif-treated animals. There was a dose-effect relationship between insufflated Rif-PLGA and burden of bacteria (H37Rv) in the lungs, reduced inflammation and lung damage than lactose or saline control, PLGA or Rif treated guinea pigs. MP targeted macrophages and not epithelial cells in mice, and drug concentration in macrophages were 20 times higher when MP were inhaled rather than drug solutions administrated. The proliposome containing pyrazinamide showed no toxicity in vitro or in vivo


The lung and spleens of guinea pigs infected H37Rv treated with 14.5 mg/ kg dose Cap MP showed lower degree of inflammation, bacterial burden, and tissue damage compared to cap injection. Cap-oleated and linoleate showed the same efficacy of cap sulfate against H37Rv using the agar proportion susceptibility test, but cap-linolenate showed a reduced efficacy. 99% killing of H37Rv intracellular in human monocytes at 2.5 mg/mL. In mice, 480 and 720 mg inhaled twice per week over 4 weeks reduced numbers of CFU in the lungs. MP containing ofloxacin had a higher uptake in RAW 264.7 (2.1 and 1.7) than for aqueous solution and ofloxacin MP. The AUC ration between lungs and plasma was also highest for MP delivered oflaxacin. NR8383 cellular uptake of ofloxacin was higher compared to free drug.


Aerosolized-gentamicin-treated mice showed significantly reduced lung H37Rv loads and fewer granulomas relative to untreated controls.

(Roy et al., 2012)


(Hirota et al., 2010)

(Suarez et al., 2001a)

(Suarez et al., 2001b)



(Schoubben et al., 2013)

(Verma et al., 2013)

(Hwang et al., 2008)

(Park et al., 2013)

(continued )

8




Table 3. Continued

Antibiotic

Drug load (%)

PA-824

75.7 ± 0.7 (wt/wt)

PA-824

75 (wt/wt)

Drug release

Combination treatment Rifampicin (R), 25 Isoniazid (H)

Rifampicin (R), Isoniazid (H), Pyrazinamide (P)

47

Drugs released at different rates

polymers (polylactide-co-glycolide (PLGA) MP (O’Hara and Hickey, 2000; Suarez et al., 2001a, 2001b; Hirota et al., 2010) and L-poly lactide (PLA; Sharma et al., 2001; Muttil et al., 2007; Palazzo et al., 2013), but amino acids (leucine; Garcia-Contreras et al., 2007; Sung et al., 2009; Verma et al., 2013), phospholipids (dipalmitoylphosphatidylcholine [DPPC] (Garcia-Contreras et al., 2010, Sung et al., 2009)), sugars (mannitol; Rojanarat et al., 2011) and fatty acids (oleate; Schoubben et al., 2013), linoleate (Schoubben et al., 2013), linolenate (Schoubben et al., 2013), hyaluronan (Hwang et al., 2008) and steric acid (Pandey and Khuller, 2005b)) are also used. While these excipients ensure suitable sizes and dispersion properties of MPs, they offer a challenge with drug loading, drug release, and future approval for drug delivery by FDA. At present, few of the listed excipients are approved for pulmonary delivery. In one reported study that investigated the use of oleate, linoleate, linolenate as excipients, capreomycin-linolenate actually decreased the efficacy of the drug (Schoubben et al., 2013). Another strategy when designing MPs for inhaled TB therapy is to employ pure or almost pure drug. This has the advantage of allowing smaller total powder doses as there is little or no diluent present. Multiple factors contribute to delivery of a therapeutic dose from the MPs once deposited in the lungs including drug load, dissolution rate, and drug half-life, all determining the efficiency of delivery and what proportion of the dose that is available at any given point in time. In Table 1, the properties of the most commonly used antibiotics in TB treatment is discussed. The listed minimal inhibitory concentration (MIC) is for extracellular bacillus of M. tuberculosis model strain H37Rv, when MIC is mentioned in the text below it refers to the concentration for H37Rv. The first-line antibiotic isoniazid has a low MIC (0.02– 0.25 mg/mL), but also a short half-life (0.5–1.6 h), in addition this antibiotic passes freely into mammalian cells and, therefore, has a high probability of reaching intracellular M. tuberculosis within a short period of time. Unfortunately, the excellent solubility and rapid elimination of isoniazid do not make it a good candidate for aerosol delivery as drug alone. Therefore, isoniazid has been

Biologic endpoint

References

The lung and spleens of H37Rv infected guinea pigs receiving the high dose of inhaled PA-824 MP exhibited a lower degree of inflammation, bacterial burden, and tissue damage than those of untreated animals. Reduction in bacterial burden in lungs and spleen in treatment with oral PA-824 cyclodextrin/lecithin suspension were consistent with the high inhaled dose. PK study in guinea pigs showed a higher AUC for 40 and 60 mg/kg Pa-824 MP compared to i.v. (20 mg/kg) and oral (40 mg/kg) treatment.


Inhaled MP were taken up by the alveolar macrophages in rats, and the intracellular drug concentrations was higher than for vascular delivery of soluble drugs. Nebulized MP treatment in H37Rv infected guinea pigs resulted in undetectable CFU in lung and spleen. Drug concentration where above MIC90 in plasma for all time points.

(Sharma et al., 2001)

(Sung et al., 2009)

(Pandey and Khuller, 2005b)

incorporated into matrices or vesicles to enhance its residence time and extend its dissolution and elimination rate sufficiently to be therapeutic in the lungs. Isoniazid proliposomes loaded with 18–30% antibiotic had a higher bactericidal effect on M. bovis inside rat alveolar cell line, NR8383 (Rojanarat et al., 2011). PLA MP loaded with 50% isoniazid, and a total drug release in 6 h, was shown to target alveolar macrophages in mice and not epithelial cells, and furthermore result in a 20 times higher intracellular drug concentration in macrophages compared to epithelial cells (Muttil et al., 2007). The MIC for rifampicin is also low (0.016–0.4 mg/mL) and the half-life (3.35 h) a little longer than for isoniazid, however its water solubility is low (1.4 mg/mL), but it still enter phagocytic cells. PLGA MPs have in general been used for pulmonary delivery of rifampicin (Suarez et al., 2001a, 2001b; Hirota et al., 2010), with drug loads ranging between 4.3% (w/w) with dissolution rate after 24 h of 75% at pH 7.4 and 52% at pH 5.3 (Suarez et al., 2001b) to 30% (w/w) with dissolution rate after 24 h of 75%, at pH 7.4, 52% pH 5.3. Rifampicin delivered with PLGA showed promising results both in vitro and in vivo (Suarez et al., 2001a, 2001b; Hirota et al., 2010), with high intracellular rifampicin concentration in macrophages, and higher bactericidal effect on intracellular BCG than for higher concentrations of free antibiotic (Hirota et al., 2010). Also the semi-synthetic derivative of rifamycin, rifabutin, has been studied as a candidate for inhaled TB therapy (Muttil et al., 2007). Rifabutin is minimally soluble (0.19 mg/mL), but has a half-life of 45 ± 17 h, and the MIC for H37Rv is low, 0.016 mg/mL (Hirata et al., 1995). The MP had 50% rifabutin and the drug release was 490% in 12 h. A mouse study showed that MPs targeted macrophages and not epithelial cells in mice, and drug concentrations in macrophages were 20 times higher when MP were inhaled rather than drug solutions administrated (Muttil et al., 2007). The second-line antibiotics capreomycin and ofloxacin has also been formulated into MPs as candidates for TB treatment, but for these two drugs the excipients employed were sugars and amino acids rather than polymers. The half-time data is not available for capreomycin, but it is soluble in waters as a disulfate


DOI: 10.3109/02652048.2014.932029


salt, with a MIC of 2.0 mg/mL. The importance of choosing the right excipient has been demonstrated in vitro, where the capreomycin-oleate and capreomycin-linolate showed same efficacy as capreomycin sulfate against H37Rv, but capreomycinlinolenate showed a reduced efficacy (Schoubben et al., 2013). Capreomycin MPs with a 73.8% drug load and leucine as excipient showed promising results in vivo, where the lung and spleens of guinea pigs infected H37Rv treated with 14.5 mg/kg dose capreomycin MP showed lower degree of inflammation, bacterial burden, and tissue damage compared to injection (Garcia-Contreras et al., 2007). The other second-line antibiotic ofloxacin has been studied using both PLA (Palazzo et al., 2013), hyaluronan (Hwang et al., 2008) as excipients and MP generated by water-in-oil emulsification using chitosan (Park et al., 2013). The water solubility of ofloxacin is 28.3 mg/mL, with a half-time of 9 h and a MIC of 0.5 mg/mL. The in vitro uptake studies of oflaxacin MP loaded with 50.0 ± 2.5% drug had a higher uptake in RAW 264.7 (2.1) than for aqueous solution. The AUC ratio between lungs and plasma was also highest for MP delivered oflaxacin (Hwang et al., 2008). The intracellular concentrations of ofloxacin in NR8383 cells were also higher for MP delivered drug compared to the free drug (Park et al., 2013). A new drug candidate in TB treatment is nitroimidazoleoxazine (PA-824) with a MIC of 0.13–0.4 mg/mL (Stover et al., 2000; Li et al., 2008), is in the pipeline and currently in phase II clinical studies as an orally administered therapy (Diacon et al., 2012). These studies testing the safety, tolerability, pharmacokinetics, and early bactericidal activity of PA-824 indicated that the lowest dose had the highest bactericidal effect and that all tested doses were safe and well tolerated (Diacon et al., 2012). However, PA-824 has a low water solubility of 10.2 ± 1.6 mg/mL (Li et al., 2008), which makes pulmonary delivery and attractive alternative to oral administration. Two guinea pig studies have reported promising results using MPs with 75% drug content, in the presence of the excipients leucine and DPPC (Sung et al., 2009; Garcia-Contreras et al., 2010). PK study in guinea pigs showed a higher AUC for 40 and 60 mg/kg PA-824 MP compared to i.v. (20 mg/kg) and oral (40 mg/kg) treatment (Garcia-Contreras et al., 2010). The lungs and spleens of H37Rv infected guinea pigs receiving a pulmonary dose of 9.7 mg/kg PA-824 exhibited a lower degree of inflammation, bacterial burden, and tissue damage than those of untreated animals. Reduction in bacterial burden in lungs and spleen in treatment with oral PA-824 cyclodextrin/lecithin suspension were consistent with that inhaled dose (Garcia-Contreras et al., 2010). There are few literature citations of the dissolution properties of inhaled drug particles with respect to the presence of excipient. However, it is evident that certain additives would inhibit dissolution and delay the appearance of drug at the airway surface or in lung tissues. This delay would reduce the instantaneous dose of drug and would require a large mass of the total formulation (drug/excipient combination) to achieve a therapeutic dose (Figure 2). Ultimately, the requirement to deliver large total masses might become impractical as there is a limit to the total mass of aerosol that can be inhaled in a single breath, or minimal number of breaths, required for patient compliance. A phase I clinical study of inhaled capreomycin sulfate dry powder therapy used dosages of 25, 75, 150 and 300 mg (Dharmadhikari et al., 2013), all were well tolerated, the most common adverse event was mild to moderate transient cough in five subjects. These doses were selected to exceed the likely routine dose as is common in tolerability studies. However, for practical reasons a nominal local dose, not exceeding 100 mg, is desirable and in the range of current practice for inhaled antibiotics for cystic fibrosis therapy (Geller et al., 2007). Figure 2 illustrates theoretically the importance of drug load and drug release in achieving the desired

9

Figure 2. The optimal drug load for inhaled TB therapy could be 100 mg, for example. The higher the % of excipient incorporated in the MP the lower the actually drug load will be (circles). Therefore, with increasing % of excipient, the higher volume of MP is needed to achieve the therapeutic dose (squares). If the drug release and dissolution rate is for example 75%, that will further increased the needed volume of delivered MP (triangles).

therapeutic dose following inhaled drug delivery. For every percentage increase in excipient the required total MP mass also increases to accommodate the desired dose, a number that is highly influenced by the drug-excipient particle dissolution kinetics where large quantities of certain excipients are employed. It is also worth remembering that the MIC for intracellular M. tuberculosis has been reported to be several times higher than that for the bacillus tested in broth (Rastogi et al., 1996; Shandil et al., 2007). So MIC for ofloxacin increases from 0.5 to 2.0 mg/ mL, and ciprofloxacin from 0.5 to 4.0 mg/mL (Shandil et al., 2007). This illustrates that the therapeutic concentration needed to treat dormant intracellular bacilli is higher than extracellular bacilli in the lungs, and to avoid contributing to the already growing sub-population of MDR and XDR M. tuberculosis strains a high dose of antibiotic is needed at all times during treatment.

Conclusion Acceleration of TB treatment by including inhaled therapy using MPs has been investigated over the last decades with encouraging results. However inhaled therapy is not currently available. The strategy behind inhaled drug therapy is to increase the local antibiotic concentrations in the lungs targeting the bacilli inside phagocytising cells and lung lesions, which may be difficult to treat with systemic concentrations of antibiotics. One strategy to deliver the drug to the periphery of the human lungs is to use drug loaded MPs. The complexity of the airway, its impact on the deposition of inhaled MPs and the role of the physicochemical properties of the inhaled MPs have been reviewed to illustrate the importance of the many factors contributing to efficacy. Also the aspects of drug loading, drug release, dissolution rate and drug pharmacokinetic half-life are essential in ensuring a constant therapeutic concentration in the lungs. As presented in Table 2, does the majority of the papers report the manufacture of MPs with a favorable da or MMAD for inhalation therapy. However, many of these MPs have very-low drug loading which raises makes their success in delivering a constant therapeutic drug concentrations to the lungs improbable. Several papers report an increased intracellular drug concentration in macrophages in comparison with the extracellular environment, but they do not evaluate this with respect to the MIC for M. tuberculosis.


10


Another, often overlooked, aspect of formulation design is the choice of excipient and its suitability for lung delivery. As illustrated in Figure 2, there is a theoretically lower limit for the drug load that would allow therapeutic concentration to be reached in the lung. However, assuming such conditions could be accommodated the choice of excipient requires safety considerations that might be an obstacle for future regulatory approval. Interactions with surfactant components, including the accrual of a protein corona, is one biophysical consideration regarding extending the residence time of particles in the lungs that may influence mechanism of disposition and, thus, enhance efficacy or toxicity and as such would require investigation. Ideally excipients should be selected that have been used previously in inhaled products. Clearly, not only does pure drug (or minimal excipient) make sense from a dose delivery standpoint it is the most likely approach to receive regulatory approval from the perspective of safety.

Declaration of interest Funding from the National Institute of Allergy and Infectious Disease is gratefully acknowledged.

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Pulmonary drug delivery systems for tuberculosis treatment.

The role of disposable inhalers in pulmonary drug delivery.

Effects of the microbubble shell physicochemical properties on ultrasound-mediated drug delivery to the brain.

The PulmoSphere™ platform for pulmonary drug delivery.

Role of Physicochemical Properties in Nanoparticle Toxicity.

Differences in physicochemical properties to consider in the design, evaluation and choice between microparticles and nanoparticles for drug delivery.

Antisolvent vapor precipitation: the future of pulmonary drug delivery particle production?

Role of Nanodiamonds in Drug Delivery and Stem Cell Therapy.

Across the pulmonary epithelial barrier: Integration of physicochemical properties and human cell models to study pulmonary drug formulations.

[Therapy of pulmonary tuberculosis].

CNS drug design: balancing physicochemical properties for optimal brain exposure.

Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy.

Drug treatment of pulmonary tuberculosis.

Adjunctive steroid therapy for managing pulmonary tuberculosis.

The evolution of peptic ulcer therapy. A role for temporal control of drug delivery.

rifampin drug delivery and osteoarticular tuberculosis therapy.

Nanostructured drug delivery for better management of tuberculosis.

Pulmonary drug delivery by surfactant.

Transport mechanisms at the pulmonary mucosa: implications for drug delivery.

Letter: Drug therapy of tuberculosis.

Drug resistance in patients with pulmonary tuberculosis.

Advances in device and formulation technologies for pulmonary drug delivery.

Study of Drug Resistant Pulmonary Tuberculosis.

Treatment of Drug Susceptible Pulmonary Tuberculosis.