Excipient-free isoniazid aerosol administration in mice: Evaporation-nucleation particle generation, pulmonary delivery and body distribution
Graphical abstract
Introduction
Tuberculosis (TB) is a dangerous infectious disease caused by the bacillus Mycobacterium tuberculosis (M-TB), which requires a lengthy treatment and is one of the top 10 causes of death in the world. (Global Tuberculosis report, 2018). The main problems in the treatment of tuberculosis are poor patient compliance and the toxicity of anti-tuberculous drugs resulting in adverse side effects in the liver and gastrointestinal tract (Das et al., 2015, Kaur et al., 2016). It is expected that the aerosol inhalation administration can diminish the burden of toxicity as, in this case, the drugs are absorbed to the systemic circulation avoiding the hepatic first-pass metabolism. Besides, the pulmonary introduction of drugs provides high concentrations in the lungs, which are the intended target site of drug delivery (Giovagnoli et al., 2017, Misra et al., 2011).
The modern era of tuberculosis treatment started in 1944 when Schatz and Waksman demonstrated in their report that a human strain of M-TB was sensitive to streptomycin (Schatz and Waksman, 1944), and the first papers on the aerosol inhalation therapy for tuberculosis were published at the turn of the 1940s (Hinshaw et al., 1947, Levaditi et al., 1948, Larroude, 1948, Prigal et al., 1950, Miller et al., 1950). The discovery of new drugs reduced the need for aerosol therapy for some time, but the evolution of multiple and extremely drug-resistant tuberculosis renewed the interest to the aerosol delivery in the 1990s.
Many papers on the clinical outcome of the aerosol delivery of anti-tuberculous drugs were published in the Soviet Union, however, that part of literature is not easily accessible for the majority in the international community. Therefore, we give here a list of Russian works on the subject. Most of those works considered the effect from a single-component aerosol inhalation. Pulmonary tuberculosis was treated by the aerosol of streptomycin (Efimov, 1958, Zarnizkaya, 1958, Lavor, 1976, Semenova, 1977, Kaliberda and Malevsky, 1983, Aksenova, 1985, Protsyuk, 1985, Pilipchuk and Protsiuk, 1986), isoniazid (Lavor, 1976, Semenova, 1977, Kaliberda and Malevsky, 1983, Aksenova, 1985, Pilipchuk and Protsiuk, 1986), ftivazide (Protsyuk, 1985, Abdurashitova, 1955), kanamycin, florimycin (Lavor, 1976, Protsyuk, 1985), ethionamide (Lavor, 1976), cycloserine (Kulik, 1967), rifampicin (Gorbach and Samtsov, 1991). There are many works where the aerosols of two or three anti-tuberculous substances were delivered to patients (Fridkin and Krasnoschekova, 1958, Gerasimov and Ganushchak, 1972, Kulik, 1974, Kulik, 1975, Kulik, 1980, Abdurashitova and Aun, 1979, Krasnova and Guryeva, 1980, Shesterina et al., 1981, Frolova, 1982, Protsyuk, 1983, Protsyuk, 1984, Agzamov et al., 1983). There are Russian works that consider possible side effects from the isoniazid and streptomycin aerosol inhalations (Otroschenko and Berezovsky, 1977, Berezovsky et al., 1984, Protsyuk et al., 1985, Gerasin et al., 1984). A review of the Soviet works published in 1950s−1970s is given by Shesterina and Krasnova (1979).
Since the middle of the 1990s, the pulmonary delivery of anti-tuberculous drugs incorporated into excipient micron-sized particles has been studied extensively. This incorporation results in the sustained release of drugs in the lungs. As the drug remains in the lung for a longer time, the frequency of administration can be reduced with respect to the oral treatment. The first and the simplest way to incorporate drugs was in using liposomes (Kurunov et al., 1995, Kurunov et al., 1998., Justo and Moraes, 2003, Pandey et al., 2004, Vyas et al., 2004, Zaru et al., 2007, Olivier et al., 2017). Apart from liposomes, porous and hollow nanoaggregates and microparticles are used as carriers for anti-tuberculous drugs in inhalable forms (Kaur et al., 2016, Misra et al., 2011, AI-Hallak et al., 2011, Hickey et al., 2016, Pham et al., 2015, Sanzhakov et al., 2013, Jawahar and Reddy, 2012, Sung et al., 2007). All these forms have some disadvantages. In the case of aerosol formed by the nebulization or pressurized metered-dose inhalers, the storage of the particle-containing medium may create problems due to the destruction of the polymer, particle agglomeration and settling, crystal growth. Liposome suspensions can suffer from the leakage of drug. The aerosol from the dry powder inhalers suffers from the low redispersibility. The use of excipient carrier particles or other kinds of excipient ingredients intended to improve product delivery or efficacy has limitations which hinder the formulation development. First of all, the excipients approved for respiratory drug administration are very limited in number (Pilcer and Amighi, 2010). The use of excipients requires higher powder doses with respect to the pure aerosol, which makes problems in reaching the high drug concentration in the target site. In addition, there are other excipient-associated problems related to the supply-chain integrity, product quality and uniformity, compatibility with manufacturing equipment and processes (Hickey et al., 2013). Therefore, the elaboration of excipient-free pulmonary drug delivery formulations for use against tuberculosis is highly desirable (Das et al., 2015, Brunaugh et al., 2017, Chan et al., 2013, Roy et al., 2012, Durham et al., 2015).
In the present paper, an excipient-free approach for the synthesis of anti-tuberculous drug aerosol is elaborated on the basis of the evaporation-nucleation technique (Rabinowitz et al., 2004, Onischuk et al., 2008, Onischuk et al., 2009, Onischuk et al., 2014, Onischuk et al., 2016). The idea of the method is in vapor generation by heating the maternal substance in the flow of air or inert gas. Then, due to vapor cooling, aerosol particles are formed in the process of nucleation from supersaturated vapor. The advantages of the evaporation-nucleation approach are as follows. No excipient is necessary, and the method does not require a specially prepared formulation. A pure substance is used as a maternal phase, which does not cause any storage-related problems. This technique allows generation of the mono-modal aerosol with a narrow size distribution. The mean diameter of particles can be varied in the wide range of 0.003–5 μm by changing the evaporation temperature. The particle number concentration can be reached as high as 108 cm−3, which is several orders of magnitude higher than in the case of nebulization or dry powder inhalers. Any substance either water-soluble or insoluble, can be used for the aerosol generation. The only limitation can be in the substance stability at the temperature of evaporation. In other words, there must be no thermal decomposition of the maternal substance at temperatures corresponding to the saturated vapor pressure of about 0.1 Torr. In our previous works we used the evaporation- nucleation approach to generate the aerosol of non-steroid anti-inflammatory drugs (Onischuk et al., 2008, Onischuk et al., 2009, Onischuk et al., 2016) and hypotensive drugs (Onischuk et al., 2014), while this paper is devoted to the evaporation-nucleation aerosol of isoniazid. To proceed with further improvements in aerosol drug delivery, it would be insufficient to rely only upon the obvious advantages of this administration method. It is important to achieve detailed understanding of the routes through which the drugs enter the systemic circulation. Evaluation of the drug-specific body distribution is an essential step towards this goal.
The lungs of laboratory animals are considered as a surrogate for the human lungs in the investigations of health effects from inhaled aerosols as the regional deposition patterns are qualitatively similar in laboratory animals and in humans. Laboratory mice are extensively used in the studies of aerosol pulmonary administration (Méndez et al., 2010, Bivas-Benita et al., 2005, Fernandes and Vanbever, 2009, Garcia-Contreras, 2011), and it is important to know the relationship between the lung delivered dose and the distribution of inhaled substance over the body, as well as the effect of inhalation conditions on the drug intake.
In the inhalation experiments with the laboratory animals, both nose-only and whole-body exposure chambers are used. In whole-body chambers, aerosol is delivered into a box where animals are immersed in the aerosol atmosphere. This approach is simple, and a large number of animals can be exposed all at once. The disadvantage with the chambers of this type is the aerosol deposition on the skin and, as a result, ingestion through the grooming, which can give additional burdens in blood and organs. The nose-only chambers are free from the skin deposition problems but are more effort-consuming. The additional effect from the skin deposition in the whole-body chambers depends on the ratio between the lung and oral permeability, design of the inhalation experiment and other aspects. Therefore, when studying the drug particle lung delivery, it is important to know the possible side effects from different inhalation routes.
The objective of this paper is to investigate the excipient-free evaporation-nucleation route of isoniazid aerosol formation; to study the delivery of the isoniazid aerosol to the laboratory mice using both whole-body and nose-only exposure chambers, to determine the drug additional intake in the whole-body chambers due to the oral consumption from the skin; and to study isoniazid distribution over the organs of the laboratory animals. To compare isoniazid distribution over the body with the lung regional deposition, the particle lung deposition efficiency was measured. To elucidate the mechanism of isoniazid absorption, we give also a comparison between the pharmacokinetics for the aerosol, per-oral and intravenous administrations.
Section snippets
Aerosol generation and inhalation equipment
We used a two-compartment horizontal laminar flow nucleation chamber for isoniazid aerosol generation (Fig. 1). To decrease the evaporation temperature and to avoid the thermal decomposition of maternal substance during evaporation, seeding particles of NaCl are used as nuclei for heterogeneous nucleation to be generated in the first compartment of nucleation chamber. The seeding particle compartment consists of a horizontal cylindrical quartz tube with an outer heater. The inner diameter
Inhalation dose
The total dose delivered by inhalation was measured in experiments with the NO chamber. The total mass of particles deposited in the respiratory tract of one mouse per time t (min) can be written aswhere α is the mean fraction of particles that were consumed by one mouse from the aerosol stream, C (g/cm3) is the particle mass concentration in the aerosol chamber, F (cm3/min) is the aerosol flow rate through the aerosol chamber. The fraction α was measured aswhere N is the number
Results and discussion
The typical size spectrum of aerosol particles used in the inhalation experiments (Fig. 3) is well fitted by the lognormal function with the standard geometric deviation . To be sure that the aerosol particles contain no contaminations (from the thermal decomposition of the maternal phase or other unwanted processes like evaporation of impurities from the construction materials of the nucleation chamber), the chromatographic analysis of particles sampled on the high-efficiency Whatman
Conclusions
Excipient-free isoniazid aerosol delivery to outbread male mice is investigated. For this purpose, the evaporation-nucleation technique is elaborated to generate the aerosol within the diameter range 10–3000 nm and particle number concentration 104 –107 cm−3. Chromatographic analysis shows that the aerosol particles are chemically identical to the original substance, which means that there is no thermal decomposition of isoniazid during evaporation in the aerosol generator.
The total particle
Funding
This work was supported by RFBR according to the research project No 17-43-540767 h_a.
Conflict of interest
The authors declare no conflict of interests.
Acknowledgment
Financial support from the Russian Foundation for Basic Research under Project No. 17-43-540767 h_a is gratefully acknowledged.
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