Toxicology Studies for Inhaled and Nasal Delivery - American

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Toxicity Studies for inhaled and Nasal Delivery Ronald Wolff Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00146 • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on May 4, 2015

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Molecular Pharmaceutics

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Toxicology Studies for Inhaled and Nasal Delivery

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R.K. Wolff 7

RK Wolff - Safety Consulting Inc, Ft Myers, FL, USA 8 9 10

Abstract 1 12 13 1 4 15 16 17 18 19 20 21 2 23 2 4

This paper reviews issues related to the toxicological testing of pharmaceuticals delivered by the inhalation or nasal route.. The purpose of the toxicology studies is to conduct studies in animals that will aid the assessment of the safety of these agents delivered to patients. Inhalation toxicology studies present some unique issues because the dosing method differs from more standard administration methods such as oral or injection administration. Also dose determination issues are more complex, particularly for inhalation administration since it is often difficult to determine the amount of material delivered to the lung both for patients and also in the animal toxicology studies.

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Keywords: inhalation toxicology, aerosols, nasal, lung, inhaled pharmaceutical, respiratory tract toxicity, excipients, MMAD, pulmonary 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

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Introduction This paper reviews issues related to the toxicological testing of inhaled and nasal pharmaceuticals. Inhalation studies, in general, are more complex than other toxicology studies using other more common routes of administration. A key difference is that the dosing method is different requiring the use of some type of aerosol generation method to produce an atmosphere that the animals can inhale into the respiratory tract. In some instances clinical inhalers can be used for animal studies, but in many cases they are unsuitable and specialized aerosol generation methods must be used. This fact in turn creates a requirement for careful characterization of the exposure atmospheres with respect to aerosol concentration and particle size in order to determine the inhaled dose. Another major issue is that of dose. Inhaled dose is more difficult to assess than that from other delivery routes. With oral, or parenteral administration in humans a known amount is generally given which is usually quite clear as the mass in a tablet or capsule, or syringe cartridge. Inhaled pharmaceuticals are usually delivered with an oral inhalation and delivered dose varies with factors such as emitted dose, particle size, expsoureinhalation flow rate and duration of breath. Animals can only breath the exposure atmospheres throught the nose and so there is nasal deposition to consider which reduces lung deposition compared to humans.. Animal exposures usually have to occur over an extended period of time to achieve high doses needed to assess

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toxicity. Inhaled dose in animals depends on exposure concentration, particle size, Local distribution within the regions of the respiratory tract also needs to be considered. For instance, humans deliver All of these factors need to be taken into consideration to assess animal doses in comparison to human doses

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Since the respiratory tract is the site of aerosol deposition, local respiratory tract toxicity must be a focus of inhalation toxicity studies. Naturally, if the pharmaceutical is absorbed from the lung into the blood and delivered throughout the body, systemic toxicity is also a major concern. Other reviews1,2,3,4,5,6 of inhalation toxicity studies should be consulted for more complete information. In this paper, the emphasis is on basic inhalation toxicology methods and also on general study designs and specific considerations to support clinical trials of inhaled or nasal therapeutics.

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Overview of Toxicology Study Types Table 1 provides high level outlines of the types of general inhalation or nasal toxicology studies

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Exposure levels Exposure duration

Table 1 Acute, subchronic, and chronic/oncogenicity studies* Acute Subchronic/chronic Chronic/oncogenicity Sufficient to grossly categorize maximum doses 3 plus control 3 plus control for subsequent studies Usually 1–4 h

Species Rodent Rat Nonrodent Dog or monkey Number of animals

1–6 h day−1; 1, 3, 6, 9 or 12 months 1–6 h day−1; 18–24 months

Rat Dog or monkey

10–20 per sex per group (+recovery animals, 5-10 per sex per group); toxicokinetic animals as needed, 3–4 per sex per group (+recovery Nonrodent 1/sex/group animals, 2–4 per sex per group) Clinical signs, clinical Histopathology, hematology, clinical End points chemistry, gross pathology chemistry, estimate of delivered (histopathology – optional) dose, special studies – optional Rodent

5–10 per sex per group

Rat and mouse

50 per sex per group; toxicokinetic animals as needed,

Histopathology, tumor identification, hematology, clinical chemistry, estimate of delivered dose

* adapted from reference 6 Guidance for subchronic and chronic inhalation studies suggests that it is desirable that the mass median aerodynamic diameter (MMAD) of the particle size distribution should generally be 3 µm or less7,8. Doses should should range from a minimal or no effect level to some effect level. Exposure concentrations are limited to approximately 2 mg/L so that particle agglomeration is minimized and particle size does not increase


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sufficiently to reduce lung deposition because of increased nasal deposition9. If the candidate compound has very low solubility, lung clearance can be impaired at high exposure concentrations and this can be problematical. High-level exposures to lowtoxicity low solubility particles can impair lung clearance and cause an overload phenomenon resulting in lung tumor formation in rats as a nonspecific response10,11., as evidenced by the data on TiO2 12 and carbon black13. Therefore, compounds that reduce lung clearance dramatically resulting in lung accumulation should be avoided.

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Inhalation Exposure Technology

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An inhalation exposure system consists of aerosol generation, aerosol delivery to the chamber, aerosol sampling, and environmental control and monitoring. 16 17 18 19 20

Inhalation Exposure Methods and Devices 21 2 23 2 4 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

Phalen 2, Cheng and Moss 14 Wong 15 have extensively reviewed the basic inhalation methods: whole-body, nose-only, head-only, lung-only, and partial-lung exposures. By far, the predominant exposure methods for inhaled pharmaceuticals are nose-only exposures for rodents and face mask exposures for non-rodents shown schematically below. The design and construction of inhalation exposure chambers have been extensively reviewed elsewhere 1, 2 and will not be dealt with in detail here. High uniformity of port-to-port aerosol concentration is a feature of all well-designed nose-only chambers as demonstrated in the flow past designs (Figure 1) of Cannon et al 16 and Jaeger (CH technologies), the Lovelace design 17, and other systems based on similar principles 14. One of the major advantages of nose-only exposures is that much less test material is required compared to that required for whole-body exposures, because nose-only chambers have much smaller chamber volumes than whole-body chambers and consequently lower volumetric flow rates. The reduction in test material needs is particularly important with materials that are difficult and/or expensive to synthesize. Flow rates should be minimally approximately 2 times the total ventilation rate of all animals in the chamber in order to avoid build-up of CO2. For 50 rats this corresponds to approximately 20 l min−1. It should be noted that the flow required for a nose only rodent chamber is not based strictly on the number of animals. Rather it is more correctly based on the species (respiratory minute volume and safety factor) and the number of ports on the chamber. This assumes that the unused ports have not been plugged. For example a 36 port chamber with 24 animals requires the same flow as a 36 port chamber with 12 animals.

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Another advantage of nose-only chambers is the fact that pelt contamination is minimized.. It was estimated that 60–80% of material deposited on the pelts of rats during whole-body exposure was ingested 18. Also,it is possible to measure respiratory

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patterns during inhalation exposure because the restraint tube can be modified to be used as a plethysmograph 19.

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Intratracheal instillation (a lung-only exposure methods) can be used as a screening method as reviewed by Driscoll et al20. One advantage of intratracheal instillation is that the amount of material delivered to the lung can be accurately determined. Also much less material is needed than for inhalation studies. Disadvantages include the need for anesthesia and a more patchy distribution of particles deposited in lung than is achieved with inhalation20. Oropharyngeal exposures have also been developed21 with similar advantages and disadvantages.

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Rodent exposure systems for inhaled pharmaceuticals have challenges. One challenge is in some cases to attempt to use human inhalers for much smaller rodents. Some rodent systems have been designed using metered dose inhalers (MDIs)22. Also because rodents are obligate nose breathers, nasal deposition is a significant issue. and keeping particle size as small as possible can be an approach to minimize nasal deposition. However, particle size reduction can be difficult since the clinical formulation should be used for the toxicology studies. Face mask exposure systems, such as shown schematically in Figure 2, are the most common large animal exposure systems for inhaled pharmaceuticals. Aerosols are generated and dispersed into a medium size plenum with tubing connected to individual face masks as shown in Figure 2. Approximately 4–8 dogs or monkeys can be exposed at a time. Training is very important and usually a minimum of 2 weeks is required for acclimation of the animals. A maximum exposure duration for many systems is approximately 1 hr, but minimal restraint systems for dogs have been developed that allow longer exposure times 23. There have been some attempts to try to produce oral breathing in dogs through the use of oropharyngeal tubes placed in the face masks. Measurements have shown that this technique produces only modest increases in lung deposition at best 24 and is not recommended.

Animal Exposure Systems for Nasal Delivery The systems for nasal delivery are simpler than those for inhalation delivery echoing the simple clinical nasal drops and spray pumps that are frequently used. For large animals (monkeys and dogs) it is frequently possible to use the same device that is used clinically or similar nasal pump sprayers. For rodents, either micropipettes or small catheters with microsyringes are used. Dose volume is a significant issue. Individual instillation volumes at a given time should be limited to approximately 5 µL per nostril for rats and mice and 40 µL per nostril for monkeys and dogs. Daily dose can be increased by multiple instillations per day. If dose volumes significantly


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larger than this are used it is likely that the dose will escape from the nose and drain into the esophagus or lungs 25,26.

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Exposure Atmosphere Generation 9 10 1 12 13 1 4 15 16 17 18 19 20 21 2 23 2 4 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

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Liquid aerosols have been generated using a variety of nebulizers. A wide range of compressed air jet nebulizers have been characterized 27,28. Different units may be chosen based primarily on desired droplet particle size and concentration output. A significant concern for many nebulizers is the increase in solute concentration that occurs over time as the reservoir volume is depleted with nebulization leading to alterations in concentration and particle size. A solution can be a continuous feed to maintain a constant level in the nebulizer reservoir for extended periods of operation. More recently there has been use of vibrating mesh nebulizers (Aerogen, Pari). These nebulizers operate by “pumping” droplets through micron size orifices. Aerosol generation is a “once through” process and so there is no recirculation of the solutions or increase in solute concentration. There are a variety of dry powder aerosol generation methods that have been reviewed by others 2, 14. The Wright Dust Feeder 29 has been used in inhalation toxicology for decades. Air jet mills such as the Jet-O-Mizer 30, and the Trost jet mill can be useful in generating dry powder aerosols with good particle size distributions particularly for materials that are ‘sticky’ and difficult to generate by other methods. Other methods of dry powder generation include fluidized beds, brush mechanisms, venturis, and turntable designs. The Vilnius generator (CH technologies) is another generator than can be useful particularly in small scale studies. Sometimes, it can be useful to combine delivery devices. Bernstein et al 311 have described combining a brush feeder with a jet mill. Lee et al32 have described a versatile and robust approach using a reservoir with a stirring motor feeding into a Venturi T-section. Rotating brush aerosol generators are used for aerosol generation over a wide range of concentrations and are commonly used for dry powder aerosol generation. Test article is packed in a cylinder that moves upward against a rotating brush, dispersing the compound into an air jet. Powder generation rate is controlled by varying diameter of the delivery cylinders and by using a range of cylinder feed rates.. Care must be taken in filling the cylinders because variation in packing density will cause variation in output. Use of small weights to tap down the test article in the cylinder helps to achieve reproducible test particle density. It should ne noted that a major difference between the Wright Dust Feeder (WDF) and Rotating Brush Generators (RBG) include the difference in packing pressure. Packing pressures of the WDF range from 200 to 5000 psi while the RBG is generally much less than this. Therefore, the WDF often compromises the structure of engineered dry powders which are generally low density..



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Using metered dose inhalers (MDIs) as aerosol generation devices for nonclinical toxicology studies22 can be done when this is the clinical device. One of the goals of toxicology studies is to expose animals to a range of doses. In inhalation toxicology studies, this is usually done by using graded constant exposure concentrations. When using MDIs, a constant concentration is virtually impossible to achieve because of the dynamic characteristics of the aerosol "puff' resulting from each individual actuation of the MDI. The concentration profile is pulsatile, with peaks created by each MDl actuation followed by a rapid decrease, until the next actuation occurs. Therefore, an average concentration is defined effectively by the number of actuations per minute. Given these constraints careful consideration of aerosol science and electromechanical challenges in setting up such systems need to be considered as described by Rothenberg et al22.. At the high exposure concentrations required for rodent studies, it is often necessary to use multiple numbers of MDls actuating into a plenum and then delivering the resultant aerosol to an exposure chamber. Usually several MDIs are fired simultaneously into the plenum sufficiently large to avoid impaction of the aerosol plume on the plenum walls. Use of a plenum also allows sufficient time for reasonably complete evaporation of the CFCs or HFAs to minimize particle size, and provides mixing of the aerosol to promote uniformity of aerosol distribution in the exposure chamber. Use of tubular plenums over 60 cm long have demonstrated less concentration variation and a fairly steady concentration can be obtained (Rothenberg et al22) The MDI banks are activated at intervals ranging from every five seconds to once a minute, with the aerosol concentration controlled by the number of MDIs activated in each bank or row and the frequency of activation. If the banks of MDIs are activated at too high a frequency the MDIs cool and freeze, causing a sudden and catastrophic reduction in output. Most MDIs should not be activated more often than ten times a minute. Prior to use, the MDIs must be shaken and inverted to resuspend the powder. This is crucial to consistent output when used by patients and in nonclinical studies. MDIs should be well shaken before they are mounted in the aerosol generator during a nonclinical study and periodically inverted to maintain steady output.

Exposure Characterization Concentrations must be monitored accurately and maintained within well-defined limits in order to conduct valid inhalation studies. Only a brief treatment will be given here; additional details are available in other reviews 33.

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Particle exposures require careful monitoring. Particle concentration is usually monitored with filter sampling that is frequently augmented with specific chemical 58

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analysis for the test compound of interest. Real-time monitoring, using methods such as optical light scattering, can be used as an adjunct. However, such methods are difficult to calibrate and should only be used to determine relative concentrations. Concentration variability should be controlled such that coefficients of variation are within approximately 20–25% for single exposures and within approximately 15% for repeated exposures. Particle size measurements are very important for characterizing the exposure to inhaled particles because respiratory tract deposition is highly dependent on the MMAD 34. Cascade impaction is currently the gold standard for making such measurements for particles of 0.5µm and larger 35. Real-time measurements using optical light scattering instruments or laser-based aerodynamic measuring methods can also be used. Such methods, particularly the light scattering ones, need to be carefully compared to results using cascade impaction. At present, most regulatory studies require sizing with cascade impaction as part of the characterization. Additional details on aerosol sampling considerations and aerosol sizing instruments can be found in other sources 33.

3.16.5. Dose 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 5 4

Background One of the most important issues in inhalation toxicology studies is delivered dose. This issue is highly important to studies of inhaled agents because there must be an understanding of the dose delivered to humans, the dose delivered to animals in toxicology studies, and an ability to interpret and compare both sets of information relative to safety. Unlike oral or intravenous administrations, total delivered dose is not easy to determine following inhalation exposure and is also not necessarily the most important determinant of toxicity. Dose distribution throughout the respiratory tract as well as total dose is of considerable importance. Total deposition is defined as the amount of material deposited throughout the entire respiratory tract – from nose or mouth down to the alveolar region of the lung. Regional deposition is defined as the amount of material deposited in specific anatomical subregions of the respiratory tract. Regional deposition of aerosols has been reviewed by a number of authors 1, 2, 34 mainly from the perspective of assessing effects from inhaled environmental or occupational agents. Phalen and Mendez 36 have provided a general review on dosimetry considerations for animal aerosol inhalation studies. Three major regions of the respiratory tract have been designated – the head region (nose, mouth, naso- and oropharynx, larynx), the tracheobronchial region (conducting airways from the trachea to terminal bronchioles), and the pulmonary region (alveoli) 34, 36.

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Dose Distributions in Animals and Humans Aerosol deposition is an important parameter in determining delivered dose. It is affected by particle characteristics, respiratory tract anatomy, and breathing patterns. It is also important to distinguish between total respiratory tract deposition and regional deposition. Variations in toxicity may reflect regional deposition pattern as well as total deposition. Frequently, a toxic effect is determined by the local concentration of a toxic agent at the target tissue site. Therefore, comparative respiratory tract deposition between experimental animals and man is of central importance in interpreting toxicology studies.

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In spite of differences in respiratory tract anatomy, ventilation, and body size, there are some general qualitative similarities between deposition in experimental animals and man. Deposition is at a minimum for particles approximately 0.5 µm in diameter, and pulmonary deposition increases from this minimum to a relative maximum for particles about 1 µm in diameter for rats and 2–4 µm in diameter for man 34, 35. There are, however, important qualitative differences in deposition among species (F. Pulmonary deposition in rats is considerably less than that in humans The deposition curve in rodents decreases dramatically for particles over 4 µm 37. Thus, deposition fraction is lower in rats than in people for particles over 4 µm MMAD at equivalent exposure concentrations. Deposition in larger species such as dogs and monkeys is similar to that in people. Therefore, toxicology studies in these species are often of more benefit in making safety assessments for people. Smaller species tend to have higher specific metabolic rates than larger species and so they have a higher ventilation rate per kilogram. This fact needs to be considered as well as deposition fraction when calculating the amount of material deposited. When these factors are taken into account, deposited pulmonary dose on a microgram per gram lung basis is usually the best dose comparator. Deposition predictions in both man and animals require additional refinements. Most current deposition models assume tidal breathing of stable aerosols and provide a good characterization under these conditions 38. Advances in simulation modeling using computational fluid dynamics 39 may aid the development of improved methods to predict the deposition pattern of aerosols with complex geometries and behaviors. Until these methods are available, it appears prudent to use measurements of lung deposition in man and animals whenever possible. Even here caution must be observed because methodologies differ and there have been differences in definitions and standards that are currently being addressed to aid dosimetric determinations.

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Dose Metrics Inhaled versus regionally deposited dose In conducting toxicology studies, it becomes important to quantitate the delivered dose so that it can be compared to projected or measured human clinical doses. Generally for inhalation toxicity testing, the two most important dose metrics are inhaled dose and dose deposited in the lung.

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Inhaled dose is the amount of material entering the respiratory tract via the nose in most animal studies and via the mouth in clinical studies. Inhaled dose is thus the dose metric that compares to delivered dose in oral or injection studies, because it represents the total dose entering the body.

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Inhaled dose (mg) = Aerosol concentration (mg/L) x volume of air breathed (L) = Aerosol concentration (mg/L) x minute volume (L/min) x exposure time (min) Inhaled dose is the first dose metric to be calculated because it is most directly associated with exposure parameters like aerosol concentration, which can be directly measured by filter sampling, and exposure time, which is known for each experiment. Minute volume of the experimental animals can be assumed from existing literature using either allometric relationships 40, 41 or measured values 18 but this assumes that the compound itself or the experimental conditions used will not alter respiration compared to normal basal breathing. Direct measurements using plethysmography during an inhalation exposure are clearly the ideal procedure, but this is feasible only in limited cases. An allometric relationship has been derived by Alexander et al 41 from a large database of measurements made in inhalation toxicology or respiratory safety pharmacology studies which should be representative of these classes of studies and so is a preferred approach to estimate minute volume using the equation given below. Respiratory minute volume = 0.608 x BW0.852 Dose distribution throughout the respiratory tract as well as total dose is of considerable importance. Total deposition is defined as the amount of material deposited throughout the entire respiratory tract, from nose or mouth down to the alveolar region of the lung. Three major regions have been designated: the head airways (nose, mouth, nasopharynx, oropharynx, larynx), the tracheobronchial region (conducting airways from the trachea to terminal bronchioles), and the pulmonary region (alveoli). Lung deposition is also an important dose estimate and it is generally



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defined as material deposited below the larynx (i.e., tracheobronchial + pulmonary deposition). Deposition fraction can be estimated from literature values 34, 37.

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Deposited dose 9

= Aerosol concentration (mg/L) x minute volume (l/min)

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x exposure period (min) x deposition fraction (tracheobronchial 1 12 13 1 4 15 16 17 18 19 20 21 2 23 2 4 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

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+ pulmonary) Generally, for inhaled particles and particularly for inhaled pharmaceuticals, the lung is the target of most interest and hence the emphasis on determination of dose deposited in the lung. Therefore, lung deposited dose is one of the most important dose metrics and interspecies dose comparisons can be made on a lung dose per unit lung weight basis. This is generally a superior metric to inhaled dose per unit body weight to compare doses for an inhaled product since it focuses on local dose at the likely target organ, the lung. Dose measurements Several different approaches have been used to determine lung burdens of test agents and to measure values in lung tissues using chemical assays, radiolabel methods, and inferences from blood level measurements. Measurements of material deposited in the lung provide the most direct determination of deposited dose. Lung tissue assays using analytical chemistry methods can be developed for the test agent in question, in many cases and this is usually the preferred method for measuring lung dose in non-clinical studies. Carbon-14 radiolabeling and/or quantitative whole-body autoradiography techniques has been used in inhalation programs in limited circumstances. This method was used to determine organ distribution following exposure to a 14C-labeled antiasthma aerosol candidate in rats 42. Logistic considerations including the need for large amounts of radiolabeled compound means that this type of specialty study is only conducted rarely. Estimates of deposited dose from blood level information can be made, but this method an indirect determination and is heavily dependent on assumptions. Pharmacokinetics must be well understood so that AUCs from blood or urine levels can be expressed as a fraction of delivered dose. It is also necessary to estimate amount deposited in head airways and cleared to the gastrointestinal tract and absorbed, so that this may be subtracted from the total. This means the method is feasible only for compounds that are not absorbed from the gastrointestinal tract or if techniques have been used such as charcoal meal administration to markedly reduce gastrointestinal absorption. However, for inhaled pharmaceuticals that result in


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substantive blood levels, such as some corticosteroids or agents intended for systemic delivery such as inhaled insulin this can be a very useful approach since the blood levels achieved are of prime interest for determining systemic pharmacological effect.

Respiratory Tract Toxicity 9 10 1

Histopathology 12 13 1 4 15 16 17 18 19 20 21 2 23 2 4 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

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Both systemic toxicity and local toxicity must be evaluated following inhalation exposure6. Systemic toxicity is evaluated with histopathologic examination of a range of organs. Since the respiratory tract receives the majority of deposited inhaled dose, possible toxicity at this site needs to be evaluated carefully, particularly for agents that react chemically with respiratory tract cells. The main end point of respiratory tract toxicity evaluation is histopathology. Histopathology of lung and nasal sections needs to be evaluated carefully including sections of all major anatomical regions. This includes alveolar, tracheobronchial, and head airway regions. Examination of possible effects on major cell types such as type I and II cells in the alveolar region, Clara cells in the terminal bronchiolar region, and the ciliated, goblet, and serous cells is needed 43 . Nasal histopathology is obviously of paramount importance for agents intended for nasal delivery. Careful histological methods should be used to make observations in the complex turbinate regions of rats by taking serial cross sections at predetermined sites to cover the major anatomical regions 44,45. Nasal pathology is also of interest for agents intended for oral inhalation in humans but its importance is diminished because the nose is not a target tissue for mouth breathing patients. An important point to note is that generally the large animal species, dog or monkey, provide results more relevant to humans than rats. As an example the larynx is a common site for nonspecific induced changes in rat inhalation studies 46. Squamous metaplasia of the laryngeal epithelium that is observed with high-level exposures, even to essentially nontoxic materials, is considered to be a defense mechanism by which sensitive epithelium is replaced by a more resistant epithelium. It signifies an early attempt at epithelial repair 47. Another instance that suggests that results from rat studies should not be over-interpreted is the experience with inhaled tobramycin (TOBI). The rat studies showed dose-related increases in respiratory tract inflammation and accumulation of macrophages without an apparent no-effect level at the doses tested, while the clinical trials showed clear lung function benefit to the patients leading to the approval of inhaled tobramycin (TOBI) 48. Also, studies of inhaled gentamicin have shown substantially greater effects in rats than in dogs at the same deposited dose. The adverse effects in rats were correlated with lung accumulation in the rats 49 while there was no evidence for lung accumulation in dogs. Importantly clinical studies in humans indicated inhaled gentamicin was well



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tolerated. These data suggest that for inhaled antibiotics, rats are more sensitive than the larger species, including humans.

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Another issue that arises in inhalation studies is interpretation of presence of alveolar macrophages which has been discussed in an excellent review by Forbes et al50. One of the important observations they make is that “Nonclinical inhalation toxicity studies, particularly those conducted with drugs designed to maximize effects in the lung through low solubility, often report an increase in AMs as the primary histopathology observation. ln the absence of any other indication of inflammation, an increase in AMs is consistent with a non-specific physiological response to the delivery of particles to the alveolus and may be considered non-adverse (Nikula et al.51). In such circumstances, development may progress into the clinic even though it is currently not possible to monitor and characterize potential macrophage accumulation in man due to the absence of clinical measurement techniques and discriminatory biomarker.” They also note that if there is evidence of other lung changes including inflammatory effects, then to consider if this has clinical relevance there must at least be a consideration if it is a result of lung accumulation of particles, and further if there is a similar likelihood of the same phenomenon in people at clinically relevant doses.

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Interspecies differences can be very important for inhaled biopharmaceuticals. An important examples is inhaled DNase (Pulmozyme). In the inhalation toxicology studies there was histologic evidence in both rat and monkeys of inflammatory changes in the lung typical of a foreign body reaction. The evidence is strong that this occurred because rat DNase is only 80% homologous with human DNase, with monkey DNase being similarly non-homologous 52,53. In the animal studies there were dose-related increase in DNase antibodies while in human use antibody formation has not been a clinical issue 52. Therefore the observed lung histopathology changes in the nonclinical studies were not considered to be relevant to humans.

Design of Inhalation and Nasal Toxicity Studies General Principles Reviews by Wolff and Dorato 6,35, Hickey 4, and McElroy et al 54 provide comprehensive information relative to nonclinical considerations for inhaled pharmaceutical development. The guiding principles for inhalation toxicology studies are generally the same as for of routes of exposure. However, there are some specific factors that come into play for inhalation studies. One factor is that generally the same formulation that will be used in the clinical trials and marketed drug product should also be used in the nonclinical animal studies.


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Another issue that occurs more frequently for inhalation studies than for other routes of exposure is the need to use a maximum feasible dose as the high dose. Maximum feasible dose varies dependent on many factors, including characteristics of the test article formulation and the inhalation exposure systems. Some rough general considerations follow, but actual conditions must be considered on a case by case basis. Maximum exposure concentration of approximately 2 mg/L are suggested otherwise particle agglomeration occurs occurs and particle size become too large, nasal deposition predominates and sufficient lung deposition cannot be achieved8. In some instances, exposure concentrations up to 3 mg/L are possible. There are also practical limits to inhalation exposure duration for animal welfare reasons: approximately 3-6 hrs for rodents, and generally 1-2 hrs for non-rodents, although as noted earlier some low restraint systems allow longer exposures. These considerations mean that the upper limit for inhalation dosing of rodents is in the range of 300-450 mg/kg for rodents and 100 mg/kg for non-rodents total mass delivered. For many practical reasons, such as the fact that there can be a substantial component of excipient in the formulations it is not always possible to achieve such high values for the active agent. For products such as antibiotics which frequently require clinical doses of 100’s of mg of active agent this means that animal dose multiples can be relatively low compared to human use, when the typical lung deposition factors of 10% for rodents and 25% for non-rodents are considered. Other situations such as low maximum concentrations of drug in nebulized solutions or dry powder formulations also limits inhaled dose, and so in some instances it may not be possible to achieve doses in animal studies that are much higher than clinical doses. There are relatively few specific guidelines for inhalation toxicity studies related to inhaled pharmaceuticals. The scope of the studies needed for a toxicology profile is similar to that of noninhaled pharmaceuticals 55, 56. The pulmonary division of FDA published a perspective on regulatory needs for development of inhaled drugs 57 and FDA has provided a draft guidance document on alternative delivery modes (including inhalation) for approved products 58.

Inhalation as an Alternative Delivery for Marketed Products When an approved drug is considered for delivery by the nasal or inhalation the regulatory pathway can be much simpler than for a new chemical entity because of the data relating to systemic toxicity that is available from the studies for the approved route of administration, be it oral, subcutaneous or intravenous. Because of the large body of pre-existing information on systemic toxicity, the focus of studies for the alternative delivery route is the respiratory tract. FDA guidance 59 recommends that two short term studies be carried out first in a rodent and non-rodent species. These studies are typically 28 days in duration. Then a 6-month study should be carried out in the most appropriate species. Carcinogenicity studies are not likely to be needed if



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there is no evidence of preneoplastic or proliferative changes observed in the 6-month inhalation study. The simpler pathway for alternative delivery compared to studies needed for a new chemical entity is shown schematically in Figure 3.

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Inhaled drug formulations usually involve the use of excipients. If the excipients are novel, i.e., have not been used in previously approved inhaled drug products then toxicological characterization of the novel excipients will be required 58, 59. The toxicology studies needed for novel excipients depend on the nature of the excipient. If the excipient is a sugar, similar to lactose which is an approved inhalation excipient, or if the excipient is an endogenous material the types of studies needed might be relatively minor. This is especially the case for endogenous compounds where the administered dose results in small increments in pre-existing levels. For instance, for mannitol and glycine in Exubera (inhaled insulin) formulation and dipalmitoyl phospatidylcholine (DPPC) in Lilly’s inhaled insulin formulation a placebo control group containing excipients was included in addition to an air breathing control group into the subchronic and chronic inhalation toxicity studies. Thus, the studies used a total of 5 groups instead of the more usual 4 groups as there were air control, placebo control, and low, mid, and high dose treatment groups. This approach, as well as literature review of these agents, was considered sufficient by regulatory agencies for approval. Similar approaches have been used for the use of distearyl phospatidylcholine (DSPC) in spray dried powder formulations. A less familiar excipient has been used in Afrezza inhaled insulin, namely fumaryl diketopiperazine, which is used to produce Technosphere particles. In this case more extensive studies were needed, including a 2-year inhalation carcinogenicity study in rats, a 6-month subcutaneous transgenic mouse study (Tg.rasH2) as well as study of fumaryl diketopiperazine pharmacokinetics 59, 60.

Summary - Interpretation of Results of Inhalation Toxicology Studies The conduct of inhalation toxicology studies will allow the identification of target organs and effects, and an indication of toxicity profile in the animal species used. A major challenge is to interpret these results in terms of possible human effects. A number of factors need to be considered in this effort and they can only be mentioned briefly in this chapter. As alluded to earlier, dosimetry is a significant issue and there needs to be a thorough analysis of the estimated dose multiple from the nonclinical studies compared to


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human doses. For local lung effects the preferred dose metric is comparison of dose deposited in lung on a lung weight basis.

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Interspecies differences need to be carefully examined to determine if histologic phenomena are species related. It has been noted above that there are rat-specific laryngeal findings and also for biological materials, homology between the animal species and humans needs to be considered. 7 8 9 10 1 12 13 1 4 15 16 17 18 19 20 21 2 23 2 4

Consideration of systemic versus local toxicity is also a significant issue. Inhaled toxicants can exert their toxic effects at the point of contact, locally in the respiratory tract, and/or they may be absorbed and possibly metabolized, and exert their action systemically or through action at distant organs. If systemic effects predominate, then information can be gained from studies from other routes of administration such as oral or intravenous. If the inhalation or nasal route is an alternative route of delivery then there is usually a wealth of information from the original route of administration. If the action is principally local, then inhalation, or nasal studies have the most relevance.

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Toxicokinetics need to be considered carefully. There are many questions to be considered. Is toxicity associated only with acute exposure or are there accumulated dose effects? Does the parent compound and/or the metabolite accumulate? Are pharmacodynamic effects related to the parent compound or metabolite? An integrated analysis of all of these factors from the toxicology studies must be considered as well as any other relevant information in order to provide an adequate safety assessment of a pharmaceutical intended for the inhalation or nasal route.

References 1. R. O. McClellan and R. F. Henderson. 'Concepts in Inhalation Toxicology,' Hemisphere. New York, 1989 2. R. F. Phalen, 'Inhalation Studies: Foundation and Techniques,' CRC Press, Boca Raton. FL, 1984. 3. M. A. Dorato and Wolff, R. K. 'Inhalation exposure technology, dosimetry, and regulatory issues.' Toxicologic Pathologv, 19.373-383, 1991 4. Hickey A.(ed.) Inhalation Aerosols – Physical and Biological Basis for Therapy. Informa Healthcare. (2007) 5. Pauluhn J. Inhalation toxicology: methodological and regulatory challenges. Exp Toxicol Pathol. 2008 Jun;60(2-3):111-24 6. Wolff R and Dorato M, 2010. Inhalation toxicology studies. Chapter in “Comprehensive Toxicology, 2nd edition, Volume 3, Toxicology Testing and Evaluation, James Lamb, ed.” Pages 225-245.

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7. Lewis TR, Morrow, PE, McClellan RO, Raabe OG, Kennedy GL, Schwetz B A, Goehl TJ, Roycroft JH, Chabra RS. Establishing aerosol concentrations for inhalation toxicology studies. Toxicol. Appl. Pharmacol. 1989 99: 377-383 8. OECD. Guidelines for the Testing of Chemicals, 403, 412, 413(2009) http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-ofchemicals-section-4-health-effects_20745788. 9. Inhalation Specialty Section of the Society of Toxicology (SOT). Recommendations for the conduct of acute inhalation limit tests. Fundam. Appl. Toxicol. 18: 321, 1992. 10. Hext PM. Current perspectives on particulate induced pulmonary tumours. Hum Exp Toxicol. 1994 Oct;13(10):700-15. Review 11. Morrow PE. Dust overloading of the lungs: update and appraisal. Toxicol Appl Pharmacol. 1992 Mar;113(1):1-12. Review 12. Lee KP, Trochimowicz HJ, Reinhardt CF. Pulmonary response of rats exposed to titanium dioxide (TiO2) by inhalation for two years. Toxicol Appl Pharmacol. 1985 Jun 30;79(2):179-92. 13. Nikula KJ, Snipes MB, Barr EB, Griffith WC, Henderson RF, Mauderly JL. Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. Fundam Appl Toxicol. 1995 Apr;25(1):80-94 14. Y.-S. Cheng and O. R. Moss in 'Concepts in Inhalation Toxicology,' eds. R. O. McClellan and R. F. Henderson, Hemisphere, New York, p.44, 1989. 15. Wong BA. Inhalation exposure systems: design, methods and operation. Toxicol Pathol. 2007 Jan;35(1):3-14 16. Cannon, W. C., Blanton, E. F., and McDonald, K. E., The flow-past chamber: an improved nose only exposure system for rodents, Am. Ind. Hyg. Assoc. J, 44: 923, 1983 17. Raabe OG, Bennick JE, Light ME, Hobbs CH, Thomas RL, Tillery MI. An improved apparatus for acute inhalation exposure of rodents to radioactive aerosols. Toxicol Appl Pharmacol. 1973 Oct;26(2):264-73. 18. Griffis LC, Wolff RK, Beethe RL, Hobbs CH, McClellan RO. Evaluation of a multitiered inhalation exposure chamber. Fundam Appl Toxicol. 1981 JanFeb;1(1):8-12 19. Dorato, M.A.; Carlson, K.H.; Copple, D.L. Pulmonary mechanics in conscious Fischer 344 rats: multiple evaluations using nonsurgical techniques. Toxicol. Appl. Pharmacol. 68 (1983) 344–353. 20. Driscoll, K.E.; Costa, D.L.; Hatch, G.; Henderson, R.; Oberdorster, G.; Salem, H.; Schlesinger, R.B. Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations. Toxicol. Sci. 55 (1) (2000) 24–35. 21. Rao, G.V.S., S. Tinkle, D. N. Weissman, J. M. Antonini, M. L. Kashon, R. Salmen, L. A. Battelli, P. A. Willard, M. D. Hoover, and A. F. Hubbs. Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx. J. Tox. Environ. Health , 66(15) (2003): 1441-1452.


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22. Rothenberg SJ, Barrett JF, Dearlove GE, Parker RM, Ball DJ, Brady JT, Yeh HC, Greenspan BJ. Characterization of a microprocessor-controlled tubular multiple metered dose inhaler aerosol generator for inhalation exposures of pharmaceuticals. J. Aerosol Med. 13(3), 157, 2000 23. Kaleta, R.; Knauss, V.; Cracknell, L.A.; Hussain, M.; Hardy, C., A Refined Method of Restraint for Dogs Used in Inhalation Studies – System Adaptation and Improvement. (2007) Society of Toxicology Annual Meeting 24. Rothenberg SJ, Hershman RJ, Beihn RM, Lief SD, Ehrhart WJ, and Dewees DL. Deposition of monodisperse aerosols in young beagle dogs. Association of Inhalation toxicologists Annual Meeting, Stockholm, Sweden, Sep 13-15, 2000.

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25. Miller MA, Stabenow JM, Parvathareddy J, Wodowski AJ, Fabrizio TP, et al. Visualization of Murine Intranasal Dosing Efficiency Using Luminescent Francisella tularensis: Effect of Instillation Volume and Form of Anesthesia. (2012) PLoS ONE 7(2): e31359. doi: 10.1371/journal.pone.0031359 26. D. S. Southam , M. Dolovich , P. M. O'Byrne , M. D. Inman. Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia. American Journal of Physiology - Lung Cellular and Molecular Physiology Published 2002 Vol. 282 no. 4, L833-L839 DOI: 10.1152/ajplung.00173.2001 27. Mercer, M. l. Tillery and H. Y. Chow, Operatng characteristics of some compressed-air nebulizers' Am. Ind. Hyg, Assoc. 1.,29:66-78, ,1968. 28. Lux C, Ahrens R, and J. Hoefer, 'Evaluations of jet nebulizers available in the US for antibiotic delivery to patients with cystic fibrosis.' J. Aerosol Med., 1993 29. Wright, B. M., A new dust feed mechanism, 1. Sci. Instruments, 27. 12, 1950 30. Cheng, Y. S., Marshall, T. c., Henderson, R. F., and Newton, G. J., Use of a jet mill for dispersing dry powder for inhalation studies, Am. Ind. Hyg. Assoc.J., 46: 449, 1985 31. Bernstein DM, O. R. Moss, H, Fleissner Aerosols: Science. Technology, and Applicalions of Airborne Particles.' eds. B. Y. H. Liu. D. Y. H. Pui, and H.. Fissan, Elsevier Science, New York. 1984. 32. Lee W, Viau A, and Banks C. Extended duration powder delivery system used in preclinical studies in rodents. Society of Toxicology Annual Meeting, Philadelphia, 2000 33. Kulkarni P, Baron P, and Willeke K. Measurement of Aerosols: Principles, Techniques, and Applications, 3rd edition. (2011) Wiley 34. Schlesinger, R. B., Comparative deposition of inhaled aerosols in experimental animals and humans: a review. Toxicol. Environ. Health. 15. 197. 1985. 35. Wolff RK, Dorato MA. 1993. Toxicologic evaluation of inhaled pharmaceuticals. Crit Rev Toxicol 23:343-369 36. Phalen RF, Mendez LB. Biomarkers. Dosimetry considerations for animal aerosol inhalation studies. 2009 Jul;14 Suppl 1:63-6. doi: 10.1080/13547500902965468 37. Raabe, O.G.; Al-Bayati, M.A.; Teague, S.V.; et al. Regional deposition of inhaled monodisperse coarse and fine particles in small laboratory animals. Ann. Occup. Hyg. 32 (1988) 53-60.

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38. Asgharian B, Wood R, Schlesinger RB. Empirical modeling of particle deposition in the alveolar region of the lungs: a basis for interspecies extrapolation. Fundam Appl Toxicol. 1995 27(2):232-8 39. Schroeter JD, Asgharian B, Price OT, McClellan GE. Computational fluid dynamics simulations of inhaled nano- and microparticle deposition in the rhesus monkey nasal passages. Inhal Toxicol. 2013 25(12):691-701. doi: 10.3109/08958378.2013.835889 40. Bide RW, Armour SJ, Yee E. Allometric respiration/body mass data for animals to be used for estimates of inhalation toxicity to young adult humans. J Appl Toxicol. 2000 20(4):273-90 41. Alexander D.J., Collins C.J., Coombs D.W., Gilkison I.S., Hardy C.J., Healey G., Karantabias G., Johnson N., Karlsson A., Kilgour J.D. and McDonald P., (2008). Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies with pharmaceuticals. Inhal. Tox., 20, 1179-1189 42. Pohland, R.C.; Beck, J.M.; Carlson, K.H.; et al. Tissue distribution of a leukotriene antagonist 14C-LY170680 following inhalation exposure in the rat. Toxicologist 11 (1991) 95. 43. Plopper, C.B.; Weir, A.; St. George, J., In: (Editors: Dungworth, D.; Kimmerle, G.; Lewkowski, J.; et al.) Inhalation Toxicology: The Design and Interpretation of Inhalation Studies and Their Use in Risk Assessment (1988) Springer-Verlag, New York, pp. 25–40. 44. Young, J. Light microscopic examination of the rat nasal passages: preparation and morphologic features, in Toxicology of the Nasal Passages. Barrow, C. S., Ed., Hemisphere Publishing, Washington. D.C., p 27, 1986. 45. Harkema JR, Carey SA, Wagner JG. The nose revisited: a brief review of the comparative structure, function, and toxicologic pathology of the nasal epithelium. Toxicol Pathol. 2006;34(3):252-69 46. Lewis DJ. Morphological assessment of pathological changes within the rat larynx. Toxicol Pathol. 1991;19(4 Pt 1):352-7 47. Gopinath, C.; et al., Atlas of Experimental Toxicological Pathology. (1987) MTP Press, Lancaster, UK . 48. FDA. Summary Basis of Approval for TOBI, NDA 50-753197 49. Conway H, Dix, KJ, Miller, RA, Wall, HG, Wolff, RK and Reed, MD. Comparison of Inhalation Toxicity studies of Gentamicin in Rat and Dogs. 2013 Inhalation Toxicol. 25: 714-724 50. Forbes B, O'Lone R, Allen PP, Cahn A, Clarke C, Collinge M, Dailey LA, Donnelly LE, Dybowski J, Hassall D, Hildebrand D, Jones R, Kilgour J, Klapwijk J, Maier CC, McGovern T, Nikula K, Parry JD, Reed MD, Robinson I, Tomlinson L, Wolfreys A. Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses. Adv Drug Deliv Rev. 2014 May;71:15-33. doi: 10.1016/j.addr.2014.02.001. Epub 2014 Feb 13 51. Nikula KJ, McCartney JE, McGovern T, Miller GK, Odin M, Pino MV, Reed MD. STP position paper: interpreting the significance of increased alveolar


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macrophages in rodents following inhalation of pharmaceutical materials. Toxicol Pathol. 2014;42(3):472-86. doi: 10.1177/0192623313507003. Epub 2013 Oct 31. 52. Green J. 1994. Pharmacological –Toxicological (Expert Report on Recombinant Human Deoxyribonuclease 1 (rhDNase; Pulmozyme TM). Hum Exp Toxicol, 13: S2 – S42. 53. Wolff RK. 1998. Safety of Inhaled Protein for Therapeutic Use. J. Aerosol Med, 11: 197-219. 54. McElroy M, Kirton C, Gliddon D, and Wolff R. 2013. Inhaled Biopharmaceutical Drug Development: Nonclinical Considerations and Case Studies. Inhalation Toxicol: 25(4): 219–232. 55. Dorato MA, Buckley LA. Toxicology testing in drug discovery and development. Curr Protoc Toxicol. 2007 Feb;Chapter 19 56. ICH M3(R2). Guidance on Nonclinical Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (2009). www.ich.org 57. DeGeorge J, et al Considerations for Toxicology Studies of Respiratory Drug Products. Reg. Toxicol. Pharmacol. 25: 189-193, 1997 58. FDA Draft Guidance for Industry and Staff: Nonclinical evaluation of reformulated drug products and products intended for administration by an alternate route. 2008. http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm079245.pdf+&cd=1&hl=en&ct=clnk&gl=us 59. Greene S, Nikula K, Poulin, D, McInally K, and Reynolds J. Assessment of longterm preclinical safety of Technosphere particles and Afrezza inhalation powder. Society of Toxicology Annual Meeting, San Diego, 2013 60. FDA. Background information for Apr 2014 Review meeting on Afrezza. 2014 http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials /Drugs/EndocrinologicandMetabolicDrugsAdvisoryCommittee/UCM390864.pdf

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Figures - Wolff 16 17 18 19 20 21 2 23 2 4 25 26

Figure 1. Schematic of Rat Nose-only Aerosol Exposure System (Slide 2) Figure 2. Schematic of Dog Face Mask Dosing System

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(Slide 3)

Figure 3. Streamlined Toxicology Profile for Alternative Delivery If the Agent is Approved by Another Route (Slide 4)

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Rat

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Non-definitive Lead Optimization Studies (Acute & 14 days, kinetics, genetic toxicology)

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Definitive Toxicology

Genetic Toxicology Battery Acute Toxicology

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Subchronic Toxicology (14-day to 90-day)

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Safety Pharmacology

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Definitive toxicokinetics Repro. and Devel. Tox. (timing based on inclusion of WCBP)

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Chronic Toxicology (6-month and 9-month)

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Toxicology of Major Metabolites

Qualification Studies

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Table of Contents Graphic Toxicology Studies for Inhaled and Nasal Delivery RK Wolff

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Toxicology Studies for Inhaled and Nasal Delivery - American

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