http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, 2013; 25(13): 714–724 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2013.843043
RESEARCH ARTICLE
Comparison of inhalation toxicity studies of gentamicin in rats and dogs Heather Conway1, Kelly J. Dix2, Jacob D. McDonald2, Rodney A. Miller3, Henry G. Wall3, Ronald K. Wolff4, and Matthew D. Reed2 Merck & Co. Inc., Summit, NJ, USA, 2Lovelace Respiratory Research Institute, Albuquerque, NM, USA, 3Experimental Pathology Laboratories, Sterling, VA, USA, and 4Safety Consulting Inc., Carbondale, CO, USA
Abstract
Keywords
Nebulized gentamicin solution was administered to rats (nose-only) and dogs (face mask) for 14 days with a 14-day recovery period. Control groups of each were exposed to saline aerosols. Mean estimated inhaled lung doses of gentamicin were 39, 123 and 245 mg/kg for rats (deposited doses 6, 17 and 34 mg/kg) over 30, 90 and 180 min, respectively. Since dogs do not tolerate exposures as long as rats, inhaled lung doses were limited to 7, 14 and 41 mg/kg (deposited doses of 1, 3 and 8 mg/kg) over 15, 30 and 60 min. Comparable doses were achieved at the low dose for rats and high dose for dogs. Serum AUCs (14 2 mg/mL*h (mean SD) at 6 mg/kg in rats and 11 7 mg/mL*h at 8 mg/kg in dogs) showed comparable exposure between the two, implying similar absorbed doses and confirming similar deposited lung doses. Rat exposures resulted in dose-related lung pathology (including low dose) manifested as upper respiratory tract irritant reactions with alveolar histiocytosis, inflammation, airway epithelial metaplasia and lymphomegaly in lung tissue. This was associated with high lung tissue gentamicin levels 24 h post-dose on Day 14 (375 33 mg/g at deposited dose of 6 mg/kg). Dose-related kidney tubular necrosis (a well-known toxicity of parenteral gentamicin) was observed, but no test-article related effects on lung histopathology in dogs (even at highest deposited dose of 8 mg/kg) and low levels of lung tissue gentamicin (42 11 mg/g) 24 h post-dose on Day 14.
Gentamicin, histiocytosis, histopathology, infectious disease, inflammation, inhaled, inhaled antibiotics, lymphomegaly, metaplasia, nebulized, respiratory
Introduction A significant body of information currently exists on the use of aerosolized antibiotics to treat respiratory disease (Gellar et al., 2011; Høiby, 2011; Naesens et al., 2011; Schuster et al., 2013). Tobramycin inhalation solution and dry powder are approved US products for management of infections in cystic fibrosis patients. Hall (1989) reviewed the use of aerosolized antibiotics and concluded that inhaled antibiotics may offer advantages over systemic or orally administered products by (1) treating at high local concentrations at the site of the infection and (2) limiting systemic side effects. Additionally, the American Academy of Pediatrics (Prober et al., 2000) noted in a review that there has been extensive use of inhaled antibiotics formulated for other purposes over several decades. Further, dry powder formulations of gentamicin have been studied in rodents and non-human primates for use against pneumonic conditions resulting from the gram negative biological threat agents tularemia and plague (Talton et al., 2010) and tuberculosis (Roy et al., 2012). Gentamicin is a broad spectrum, generic aminoglycoside antibiotic currently formulated for parenteral and topical administration and is indicated for some cases of severe
Address for correspondence: Dr Matthew D. Reed, LRRI, Preclinical Drug Development, 2425 Ridgecrest Dr SE, Albuquerque, NM 87108, USA. Tel: 505-348-9451. Fax: 505-348-4983. E-mail: [email protected]
History Received 3 July 2013 Revised 5 September 2013 Accepted 6 September 2013 Published online 20 November 2013
pneumonia (Rotstein et al., 2008). Gentamicin consists of a mixture of three active components produced by the gram positive species Micromonosproa and isolated by Roche in the early 1960’s (Weinstein et al., 1963). Susceptible bacterial species include pseudomonas, staphylococcus, tularemia and plague among others. Although extremely effective when administered for the correct indication, gentamicin has rate limiting nephro- and ototoxicity associated with prolonged systemic administration (Zhanel et al., 2012). There have been several clinical investigations related to aerosolized treatment using gentamicin (Crowther Labiris et al., 1999; Murray et al., 2010; Palmer et al., 1998; Twiss et al., 2005). Inhaled gentamicin administered using 80 mg in the nebulizer has been shown to result in relatively high levels in the sputum of intubated patients; these levels have been generally substantially higher than the minimal inhibitory concentrations of sensitive gram negative organisms with low or undetectable serum concentrations (Palmer et al., 1998). This aerosol delivery system resulted in serum concentrations indicating low potential for systemic toxicity, since they were generally undetectable, and thus they were substantially below the ‘‘cautionary limit’’ for systemic toxicity of 12 mg/mL (Gentamicin Sulfate Injection, 2011). There were additional indications of efficacy such as reduced sputum volume and eradication of pathogens, and minimal safety issues were identified. These and other data suggest the possibility of additional preclinical and clinical investigation
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Comparing gentamicin inhalation toxicity studies
DOI: 10.3109/08958378.2013.843043
of inhaled gentamicin to aid in the treatment of respiratory infectious by increasing local lung concentrations while limiting systemic exposure and potential side effects. The purpose of the current program was to conduct 14-day inhalation toxicology studies in rats and dogs with nebulized, preservative-free gentamicin solutions under Good Laboratory Practice regulations. The studies were performed at up to maximal feasible exposure concentrations to support safe use in clinical studies and potential FDA licensure.
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prepared at 200 mg/mL concentrations of gentamicin activity and Aerotech II jet nebulizers (CIS-US INC., Bedford, MA) were used to generate the inhalation atmosphere. Target aerosol concentrations were 1.5–2 mg/L gentamicin and target particle size was 2-3 mm MMAD. Control exposures were nebulized from solutions of 0.9% saline. Stability of gentamicin was confirmed in nebulizer solutions, stock solutions (used to create linearity standards for quantitation) and spiked filters utilizing a qualified content method. Rat exposure system
Methods
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Animals All studies were performed under Institutional Animal Care and Use Committee approval and Good Laboratory Practice regulations. A total of 116 male and 116 female CD rats, approximately 9–10 weeks old at the beginning of the 14-day exposure, were used on study (Charles River Laboratories, Raleigh, NC). A total of 16 male and 16 female beagle dogs (Ridglan Farms, Mt. Horeb, WI) approximately 6–7 months of age were used at the beginning of the 14-day exposure. The animal allocations to the control, low, mid and high exposure groups, as well as designations for toxicokinetics (TK), are shown in Table 1 for rats and Table 2 for dogs. All animals were conditioned to inhalation exposures conditions and apparatuses on at least three occasions prior to placement on study to ease environmental stress effects. A single group of saline control exposure rats and dogs were used for each study and exposed for a duration equivalent to the high exposure group to conserve animals and in accordance with US FDA recommendations (proprietary pre-Investigational New Drug Applications meeting conducted prior to the study start). Aerosol generation Preservative-free solutions of gentamicin sulfate (Fujian Fukang Pharmaceutical Co. Ltd., Fuzhou, China) were
Nose-only exposures were conducted in a ‘‘flow-past-type’’ cylindrical inhalation chamber (Cannon et al., 1983) placed inside a steel-framed Plexiglas secondary containment box. The chamber contained 48 rodent ports; each was compatible with a single nose-only exposure tube, aerosol concentration sampling device (e.g. filter) or oxygen monitor. The total air flow through the exposure system was balanced to achieve individual rodent port flows of 500 mL/ min (port flow approximated based on total chamber flow). Measured flows included sample flow rate, nebulizer flow rate, dilution flow rate and chamber exhaust flow. The exposure chamber had a slightly higher exhaust flow rate than inlet flow rate. Dog exposure system The exposure system consisted of a single, cylindrical, Plexiglas inhalation chamber (volume of 23.7 L, 14.61-cm radius, 35.56-cm height). The chamber was supplied with two Aerotech II nebulizers operated at 30 psi (gentamicin low and mid) and 40 psi (gentamicin high). Nebulized test article and nebulizer air supply were diluted with 10 L/min HEPA-filtered dilution air. The flow through the system was 36 L/min. Measured flows included sample flow rate, nebulizer flow rate, dilution flow rate and chamber exhaust flow. The exposure chamber had a slightly higher exhaust flow rate than inlet flow rate. Six equally spaced corrugated plastic exposure lines of 1 m were connected from the
Table 1. Number of rats used in each of the experimental group assignments. Main study Group Saline control Low (gentamicin) Mid (gentamicin) High (gentamicin)
TKa – Day 1
Recovery
TKa – Day 14
Exposure duration (h)
Male
Female
Male
Female
Male
Female
Male
Female
3 0.5 1.5 3
10 10 10 10
10 10 10 10
5 NA NA 5
5 NA NA 5
3 9 9 9
3 9 9 9
3 9 9 9
3 9 9 9
a
Serum, lung and kidney samples taken from three rats per gender at pre-dose and 3, 6 and 24 h post-dose on Days 1 and 14.
Table 2. Number of dogs in each of the experimental group assignments. Main study Group Saline control Low (gentamicin) Mid (gentamicin) High (gentamicin) a
Exposure duration (h) 1 0.25 0.5 1
Male 3 3 3 3
Female 3 3 3 3
Recovery Male 2 NA NA 2
TK – Day 1
TK – Day 14
Female
Male
Female
Male
Female
2 NA NA 2
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
Serum TK samples taken from Main Study and Recovery dogs pre-dose, as well as immediately (0) and 3, 6, 12 and 24 h post-dose on Days 1 and 14. Lung and kidney samples taken 24 h post-Day14 dosing.
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chamber to a plastic ‘‘Y’’. The ‘‘Y’’ is additionally connected at one end to a single inlet neoprene mask (mask covers the snout and is affixed to the dog by velcro straps) and an exhaust line connected to the institutional exhaust system on the other. Each exhaust line flows through an inline filter followed by a critical orifice designed to allow not more than 6 L/min air flow from the chamber to institutional exhaust. The dog was allowed to pull (breathe) test article from the Y. Since five to six dogs were exposed to control or test article per exposure interval, one or two lines were plugged at the Y. An additional sampling probe inserted through a reference sampling port pulls 0.5 L/min from the center of the chamber in order to monitor chamber concentrations during exposures. Exposure atmosphere monitoring The test article was sampled from the chamber onto Pallflex (Pall Corp., Port Washington, NY) filters (for concentration measurements) or Mercer-style, seven-stage cascade impactor (InTox Products, Moriarty, NM) substrates (for particle sizing). Aerosol concentration was determined by taking filter samples directly from the chamber. For each exposure day, exposure filters were extracted with water and analyzed chemically (high-pressure liquid chromatography with ultraviolet absorptiondetection [Agilent Technologies, Santa Clara, CA]) to monitor and define gentamicin aerosol concentrations during exposures. Chromatography was performed on a Phenomenex Luna 3 mL C18 (2), 75 4.60 mm (Phenomenex, Torrance, CA). A flow rate of 1.0 mL/min and an injection volume of 50 mL were used for each sample. Mobile phase A was 2 mM hexane sulfonate, 20 mM sodium sulfate, 0.005% acetic acid in water. Mobile phase B was 100% methanol. A linear mobile phase composition gradient changed from 95:5 A:B at time ¼ 0 min to 5:95 A:B at time ¼ 6 min. After 6 min, the mobile phase was returned to initial conditions and equilibrated for 2 min for a total sample run time of approximately 8 min. Post-column, the effluent was mixed with a reaction solution of 0.33 mg/mL o-Pthaldialdehyde, 0.66 mg/mL mercaptoethanol and 0.133 M borate buffer (pH ¼ 10.4). After derivitazation, the gentamicin mixture was detected by UV at ‘A ¼ 330 nm. Aerosol particle size measurements were made three times throughout the duration of the study (approximately at the beginning, middle and end of the exposure period) by Mercer-style impactor stage assessments. Chemical detection was the definitive measurement for particle size. Dose determination Gentamicin doses for rats and dogs were determined using the equations and assumptions listed below: Inhaled Dose ðmgÞ ¼ Empirically determined aerosol concentration ðmg=LÞ published ðdogsÞ or allometrically ðratsÞ converted average minute volumes exposure time ðminÞ:
Inhal Toxicol, 2013; 25(13): 714–724
Inhaled Deposited Dose ðmgÞ ¼ Inhaled Dose published ðdogsÞ or mathematically processed ðratÞ pulmonary deposition fraction: Times of exposure and aerosol concentrations were determined empirically. Inhaled and Inhaled Deposited Doses were divided by approximated average body weights for rats (300 g) and beagle dogs (8 kg) to determine aerosol dosing on a mg/kg basis. Minute volume for rats (0.188 L/min for a 300 g rat) was determined by the allometric conversion method of Bide et al. (2000). Minute volume for beagle dogs (4 L/min) was determined using empirically generated data as measured by Mauderly (1972, 1979). Pulmonary deposition for rats (12.7%) was determined based on empirical particle size data and derivations using the Multiple Path Particle Dosimetry model (Anjilvel & Asgharian, 1995; RIVM, 2002). Pulmonary deposition for dogs (20%) was determined based on empirical particle size data and deposition fraction as determined by Schlesinger (1985). Biological endpoints The following list summarizes the biological endpoints. (a) Clinical observations were made daily. (b) Body weights were measured weekly and prior to necropsy (fasted). (c) Hematology, coagulation and clinical chemistry parameters were measured using standard panels at the end of the 14-day exposure period for rats and prior to treatment and at the end of the 14-day exposure period for dogs, as well as at the end of the recovery period for both rats and dogs. Blood was collected by syringe from the descending vena cava during necropsy (rats) or by jugular stick (dogs) prior to euthanasia. Standard tubes for clotting (serum chemistry, room temperature), hematology (calcium EDTA, on ice), or clotting parameters (sodium citrate on ice) were used for collections. Clinical chemistry analyses were performed within 24 h of collection utilizing Hitachi 911 (Roche Diagnostics, Indianapolis, IN). Hematology samples were analyzed within 24 h of collection utilizing an AdviaÔ 120 (Bayer Corporation, Tarrytown, NY) and clotting samples were analyzed within 24 h of collection utilizing a Amelung KC 4AÔ Micro (Trinity Biotech, St. Louis, MO). (d) Toxicokinetics – Concentrations of gentamicin C1, C1a, C2 and C2a were measured using validated LC-MS/MS methods in serum, kidney and lung according to the method of Ishii et al. (2008) with proprietary modification. Blood samples were collected at sacrifice (rat) or by jugular vein (dog) and processed to serum. Tissue samples were collected at necropsy. Samples were frozen at 80 C until analyses. Total gentamicin concentrations were expressed as a sum of these subcomponents, and the sum was used in all toxicokinetic analyses, WinNonlin Enterprise Version 5.0.1 software (Pharsight Inc., Mountain View, CA). Rats – Serum, lung, and kidney samples were taken predose, end of dosing (0 h), and 3, 6 and 24 h after the end of dosing on Days 1 and 14.
Comparing gentamicin inhalation toxicity studies
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Dogs – Serum samples were taken on Days 1 and 14 predose, end of dosing (0 h), and 3, 6 and 24 h after the end of dosing. Lung and kidney samples were taken at necropsy, 24 h after the last dosing on Day 14. Limit of quantitation (LOQ) for total gentamicin was 7.4 ng/mL for serum samples and 750 ng/g for lung and kidney samples. (e) Necropsy and histopathology – Animals were euthanized by overdose of barbiturate-based sedative (dogs and rats) and exsanguination (dogs only). A full standard list of tissues was taken (Bregman et al., 2003) and fixed in 10% neutral buffered formalin. Tissue sections were cut 4–6 mm thick, mounted on standard glass slides, stained with hematoxylin and eosin (H&E), and read by a board certified pathologist. Gradable findings were recorded on a 1–5 scale where, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked and 5 ¼ severe. Non-gradable findings were recorded as ‘‘not present’’ (NP). (f) Statistical analyses – For endpoints measured only at scheduled sacrifice and for body weight measurements, analyses were performed utilizing standard parameters in the Path-Tox data acquisition system (Path-Tox 4.2.2; Xybion, Cedar Knolls, NJ). In brief, data sets consistent with the number of animals in each exposure group were tested for homogeneity with Bartlett’s test. For homogenous data, Dunnett’s t test was performed, and for nonhomogenous data, the modified t test was performed to determine statistical difference from control values. For clinical chemistry and hematology parameter data sets (consistent with the number of animals in each exposure group) a one-way analysis of variance (ANOVA) was used and when a significant treatment effect was noted (p50.05), Dunnett’s multiple comparison test was performed to assess differences between treated and control groups. When data exhibited either skewed distributions or variability scaled linearly with mean values, a logarithmic transformation was applied prior to the analysis. Statistical calculations were performed using the SASÕ software system, Version 9.1 (Cary, NC). All reported p values were two-sided.
For all analyses, multivariate and univariate, statistical significance was considered as p 0.05.
Results Test article The stability of gentamicin, assessed by quantitatively measuring content prior to and after storage, was within 15% of an initial analysis under all conditions. Analysis of the peak profile in filter extracts from filters collected for test atmosphere monitoring showed that the composition of the mixture did not change (degrade) over the course of an exposure period (3 h). This indicated that the test article composition was stable under the conditions employed for the study. Exposure and dose data The mean exposure data are summarized in Tables 3 and 4 for rats and dogs, respectively. Collected data indicated that the exposures were conducted over appropriate doses ranging from near the clinical range at the lowest level and at higher multiples in the mid- and high-dose ranges. Exposure concentrations were consistent over the 14-day exposures with coefficients of variation of 10%–12% for the rat exposures and 10%–15% for the dog exposures. The particle size range of 1.9–2.5 mm MMAD for both rats and dogs was highly respirable and appropriate for comparison with clinical exposures (Schlesinger, 1985). Body weight Rats – Body weight changes were consistent with short-term inhalation toxicology studies and were generally unremarkable. No rodent mean weight gain was observed in any exposure group at the end of exposures. The only exposurerelated body weight decreases were observed in high level females at the end of exposure. Weights tracked lower than that of control animals reaching statistical significance just prior to sacrifice (9% lower than control). Dogs – There were no apparent changes in body weight in gentamicin exposed dogs as compared to controls.
Table 3. Mean exposure data for rats with inhaled doses of 39, 123 and 245 mg/kg in the low, mid and high dose groups, respectively.
Rat group Saline control Low Mid High
Exposure duration (h)
Exposure concentration (mg/L)
Particle size MMAD (GSD)
Estimated inhaled dose (mg/kg)
Estimated lung dose (mg/kg)
3.0 0.5 1.5 3.0
– 2.09 2.18 2.17
– 2.1 mm (1.8) 1.9 mm (1.9) 2.5 mm (1.7)
– 39 123 245
– 6 17 34
Table 4. Mean exposure data for dogs at inhaled doses of 7, 14 and 41 mg/kg at the low, mid and high doses.
Dog group Saline control Low Mid High
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Exposure duration (hr)
Exposure concentration (mg/L)
Particle size MMAD (GSD)
Estimated inhaled dose (mg/kg)
Estimated lung dose (mg/kg)
1.0 0.25 0.5 1.0
– 1.19 1.17 1.72
– 2.5 mm (1.6) 1.9 mm (1.7) 2.1 mm (1.6)
– 7 14 41
– 1 3 8
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Hematology, coagulation, and clinical chemistry
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Rats – Hematology and clinical chemistry parameters were generally unremarkable among exposed animals. Multiple, mostly small statistically significant effects versus control were observed, Tables 5 and 6. Most changes either lacked gender agreement or lacked clear evidence of an exposure– response relationship. Although whole cell counts were unaffected by exposure, neutrophils were increased in the male mid level (69% over control values) and high level (73%) rats. This change may have been related to the pulmonary inflammation induced by gentamicin (see below). Overall, exposure-related responses in clinical chemistry parameters were likely indicative of effects of the gentamicin on the lung (inflammation indicated by the protein panel changes) and the kidney (protein panel, NA-S, K-S, CL-S, CA-S, PHOS, BUN Table 5. Significant effects of exposure on hematology and clotting parameters of ratsa. Terminal sacrifice
RBC HGB HCT MCHC Reticulocytes Platelets Neutrophils Lymphocytes
Females
Males
#M (6%)
"H (6%) "H (7%)
#M (6%) "M, H (3%)
Recovery sacrifice Females
Males #H (7%) #H (7%) #H (7%)
"M (3%) #H (48%)
"H (58%) "H (34%)
Rats – Organ weight changes were generally unremarkable except for changes in kidney and lung weights. Treatmentrelated statistically significant increases in absolute kidney weights were observed in mid level (12% over control) and high level (18%) females and low level (10%), mid level (14%), and high level (19%) males. Treatment-related statistically significant increases in absolute lung weights were observed in females from all exposure groups [low level (16%), mid level (22%) and high level (65%) levels.]. Male changes were similar in scope and magnitude [mid level (26%) and high level (60%).]. Both kidney and lung weights returned to control values after the recovery period. Dogs – There were no exposure-related increases or decreases in organ weights in dogs. Histopathology By far the dominant histopathological effects seen in these studies were in the kidney and respiratory tract. These effects are detailed below. Respiratory tract histopathology
Arrows indicate direction of response [increase (") or decreases (#)]. Letters indicate the exposure groups differing significantly (p50.05 for difference between exposure groups and control) from controls (L ¼ low, M ¼ mid, H ¼ high). Values in parentheses are the largest percentage differences between exposed and control group means or medians, and immediately follow the indicator for the group having the greatest difference.
Table 6. Significant effects of exposure on clinical chemistry parameters of ratsa.
a
Organ weights
"M, H (73%) #H (38%)
a
NA-S CL-S PHOS CA K-S BUN CRE-S BUN/CRE GLU CHOL ALP ALT TP ALB GLOB AG-RATIO
and CRE-S), see below. There were no statistical difference from control values in coagulation parameters. Dogs – No gentamicin exposure-related responses were observed in hematology, coagulation and clinical chemistry parameters.
Females
Males
"H (2%)
"H (3%) "M, H (3%) #M (12%) #M, H (6%) #L, M (23%) "M, H (38%) "M, H (33%)
"H (25%) #H (6%) "H (64%) "M (17%) "H (45%) "M (27%)
"H (44%) #H (24%) "H (150%) #M, H (13%) #M, H (10%) #L, M, H (17%) "M, H (12%)
#M, H (9%) #L, M, H (13%) #M (7%)
Arrows indicate direction of response [increase (") or decreases (#)]. Letters indicate the exposure groups differing significantly (p50.05 for difference between exposure groups and control) from controls (L ¼ low, M ¼ mid, H ¼ high). Values in parentheses are the largest percentage differences between exposed and control group means or medians, and immediately follow the indicator for the group having the greatest difference. No changes were observed after the recovery period.
Micrographs of lung histopathology are shown in Figure 1 and tabular findings are shown in Tables 7 and 8. Rats – Alveolar histiocytosis and chronic interstitial inflammation were seen in all groups exposed to inhaled gentamicin in a dose-related manner. Findings ranged from near minimal at the low level to generally mild at the mid level and mildmoderate at the high level. Bronchiolar epithelial hyperplasia was seen starting at the mid exposure level and more prominently at the high level. Alveolar epithelial hyperplasia was characterized by proliferation of alveolar type II cells in areas of inflammation and was also seen starting at the mid level with greater severity at the high level. Effects in both genders were similar. Other effects seen in most gentamicin-exposed rats were dose-related mean minimal to mild nasal olfactory epithelial necrosis and minimal to moderate squamous metaplasia of laryngeal epithelium. The laryngeal lesions are a rat-specific response which can occur with any virtually inhaled material (Burger et al., 1989; Lewis, 1991). Dogs – There were no treatment related effects in the lungs of dogs. Treatment-related gross necropsy observations were limited to a pale thickened area on the lumen surface of the larynx of one high level male necropsied at the end of the exposure. Microscopically, the only treatment-related finding in the respiratory tract was mild subacute inflammation in the larynx of the same dog. The laryngeal microscopic lesion was further characterized by discontinuities in the pattern of submucosal connective tissue and muscle layers of the larynx due to infiltrates of macrophages and degenerative changes in skeletal muscle fibers in the wall of the larynx.
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Figure 1. Lung histopathology. Table 7. Rat – lung histopathology findings. Control Males (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar epithelium Hyperplasia, bronchiolar epithelium Inflammation, interstitial, chronic Females (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar epithelium Hyperplasia, bronchiolar epithelium Inflammation, interstitial, chronic
Low
Mid
High
(10) 7 (1.1)* – – –
10 3 2 9
(10) (2.0)* (1.0)* (1.5)* (1.0)*
10 9 9 10
(10) (2.1)* (1.9)* (1.6)* (2.0)*
10 10 10 10
(10) (3.0)* (2.5)* (2.6)* (3.3)*
(10) 8 (1.0)* – – –
(10) 10 (2.0)* – – 4 (1.0)*
10 10 9 10
(10) (2.0)* (1.8)* (1.4)* (2.0)*
10 10 10 10
(10) (3.2)* (2.7)* (2.3)* (3.1)*
()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe. Table 8. Dog – lung histopathology findings.
Males (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar or bronchiolar epithelium Infiltrate, lymphocytic, perivascular Inflammation, interstitial, chronic Females (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar or bronchiolar epithelium Infiltrate, lymphocytic, perivascular Inflammation, interstitial, chronic
Control
Low
Mid
High
(6) – – 5 (1.0) * 4 (1.5) *
(6) – – 1 (1.0)* –
(6) – – 1 (1.0)* 2 (1.0)*
(6) 1 (1.0)* – 3 (1.0)* 4(1.25)*
(6) – – 3 (1.0) * –
(6) 1 (1.0) * – – 1 (1.0)*
(6) – – 5 (1.0)* –
(6) – – 1 (1.0)* 1 (1.0)*
()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe.
Kidney histopathology Rats – Kidney tubular necrosis was present to a minimal degree in low level male rats and a few low level female rats at the end of the 14-day exposure period. Average severity increased to moderate in the high dose level at the end of the exposure period. There were regenerative renal tubular
changes observed in the male recovery high level rats; however, no treatment-related changes were observed in the female high level recovery rats. These findings are summarized in Table 9. Dogs – Kidney effects were limited to the high dose group only. Minimal to mild multifocal tubular regeneration occurred in the kidneys of all male dogs (two per group) in
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the high recovery group. This observation was considered as a treatment-related effect with delayed onset since that effect was not observed in the kidneys of dogs necropsied at the end of the exposure period. Table 9. Rat – kidney histopathology findings. Males
Control
Low
Mid
High
(No. examined) Renal tubule necrosis Females (No. examined) Renal tubule necrosis
(10) – Control (10)
(10) 9 (1.0)* Low (10) 2 (1.0)*
(10) 10 (2.3)* Mid (10) 10 (2.3)*
(10) 10 (2.7)* High (10) 10 (2.7)*
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()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe.
Toxicokinetics Serum, lung and kidney concentration–time course responses were comparable between genders in rats and dogs, respectively, after acute and repeated gentamicin exposures. Serum kinetics are shown in Figure 2 for rats and Figure 3 for dogs on the first and last day of the 14-day exposure. Genderpooled serum, lung and kidney parameters for each species are presented in Tables 10 and 11. Similar serum kinetics were observed at 6 and 8 mg/kg deposited doses in rats and dogs, which are highlighted (see Table 10). Low serum levels, many below quantifiable levels, did not allow for appropriate assessment of gentamicin exposure Area Under the Curve (AUC) and half-life (T1/2) in the single (1 day) rat low exposure level (6 mg/kg), as well as the single and repeat (14 days) exposure low (1 mg/kg) and single day mid level (3 mg/kg) in dogs. However, the 6 and
Figure 2. Mean serum concentrations (þSE) of gentamicin in rats on the first and last day of inhalation exposure.
Figure 3. Mean serum concentrations (þSE) of gentamicin in dogs on the first and last day of inhalation exposure.
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Table 10. Toxicokinetic parameters. Rat Exposure group
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1st Exposure Day mg/kg IHDDa Serum Cmax (mg/mL) AUC (mg h/mL) T1/2 (h) Lung Cmax (mg/g) AUC (mg h/g) T1/2 (h) Kidney Cmax (mg/g) AUC (mg h/g) T1/2 (h) Day 14 Exposure Serum Pre-exposure (mg/ml) Cmax (mg/mL) AUC (mg h/mL) T1/2 (h) Lung Pre-exposure (mg/g) Cmax (mg/g) AUC (mg h/g) T1/2 (h) Kidney Pre-exposure (mg/g) Cmax (mg/g) AUC (mg h/g) T1/2 (h)
Dog
Low
Mid
High
Low
Midb
High
6 3.15 ND ND 840 5080 8.27 67.8 1400 ND
17 10.6 58.1 3.83 2390 13 200 9.19 142 2990 ND
34 19.8 70.8 4.16 4230 16 400 14.1 269 5420 ND
1 0.627 ND ND
3 0.993 ND ND
8 3.81 12.6 3.99
0.0371c 3.34 14.2 7.44 274 682 9570 ND 189 236 5070 ND
0.126c 8.74 37.3 5.45 411 1220 17 700 ND 350 505 10 800 ND
0.772c 29 91 6.84 393 1060 14 700 ND 384 534 11 200 ND
ND 0.305 ND ND
0.036 1.65 5.3 2.7
0.046 3.8 11.3 7.28
ND ¼ Non-discernible due to insufficient data in the terminal elimination phase. Values represent average of male and female with ‘‘0’’ used as baseline for males. a Inhaled Deposited Dose (IHDD). b n was 5 total (2 females and 3 males; no sample obtained from one female); pre-exposure serum concentrations only present in female animals. c n was 2/gender.
Table 11. Terminal exposure and recovery lung and kidney tissue levels. Lung and kidney tissue levels (mg/g) of gentamicin in rats and dogs Lung
Rat Low Mid High Dog Low Mid High
(6 mg/kg)
(8 mg/kg)
Kidney
First/24 h
Last/24 h
Recovery
First/24 h
Last 24 h
Recovery
75 149 241
375 797 591
NA NA 350
67.9 120.2 187
139 455 432
NA NA 95.5
NA NA NA
9.4 21.4 41.6
NA NA 39
NA NA NA
186 360 687
8 mg/kg dose-matched tissue maximum concentration (Cmax) levels were similar in both species. Further, dosematched Day 14 Cmax, T1/2 and AUC were comparable among species and mirrored the single-day dog data. Dose proportionality was observed in TK parameters at the higher dose levels achieved in single-day exposed rats and in repeat exposure rats and dogs. Very low pre-exposure levels of gentamicin were evident in the serum of repeat exposure rats and dogs prior to Day 14 exposures. TK parameters were only calculated in lung and kidney in terminally time-sacrificed rodents. Due to slow tissue clearance and tissue retention, T1/2 was only calculable from the lungs of rodents exposed for one day. Dose proportionality was evident in single-day exposure lung T1/2, Cmax and AUC;
NA NA 481
and kidney Cmax and AUC in both species. After repeated inhalation exposure, however, the kidney and lung tissue levels, Cmax and AUC were comparable, suggesting that a plateau had been reached. Lung and kidney tissue burdens of gentamicin assessed 24 h after the first exposure (rats only), the Day 14 exposure (rats and dogs), and at recovery (rats and dogs high exposure groups only) reflect mechanisms consistent with tissue retention and slow clearance. The levels in rat lungs and kidneys after 14 days of exposure were substantially higher than the Day 1 levels, suggesting an accumulation (see Figure 4). However, at 24 h after the 14-day exposure, dog lung levels were modest, suggesting effective clearance and lack of accumulation. This difference in rat and dog lung
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Figure 4. Lung and kidney levels of gentamicin in rats and dogs 24 h after end of 14-day exposure.
effects results in a nine-fold greater gentamicin lung tissue burden observed in 6 mg/kg rats versus 8 mg/kg dogs sacrificed 24 h after the last exposure despite the similar lung deposition (Table 11). The high levels found in both rat and dog kidneys 24 h after the 14-day exposure show substantial accumulation in both species for this tissue. However, at similar doses (6 mg/kg rats versus 8 mg/kg dogs), the accumulation levels in dogs are higher than those in rats. The tissue concentrations at recovery are consistent with long-term components of retention in both tissues for both species since levels were relatively high as compared to those observed at the end of the 14-day exposure period (see Table 11).
Discussion The systemic findings in these studies of inhaled gentamicin are consistent with observations in other studies of aminoglycoside antibiotics delivered by the injectable route (Zhanel et al., 2012). The kidney changes observed in the current studies are expected and completely consistent with the known actions of IV and IM gentamicin and other parenteral aminoglycoside antibiotics. Nephrotoxicity is one of the major adverse side effects of gentamicin both in humans (Gentamicin Sulfate Injection, 2011; Schentag et al., 1981) and animals (Gilbert et al., 1978). The observance of accumulation of gentamicin in rat kidney is consistent with observations made earlier by other investigators, who also
noted tissue damage along with accumulation (Luft & Kleit, 1974; Schentag et al., 1978). To avoid nephrotoxicity in patients, it is advised to avoid Cmax levels of greater than 12 mg/mL and trough blood concentrations of greater than 2 mg/mL (Gentamicin Sulfate Injection, 2011). It is usually only high serum concentrations above these levels that can occur in renally impaired patients or overdose situations that lead to kidney damage in patients. Observations of lung toxicity at high inhaled doses in rats have been made for other inhaled aminoglycosides, namely tobramycin in the summary basis of approval for NDA 50753 (FDA, 1997). The pattern of toxicity seen with inhaled tobramycin at high doses was generally similar to that seen with inhaled gentamicin. Chronic nephropathy was observed at a greater frequency in rats from the high dose group compared to control. There was hyperplasia of bronchiolar epithelium and infiltration of macrophages at the mid and high doses. Only macrophage infiltration was observed at the low dose and since it was an adaptive response, such as can be observed from inhaling any particles, the low dose was a NOAEL. However, it should be noted that the serum concentrations of tobramycin in the rats (3.7, 9.6 and 20.0 mg/ mL: low, mid and high dose groups, respectively), were much higher than what has been measured in humans after dosing via inhalation with mean values typically in the range of 1 mg/ mL or less (FDA, 1997; Newhouse et al., 2003). Blood levels in patients are also low after use of aerosolized gentamicin (Ilowite et al., 1987).
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In the current toxicology studies in rats, Cmax levels of gentamicin were also quite high with mean values of 3.1, 11.3 and 20.0 mg/mL in the low, mid and high dose groups, respectively. Differences in the two toxicology programs were that the gentamicin studies were performed at somewhat higher inhaled doses (39, 123 and 245 mg/kg for gentamicin and 12, 37 and 112 mg/kg for tobramycin) and a shorter duration than the tobramycin study (14 days versus 6 months). Also, a NOAEL was not identified in the gentamicin rat studies. No previous reported studies were found of inhaled gentamicin in dogs. However, there have been several reports of use of aerosolized gentamicin in another large species, namely humans (Baran et al., 1975; Crowther Labiris et al., 1999; Hall, 1989; Ilowite et al., 1987: Klastersky et al., 1972; Murray et al., 2010; Palmer et al., 1998; Prober et al., 2000; Twiss et al., 2005). These studies showed that aerosolized gentamicin was generally well tolerated, and there were varying degrees of clinical efficacy. Determining pulmonary deposited doses in these studies is fraught with difficulties, but some of the studies showing some clinical benefit were estimated to result in daily deposited doses of approximately 50 mg (Palmer et al., 1998). This dose of approximately 1 mg/kg is in the same range as the low dose in the current dog study. A striking feature of the current toxicity studies is the more profound lung effects noted in rats as compared to dogs. Some of these observations result from the fact that it was possible to achieve higher doses in the rat study as compared to the dog study. This occurred primarily because it was possible that longer exposures (up to 3 h) could be tolerated by rats in the nose-only exposure systems, whereas 1 h was the maximum time that the dogs could tolerate in a face mask exposure system. Also, it was possible to achieve somewhat higher exposure concentrations in the rat exposure chambers compared to the dog chambers. The net result was that inhaled doses of 37, 123 and 245 mg/kg were achieved in the rat study, whereas inhaled doses were 7, 14 and 41 mg/kg in the dogs. Notably, there was dose overlap between the low dose in rats and the high dose in dogs. It is also noteworthy that this was a clear evidence of accumulation of gentamicin in the lungs of rats and some toxicity even at the low dose, whereas this was not the case for dogs even at the high dose. The low dose group in the rat study and the high dose group did have similar inhaled doses of 37 and 41 mg/ kg, respectively and deposited lung doses of 6 and 8 mg/kg, respectively. At these similar doses, it was clear that there were lung effects in the rat study while there were no lung effects in the dog study. A further confirmation that deposited lung doses were similar is the fact that serum concentrations were also similar. Mean Cmax levels of 3.1 mg/mL were achieved in the rat low dose group as compared to 3.8 mg/mL in the dog high dose group. AUCs were 14 and 11.3 mg. h/mL in the rats and dogs, respectively. Because oral absorption is very low for gentamicin, these blood levels can be attributed primary to lung absorption. The results further reinforce the view that lung deposition was similar in the two species. In the present study, there was accumulation of gentamicin in rat lungs, whereas this was not a feature of the results in dog lungs. The accumulation of gentamicin even at the low
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dose, in this study, led to retained tissue levels in rats in the range of a few hundred mg/g. An accumulation of gentamicin in kidney tissues at these same levels gave rise to cell toxicity, and it appears that this is also the case for lung tissue and the reason for the adverse lung histopathology seen in rats.
Conclusions Adverse kidney effects were observed in both rats and dogs as have been observed previously in toxicology studies using injection administration. Adverse lung effects were seen in rats only. Rats are a more sensitive species for effects of inhaled gentamicin than dogs. The lowest rat deposited lung dose at which adverse effects were seen was comparable to or somewhat lower than the highest deposited dog lung dose, which showed no adverse effects. The relevance of these findings to possible clinical effects depends on a dose comparison. Data from Palmer et al. (1998) found that approximately 30% of an 80 mg nebulizer dose was delivered to the lungs of intubated patients, who were dosed three times per day. If this 72 mg total dose is deposited in 60 kg subjects, the deposited lung dose is 1.2 mg/kg/day. This dose was well tolerated in the Palmer study and suggests that, if effects in man are like those in dogs, deposited lung doses up to 8 mg/kg could be acceptable. Effects in humans are more likely to be similar to those in dogs than rats, given the similarities in respiratory tract anatomy of dogs to humans (Tyler & Julian, 1992); the large amount of clinical experience with inhaled gentamicin in humans; and the fact that generally toxicology effects in large species are more predictive of effects in humans than rodents (Tomazewski, 2004). However, the effects in rats, though modest at the low deposited dose of 6 mg/kg and possibly due to rat sensitivity, are cautionary. Since it has been shown that 1.2 mg/kg deposited lung dose of gentamicin has been acceptable in humans, clinical studies might start at these levels and then escalate higher with careful monitoring if higher doses are needed for therapeutic efficacy. Publication of additional studies with other antibiotics would be useful to determine if this is a general effect of increased rodent sensitivity to inhaled antibiotics delivered at high doses.
Acknowledgements Thanks to the aerosol technology group at LRRI for excellent aerosol generation and characterization.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by Nektar Therapeutics with rights subsequently transferred to Novartis. R. Wolff and H. Conway were responsible for the study as employees of Nektar at the time. Matthew D. Reed supervised the study conducted at LRRI.
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Naesens R, Vlieghe E, Verbrugghe W, et al. (2011). A retrospective observational study on the efficacy of colistin by inhalation as compared to parenteral administration for the treatment of nosocomial pneumonia associated with multidrug-resistant Pseudomonas aeruginosa. BMC Infect Dis 11:317. National Institute for Public Health and the Environment, RIVM. (2002). Multiple path particle dosimetry model (MPPD v 1.0): a model for human and rat airway particle dosimetry. RIVA Report 65001(0030). Bilthoven, The Netherlands. Newhouse MT, Hirst PH, Duddu SP, et al. (2003). Inhalation of a dry powder tobramycin PulmoSphere formulation in healthy volunteers. Chest 124:360–6. Palmer LB, Smaldone GC, Simon SR, et al. (1998). Aerosolized antibiotics in mechanically ventilated patients: delivery and response. Critical Care Med 26:31–9. Prober CG, Walson PD, Jones J; the Committee on Infectious Diseases and Committee on Drugs. (2000). Precautions regarding the use of aerosolized antibiotics. Pediatrics 106:89–95. Rotstein C, Evans G, Born A, et al. (2008). Clinical practice guidelines for hospital-acquired pneumonia and ventilator-associated pneumonia in adults. Can J Infect Dis Med Microbiol 19:19–53. Roy CJ, Sivasubramani SK, Dutta NK, et al. (2012). Aerosolized gentamicin reduces the burden of tuberculosis in a murine model. Antimicrob Agents Chemother 56:883–6. Schentag JJ, Cumbo TJ, Jusko WJ, Plant ME. (1978). Gentamicin tissue accumulation and nephrotoxic reactions. J Am Med Assoc 240: 2067–9. Schentag JJ, Plaut ME, Cerra FB. (1981). Comparative nephrotoxicity of gentamicin and tobramycin: pharmacokinetic and clinical studies in 201 patients. Antimicro Agents Chemother 19:859–66. Schlesinger RB. (1985). Comparative deposition of inhaled aerosols in experimental animals and humans: a review. J Toxicol Environ Health 15:197–214. Schuster A, Haliburn C, Do¨ring G, Goldman MH. (2013). Safety, efficacy and convenience of colistimethate sodium dry powder for inhalation (Colobreathe DPI) in patients with cystic fibrosis: a randomised study. Thorax 68:344–50. Talton JD, Eppler B, Kirk JF, et al. (2010). Inhaled gentamicin: making a dry powder formulation to satisfy a clinical need. Proc Resp Drug Del 2:63–72. Tomazewski JE. (2004). Multi-species toxicology approaches for oncology drugs: the US perspective. Eur J Cancer 40:907–13. Twiss J, Byrnes C, Johnson R, Holland D. (2005). Nebulised gentamicin-suitable for childhood bronchiectasis. Int J Pharm 13: 295:113–19. Tyler WS, Julian MD. (1992). Gross and subgross anatomy of lungs, pleura, connective tissue septa, distal airways, and structural units. In: Parent RA. (ed.) Treatise on pulmonary biology: comparative biology of the normal lung. Boca Raton: CRC Press, 37–48. Weinstein MJ, Luedemann GM, Oden EM, et al. (1963). Gentamicin, a new antibiotic complex from micromonospora. J Med Chem 6: 463–4. Zhanel GG, Lawson CD, Zelenitsky S, et al. (2012). Comparison of the next-generation aminoglycoside plazomicin to gentamicin, tobramycin and amikacin. Expert Rev Anti Infect Ther 10:459–73.
RESEARCH ARTICLE
Comparison of inhalation toxicity studies of gentamicin in rats and dogs Heather Conway1, Kelly J. Dix2, Jacob D. McDonald2, Rodney A. Miller3, Henry G. Wall3, Ronald K. Wolff4, and Matthew D. Reed2 Merck & Co. Inc., Summit, NJ, USA, 2Lovelace Respiratory Research Institute, Albuquerque, NM, USA, 3Experimental Pathology Laboratories, Sterling, VA, USA, and 4Safety Consulting Inc., Carbondale, CO, USA
Abstract
Keywords
Nebulized gentamicin solution was administered to rats (nose-only) and dogs (face mask) for 14 days with a 14-day recovery period. Control groups of each were exposed to saline aerosols. Mean estimated inhaled lung doses of gentamicin were 39, 123 and 245 mg/kg for rats (deposited doses 6, 17 and 34 mg/kg) over 30, 90 and 180 min, respectively. Since dogs do not tolerate exposures as long as rats, inhaled lung doses were limited to 7, 14 and 41 mg/kg (deposited doses of 1, 3 and 8 mg/kg) over 15, 30 and 60 min. Comparable doses were achieved at the low dose for rats and high dose for dogs. Serum AUCs (14 2 mg/mL*h (mean SD) at 6 mg/kg in rats and 11 7 mg/mL*h at 8 mg/kg in dogs) showed comparable exposure between the two, implying similar absorbed doses and confirming similar deposited lung doses. Rat exposures resulted in dose-related lung pathology (including low dose) manifested as upper respiratory tract irritant reactions with alveolar histiocytosis, inflammation, airway epithelial metaplasia and lymphomegaly in lung tissue. This was associated with high lung tissue gentamicin levels 24 h post-dose on Day 14 (375 33 mg/g at deposited dose of 6 mg/kg). Dose-related kidney tubular necrosis (a well-known toxicity of parenteral gentamicin) was observed, but no test-article related effects on lung histopathology in dogs (even at highest deposited dose of 8 mg/kg) and low levels of lung tissue gentamicin (42 11 mg/g) 24 h post-dose on Day 14.
Gentamicin, histiocytosis, histopathology, infectious disease, inflammation, inhaled, inhaled antibiotics, lymphomegaly, metaplasia, nebulized, respiratory
Introduction A significant body of information currently exists on the use of aerosolized antibiotics to treat respiratory disease (Gellar et al., 2011; Høiby, 2011; Naesens et al., 2011; Schuster et al., 2013). Tobramycin inhalation solution and dry powder are approved US products for management of infections in cystic fibrosis patients. Hall (1989) reviewed the use of aerosolized antibiotics and concluded that inhaled antibiotics may offer advantages over systemic or orally administered products by (1) treating at high local concentrations at the site of the infection and (2) limiting systemic side effects. Additionally, the American Academy of Pediatrics (Prober et al., 2000) noted in a review that there has been extensive use of inhaled antibiotics formulated for other purposes over several decades. Further, dry powder formulations of gentamicin have been studied in rodents and non-human primates for use against pneumonic conditions resulting from the gram negative biological threat agents tularemia and plague (Talton et al., 2010) and tuberculosis (Roy et al., 2012). Gentamicin is a broad spectrum, generic aminoglycoside antibiotic currently formulated for parenteral and topical administration and is indicated for some cases of severe
Address for correspondence: Dr Matthew D. Reed, LRRI, Preclinical Drug Development, 2425 Ridgecrest Dr SE, Albuquerque, NM 87108, USA. Tel: 505-348-9451. Fax: 505-348-4983. E-mail: [email protected]
History Received 3 July 2013 Revised 5 September 2013 Accepted 6 September 2013 Published online 20 November 2013
pneumonia (Rotstein et al., 2008). Gentamicin consists of a mixture of three active components produced by the gram positive species Micromonosproa and isolated by Roche in the early 1960’s (Weinstein et al., 1963). Susceptible bacterial species include pseudomonas, staphylococcus, tularemia and plague among others. Although extremely effective when administered for the correct indication, gentamicin has rate limiting nephro- and ototoxicity associated with prolonged systemic administration (Zhanel et al., 2012). There have been several clinical investigations related to aerosolized treatment using gentamicin (Crowther Labiris et al., 1999; Murray et al., 2010; Palmer et al., 1998; Twiss et al., 2005). Inhaled gentamicin administered using 80 mg in the nebulizer has been shown to result in relatively high levels in the sputum of intubated patients; these levels have been generally substantially higher than the minimal inhibitory concentrations of sensitive gram negative organisms with low or undetectable serum concentrations (Palmer et al., 1998). This aerosol delivery system resulted in serum concentrations indicating low potential for systemic toxicity, since they were generally undetectable, and thus they were substantially below the ‘‘cautionary limit’’ for systemic toxicity of 12 mg/mL (Gentamicin Sulfate Injection, 2011). There were additional indications of efficacy such as reduced sputum volume and eradication of pathogens, and minimal safety issues were identified. These and other data suggest the possibility of additional preclinical and clinical investigation
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of inhaled gentamicin to aid in the treatment of respiratory infectious by increasing local lung concentrations while limiting systemic exposure and potential side effects. The purpose of the current program was to conduct 14-day inhalation toxicology studies in rats and dogs with nebulized, preservative-free gentamicin solutions under Good Laboratory Practice regulations. The studies were performed at up to maximal feasible exposure concentrations to support safe use in clinical studies and potential FDA licensure.
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prepared at 200 mg/mL concentrations of gentamicin activity and Aerotech II jet nebulizers (CIS-US INC., Bedford, MA) were used to generate the inhalation atmosphere. Target aerosol concentrations were 1.5–2 mg/L gentamicin and target particle size was 2-3 mm MMAD. Control exposures were nebulized from solutions of 0.9% saline. Stability of gentamicin was confirmed in nebulizer solutions, stock solutions (used to create linearity standards for quantitation) and spiked filters utilizing a qualified content method. Rat exposure system
Methods
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Animals All studies were performed under Institutional Animal Care and Use Committee approval and Good Laboratory Practice regulations. A total of 116 male and 116 female CD rats, approximately 9–10 weeks old at the beginning of the 14-day exposure, were used on study (Charles River Laboratories, Raleigh, NC). A total of 16 male and 16 female beagle dogs (Ridglan Farms, Mt. Horeb, WI) approximately 6–7 months of age were used at the beginning of the 14-day exposure. The animal allocations to the control, low, mid and high exposure groups, as well as designations for toxicokinetics (TK), are shown in Table 1 for rats and Table 2 for dogs. All animals were conditioned to inhalation exposures conditions and apparatuses on at least three occasions prior to placement on study to ease environmental stress effects. A single group of saline control exposure rats and dogs were used for each study and exposed for a duration equivalent to the high exposure group to conserve animals and in accordance with US FDA recommendations (proprietary pre-Investigational New Drug Applications meeting conducted prior to the study start). Aerosol generation Preservative-free solutions of gentamicin sulfate (Fujian Fukang Pharmaceutical Co. Ltd., Fuzhou, China) were
Nose-only exposures were conducted in a ‘‘flow-past-type’’ cylindrical inhalation chamber (Cannon et al., 1983) placed inside a steel-framed Plexiglas secondary containment box. The chamber contained 48 rodent ports; each was compatible with a single nose-only exposure tube, aerosol concentration sampling device (e.g. filter) or oxygen monitor. The total air flow through the exposure system was balanced to achieve individual rodent port flows of 500 mL/ min (port flow approximated based on total chamber flow). Measured flows included sample flow rate, nebulizer flow rate, dilution flow rate and chamber exhaust flow. The exposure chamber had a slightly higher exhaust flow rate than inlet flow rate. Dog exposure system The exposure system consisted of a single, cylindrical, Plexiglas inhalation chamber (volume of 23.7 L, 14.61-cm radius, 35.56-cm height). The chamber was supplied with two Aerotech II nebulizers operated at 30 psi (gentamicin low and mid) and 40 psi (gentamicin high). Nebulized test article and nebulizer air supply were diluted with 10 L/min HEPA-filtered dilution air. The flow through the system was 36 L/min. Measured flows included sample flow rate, nebulizer flow rate, dilution flow rate and chamber exhaust flow. The exposure chamber had a slightly higher exhaust flow rate than inlet flow rate. Six equally spaced corrugated plastic exposure lines of 1 m were connected from the
Table 1. Number of rats used in each of the experimental group assignments. Main study Group Saline control Low (gentamicin) Mid (gentamicin) High (gentamicin)
TKa – Day 1
Recovery
TKa – Day 14
Exposure duration (h)
Male
Female
Male
Female
Male
Female
Male
Female
3 0.5 1.5 3
10 10 10 10
10 10 10 10
5 NA NA 5
5 NA NA 5
3 9 9 9
3 9 9 9
3 9 9 9
3 9 9 9
a
Serum, lung and kidney samples taken from three rats per gender at pre-dose and 3, 6 and 24 h post-dose on Days 1 and 14.
Table 2. Number of dogs in each of the experimental group assignments. Main study Group Saline control Low (gentamicin) Mid (gentamicin) High (gentamicin) a
Exposure duration (h) 1 0.25 0.5 1
Male 3 3 3 3
Female 3 3 3 3
Recovery Male 2 NA NA 2
TK – Day 1
TK – Day 14
Female
Male
Female
Male
Female
2 NA NA 2
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
Serum TK samples taken from Main Study and Recovery dogs pre-dose, as well as immediately (0) and 3, 6, 12 and 24 h post-dose on Days 1 and 14. Lung and kidney samples taken 24 h post-Day14 dosing.
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chamber to a plastic ‘‘Y’’. The ‘‘Y’’ is additionally connected at one end to a single inlet neoprene mask (mask covers the snout and is affixed to the dog by velcro straps) and an exhaust line connected to the institutional exhaust system on the other. Each exhaust line flows through an inline filter followed by a critical orifice designed to allow not more than 6 L/min air flow from the chamber to institutional exhaust. The dog was allowed to pull (breathe) test article from the Y. Since five to six dogs were exposed to control or test article per exposure interval, one or two lines were plugged at the Y. An additional sampling probe inserted through a reference sampling port pulls 0.5 L/min from the center of the chamber in order to monitor chamber concentrations during exposures. Exposure atmosphere monitoring The test article was sampled from the chamber onto Pallflex (Pall Corp., Port Washington, NY) filters (for concentration measurements) or Mercer-style, seven-stage cascade impactor (InTox Products, Moriarty, NM) substrates (for particle sizing). Aerosol concentration was determined by taking filter samples directly from the chamber. For each exposure day, exposure filters were extracted with water and analyzed chemically (high-pressure liquid chromatography with ultraviolet absorptiondetection [Agilent Technologies, Santa Clara, CA]) to monitor and define gentamicin aerosol concentrations during exposures. Chromatography was performed on a Phenomenex Luna 3 mL C18 (2), 75 4.60 mm (Phenomenex, Torrance, CA). A flow rate of 1.0 mL/min and an injection volume of 50 mL were used for each sample. Mobile phase A was 2 mM hexane sulfonate, 20 mM sodium sulfate, 0.005% acetic acid in water. Mobile phase B was 100% methanol. A linear mobile phase composition gradient changed from 95:5 A:B at time ¼ 0 min to 5:95 A:B at time ¼ 6 min. After 6 min, the mobile phase was returned to initial conditions and equilibrated for 2 min for a total sample run time of approximately 8 min. Post-column, the effluent was mixed with a reaction solution of 0.33 mg/mL o-Pthaldialdehyde, 0.66 mg/mL mercaptoethanol and 0.133 M borate buffer (pH ¼ 10.4). After derivitazation, the gentamicin mixture was detected by UV at ‘A ¼ 330 nm. Aerosol particle size measurements were made three times throughout the duration of the study (approximately at the beginning, middle and end of the exposure period) by Mercer-style impactor stage assessments. Chemical detection was the definitive measurement for particle size. Dose determination Gentamicin doses for rats and dogs were determined using the equations and assumptions listed below: Inhaled Dose ðmgÞ ¼ Empirically determined aerosol concentration ðmg=LÞ published ðdogsÞ or allometrically ðratsÞ converted average minute volumes exposure time ðminÞ:
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Inhaled Deposited Dose ðmgÞ ¼ Inhaled Dose published ðdogsÞ or mathematically processed ðratÞ pulmonary deposition fraction: Times of exposure and aerosol concentrations were determined empirically. Inhaled and Inhaled Deposited Doses were divided by approximated average body weights for rats (300 g) and beagle dogs (8 kg) to determine aerosol dosing on a mg/kg basis. Minute volume for rats (0.188 L/min for a 300 g rat) was determined by the allometric conversion method of Bide et al. (2000). Minute volume for beagle dogs (4 L/min) was determined using empirically generated data as measured by Mauderly (1972, 1979). Pulmonary deposition for rats (12.7%) was determined based on empirical particle size data and derivations using the Multiple Path Particle Dosimetry model (Anjilvel & Asgharian, 1995; RIVM, 2002). Pulmonary deposition for dogs (20%) was determined based on empirical particle size data and deposition fraction as determined by Schlesinger (1985). Biological endpoints The following list summarizes the biological endpoints. (a) Clinical observations were made daily. (b) Body weights were measured weekly and prior to necropsy (fasted). (c) Hematology, coagulation and clinical chemistry parameters were measured using standard panels at the end of the 14-day exposure period for rats and prior to treatment and at the end of the 14-day exposure period for dogs, as well as at the end of the recovery period for both rats and dogs. Blood was collected by syringe from the descending vena cava during necropsy (rats) or by jugular stick (dogs) prior to euthanasia. Standard tubes for clotting (serum chemistry, room temperature), hematology (calcium EDTA, on ice), or clotting parameters (sodium citrate on ice) were used for collections. Clinical chemistry analyses were performed within 24 h of collection utilizing Hitachi 911 (Roche Diagnostics, Indianapolis, IN). Hematology samples were analyzed within 24 h of collection utilizing an AdviaÔ 120 (Bayer Corporation, Tarrytown, NY) and clotting samples were analyzed within 24 h of collection utilizing a Amelung KC 4AÔ Micro (Trinity Biotech, St. Louis, MO). (d) Toxicokinetics – Concentrations of gentamicin C1, C1a, C2 and C2a were measured using validated LC-MS/MS methods in serum, kidney and lung according to the method of Ishii et al. (2008) with proprietary modification. Blood samples were collected at sacrifice (rat) or by jugular vein (dog) and processed to serum. Tissue samples were collected at necropsy. Samples were frozen at 80 C until analyses. Total gentamicin concentrations were expressed as a sum of these subcomponents, and the sum was used in all toxicokinetic analyses, WinNonlin Enterprise Version 5.0.1 software (Pharsight Inc., Mountain View, CA). Rats – Serum, lung, and kidney samples were taken predose, end of dosing (0 h), and 3, 6 and 24 h after the end of dosing on Days 1 and 14.
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Dogs – Serum samples were taken on Days 1 and 14 predose, end of dosing (0 h), and 3, 6 and 24 h after the end of dosing. Lung and kidney samples were taken at necropsy, 24 h after the last dosing on Day 14. Limit of quantitation (LOQ) for total gentamicin was 7.4 ng/mL for serum samples and 750 ng/g for lung and kidney samples. (e) Necropsy and histopathology – Animals were euthanized by overdose of barbiturate-based sedative (dogs and rats) and exsanguination (dogs only). A full standard list of tissues was taken (Bregman et al., 2003) and fixed in 10% neutral buffered formalin. Tissue sections were cut 4–6 mm thick, mounted on standard glass slides, stained with hematoxylin and eosin (H&E), and read by a board certified pathologist. Gradable findings were recorded on a 1–5 scale where, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked and 5 ¼ severe. Non-gradable findings were recorded as ‘‘not present’’ (NP). (f) Statistical analyses – For endpoints measured only at scheduled sacrifice and for body weight measurements, analyses were performed utilizing standard parameters in the Path-Tox data acquisition system (Path-Tox 4.2.2; Xybion, Cedar Knolls, NJ). In brief, data sets consistent with the number of animals in each exposure group were tested for homogeneity with Bartlett’s test. For homogenous data, Dunnett’s t test was performed, and for nonhomogenous data, the modified t test was performed to determine statistical difference from control values. For clinical chemistry and hematology parameter data sets (consistent with the number of animals in each exposure group) a one-way analysis of variance (ANOVA) was used and when a significant treatment effect was noted (p50.05), Dunnett’s multiple comparison test was performed to assess differences between treated and control groups. When data exhibited either skewed distributions or variability scaled linearly with mean values, a logarithmic transformation was applied prior to the analysis. Statistical calculations were performed using the SASÕ software system, Version 9.1 (Cary, NC). All reported p values were two-sided.
For all analyses, multivariate and univariate, statistical significance was considered as p 0.05.
Results Test article The stability of gentamicin, assessed by quantitatively measuring content prior to and after storage, was within 15% of an initial analysis under all conditions. Analysis of the peak profile in filter extracts from filters collected for test atmosphere monitoring showed that the composition of the mixture did not change (degrade) over the course of an exposure period (3 h). This indicated that the test article composition was stable under the conditions employed for the study. Exposure and dose data The mean exposure data are summarized in Tables 3 and 4 for rats and dogs, respectively. Collected data indicated that the exposures were conducted over appropriate doses ranging from near the clinical range at the lowest level and at higher multiples in the mid- and high-dose ranges. Exposure concentrations were consistent over the 14-day exposures with coefficients of variation of 10%–12% for the rat exposures and 10%–15% for the dog exposures. The particle size range of 1.9–2.5 mm MMAD for both rats and dogs was highly respirable and appropriate for comparison with clinical exposures (Schlesinger, 1985). Body weight Rats – Body weight changes were consistent with short-term inhalation toxicology studies and were generally unremarkable. No rodent mean weight gain was observed in any exposure group at the end of exposures. The only exposurerelated body weight decreases were observed in high level females at the end of exposure. Weights tracked lower than that of control animals reaching statistical significance just prior to sacrifice (9% lower than control). Dogs – There were no apparent changes in body weight in gentamicin exposed dogs as compared to controls.
Table 3. Mean exposure data for rats with inhaled doses of 39, 123 and 245 mg/kg in the low, mid and high dose groups, respectively.
Rat group Saline control Low Mid High
Exposure duration (h)
Exposure concentration (mg/L)
Particle size MMAD (GSD)
Estimated inhaled dose (mg/kg)
Estimated lung dose (mg/kg)
3.0 0.5 1.5 3.0
– 2.09 2.18 2.17
– 2.1 mm (1.8) 1.9 mm (1.9) 2.5 mm (1.7)
– 39 123 245
– 6 17 34
Table 4. Mean exposure data for dogs at inhaled doses of 7, 14 and 41 mg/kg at the low, mid and high doses.
Dog group Saline control Low Mid High
717
Exposure duration (hr)
Exposure concentration (mg/L)
Particle size MMAD (GSD)
Estimated inhaled dose (mg/kg)
Estimated lung dose (mg/kg)
1.0 0.25 0.5 1.0
– 1.19 1.17 1.72
– 2.5 mm (1.6) 1.9 mm (1.7) 2.1 mm (1.6)
– 7 14 41
– 1 3 8
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Hematology, coagulation, and clinical chemistry
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Rats – Hematology and clinical chemistry parameters were generally unremarkable among exposed animals. Multiple, mostly small statistically significant effects versus control were observed, Tables 5 and 6. Most changes either lacked gender agreement or lacked clear evidence of an exposure– response relationship. Although whole cell counts were unaffected by exposure, neutrophils were increased in the male mid level (69% over control values) and high level (73%) rats. This change may have been related to the pulmonary inflammation induced by gentamicin (see below). Overall, exposure-related responses in clinical chemistry parameters were likely indicative of effects of the gentamicin on the lung (inflammation indicated by the protein panel changes) and the kidney (protein panel, NA-S, K-S, CL-S, CA-S, PHOS, BUN Table 5. Significant effects of exposure on hematology and clotting parameters of ratsa. Terminal sacrifice
RBC HGB HCT MCHC Reticulocytes Platelets Neutrophils Lymphocytes
Females
Males
#M (6%)
"H (6%) "H (7%)
#M (6%) "M, H (3%)
Recovery sacrifice Females
Males #H (7%) #H (7%) #H (7%)
"M (3%) #H (48%)
"H (58%) "H (34%)
Rats – Organ weight changes were generally unremarkable except for changes in kidney and lung weights. Treatmentrelated statistically significant increases in absolute kidney weights were observed in mid level (12% over control) and high level (18%) females and low level (10%), mid level (14%), and high level (19%) males. Treatment-related statistically significant increases in absolute lung weights were observed in females from all exposure groups [low level (16%), mid level (22%) and high level (65%) levels.]. Male changes were similar in scope and magnitude [mid level (26%) and high level (60%).]. Both kidney and lung weights returned to control values after the recovery period. Dogs – There were no exposure-related increases or decreases in organ weights in dogs. Histopathology By far the dominant histopathological effects seen in these studies were in the kidney and respiratory tract. These effects are detailed below. Respiratory tract histopathology
Arrows indicate direction of response [increase (") or decreases (#)]. Letters indicate the exposure groups differing significantly (p50.05 for difference between exposure groups and control) from controls (L ¼ low, M ¼ mid, H ¼ high). Values in parentheses are the largest percentage differences between exposed and control group means or medians, and immediately follow the indicator for the group having the greatest difference.
Table 6. Significant effects of exposure on clinical chemistry parameters of ratsa.
a
Organ weights
"M, H (73%) #H (38%)
a
NA-S CL-S PHOS CA K-S BUN CRE-S BUN/CRE GLU CHOL ALP ALT TP ALB GLOB AG-RATIO
and CRE-S), see below. There were no statistical difference from control values in coagulation parameters. Dogs – No gentamicin exposure-related responses were observed in hematology, coagulation and clinical chemistry parameters.
Females
Males
"H (2%)
"H (3%) "M, H (3%) #M (12%) #M, H (6%) #L, M (23%) "M, H (38%) "M, H (33%)
"H (25%) #H (6%) "H (64%) "M (17%) "H (45%) "M (27%)
"H (44%) #H (24%) "H (150%) #M, H (13%) #M, H (10%) #L, M, H (17%) "M, H (12%)
#M, H (9%) #L, M, H (13%) #M (7%)
Arrows indicate direction of response [increase (") or decreases (#)]. Letters indicate the exposure groups differing significantly (p50.05 for difference between exposure groups and control) from controls (L ¼ low, M ¼ mid, H ¼ high). Values in parentheses are the largest percentage differences between exposed and control group means or medians, and immediately follow the indicator for the group having the greatest difference. No changes were observed after the recovery period.
Micrographs of lung histopathology are shown in Figure 1 and tabular findings are shown in Tables 7 and 8. Rats – Alveolar histiocytosis and chronic interstitial inflammation were seen in all groups exposed to inhaled gentamicin in a dose-related manner. Findings ranged from near minimal at the low level to generally mild at the mid level and mildmoderate at the high level. Bronchiolar epithelial hyperplasia was seen starting at the mid exposure level and more prominently at the high level. Alveolar epithelial hyperplasia was characterized by proliferation of alveolar type II cells in areas of inflammation and was also seen starting at the mid level with greater severity at the high level. Effects in both genders were similar. Other effects seen in most gentamicin-exposed rats were dose-related mean minimal to mild nasal olfactory epithelial necrosis and minimal to moderate squamous metaplasia of laryngeal epithelium. The laryngeal lesions are a rat-specific response which can occur with any virtually inhaled material (Burger et al., 1989; Lewis, 1991). Dogs – There were no treatment related effects in the lungs of dogs. Treatment-related gross necropsy observations were limited to a pale thickened area on the lumen surface of the larynx of one high level male necropsied at the end of the exposure. Microscopically, the only treatment-related finding in the respiratory tract was mild subacute inflammation in the larynx of the same dog. The laryngeal microscopic lesion was further characterized by discontinuities in the pattern of submucosal connective tissue and muscle layers of the larynx due to infiltrates of macrophages and degenerative changes in skeletal muscle fibers in the wall of the larynx.
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Figure 1. Lung histopathology. Table 7. Rat – lung histopathology findings. Control Males (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar epithelium Hyperplasia, bronchiolar epithelium Inflammation, interstitial, chronic Females (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar epithelium Hyperplasia, bronchiolar epithelium Inflammation, interstitial, chronic
Low
Mid
High
(10) 7 (1.1)* – – –
10 3 2 9
(10) (2.0)* (1.0)* (1.5)* (1.0)*
10 9 9 10
(10) (2.1)* (1.9)* (1.6)* (2.0)*
10 10 10 10
(10) (3.0)* (2.5)* (2.6)* (3.3)*
(10) 8 (1.0)* – – –
(10) 10 (2.0)* – – 4 (1.0)*
10 10 9 10
(10) (2.0)* (1.8)* (1.4)* (2.0)*
10 10 10 10
(10) (3.2)* (2.7)* (2.3)* (3.1)*
()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe. Table 8. Dog – lung histopathology findings.
Males (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar or bronchiolar epithelium Infiltrate, lymphocytic, perivascular Inflammation, interstitial, chronic Females (No. Examined) Alveolar histiocytosis Hyperplasia, alveolar or bronchiolar epithelium Infiltrate, lymphocytic, perivascular Inflammation, interstitial, chronic
Control
Low
Mid
High
(6) – – 5 (1.0) * 4 (1.5) *
(6) – – 1 (1.0)* –
(6) – – 1 (1.0)* 2 (1.0)*
(6) 1 (1.0)* – 3 (1.0)* 4(1.25)*
(6) – – 3 (1.0) * –
(6) 1 (1.0) * – – 1 (1.0)*
(6) – – 5 (1.0)* –
(6) – – 1 (1.0)* 1 (1.0)*
()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe.
Kidney histopathology Rats – Kidney tubular necrosis was present to a minimal degree in low level male rats and a few low level female rats at the end of the 14-day exposure period. Average severity increased to moderate in the high dose level at the end of the exposure period. There were regenerative renal tubular
changes observed in the male recovery high level rats; however, no treatment-related changes were observed in the female high level recovery rats. These findings are summarized in Table 9. Dogs – Kidney effects were limited to the high dose group only. Minimal to mild multifocal tubular regeneration occurred in the kidneys of all male dogs (two per group) in
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the high recovery group. This observation was considered as a treatment-related effect with delayed onset since that effect was not observed in the kidneys of dogs necropsied at the end of the exposure period. Table 9. Rat – kidney histopathology findings. Males
Control
Low
Mid
High
(No. examined) Renal tubule necrosis Females (No. examined) Renal tubule necrosis
(10) – Control (10)
(10) 9 (1.0)* Low (10) 2 (1.0)*
(10) 10 (2.3)* Mid (10) 10 (2.3)*
(10) 10 (2.7)* High (10) 10 (2.7)*
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()* – Average Severity; Severity Scale based on 0 ¼ no findings, 1 ¼ minimal, 2 ¼ mild, 3 ¼ moderate, 4 ¼ marked, 5 ¼ severe.
Toxicokinetics Serum, lung and kidney concentration–time course responses were comparable between genders in rats and dogs, respectively, after acute and repeated gentamicin exposures. Serum kinetics are shown in Figure 2 for rats and Figure 3 for dogs on the first and last day of the 14-day exposure. Genderpooled serum, lung and kidney parameters for each species are presented in Tables 10 and 11. Similar serum kinetics were observed at 6 and 8 mg/kg deposited doses in rats and dogs, which are highlighted (see Table 10). Low serum levels, many below quantifiable levels, did not allow for appropriate assessment of gentamicin exposure Area Under the Curve (AUC) and half-life (T1/2) in the single (1 day) rat low exposure level (6 mg/kg), as well as the single and repeat (14 days) exposure low (1 mg/kg) and single day mid level (3 mg/kg) in dogs. However, the 6 and
Figure 2. Mean serum concentrations (þSE) of gentamicin in rats on the first and last day of inhalation exposure.
Figure 3. Mean serum concentrations (þSE) of gentamicin in dogs on the first and last day of inhalation exposure.
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Table 10. Toxicokinetic parameters. Rat Exposure group
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1st Exposure Day mg/kg IHDDa Serum Cmax (mg/mL) AUC (mg h/mL) T1/2 (h) Lung Cmax (mg/g) AUC (mg h/g) T1/2 (h) Kidney Cmax (mg/g) AUC (mg h/g) T1/2 (h) Day 14 Exposure Serum Pre-exposure (mg/ml) Cmax (mg/mL) AUC (mg h/mL) T1/2 (h) Lung Pre-exposure (mg/g) Cmax (mg/g) AUC (mg h/g) T1/2 (h) Kidney Pre-exposure (mg/g) Cmax (mg/g) AUC (mg h/g) T1/2 (h)
Dog
Low
Mid
High
Low
Midb
High
6 3.15 ND ND 840 5080 8.27 67.8 1400 ND
17 10.6 58.1 3.83 2390 13 200 9.19 142 2990 ND
34 19.8 70.8 4.16 4230 16 400 14.1 269 5420 ND
1 0.627 ND ND
3 0.993 ND ND
8 3.81 12.6 3.99
0.0371c 3.34 14.2 7.44 274 682 9570 ND 189 236 5070 ND
0.126c 8.74 37.3 5.45 411 1220 17 700 ND 350 505 10 800 ND
0.772c 29 91 6.84 393 1060 14 700 ND 384 534 11 200 ND
ND 0.305 ND ND
0.036 1.65 5.3 2.7
0.046 3.8 11.3 7.28
ND ¼ Non-discernible due to insufficient data in the terminal elimination phase. Values represent average of male and female with ‘‘0’’ used as baseline for males. a Inhaled Deposited Dose (IHDD). b n was 5 total (2 females and 3 males; no sample obtained from one female); pre-exposure serum concentrations only present in female animals. c n was 2/gender.
Table 11. Terminal exposure and recovery lung and kidney tissue levels. Lung and kidney tissue levels (mg/g) of gentamicin in rats and dogs Lung
Rat Low Mid High Dog Low Mid High
(6 mg/kg)
(8 mg/kg)
Kidney
First/24 h
Last/24 h
Recovery
First/24 h
Last 24 h
Recovery
75 149 241
375 797 591
NA NA 350
67.9 120.2 187
139 455 432
NA NA 95.5
NA NA NA
9.4 21.4 41.6
NA NA 39
NA NA NA
186 360 687
8 mg/kg dose-matched tissue maximum concentration (Cmax) levels were similar in both species. Further, dosematched Day 14 Cmax, T1/2 and AUC were comparable among species and mirrored the single-day dog data. Dose proportionality was observed in TK parameters at the higher dose levels achieved in single-day exposed rats and in repeat exposure rats and dogs. Very low pre-exposure levels of gentamicin were evident in the serum of repeat exposure rats and dogs prior to Day 14 exposures. TK parameters were only calculated in lung and kidney in terminally time-sacrificed rodents. Due to slow tissue clearance and tissue retention, T1/2 was only calculable from the lungs of rodents exposed for one day. Dose proportionality was evident in single-day exposure lung T1/2, Cmax and AUC;
NA NA 481
and kidney Cmax and AUC in both species. After repeated inhalation exposure, however, the kidney and lung tissue levels, Cmax and AUC were comparable, suggesting that a plateau had been reached. Lung and kidney tissue burdens of gentamicin assessed 24 h after the first exposure (rats only), the Day 14 exposure (rats and dogs), and at recovery (rats and dogs high exposure groups only) reflect mechanisms consistent with tissue retention and slow clearance. The levels in rat lungs and kidneys after 14 days of exposure were substantially higher than the Day 1 levels, suggesting an accumulation (see Figure 4). However, at 24 h after the 14-day exposure, dog lung levels were modest, suggesting effective clearance and lack of accumulation. This difference in rat and dog lung
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Figure 4. Lung and kidney levels of gentamicin in rats and dogs 24 h after end of 14-day exposure.
effects results in a nine-fold greater gentamicin lung tissue burden observed in 6 mg/kg rats versus 8 mg/kg dogs sacrificed 24 h after the last exposure despite the similar lung deposition (Table 11). The high levels found in both rat and dog kidneys 24 h after the 14-day exposure show substantial accumulation in both species for this tissue. However, at similar doses (6 mg/kg rats versus 8 mg/kg dogs), the accumulation levels in dogs are higher than those in rats. The tissue concentrations at recovery are consistent with long-term components of retention in both tissues for both species since levels were relatively high as compared to those observed at the end of the 14-day exposure period (see Table 11).
Discussion The systemic findings in these studies of inhaled gentamicin are consistent with observations in other studies of aminoglycoside antibiotics delivered by the injectable route (Zhanel et al., 2012). The kidney changes observed in the current studies are expected and completely consistent with the known actions of IV and IM gentamicin and other parenteral aminoglycoside antibiotics. Nephrotoxicity is one of the major adverse side effects of gentamicin both in humans (Gentamicin Sulfate Injection, 2011; Schentag et al., 1981) and animals (Gilbert et al., 1978). The observance of accumulation of gentamicin in rat kidney is consistent with observations made earlier by other investigators, who also
noted tissue damage along with accumulation (Luft & Kleit, 1974; Schentag et al., 1978). To avoid nephrotoxicity in patients, it is advised to avoid Cmax levels of greater than 12 mg/mL and trough blood concentrations of greater than 2 mg/mL (Gentamicin Sulfate Injection, 2011). It is usually only high serum concentrations above these levels that can occur in renally impaired patients or overdose situations that lead to kidney damage in patients. Observations of lung toxicity at high inhaled doses in rats have been made for other inhaled aminoglycosides, namely tobramycin in the summary basis of approval for NDA 50753 (FDA, 1997). The pattern of toxicity seen with inhaled tobramycin at high doses was generally similar to that seen with inhaled gentamicin. Chronic nephropathy was observed at a greater frequency in rats from the high dose group compared to control. There was hyperplasia of bronchiolar epithelium and infiltration of macrophages at the mid and high doses. Only macrophage infiltration was observed at the low dose and since it was an adaptive response, such as can be observed from inhaling any particles, the low dose was a NOAEL. However, it should be noted that the serum concentrations of tobramycin in the rats (3.7, 9.6 and 20.0 mg/ mL: low, mid and high dose groups, respectively), were much higher than what has been measured in humans after dosing via inhalation with mean values typically in the range of 1 mg/ mL or less (FDA, 1997; Newhouse et al., 2003). Blood levels in patients are also low after use of aerosolized gentamicin (Ilowite et al., 1987).
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In the current toxicology studies in rats, Cmax levels of gentamicin were also quite high with mean values of 3.1, 11.3 and 20.0 mg/mL in the low, mid and high dose groups, respectively. Differences in the two toxicology programs were that the gentamicin studies were performed at somewhat higher inhaled doses (39, 123 and 245 mg/kg for gentamicin and 12, 37 and 112 mg/kg for tobramycin) and a shorter duration than the tobramycin study (14 days versus 6 months). Also, a NOAEL was not identified in the gentamicin rat studies. No previous reported studies were found of inhaled gentamicin in dogs. However, there have been several reports of use of aerosolized gentamicin in another large species, namely humans (Baran et al., 1975; Crowther Labiris et al., 1999; Hall, 1989; Ilowite et al., 1987: Klastersky et al., 1972; Murray et al., 2010; Palmer et al., 1998; Prober et al., 2000; Twiss et al., 2005). These studies showed that aerosolized gentamicin was generally well tolerated, and there were varying degrees of clinical efficacy. Determining pulmonary deposited doses in these studies is fraught with difficulties, but some of the studies showing some clinical benefit were estimated to result in daily deposited doses of approximately 50 mg (Palmer et al., 1998). This dose of approximately 1 mg/kg is in the same range as the low dose in the current dog study. A striking feature of the current toxicity studies is the more profound lung effects noted in rats as compared to dogs. Some of these observations result from the fact that it was possible to achieve higher doses in the rat study as compared to the dog study. This occurred primarily because it was possible that longer exposures (up to 3 h) could be tolerated by rats in the nose-only exposure systems, whereas 1 h was the maximum time that the dogs could tolerate in a face mask exposure system. Also, it was possible to achieve somewhat higher exposure concentrations in the rat exposure chambers compared to the dog chambers. The net result was that inhaled doses of 37, 123 and 245 mg/kg were achieved in the rat study, whereas inhaled doses were 7, 14 and 41 mg/kg in the dogs. Notably, there was dose overlap between the low dose in rats and the high dose in dogs. It is also noteworthy that this was a clear evidence of accumulation of gentamicin in the lungs of rats and some toxicity even at the low dose, whereas this was not the case for dogs even at the high dose. The low dose group in the rat study and the high dose group did have similar inhaled doses of 37 and 41 mg/ kg, respectively and deposited lung doses of 6 and 8 mg/kg, respectively. At these similar doses, it was clear that there were lung effects in the rat study while there were no lung effects in the dog study. A further confirmation that deposited lung doses were similar is the fact that serum concentrations were also similar. Mean Cmax levels of 3.1 mg/mL were achieved in the rat low dose group as compared to 3.8 mg/mL in the dog high dose group. AUCs were 14 and 11.3 mg. h/mL in the rats and dogs, respectively. Because oral absorption is very low for gentamicin, these blood levels can be attributed primary to lung absorption. The results further reinforce the view that lung deposition was similar in the two species. In the present study, there was accumulation of gentamicin in rat lungs, whereas this was not a feature of the results in dog lungs. The accumulation of gentamicin even at the low
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dose, in this study, led to retained tissue levels in rats in the range of a few hundred mg/g. An accumulation of gentamicin in kidney tissues at these same levels gave rise to cell toxicity, and it appears that this is also the case for lung tissue and the reason for the adverse lung histopathology seen in rats.
Conclusions Adverse kidney effects were observed in both rats and dogs as have been observed previously in toxicology studies using injection administration. Adverse lung effects were seen in rats only. Rats are a more sensitive species for effects of inhaled gentamicin than dogs. The lowest rat deposited lung dose at which adverse effects were seen was comparable to or somewhat lower than the highest deposited dog lung dose, which showed no adverse effects. The relevance of these findings to possible clinical effects depends on a dose comparison. Data from Palmer et al. (1998) found that approximately 30% of an 80 mg nebulizer dose was delivered to the lungs of intubated patients, who were dosed three times per day. If this 72 mg total dose is deposited in 60 kg subjects, the deposited lung dose is 1.2 mg/kg/day. This dose was well tolerated in the Palmer study and suggests that, if effects in man are like those in dogs, deposited lung doses up to 8 mg/kg could be acceptable. Effects in humans are more likely to be similar to those in dogs than rats, given the similarities in respiratory tract anatomy of dogs to humans (Tyler & Julian, 1992); the large amount of clinical experience with inhaled gentamicin in humans; and the fact that generally toxicology effects in large species are more predictive of effects in humans than rodents (Tomazewski, 2004). However, the effects in rats, though modest at the low deposited dose of 6 mg/kg and possibly due to rat sensitivity, are cautionary. Since it has been shown that 1.2 mg/kg deposited lung dose of gentamicin has been acceptable in humans, clinical studies might start at these levels and then escalate higher with careful monitoring if higher doses are needed for therapeutic efficacy. Publication of additional studies with other antibiotics would be useful to determine if this is a general effect of increased rodent sensitivity to inhaled antibiotics delivered at high doses.
Acknowledgements Thanks to the aerosol technology group at LRRI for excellent aerosol generation and characterization.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This work was supported by Nektar Therapeutics with rights subsequently transferred to Novartis. R. Wolff and H. Conway were responsible for the study as employees of Nektar at the time. Matthew D. Reed supervised the study conducted at LRRI.
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