Subchronic toxicity and toxicokinetics of long-term intranasal administration of bencycloquidium bromide: A 91-day study in dogs

Regulatory Toxicology and Pharmacology 59 (2011) 343–352

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Subchronic toxicity and toxicokinetics of long-term intranasal administration of bencycloquidium bromide: A 91-day study in dogs Juan Li a, Haixia He a,d,e, Yuanda Zhou a,⇑, Pei Yuan b, Xiaoping Chen c a

Department of Clinical Pharmacology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China Medical Engineering Research Center, Chongqing Medical University, Chongqing 400016, PR China c Beijing Shiqiao Biological & Pharmaceutical Co. Ltd., Beijing 100190, PR China d Herbal Medicine EBM Research Center, Korea Institute of Oriental Medicine, 483 Expo-ro, Yusung-gu, Daejeon 305-811, Republic of Korea e Department of Herbal Pharmaceutical Development, Nambu University, 864-1, Wolgye-dong, Gwangsan-gu, Gwangju 506-706, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 29 June 2010 Available online 2 December 2010 Keywords: Bencycloquidium bromide Subchronic toxicity Toxicokinetics Dogs

a b s t r a c t The subchronic toxicity and toxicokinetics of Bencycloquidium bromide (BCQB) were evaluated after 91day intranasal administration in dogs at daily dose levels of 2.5, 5.0, and 10.0 mg/kg. Following repeated exposure to medium- or high-dose of BCQB, apparent changes were observed in the levels of blood glucose, creatinine or blood urea nitrogen in both male and female dogs. The no-observed-adverse-effect level (NOAEL) of BCQB was considered to be 2.5 mg/kg/day under the present study conditions. There were no significant gender differences in most indexes of subchronic toxicity throughout the experimental period with the exception of food consumption and body weight, or in the parameters of plasma toxicokinetics after either single-dose or repeated administrations of BCQB at each dosage. In dog, BCQB did not accumulate in blood plasma, while much higher concentrations of BCQB residues were found in most tissues examined (especially the kidney) following 91-day repeated exposure relative to a single dose. In all tissues except the reproductive organs, BCQB concentrations reverted to low levels by 2 weeks postdosing. The results indicate that blood glucose levels and renal function should be closely monitored when BCQB is used in long-term therapy. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Rhinorrhea is one of the most troublesome symptoms described by allergic and non-allergic patients alike, causing significant discomfort and interference with daily life. Since nasal secretion originating from serous and seromucous cells is primarily mediated by the cholinergic nervous system (Naclerio and Baroody, 1995), anticholinergic medications would be a good choice for the treatment of rhinorrhea. Ipratropium bromide, a cholinergic receptor antagonist, can reduce nasal secretions induced by methacholine (Wagenmann et al., 1994). In previous studies (Kaiser et al., 1995; Georgitis et al., 1994; Grossman et al., 1995), it was shown that ipratropium bromide nasal spray provided effective relief from rhinorrhea with minimal side effects. Moreover, ipratropium bromide is equally or more effective than beclomethasone, and the coadministration of these two agents was superior to either one alone (Dockhorn et al., 1999). Bencycloquidium bromide (BCQB, 3-{(2-cyclopentyl-2-hydroxy-2-phenyl) ethoxy}-1-methyl-1-azabicyclo[2,2,2]octane bromide (chemical structure, see Fig. 1), an analog of ipratropium bromide, is a novel compound developed by Beijing Shiqiao Biological & Pharmaceutical Co. Ltd. that is currently in Phase II clinical ⇑ Corresponding author. Fax: +86 23 68898185. E-mail address: [email protected] (Y. Zhou). 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.11.006

trial in China. In our previous study (Li and Zhou, 2007b; Li et al., 2008), BCQB demonstrated good efficacy in the treatment of symptoms of allergic rhinitis such as pruritus, sneezing, and especially hypersecretion in rats and guinea pigs. Furthermore, it was observed that BCQB had much higher affinities to muscarinic M1 and M3 receptors than to the muscarinic M2 receptor (Li et al., 2010), while ipratropium bromide showed less selectivity among muscarinic M1, M2, and M3 receptors (Haddad et al., 1999). Similar to ipratropium bromide, BCQB did not cause systemic anticholinergic side effects when administered topically because of its quaternary structure. Thus, BCQB is a promising drug candidate for the treatment of rhinorrhea with few side effects. Since BCQB is being developed for the treatment of chronic allergic rhinitis, chronic non-allergic rhinitis, and acute rhinitis resulting from the common cold (Zhao, 2004), there is merit in characterizing the toxicity and toxicokinetics (TK) of BCQB after long-term intranasal administration in animals. Long-term toxicity is an important part of safety assessment in drug development, while TK is helpful in assessing the relationship between dose and exposure, and in reducing uncertainties inherent in safety assessment. Given a lack of available information about the safety of long-term use of BCQB in animals or humans, the present study was performed to assess the subchronic toxicity and TK of BCQB, specifically after 91-day repeated intranasal administrations in

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dogs at daily dose levels of 2.5, 5.0, and 10.0 mg/kg. Blood or tissue samples were collected at various time points for the analysis of hematology, clinical chemistry, pathology, TK parameters, and BCQB residue content in plasma and tissues. As an evaluation of preclinical safety, this study will provide guidance in the design of further preclinical toxicity studies as well as clinical trials involving BCQB. 2. Materials and methods 2.1. Chemicals and reagents The reference substance BCQB (purity: 99.5%, Lot: 031120) and 1-ethyl-BCQB (internal standard, purity: 98%, Lot: 041205), as well as BCQB (purity: 99%, Lot: BQB-O7A) for animal studies, were kindly supplied by Beijing Shiqiao Biopharmaceutical Company (Beijing, China). Methanol (HPLC grade) was obtained from Merck (Darmstadt, Germany). All other chemicals used were of analytical reagent grade and purchased from Chongqing Dongfang Chemical Reagent Co. (Chongqing, China). C18-cartridge solid-phase extraction columns were supplied by Supelco (Bellefonte, PA, USA). A Milli-QÒ (Millipore, USA) water purification system was used to obtain the purified and deionized water for LC–MS analysis. 2.2. Animals Sixty-six male and female beagle dogs (Certificate No. SCXK (Chuan)-2008-01), aged 7–9 months and weighing 7.8–9.0 kg (males) or 7.3–8.3 kg (females), were purchased from the Sichuan Yang She Institute (Sichuan, China). They were housed in a light cycle-controlled room (light: 07:00–19:00, dark: 19:00–07:00) at a temperature of 22 ± 2 °C and relative humidity of 55 ± 12%, with free access to water. Food was restricted to 400 g per day, and any remaining, unconsumed food was weighed to calculate net food intake per animal. The study was performed in compliance with the Guide for the Care and Use of Laboratory Animals as adopted by Chongqing Medical University (Chongqing, China). Animals were allowed a 2-week quarantine and acclimation period prior to initiation of the study. 2.3. Dosing The high-, medium-, and low-doses were selected as 10.0, 5.0, and 2.5 mg/kg body weight, respectively, based on the results of acute toxicity testing (which identified the approximate lethal dose to be 75–90 mg/kg body weight) and preliminary studies in beagle dogs. BCQB dosing solutions were prepared at concentrations of 100, 50, and 25 mg/ml in 0.01% benzalkonium chloride. Dogs were randomized into four groups: three test groups (n = 18 dogs each) and a vehicle group as control (n = 12 dogs). BCQB was administered into both nasal cavities in each dog based on individual daily body weights (each side with half of the specified dosage) once daily for 91 days, to evaluate the subchronic toxicity and TK parameters of BCQB.

final administration, and at the end of the recovery period. The animals were fasted for 24 h before blood sampling from the forelimb vein. A portion of each blood sample was treated with EDTA-2 K and analyzed for hematology indexes such as the leukocyte count, erythrocyte count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet, neutrophil, and lymphocyte count, and reticulocyte ratio with a hematology analyzer (SE9000, Sysmex, Japan). Another portion of blood sample was treated with sodium citrate and used to determine the prothrombin time (PT) with an autocoagulometer (CA-1500, Sysmex, Japan). Moreover, an autoanalyzer (7170, Hitachi, Japan) was used to examine the serum obtained from another portion of the blood sample for the content of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, glucose, total protein, albumin, blood urea nitrogen, creatinine, triglycerides, total cholesterol, total bilirubin, creatinekinase, c-glutamyltranspeptidase (c-GTP), chloride, sodium, and potassium. For TK evaluations, blood samples were collected at 0.017, 0.050, 0.083, 0.170, 0.330, 0.670, 1, 2, 4, 8, 12, and 24 h after the first or final administration. Additional blood samples were collected at the end of a 2-week recovery period for the analysis of BCQB residues in plasma. Necropsy was carried out 24 h after the final administration and at the end of the recovery period. At each time point, three male and three female dogs from each group were euthanized by exsanguination, and all organs and tissues were macroscopically examined for gross pathology. Following necropsy, the brain, heart, liver, spleen, lung, kidneys, adrenal glands, thymus, testes, epididymis, uterus, and ovaries were individually isolated and weighed to calculate the ratios of organ weight to body weight. These and the following organs were then fixed in 10% neutral buffered formalin: hypophysis, thyroids, trachea, gall bladder, pancreas, prostate, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, skeletal muscle, mesenteric lymph node, submaxillary lymphnode, aorta, sciatic nerve, skin, subcutis, mammary gland, sternum, salivary gland, thoracic spinal cord, urinary bladder, and nose. Parts of following tissues were collected for the analysis of BCQB residues: heart, liver, spleen, lung, kidney, brain, stomach, trachea, small intestine, fat, and reproductive tissues (testes and uterus). Furthermore, the above tissues were collected at 24 h after the first administration from additional eighteen dogs (three male and three female dogs for each dosage of BCQB) for the analysis of BCQB residues compared with those at 24 h after the final administration. 2.5. Sample preparation for TK study Plasma was separated by centrifugation at 4000 rpm for 10 min and stored at 20 °C until analyzed. All frozen standards and samples were allowed to thaw at room temperature and homogenized by vortexing. Thirty microliters of internal standard (I.S., 1.5 lg/ ml) was added to 30 ll blood plasma. The sample mixture was mixed with 100 ll of trichloracetic acid and vortex-mixed for

2.4. Study design During the administration and recovery period, clinical signs and mortality were observed daily. Body weight and food consumption for each dog was measured daily and subjected to statistical analysis once a week. Hematology, clinical chemistry, ophthalmic findings (including ocular fundus examinations, slit lamp examinations and macroscopic observations), electrocardiography, and urinalysis (for glucose, pH, specific gravity, protein, bilirubin, ketone, nitrite, and urobilinogen) were conducted prior to drug administration, post-

Fig. 1. Chemical structure of bencycloquidium bromide.

J. Li et al. / Regulatory Toxicology and Pharmacology 59 (2011) 343–352

3 min, after which the precipitate was removed by centrifugation at 15,000 rpm for 10 min. Five microliters of supernatant were injected into the LC–ESI-MS system. The ratio of the peak area of the sample relative to that of the internal standard was calculated for quantitative analysis. Tissue samples collected at different time-points were immediately weighed and rinsed with saline (0.9% NaCl) to remove blood or content, and then homogenized in saline at 4 ml per gram tissue. Tissue homogenates were centrifuged at 4000 rpm for 10 min, and the supernatants were stored at 20 °C until analyzed. For analysis, solid-phase extraction (SPE) and liquid–liquid extraction (LLE) methods were used to prepare samples as described in Xu et al. (2008). Mixtures of fat tissue homogenate and I.S. were extracted by LLE with 4-fold volume of cyclohexane. After being centrifuged at 15,000 rpm for 10 min, a 5-ll aliquot of the aqueous phase solution was injected into the LC–ESI-MS system. All other tissue mixtures (homogenate and I.S.) were subjected to SPE and vortex-mixed with 4-fold volume of methanol for 3 min, and the resultant precipitate was removed by centrifugation at 4000 rpm for 10 min. In a 45 °C water bath, the supernatant was subsequently evaporated to approximately 0.5 ml for most samples (or evaporated to dryness and reconstituted in 1 ml aqueous solution for trachea sample). Samples were then loaded onto a pre-conditioned C18 solid-phase extraction column. BCQB and I.S. were eluted from the columns using an organic solvent mixture consisting of methanol – triethylamine – acetic acid (99:0.5:0.5; v/v/v). The combined extracts were evaporated to dryness under a gentle stream of nitrogen at 45 °C, after which the residue was reconstituted in mobile phase. Following centrifugation at 4000 rpm for 5 min, 5 ll of supernatant were injected into the LC–ESI-MS system. For all extracted tissue samples, the ratio of the peak area of the sample relative to that of the internal standard was calculated for quantitative analysis. 2.6. LC–ESI-MS analysis of BCQB The concentrations of BCQB in plasma and tissue samples were analyzed by LC–ESI-MS assay. A Shimadzu LC-2010AHT system (Shimadzu Corporation, Kyoto, Japan) was utilized for analysis, which included a vacuum degasser, temperature controlled column compartment, autosampler, and a Shimadzu LCMS-2010A single quadrupole mass spectrometer equipped with an electrospray source. Signal acquisition and peak integration were performed using the LCMS solution 3.4 version software supplied by Shimadzu. A Luna C18 column (3 lm, 100 mm  2.0 mm, 100A) and a Luna C18 pre-column (3 lm, 10 mm  2.0 mm) from Phenomenex Corporation (USA) were used for separation of analytes. The mobile phase was delivered at a flow rate of 0.2 ml/min and consisted of 8 mM ammonium acetate buffer solution (containing 1% formic acid) – methanol (47:53; v/v). The column temperature was maintained at 35 °C. The MS was operated in an electrospray mode with positive ion detection at an electrospray voltage of 1.4 kV, a heated block temperature of 200 °C and a nebulizer gas flow rate of 1.5 L/min. Selected-ion monitoring was accomplished at [M]+ m/z 330.1 for BCQB and [M]+ m/z 344.5 for I.S (1-ethyl-BCQB).

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the fitting parameters, and dispersion of the residual under equal-weight scheme (Chen et al., 1995). All data are expressed as mean ± standard deviation (SD). The data of BCQB residues in tissues and ratios of organ weight to body weight were analyzed by ANOVA with LSD or Dunette’s test (SPSS 12.0 software, USA). Other data were subjected to variance analysis using the Repeated Measures of General Linear Model (SPSS 12.0 software, USA), and inter-group comparisons were made using the Multivariate of General Linear Model. P-values of <0.05 are considered statistically significant.

3. Results 3.1. Subchronic toxicity 3.1.1. Mortality and clinical observations No deaths were observed in any group during the administration or recovery periods. Compared to controls, the test group exhibited no treatment-related changes in clinical signs such as external appearance, behavior, mental state, and daily activities. With regard to food consumption (Fig. 2B), female dogs in medium- and high-dose groups showed a significant decrease during weeks 2 and 3 compared to their initial food intakes (P < 0.05 or P < 0.01), or compared to the control dogs (P < 0.05 or P < 0.01). The food consumption of male dogs in all test groups was almost identical to that in the control group throughout the administration period (Fig. 2A). As a result of the changes in food consumption, female dogs in the medium-dose group showed a significant reduction in body weight during weeks 3 and 4 compared to day 1 (P < 0.05). Compared to control animals, female dogs in the medium- and highdose groups had significantly lower body weights during weeks 3 to 6 (Fig. 3B, P < 0.05 or P < 0.01). Male dogs tested for all dosages on the other hand showed a tendency towards increased body weights that are not significantly different from those in the control group (Fig. 3A).

2.7. Toxicokinetic and statistical analyses The following toxicokinetic parameters are estimated using the 3P97 program (Chinese Pharmacology Society): area under the plasma concentration–time curve from time zero to 24 h (AUC0–24), maximum plasma concentration (Cmax), the time to Cmax (Tmax), elimination half-life (T1/2), mean residence time (MRT) and clearance (CL). An appropriate compartment model for TK was chosen on the bases of the lowest Akaike’s information criterion (AIC) value, lowest weighed squared residuals, lowest standard errors of

Fig. 2. Food consumptions of male dogs (A) and female dogs (B) during the administration period.

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Fig. 3. Body weights of male dogs (A) and female dogs (B) during the administration period.

3.1.2. Clinical chemistry and hematology In the hematology analysis (Table 1), no treatment-related changes were observed in female dogs. However, the platelet count of male dogs following 91 days exposure to the medium dose of BCQB was significantly decreased relative to that of control dogs

(P < 0.05). This change was transient as there was no longer any significant difference between the three test groups and the control group at the end of a 2-week recovery period. In addition, there were no apparent changes in all indexes of hematology at each dose of BCQB for both male and female dogs after 91-day repeated dosing compared to the values obtained prior to administration (data not shown, P > 0.05), and no significant differences were observed between the male and female dogs during the experimental period. With regard to blood chemistry (Table 2), both female and male dogs showed a trend towards decreased blood glucose at each dose of BCQB compared to control animals, and levels of blood glucose in medium- and high-dose groups were significantly reduced in both sexes of dogs after 91-day repeated dosing compared to the pre-dosing levels (P < 0.05). In addition, trends toward increased levels of creatinine (in female dogs of the medium- and high-dose groups) and blood urea nitrogen (in both male and female dogs of the high-dose group) were observed compared to control animals following 91-day repeated dosing. Levels of ALT in male dogs were significantly elevated in the low- and medium-dose groups (P < 0.05) relative to control animals following 91-day repeated dosing. Compared to the values obtained pre-administration, levels of creatinine (in the medium- and high-dose groups) and blood urea nitrogen (in the high-dose group) were significantly elevated (P < 0.05 or P < 0.01) in both sexes of dogs after 91-day repeated dosing. After a 2-week recovery period, all of the above indexes were restored and there was no longer any significant difference between the three test groups and the control group. No treatment-related changes were observed in the other indexes examined and no significant gender differences were observed in all indexes of blood chemistry during the experimental period. 3.1.3. Urinalysis, ophthalmoscopy, and electrocardiography With regard to urinalysis and ophthalmoscopy, no obvious abnormity was observed throughout the experimental period (data

Table 1 Hematological findings in dogs treated intranasally with BCQB for 91 days. Dose

Control

2.5 mg/kg

5 mg/kg

10 mg/kg

Males No. of animals examined Leukocytes (109/L) Erythrocytes (109/L) Hemoglobin (g/L) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/L) Reticulocyte (%) Platelets (109/L) PT (sec) Neutrophil (%) Lymphocyte (%)

6 16.27 ± 3.35 6.05 ± 0.52 140.5 ± 12.76 38.52 ± 4.88 63.58 ± 3.41 23.18 ± 0.58 365.83 ± 13.76 1.65 ± 0.55 617 ± 202.93 9.50 ± 1.33 15.68 ± 5.05 59.47 ± 19.88

6 16.72 ± 3.32 5.99 ± 0.35 142 ± 9.40 38.52 ± 3.95 64.25 ± 3.08 23.70 ± 0.61 369.50 ± 15.88 1.55 ± 0.68 547 ± 193.54 9.33 ± 1.85 14.47 ± 3.29 64.43 ± 21.52

6 15.75 ± 2.93 6.19 ± 0.51 144.83 ± 16.40 40.05 ± 5.84 64.18 ± 4.32 23.03 ± 1.09 361.50 ± 15.27 1.55 ± 0.67 384 ± 151.60* 9.63 ± 1.71 13.37 ± 3.40 68.42 ± 10.83

6 14.27 ± 3.89 5.64 ± 0.74 136.83 ± 18.1 34.92 ± 4.56 64.72 ± 4.25 23.30 ± 1.40 360.83 ± 9.72 1.50 ± 0.81 509 ± 216.71 9.45 ± 1.51 13.73 ± 4.61 71.6 ± 14.18

Females No. of animals examined Leukocytes (109/L) Erythrocytes (109/L) Hemoglobin (g/L) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/L) Reticulocyte (%) Platelets (109/L) PT (sec) Neutrophil (%) Lymphocyte (%)

6 15.43 ± 3.74 6.24 ± 0.50 146 ± 12.63 38.35 ± 3.29 64.1 ± 3.57 23.37 ± 0.77 365.67 ± 10.56 1.88 ± 0.51 560.67 ± 175.83 9.45 ± 1.13 14.73 ± 6.78 67.6 ± 12.64

6 17.3 ± 4.62 5.80 ± 0.54 139..83 ± 14.52 38.98 ± 5.73 64.35 ± 4.78 23.08 ± 1.45 366.50 ± 14.07 1.73 ± 0.42 615 ± 108.75 9.37 ± 1.51 14.82 ± 3.16 66.97 ± 14.76

6 16.52 ± 6.68 6.14 ± 0.34 145.5 ± 11.15 39.83 ± 4.24 64.87 ± 3.77 23.70 ± 0.89 366.17 ± 13.96 1.93 ± 0.78 520.67 ± 171.10 9.30 ± 1.15 12.17 ± 3.87 72.97 ± 11.95

6 18.45 ± 7.65 5.99 ± 0.29 140.5 ± 13.44 38.68 ± 4.93 64.43 ± 5.90 23.40 ± 1.35 364.33 ± 15.03 1.87 ± 0.70 525.33 ± 188.94 9.58 ± 0.87 13.12 ± 4.41 72.83 ± 13.40

No significantly difference from the pre-dosing value. P < 0.05 (significantly difference from control).

*

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J. Li et al. / Regulatory Toxicology and Pharmacology 59 (2011) 343–352 Table 2 Biochemical findings in dogs treated intranasally with BCQB for 91 days.

* # ##

Dose

Control

2.5 mg/kg

5 mg/kg

10 mg/kg

Males No. of animals examined Total protein (g/L) Albumin (g/L) Total bilirubin (lmol/L) AST (IU/L) ALT (IU/L) c-GTP (IU/L) Alkaline phosphatase (IU/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) Creatine kinase (IU/L) Glucose (mmol/L) Blood urea nitrogen (mmol/L) Creatinine (lmol/L) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L)

6 63.27 ± 4.92 28.98 ± 1.96 1.04 ± 0.70 35.33 ± 15.42 18.67 ± 4.72 3.27 ± 1.50 90.93 ± 13.94 3.52 ± 0.84 0.43 ± 0.12 200 ± 57.32 4.28 ± 0.50 5.47 ± 2.25 79.22 ± 8.38 148.57 ± 1.76 5.42 ± 0.68 108.17 ± 1.67

6 62.67 ± 4.16 28.55 ± 2.89 0.84 ± 0.45 32.67 ± 14.39 27.33 ± 3.14* 2.77 ± 0.52 126.15 ± 58.85 3.88 ± 1.45 0.42 ± 0.20 202.67 ± 51.60 3.60 ± 1.40 5.03 ± 3.18 77.55 ± 8.32 147.48 ± 2.43 5.12 ± 0.40 107.02 ± 1.87

6 60.93 ± 1.77 27.72 ± 2.17 0.94 ± 0.80 40.33 ± 14.39 28.33 ± 10.35* 3.67 ± 1.50 131.37 ± 47.36 3.90 ± 0.94 0.57 ± 0.07 288.17 ± 167.29 3.62 ± 0.78# 5.37 ± 3.12 81.15 ± 6.50# 147.76 ± 3.52 5.47 ± 0.50 107.67 ± 1.40

6 61.75 ± 5.72 26.40 ± 2.87 0.91 ± 0.81 35.33 ± 11.81 20.33 ± 4.13 2.88 ± 0.90 83.85 ± 24.32 3.68 ± 0.94 0.45 ± 0.15 311 ± 159.37 3.86 ± 0.75# 6.09 ± 2.59# 78.12 ± 12.00# 146.95 ± 3.28 5.52 ± 0.52 107.32 ± 0.59

Females No. of animals examined Total protein (g/L) Albumin (g/L) Total bilirubin (lmol/L) AST (IU/L) ALT (IU/L) c-GTP (IU/L) Alkaline phosphatase (IU/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) Creatinekinase (IU/L) Glucose (mmol/L) Blood urea nitrogen (mmol/L) Creatinine (lmol/L) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L)

6 59.4 ± 5.52 27.85 ± 3.05 0.82 ± 0.65 30.5 ± 5.75 26.17 ± 9.66 3.98 ± 1.28 88.57 ± 34.40 3.01 ± 0.74 0.49 ± 0.18 228.33 ± 86.67 4.10 ± 0.69 5.16 ± 3.33 76.65 ± 9.82 148.85 ± 3.74 5.39 ± 0.14 108.1 ± 1.19

6 58.92 ± 4.11 29.57 ± 4.27 1.10 ± 1.12 34.17 ± 21.18 25.5 ± 5.17 3.15 ± 1.17 95.75 ± 28.34 3.65 ± 0.68 0.46 ± 0.19 178.33 ± 69.47 3.66 ± 0.68 5.07 ± 2.71 75.48 ± 6.07 146.22 ± 2.74 4.85 ± 0.79 106.42 ± 4.13

6 62.65 ± 6.87 30.08 ± 1.85 1.33 ± 1.00 38.5 ± 12.28 25.0 ± 3.29 3.27 ± 1.04 97.02 ± 27.90 3.30 ± 0.78 0.46 ± 0.23 275.67 ± 145.70 3.30 ± 0.19# 5.04 ± 2.77 81.42 ± 10.97## 149.13 ± 4.21 5.33 ± 0.35 108.35 ± 1.17

6 61.45 ± 3.61 27.97 ± 2.40 0.79 ± 0.65 34.5 ± 10.31 24.3 ± 6.59 3.15 ± 0.72 88.68 ± 22.42 3.40 ± 0.54 0.36 ± 0.11 220.5 ± 54.66 3.52 ± 1.05# 6.02 ± 2.12# 83.37 ± 6.71## 148.55 ± 3.39 5.31 ± 0.46 108.57 ± 1.47

P < 0.05 (significantly difference from control). P < 0.05 (significantly difference from the pre-dosing value). P < 0.01 (significantly difference from the pre-dosing value).

Table 3 Electrocardiogram findings in dogs treated intranasally with BCQB for 91 days. Dose

Control

2.5 mg/kg

5 mg/kg

10 mg/kg

Males No. of animals examined P wave (mv) R wave (mv) QRS complex (s) ST (mv) T wave (mv) PR interval (s) QT interval (s) HR (beat/min)

6 0.21 ± 0.02 0.93 ± 0.42 0.063 ± 0.009 0.003 ± 0.008 0.245 ± 0.056 0.077 ± 0.006 0.212 ± 0.016 125.0 ± 15.5

6 0.18 ± 0.07 0.82 ± 0.63 0.063 ± 0.006 0.005 ± 0.008 0.200 ± 0.130 0.077 ± 0.005 0.212 ± 0.018 125.7 ± 30.0

6 0.17 ± 0.06 0.66 ± 0.26 0.061 ± 0.006 0 0.188 ± 0.086 0.082 ± 0.004 0.203 ± 0.027 127.2 ± 30.2

6 0.21 ± 0.06 0.93 ± 0.74 0.063 ± 0.005 0 0.212 ± 0.147 0.079 ± 0.005 0.215 ± 0.012 128.8 ± 19.3

Females No. of animals examined P wave (mv) R wave (mv) QRS complex (s) ST (mv) T wave (mv) PR interval (s) QT interval (s) HR (beat/min)

6 0.205 ± 0.056 0.83 ± 0.160 0.061 ± 0.004 0 0.238 ± 0.101 0.080 ± 0.012 0.225 ± 0.021 121.3 ± 29.1

6 0.232 ± 0.053 0.95 ± 0.519 0.062 ± 0.008 0.003 ± 0.008 0.222 ± 0.123 0.079 ± 0.007 0.213 ± 0.030 113.8 ± 15.6

6 0.207 ± 0.055 0.97 ± 0.367 0.060 ± 0.003 0.003 ± 0.008 0.200 ± 0.071 0.081 ± 0.002 0.205 ± 0.008 118.8 ± 26.5

6 0.233 ± 0.075 1.04 ± 0.489 0.062 ± 0.007 0 0.172 ± 0.060 0.075 ± 0.009 0.210 ± 0.015 125.3 ± 35.6

No significantly difference from control.

not shown). Electrocardiography revealed no treatment-related changes in any dogs of either sex following 91-day repeated dosing (Table 3), or after the 2-week recovery period (data not shown).

Moreover, no significant differences were observed in the indexes of urinalysis and electrocardiography between the male and female dogs during the experiment period.

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3.1.4. Necropsy and histopathology Macroscopic examination upon necropsy suggested no treatment-related changes. With regard to ratios of organ weight to body weight (Table 4), spleen-to-body weight ratios of male dogs in the medium-dose group were significantly increased compared to the control dogs (P < 0.05), while the difference was no longer significant after 2 weeks of recovery. Moreover, the ratios of kidney-to-body weights showed decreasing trends in both male dogs (of all treatment groups) and female dogs (of the high-dose group) after the 91-day treatment regimen, persisting even after a 2-week recovery period. With regard to histopathology, no abnormity was observed in any of the animals. 3.2. LC–ESI-MS method validation The calibration curves were constructed by plotting the peakarea ratios of BCQB-to-I.S. versus the concentrations of BCQB. All the standard curves in dog plasma and various tissues can satisfy the requirement of analytic method (data not shown). The lower limit of quantification (LLOQ) was defined as the lowest concentration with adequate accuracy (within ± 20.0% deviation of the true value) and precision (RSD within 20.0%), and this value was established using five samples independent of standards. The LLOQ of the current assay was determined to be 5 ng/ml for both plasma and tissue samples, which was sufficient for the study of toxicokinetics following single or repeated intranasal administration of BCQB. The accuracy, intra-run and inter-run precisions of the method were determined by analyzing five replicates of QC samples at three different concentrations of BCQB along with one standard curve on each of three runs. All of the results were less than 10% (data not shown). The extraction recoveries of BCQB at three concentration levels were tested by comparing the peak areas of the analytes extracted from plasma or tissue samples with those found by direct injection of standard substance dissolved in the supernatant of the processed blank plasma at the same concentration. The results showed that the mean recoveries of the analytes in plasma and tissues were in the range of 89–101%. The stability of BCQB in plasma was studied under a variety of storage and han-

dling conditions using three aliquots at each QC level. The results showed that there was no significant degradation occurred (RE within ± 15.0%) during three freeze – thaw cycles ( 20 °C) and at room temperature (for at least 8 h) in the BCQB plasma and tissue samples. BCQB in plasma and tissues at 20 °C was stable (RE within ± 15.0%) for at least 30 days. The post-preparative samples of BCQB in the autosampler were stable (RE within ± 15.0%) for at least 24 h. 3.3. Toxicokinetic parameters of BCQB in blood BCQB was intranasally administered to dogs at daily dose levels of 2.5, 5.0, and 10.0 mg/kg for 91 days. The plasma concentration– time profiles of BCQB at days 1 and 91 after intranasal administration are shown in Fig. 4. Evaluation of individual BCQB plasma concentration–time curves reveals a biphasic pattern of drug disposition, so a two-compartment model was best suited to fit the data on days 1 and 91. The estimated TK parameters in plasma from days 1 and 91 are illustrated in Table 5. After single or repeated intranasal administration, BCQB was rapidly absorbed in dogs of both sexes, and the plasma concentration of BCQB reached its peak within 10 min (Table 5). Tmax of BCQB was not significantly different between male and female dogs after a single dose or 91-day intranasal administration (P > 0.05) for each of three different dosages tested, although a shorter Tmax was observed in female dogs following 91-day repeated exposure to high–dose of BCQB relative to a single-dose treatment (P < 0.05). Maximum plasma concentrations (Cmax) after the first or final administration increased proportionally with dose in a linear fashion in both male and female dogs. In male dogs, the correlation coefficients between dose and Cmax were 0.9974 and 0.9989 for the single dosing and repeated dosing regimens, respectively, while they were 0.9970 and 0.9991 in female dogs. Correspondingly, plasma AUC0–24 showed excellent correlation with dose. In male dogs, the correlation coefficients between dose and AUC0–24 were 0.9904 and 0.9830 for single and repeated dosing, respectively, while they were 0.9843 and 0.9778 in female dogs. There were no significant differences in Cmax or AUC0–24 of BCQB between single and 91-day administrations for any dose (P > 0.05). In addition, Cmax

Table 4 Relative organ weights findings in dogs treated intranasally with BCQB for 91 days.

*

Dose

Control

2.5 mg/kg

5 mg/kg

10 mg/kg

Males No. of animals examined Heart (g/kg BW) Liver (g/kg BW) Spleen (g/kg BW) Lung (g/kg BW) Kidneys (g/kg BW) Adrenals (g/kg BW) Brain (g/kg BW) Thymus (g/kg BW) Testes (g/kg BW) Epididymis (g/kg BW)

3 8.57 ± 0.61 37.2 ± 7.14 1.76 ± 0.078 12.05 ± 0.21 13.20 ± 0.85 0.32 ± 0.028 9.88 ± 4.14 0.895 ± 0.205 1.36 ± 0.338 0.36 ± 0.057

3 8.97 ± 1.03 34.8 ± 4.03 2.03 ± 0.39 10.56 ± 2.74 11.11 ± 1.00 0.28 ± 0.028 9.43 ± 3.78 0.90 ± 0.028 1.33 ± 0.045 0.29 ± 0.014

3 8.60 ± 0.71 38.3 ± 4.17 2.35 ± 0.11* 10.48 ± 2.86 11.50 ± 0.42 0.36 ± 0.133 10.47 ± 4.57 0.695 ± 0.021 1.51 ± 0.547 0.40 ± 0.135

3 8.52 ± 1.44 36.9 ± 1.41 1.88 ± 0.46 10.70 ± 0.85 11.66 ± 0.48 0.35 ± 0.071 10.56 ± 3.87 0.75 ± 0.170 1.62 ± 0.184 0.37 ± 0.049

Females No. of animals examined Heart (g/kg BW) Liver (g/kg BW) Spleen (g/kg BW) Lung (g/kg BW) Kidneys (g/kg BW) Adrenals (g/kg BW) Brain (g/kg BW) Thymus (g/kg BW) Ovaries (g/kg BW) Uterus (g/kg BW)

3 8.03 ± 0.106 36.55 ± 1.34 2.36 ± 0.346 10.51 ± 1.97 10.44 ± 1.92 0.45 ± 0.042 11.57 ± 6.13 0.425 ± 0.318 0.202 ± 0.003 0.437 ± 0.320

3 7.91 ± 0.156 34.00 ± 2.97 2.37 ± 0.467 9.83 ± 0.38 10.32 ± 0.40 0.45 ± 0.156 10.14 ± 3.91 0.405 ± 0.148 0.129 ± 0.039 0.430 ± 0.099

3 7.71 ± 0.134 36.45 ± 0.35 2.04 ± 0.537 8.36 ± 1.78 10.90 ± 0.14 0.41 ± 0.014 10.39 ± 0.59 0.480 ± 0.127 0.206 ± 0.062 0.257 ± 0.194

3 7.81 ± 0.559 34.05 ± 1.48 1.86 ± 0.651 13.02 ± 6.34 9.66 ± 1.78 0.44 ± 0.113 10.41 ± 3.95 0.420 ± 0.170 0.294 ± 0.080 0.434 ± 0.218

P < 0.05 (significantly difference from control).

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Fig. 4. Mean plasma concentration–time profiles of BCQB in female dogs at doses of 10, 5, 2.5 mg/kg (A, C, and E, respectively) and in male dogs at doses of 10, 5, 2.5 mg/kg (B, D, and F, respectively) after a single or 91-day intranasal administration.

Table 5 Toxicokinetic parameters of BCQB after a single or 91-day intranasal administration in the plasma of dogs (n = 6). Day 1

Dose (mg/kg) 2.5 5.0 10.0

91

2.5 5.0 10.0

*

Sex

T1/2b (h)

Cmax (ng/ml)

Tmax (h)

AUC0–24 (ng h/ml)

MRT (h)

CL (L/kg/h)

M F M F M F

26.87 ± 11.83 16.75 ± 2.81 20.62 ± 7.84 18.50 ± 5.84 16.37 ± 14.54 13.32 ± 10.73

491.39 ± 105.08 451.68 ± 85.11 1679.86 ± 577.01 1675.58 ± 539.08 4930.93 ± 896.48 5108.00 ± 677.57

0.094 ± 0.061 0.102 ± 0.044 0.055 ± 0.015 0.062 ± 0.005 0.052 ± 0.035 0.069 ± 0.027

738.60 ± 124.16 710.33 ± 72.29 1197.78 ± 221.47 1145.97 ± 193.22 2984.86 ± 757.11 3322.50 ± 947.18

7.61 ± 0.61 7.64 ± 0.94 5.87 ± 1.48 6.08 ± 1.17 3.51 ± 0.93 3.30 ± 0.94

2.08 ± 0.99 2.58 ± 0.25 3.15 ± 0.95 3.25 ± 0.70 3.18 ± 0.33 3.20 ± 1.01

M F M F M F

17.58 ± 8.35 27.31 ± 15.54 10.60 ± 6.56 22.21 ± 14.70 14.88 ± 15.57 10.00 ± 6.34

481.33 ± 389.56 493.35 ± 159.56 1758.89 ± 629.95 1847.13 ± 224.13 4877.22 ± 1357.95 5092.77 ± 1874.31

0.059 ± 0.042 0.105 ± 0.032 0.058 ± 0.035 0.042 ± 0.020 0.028 ± 0.023 0.026 ± 0.011*

663.86 ± 196.45 543.14 ± 170.29 1005.35 ± 239.02 880.58 ± 165.04 2807.05 ± 677.53 3087.21 ± 1152.05

8.20 ± 1.52 6.35 ± 2.26 5.15 ± 0.80 5.35 ± 0.79 3.46 ± 1.51 3.30 ± 1.09

2.88 ± 1.11 3.10 ± 1.49 4.90 ± 0.94* 4.53 ± 1.13 4.05 ± 1.30 3.75 ± 1.58

P < 0.05 (significantly difference from a single administration at each dosage).

and AUC0–24 values at each tested dosage were not significantly different between male and female dogs (P > 0.05). After 91-day repeated doses, plasma clearance (CL) was found to be increased in both male and female dogs, especially in male dogs at the medium dose (compared to that after a single dose, P < 0.05). Accordingly, the elimination half-life (T1/2) of BCQB was decreased in male dogs at each of high-, medium-, and low-dose and in female dogs at high dose after 91-day repeated administrations compared to that after a single dose, although the difference did not reach statistical significance (P > 0.05). With regard to the plasma mean residence time (MRT) of BCQB, a robust inverse correlation with dose was observed after a single dose or 91-day repeated doses. No significant difference was observed between single and repeated dosing, or between male and female dogs (P > 0.05).

3.4. BCQB residues in blood and tissues Concentrations of BCQB in plasma and various tissues at 24 h after administration of a single dose or 91-day repeated doses are presented in Table 6. BCQB concentrations were found to be much higher after repeated exposure relative to a single dose in most tissues examined at each dosage tested (P < 0.01 or P < 0.05). Irrespective of gender, the highest concentrations were found in tissues of the reproductive system (testes and uterus) and second in the digestive system (stomach and small intestine) after a single dose, while 91-day repeated doses led to highest concentrations of BCQB in kidney and second in the tissues of the reproductive system (testes and uterus), again in both male and female dogs. The lowest concentrations of BCQB residues were found

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Table 6 Concentrations of BCQB residues in dog plasma and tissues after a single or 91-day intranasal administration (n = 3). Concentration of BCQB 24 h after intranasal administration (ng/g or ng/ml) 10.0 mg/kg

* **

5.0 mg/kg

2.5 mg/kg

Day 1

Day 91

Day 1

Day 91

Day 1

Day 91

Males Plasma Kidney Small intestine Trachea Heart Stomach Spleen Lung Adipose Liver Brain Testes

28.39 ± 7.97 1244.18 ± 154.61 2530.78 ± 259.85 2300.86 ± 636.68 1499.55 ± 69.27 2790.86 ± 101.51 1934.21 ± 528.15 1359.87 ± 99.44 1248.99 ± 49.10 422.25 ± 77.53 44.66 ± 24.89 5236.92 ± 959.94

27.48 ± 14.89 14755.10 ± 1247.34** 9900.98 ± 520.27** 8786.46 ± 645.31** 4069.22 ± 533.51** 10246.66 ± 594.74** 8167.59 ± 682.83** 6836.00 ± 527.08** 5710.23 ± 626.75** 2311.35 ± 620.25** 49.27 ± 29.22 11563.26 ± 477.98**

33.16 ± 24.89 819.99 ± 72.03 1201.49 ± 92.24 987.41 ± 109.02 694.02 ± 46.62 1209.72 ± 98.52 975.08 ± 40.38 878.90 ± 82.09 847.57 ± 102.91 232.35 ± 45.07 32.83 ± 11.14 2808.59 ± 445.32

14.02 ± 3.54 12038.01 ± 889.56** 7184.56 ± 509.27** 4930.80 ± 678.10** 1910.07 ± 517.43* 7243.74 ± 767.77** 1221.21 ± 169.61 2492.16 ± 711.19* 3019.71 ± 185.22** 857.67 ± 93.31** 36.75 ± 9.23 9253.68 ± 334.92**

18.74 ± 7.30 451.03 ± 43.81 824.99 ± 48.14 832.70 ± 45.12 366.15 ± 86.58 874.58 ± 66.68 677.32 ± 53.64 604.26 ± 43.9 298.39 ± 102.42 173.22 ± 43.37 34.59 ± 14.33 1371.03 ± 193.27

16.16 ± 2.68 5432.24 ± 893.50** 1041.17 ± 82.92* 3314.18 ± 296.28** 1014.33 ± 68.29** 1304.16 ± 94.09** 757.29 ± 126.77 872.90 ± 59.00** 1027.93 ± 92.01** 780.75 ± 73.28** 34.01 ± 15.24 3485.25 ± 416.45**

Females Plasma Kidney Small intestine Trachea Heart Stomach Spleen Lung Adipose Liver Brain Uterus

31.16 ± 15.59 631.69 ± 86.43 1640.26 ± 218.49 1727.83 ± 115.83 1160.56 ± 53.57 2915.10 ± 70.99 1582.25 ± 38.53 1171.65 ± 81.21 1422.42 ± 54.45 587.48 ± 102.40 63.66 ± 12.36 5866.65 ± 563.92

26.82 ± 16.98 9283.99 ± 2076.79** 4941.09 ± 329.66** 4428.22 ± 506.49** 3271.30 ± 226.41** 6472.02 ± 101.03** 4068.65 ± 541.19** 3331.12 ± 243.80** 4157.53 ± 441.96** 3527.92 ± 543.54** 64.83 ± 10.77 7742.02 ± 407.30**

25.66 ± 9.93 521.24 ± 62.62 937.03 ± 91.50 809.23 ± 10.46 545.03 ± 53.30 1014.03 ± 68.74 759.56 ± 56.85 659.56 ± 105.26 682.57 ± 55.17 252.77 ± 41.76 60.04 ± 11.52 3842.13 ± 743.44

12.83 ± 3.36 7992.23 ± 880.20** 2468.71 ± 205.51** 2841.55 ± 297.23** 1352.19 ± 80.23** 3607.25 ± 426.20** 1192.44 ± 77.15** 1465.48 ± 122.36** 2400.92 ± 450.60** 2090.57 ± 418.71** 68.55 ± 10.74 4670.46 ± 253.10

17.30 ± 2.67 349.44 ± 29.83 740.28 ± 60.78 611.93 ± 41.86 171.86 ± 43.39 771.31 ± 61.88 423.93 ± 34.37 475.17 ± 54.34 357.86 ± 65.03 230.94 ± 54.71 30.78 ± 14.17 2279.94 ± 673.97

19.20 ± 14.20 3488.83 ± 652.32** 1284.15 ± 83.28** 1299.20 ± 62.86** 251.39 ± 65.94 1915.15 ± 275.66** 591.57 ± 65.81* 910.23 ± 67.96** 460.59 ± 51.99 737.59 ± 55.50** 65.39 ± 19.37 2401.81 ± 386.93

P < 0.05 (significantly difference from a single administration at each dosage). P < 0.01 (significantly difference from a single administration at each dosage).

in brain and plasma irrespective of gender or dosing regimen, being substantially lower compared to other tissues at each dosage tested (P < 0.01). Table 7 shows the concentrations of BCQB in plasma and various tissues after a 2-week recovery period. The concentrations of BCQB remained much higher in testes and uterus compared to other tissues at this time-point (P < 0.01), while levels were too low to be detected in brain and plasma.

4. Discussion BCQB is being developed for the treatment of rhinorrhea associated with all kinds of rhinitis such as chronic allergic rhinitis, chronic non-allergic rhinitis, and acute rhinitis. Our previous pharmacodynamic studies showed that this compound was efficacious against the symptoms of rhinitis (Li and Zhou, 2007b; Li et al., 2008). Since long-term use of BCQB will be utilized in clinical therapy, the subchronic toxicity of BCQB was investigated to better understand its safety after repeated administrations. Furthermore, concomitant analyses of toxicokinetics and residue content in tissues and plasma were performed to assess the relationship between dose and exposure. The food consumption by, and the body weight of, female dogs in the high- and medium-dose groups were found to be decreased in the first several weeks, while both indexes showed a trend towards recovery over the remaining weeks of the study. In addition, no significant difference was observed in male dogs between the test and control groups. We speculate the reduction in food consumption and body weight observed in female dogs during the initial weeks may be the result of antagonistic actions of BCQB on muscarinic receptors located at the stomach and intestine, since BCQB can hardly penetrate the blood brain barrier (because of its quaternary structure) and the blocking action may retard gastroin-

testinal peristalsis (which was observed in our previous study in mice, Li and Zhou, 2007a). Female dogs may be more susceptible to this effect when initially exposed to BCQB but may develop tolerance with continued exposure over several weeks. The changes observed in food consumption and body weight of female dogs may belong to the transient pharmacological actions. Of course, further studies should be conducted to investigate the gender differences in food consumption and body weight. With regard to clinical chemistry and hematology, some indexes showed obvious changes after repeated dosing compared to the values in the control group or obtained prior to drug administration. The changes in ALT and platelet counts are believed to have little or no toxicological significance since they appeared to be dose-independent and were largely reversible after a 2-week recovery period. Of note, blood glucose levels in both sexes of dogs showed a decreasing trend at each dose of BCQB compared to those in control group, while these values were significantly decreased in both male and female dogs of the medium- and high-dose groups relative to the pre-dosing levels. Similarly in rats, 6 months of repeated exposure to a BCQB dose of 3 mg/kg led to a significant reduction in blood glucose levels (Li et al., 2009); hence, the effects of BCQB on blood glucose levels need to be considered when used in long-term therapy. In the analysis of renal function, we found that creatinine levels in female dogs showed a tendency to increase compared to control animals following repeated exposure to medium- or high-dose of BCQB, and were significantly elevated at these dosages relative to the pre-dosing levels in both sexes of dogs. In addition, levels of blood urea nitrogen exhibited a tendency to increase compared to control animals and were significantly elevated relative to predosing levels following repeated exposure to high-dose of BCQB in both male and female dogs. Creatinine levels were also reported to be elevated in rats of both sexes following repeated exposure to a BCQB dose of 3 mg/kg for 3 months (Li et al., 2009). Moreover, the

J. Li et al. / Regulatory Toxicology and Pharmacology 59 (2011) 343–352 Table 7 Concentrations of BCQB residues in dog plasma and tissues 2 weeks after the last administration (n = 3). Organ

Males Plasma Kidney Small intestine Trachea Heart Stomach Spleen Lung Adipose Liver Brain Testes Females Plasma Kidney Small intestine Trachea Heart Stomach Spleen Lung Adipose Liver Brain Uterus

Concentration of BCQB 2 weeks after the last administration (ng/g) 10.0 mg/kg

5.0 mg/kg

2.5 mg/kg

– 125.11 ± 10.08** 44.06 ± 7.65** 127.02 ± 7.89** 101.15 ± 4.07** 83.02 ± 7.10** 96.41 ± 4.92** 153.33 ± 18.29** 173.99 ± 19.00** 68.98 ± 21.63** – 683.06 ± 63.55

–

– 52.55 ± 7.85** 27.20 ± 5.34** 32.28 ± 2.40** 42.88 ± 7.82** 44.05 ± 8.43** 26.10 ± 2.30** 45.39 ± 8.85** 27.04 ± 3.44** 22.07 ± 3.44** – 233.01 ± 12.39

–

–

74.51 ± 8.94** 42.99 ± 3.85** 148.53 ± 14.50** 116.83 ± 7.98** 125.12 ± 14.14** 113.79 ± 10.21** 157.60 ± 16.94** 205.61 ± 11.95** 20.60 ± 6.26** – 809.66 ± 99.13

39.29 ± 10.77** 24.58 ± 3.62** 107.81 ± 13.07** 95.31 ± 7.65** 98.88 ± 6.84** 34.97 ± 5.90** 87.27 ± 10.47** 98.04 ± 7.62** 12.03 ± 2.89** – 524.07 ± 127.51

62.03 ± 8.63** 44.79 ± 4.94** 59.11 ± 9.77** 25.20 ± 5.58** 51.96 ± 13.11** 29.92 ± 4.30** 68.06 ± 6.77** 66.22 ± 6.77** 29.29 ± 3.94** – 479.74 ± 118.90

– 36.92 ± 12.31** 39.58 ± 10.59** 51.01 ± 8.54** 28.84 ± 3.45** 81.72 ± 13.44** 19.79 ± 7.38** 40.86 ± 10.89** 23.91 ± 4.94** 9.70 ± 1.96** – 395.40 ± 116.41

**

P < 0.01 (significantly difference from the concentrations of BCQB in testes or uterus at each dosage).

concentration of BCQB residues in kidney after repeated doses was much higher than that following a single dose (by 9.98-fold or more), while a decreasing trend was observed in the ratio of kidney-to-body weight after 91-day repeated doses of BCQB in dogs of both sexes. These results suggest that the functional changes in the kidney of dogs after repeated drug exposure may arise from the bioaccumulation of BCQB in this organ. Thus, although no treatment-related changes were observed by urinalysis, and no organic changes were observed in the kidney after 91-day repeated exposure, renal functions should nevertheless be closely monitored during long-term therapy with BCQB, in light of findings presented here in dogs as well as in previous studies in rats by Xu et al. (2008) and Li et al. (2009). In the current study, no apparent change in the electrocardiogram or heart rate was observed after 91-day repeated administrations, in agreement with results obtained in rats following 6-month repeated exposure to BCQB (Li et al., 2009). In another study (Li et al., 2010), we previously found BCQB to demonstrate greater selectivity towards muscarinic M1 and M3 receptors than to M2 receptor (a selectivity of 14-fold for M1/M2 and 29-fold for M3/M2), while ipratropium bromide reportedly showed less selectivity among the three subtypes of muscarinic receptors (Haddad et al., 1999). Recently, long-term use of inhaled ipratropium bromide has been associated with an increased risk of adverse cardiovascular (CV) outcomes in patients with COPD (Ogale et al., 2010), which may be related to its action on the muscarinic M2 receptor. The minimal effects of BCQB on cardiac function is consistent with a relative lack of activation of muscarinic M2 receptor and provide encouraging data in support of cardiac safety associated with prolonged BCQB use. Following a single intranasal administration, BCQB was absorbed quickly and eliminated slowly in the plasma of dogs (maximal plasma concentration of BCQB was achieved within 10 min,

351

while the plasma elimination half-life (T1/2) of BCQB was 10 h or more), consistent with the characteristics of absorption and elimination in the plasma of rats (Xu et al., 2007). The slow elimination in plasma may be the result of BCQB binding to tissues thus leading to slow release, since the levels of BCQB were much higher in tissues than in plasma at 24 h post-administration as shown in Table 6. After 91-day repeated doses, the parameters of Cmax and AUC0–24 were found to increase proportionally with dose, the values of which were not significantly different from those following a single-dose administration. Additionally, in most plasma samples, lower levels of BCQB residues at 24 h after the final administration were observed relative to the levels at 24 h after the first administration. These results suggest that BCQB does not accumulate in the plasma of dogs. We also observed a trend towards increasing clearance (CL) with repeated BCQB dosing at each dosage tested, while Tmax and T1/2 tended to decrease with the administration of high-dose BCQB, possibly as a result of induction of hepatic enzymes following repeated and prolonged exposure to high-dose BCQB. In this study, we found high concentrations of BCQB residues in the reproductive system that remained high even after a 2-week recovery period, although no pathological changes were observed in those tissues. According to findings by Lianbing Li and her coworkers (2007), a BCQB dose of 972 lg/kg negatively affected the production of sperm in male rats, while other studies demonstrated that mAChR antagonists could impair fertility in male animals (Ban et al., 2002; Sato et al., 2005). Thus, the possible toxic effects of BCQB on the reproductive system warrant extra attention when this compound is used for long-term therapy in male patients. Concentrations of BCQB residues were also higher in the gastrointestinal tract and respiratory system relative to other tissues, possibly due to the route of administration being intranasal. As a result of the intranasal administration, a small portion of BCQB may be swallowed into the gastrointestinal tract or deposited in the trachea and lung, leading to localized high concentrations of BCQB. However, no abnormity was observed by histopathology, and BCQB concentrations in these tissues were significantly reduced after the recovery period, although additional studies are needed to further investigate the actions and effects of BCQB on these tissues, if any. BCQB deposition in adipose tissue was also observed, as would be expected from the amphipathic properties of a quaternary ammonium compound (Friedle et al., 2008). The lowest concentration of BCQB residues was found in the brain, consistent with findings in our previous study indicating little effect of BCQB on the behavior of mice. Based on comprehensive consideration of the results in the present study, the no-observed-adverse-effect level (NOAEL) was considered to be 2.5 mg/kg/day. Since no apparent changes were observed at this dose, while significant changes in the levels of blood glucose and creatinine were observed after 91-day repeated dosing relative to pre-dosing levels in both male and female dogs of the medium- and high-dose groups. Moreover, levels of creatinine in female dogs showed a trend to increase compared to control animals following repeated exposure to medium- or highdose of BCQB, while blood glucose levels in both sexes of dogs showed a decreasing trend at these dosages compared to control animals after repeated exposure. Although blood glucose levels in the low-dose group tended to be lower than those in the control group after repeated doses, the pre-dosing levels of blood glucose in the low-dose group were also lower relative to those in control group (no significant difference) and the descending amplitude in the low-dose group was not as pronounced as in the medium- or high-dose groups after repeated exposure relative to their initial values (where significant decreases were observed, P < 0.05). In addition, similar changes in blood glucose and creatinine levels

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were also observed in rats in response to repeated dosing, as reported in our previous study (Li et al., 2009). In summary, three major findings are noted in this study. First, there were no significant gender-specific differences in most indexes of subchronic toxicity throughout the experimental period regardless of dosage with the exception of food consumption and body weight. The NOAEL was considered to be 2.5 mg/kg/day, under the present study conditions. Our results suggest a need to closely monitor the blood glucose levels and renal function when BCQB is used in long-term therapy. Second, there was no significant gender-specific difference in the plasma pharmacokinetic parameters of BCQB after either singledose or 91-day intranasal administrations regardless of dosage. Good dose-dependence was observed for the parameters of AUC0–24 and Cmax in the plasma of both male and female dogs, and no significant differences were observed in these two parameters between single dose and 91-day repeated exposure for any dosage in both sexes of dogs. Additionally, lower levels of BCQB residues in most plasma samples after the final administration were observed relative to the levels after the first administration. These results suggest that BCQB does not accumulate in the plasma of dogs. Third, much higher concentrations of BCQB residues were found in most tissues examined at each dosage tested after repeated exposure compared to a single dose, which could be decreased to low levels of BCQB concentrations in these tissues with the exception of the reproductive organs. Since the level of BCQB residues in kidney after 91-day repeated doses was about 10-fold higher than that following a single-dose treatment at each dosage tested, and because BCQB-induced functional changes in kidney have been observed in dogs in the present study, much attention should be paid to the potential influence on renal functions when BCQB is used in long-term therapy. Of course, further studies should be carried out to investigate the possible toxic action of BCQB on this tissue and other tissues. As part of the preclinical safety evaluation of BCQB, these findings not only lay the groundwork for additional studies to further investigate preclinical toxicity associated with chronic BCQB use, but also provide guidance in the design of clinical trials to ensure safety of prolonged BCQB use in clinical therapy. Conflict of interest statement Dr. Xiaoping Chen is the director of Beijing Shiqiao Biological & Pharmaceutical Co. Ltd. The opinions expressed in this article are those of the authors, and not necessarily represent the views of the sponsors. Acknowledgments This work was supported by the Innovation Fund for Technology Based Firms of China’s Science and Technology Ministry (06C26211100623), and was partly funded by Beijing Shiqiao Biological & Pharmaceutical Co. Ltd. The authors express their

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