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Subchronic toxicity and toxicokinetics of LZB, a new proton pump inhibitor, after 13-week repeated oral administration in dogs
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Regulatory Toxicology and Pharmacology 50 (2008) 75–86 www.elsevier.com/locate/yrtph
Subchronic toxicity and toxicokinetics of LZB, a new proton pump inhibitor, after 13-week repeated oral administration in dogs Yu Mao a, Xiaodong Zhang b
a,1
, Li Li b, Bojun Yuan
a,*
, Guocai Lu
a,*
a Center of Evaluation for Drug Safety, Second Military Medical University, Shanghai 200433, China Drug Metabolism Pharmacokinetics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Received 18 June 2007 Available online 25 October 2007
Abstract The subchronic toxicity and toxicokinetics of a novel proton pump inhibitor, pymeprazole (LZB), were investigated in beagle dogs by daily oral administration for 13 consecutive weeks. Three test groups received doses of 30, 100 and 300 mg/kg/day of LZB. Rabeprazole of 60 mg/kg/day was used as positive control. The 13-week repeated oral doses of LZB resulted in objective signs of mild gastrointestinal disturbance for high-dose group animals. One individual dog of high-dose group was found to be lethargy and astasia at the last month of administration; for hematology, mild anemia was observed at high-dose females; for clinical chemistry, higher cholest, trigly and gastrin were observed at high-dose females, higher ASAT, ALAT, cholesterol, triglyceride and gastrin at high-dose males were also observed; for histopathology, the primary effects of LZB were related to gastric mucosa of high-dose group seen by H and E or Grimelius stain. Impairment of surface epithelium was observed by SEM. The treat-related effects basically were reversible for a 4-week drug-free period. As for positive control group, 13-week oral administration of rabeprazole resulted in more severe toxicity than high-dose group of LZB although much lower dose was employed. The accumulation of LZB after 13-week oral administration was not notable at the toxic dose of 300 mg/kg/day. The toxic dose was considered to be 100 mg/kg/day and the no-observed-adverse-effect level (NOAEL) to be 30 mg/kg/day, which is much higher than other PPIs. The toxicological target could be stomach, liver, hematological system and nervous system. 2007 Elsevier Inc. All rights reserved. Keywords: LZB; Subchronic toxicity; Toxicokinetics; 13-week oral administration; Beagle dogs
1. Introduction Proton pump inhibitors (PPIs) are now the mainstay of treatment of peptic ulcers, reflux esophagitis and the Zollinger–Ellison syndrome, and have overtaken histamine-2receptor antagonists due to their superior effectiveness and faster onset of action. Current PPIs are mainly substituted pyridyl methyl sulfinyl benzimidazole or imidazopyridine prodrug that selectively inhibits the gastric acid secretion. After absorption into the circulation, PPIs readily permeate the basolateral parietal cell membrane. When the parietal cell is activated and H+/K+-ATPase is exposed *
1
Corresponding authors. Fax: +86 21 25074386. E-mail address: [email protected] (B. Yuan). Co-first author.
0273-2300/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2007.10.006
in the canaliculi, PPIs are activated by the acid environment. The activated form of the PPIs, sulfenamides, has a reactive sulfhydryl group that bonds with the cysteine residues of H+/K+-ATPase. The covalent bond permanently deactivate the proton pump-associated ATPase. Acid secretion can only occur when new pumps are synthesized (Jai et al., 2006). From the result of toxicity study in animals (Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b), the main toxicity of PPIs were vomit, some clinical parameters changes and stomach lesions. The most common clinical adverse effects associated with PPIs are gastrointestinal disturbances, upper respiratory infection, dizziness, asthenia, back pain, reversible hypergastrinemia, enterochromaffin-like (ECL) cell proliferation, hindrance of nutrition absorption, atrophic gastri-
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Y. Mao et al. / Regulatory Toxicology and Pharmacology 50 (2008) 75–86
Fig. 1. Chemical structure of LZB.
The food and water used in the study was assayed for chemical as well as for microbiological contaminants and the levels were found to be under acceptable limits. Water was supplied ad libitum. Beagle dogs (4/sex/group) with LZB administered orally at dose levels of 0 (control), 30, 100, 300 mg/kg/day and rabeprazole at 60 mg/kg/day (positive control). Half animals were sacrificed after 13-week of treatment, the remained half (2/sex/group) were kept for a 4-week recovery period and then killed.
2.3. Selection of doses
tis, interstitial inflammation of liver, and so on (Valuck and Ruscin, 2004; Marchetti et al., 2003; Bardhan et al., 2005; Gerald et al., 1986; Byrne and Murray, 1999; Feret et al., 1999; Garnett, 1998; Freston, 1997; Fitton and Wiseman, 1996). Deaths have been reported with proton pump inhibitor use (Watson et al., 2005). LZB (recently developed by Research laboratory of Haoseng Pharmaceutical Manufacturing Company, Ltd., Shanghai, China) is a novel reversible proton pump inhibitor. The structure was shown in Fig. 1. It has demonstrated low acute toxicity for dogs and rats and is absorbed quickly following oral administration for rats (unpublished data). LZB is acid labile and white to offwhite powder with a purity of 99.5%, which is made to enteric capsule, and is now being considered for evaluation in phase I clinical trial. The purpose of this study is to determine the subchronic toxicity and toxicokinetics of LZB during 13-week oral administration at doses of 0 (to serve as a control), 30, 100 and 300 mg/kg/day. The toxic effects of LZB and rabeprazole (serve as positive control drug) were also compared. 2. Materials and methods 2.1. Test substance LZB and rabeprazole enteric capsule were supplied by Research Laboratory of Haoseng Pharmaceutical Manufacturing Company, Ltd. Other chemicals were of reagent grade or high-performance liquid chromatographic (HPLC) grade and therefore were used without further purification. LZB and rabeprazole were stored refrigerated at 2–8 C, protected from light and wet. The appropriate amount of LZB enteric capsules required for daily administration was calculated and was adjusted every two weeks based on the most recently recorded body weight. The control animals received empty enteric capsules of the same size and number as those given to the mid-dose group animals. Dogs were dosed daily (6 days per week), at approximately 8:00 AM.
2.2. Animal Twenty male (weighing 6.0–7.5 kg) and 20 female (weighing 5.5– 7.0 kg) purebred beagle dogs of 5.5–6.5 months of age were purchased from ZhongKe experimental animal Co. Ltd. (Suzhou, China). After quarantine and acclimation periods of 30 days, oral studies were performed (when male dogs weighed 7.0–8.5 kg and female dogs weighed 6.0–8.0 kg). Each animal was identified by an ear tattoo as well as kennel number and housed in individual kennel. Room temperature and humidity were monitored continually, with target ranges for room temperature of 20 ± 3 C, relative humidity 30–70% and a photocycle of 12 h. Diet (500 g of dry feed) (Japan Oriental Yeast Company, Tokyo, Japan) was provided to the animals at 10:00 each morning and removed at 15:00.
In the previous acute safety study for LZB in rat and dogs, all animals survived the 2-week observation period following a single oral administration of 5000 mg/kg and 2000 mg/kg in rats and dogs, respectively. No abnormal signs, clinical test parameters, or necropsy findings were observed except that vomit was observed at the dog of 2000 mg/kg at the first day after administration. It can be concluded that LZB has a low order of acute toxicity and that the oral MTD (maximum tolerated dose) >2000 mg/kg for dogs. Otherwise, in a 21-day repeated oral dose pretest (data not shown), slight vomiting were observed in dogs at doses of 300 mg/kg of LZB in both sexes, while severe anorexia, vomiting and diarrhea were observed in positive controls at dose of 100 mg/kg of rabeprazole in both sexes. According to the results of acute toxicity study and pretests, a dose of 300 mg/kg/day of LZB (900 times as rabeprazole’s clinical dose, rabeprazole enteric tablet, 20 mg/day) was selected for the highest dose in this study. Dose of 100 and 30 mg/kg/day (300 and 90 times as rabeprazole’s clinic dose) were selected as middle and low dose, using a common ratio of 3. Dose of 60 mg/kg/day (180 times as rabeprazole’s clinic dose, two times as low-dose of LZB) of rabeprazole was selected as positive control.
2.4. Experimental design The purpose of this study was to determine the subchronic toxicity, and the potential for recovery from any adverse effects, associated with repeated exposure to LZB when administered orally to beagle dogs 6 days per week for a period of 13-week, following an additional 4 weeks to determine the reversibility, persistence, or delayed occurrence of toxic effects. This nonclinical laboratory study was carried out in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification [H] GPT2-1 issued by China Food and Drug Administration on March 2005) and the Organization for Economic Cooperation and Development under the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. This experiment was conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAA LAC), and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996).
2.5. Clinical observations Observations for mortality and viability were recorded from the beginning of the quarantine period. Each animal was examined at least twice daily for any change in behavior, reaction to treatment or ill. Observations included, but were not limited to, changes in skin and fur; eyes and mucous membranes; respiratory, circulatory, autonomic, and central nervous systems; somatic motor system; and behavior patterns. Ophthalmoscopic examinations were performed on each animal every two weeks from the quarantine period to the end of recovery for abnormalities of the eyes, at least 20 min after the instillation of 0.5% tropicamide solution, using a binocular indirect ophthalmoscope. The observation area included the cornea, conjunctiva, sclera, iris, lens and fundus.
2.6. Body weights, weight gain, and food consumption Food consumption and body weight were measured every two weeks. Dose administration was based on the most recent individual body weights.
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2.7. Clinical test parameters In the pre-administration (twice, d01 and d02), after 6-week (d45), 13week (d90) oral administration and 4-week recovery (d120), the followings were checked and measured carefully. (1) Urinanalysis was carried out for items including glucose, pH, specific gravity, protein, bilirubin, ketone, occult blood and sediment, nitrite, and urobilinogen. (2) Hematological examination was performed for the items such as erythrocyte count (RBC), hemoglobin concentration (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (Plat), total leukocyte count (WBC), differential leukocyte count, reticulocyte count (Ret%), and prothrombin time (PT). (3) Biochemical examination was carried out for the items including alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), glucose (Glu), total protein (Prot), albumin (Alb), urea nitrogen (BUN), creatinine (Cr), triglyceride (TG), total cholesterol (Tch), total bilirubin (Tbil), gamma-glutamyl transferase (GGT), calcium (Ca), phosphorus (P), creatine phosphokinase (CPK), lactate dehydrogenase (LDH), chloride (Cl), sodium (Na), potassium (K) and gastrin. (4) Erythrocyte acetylcholinesterase (AChE) was determined according to Whittaker’s method (Whittaker, 1985) with slight modification. (5) Necropsy findings were performed after 13-week treatment and 4-week recovery. (6) Organ weights of brain, hypophysis, heart, liver, spleen, kidneys, adrenal glands, prostate, testes and ovaries were measured. (7) Histopathologic findings were recorded including brain, pituitary gland, thyroid (including the parathyroid), trachea, heart, pancreas, spleen, adrenal glands, testes, prostate, epididymides, ovaries, uterus, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, submaxillary lymph nodes, aorta, eyes, skeletal muscle, sciatic nerve, mammary gland, sternum, salivary glands, abnormal lesion, spinal cord, urinary bladder, lung (including the bronchi), liver, kidneys, stomach, thymus and bone marrow. All results were compared with those of the control group. (8) SEM for gastric mucosa. The scanning electronic microscopy (SEM) analyses were carried out using an accelerating voltage of 20 kV after they were gold sputtered.
2.8. Toxicokinetics Blood samples were obtained in the 1st day, 45th day (6-week) and 89th day (13-week) of administration for plasma LZB level determinations at 0 (1st day), 0.5, 1, 2, 4, 8 and 24 h after dosing. On each occasion, approximately 2.0 ml of blood was drawn from the jugular vein and collected into tubes containing heparin. Blood samples were centrifuged immediately and a 50-ll aliquot of plasma sample was stored in a 80 C freezer until analysis. Each plasma sample (50 ll) was mixed with 10 ll IS (internal standard) (rabeprazole 100 ng/ml) spiking solution, and then extracted with 1000 ll EtOAc (ethyl acetate). After centrifugation at 16,060g, the supernatant (850 ll) was evaporated to dryness at 40 C under a stream of N2. The residue was reconstituted in 250 ll CH3CN/H2O (296:204, v/v, containing 0.02% HCOONH4) and centrifuged, and 60 ll of the resulting supernatant were subjected to LC/MS/MS (liquid chromatography/mass spectrometry) analysis. LZB and IS were separated on a Zorbax Eclipse SB C18 column (50 mm · 2.1 mm, maintained at 25 C; Chadds Ford, PA, USA) with a 0.2 mm pre-column filter (Upchurch Scientific, Oak Harbor, WA, USA). Samples were separated under isocratic conditions, with CH3CN/H2O (296:204, v/v, containing a mass fraction of 0.02% HCOONH4) used as the mobile phase at a flow rate of 0.2 ml/min. A ThermoFinnigan TSQ Quantum triple–quadrupole mass spectrometer (San Jose, CA, USA) was interfaced via an electrospray ionization (ESI) probe with an Agilent 1100 series liquid chromatograph. The Agilent Chemstation and Finnigan Xcalibur software packages were used to control the LC/MS/MS system, as well as for data acquisition and processing. The mass spectrometer was operated in the positive ESI mode with selected reaction monitoring for all the analytes. The instrumental parameters were tuned to maximize generation of the protonated analyte molecules ([M+H]+) and characteristic
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fragment ions. The precursor-to-product ion transitions were monitored at m/z 391 fi 242 for LZB and m/z 360 fi 242 for IS. An on-line motorized six-port divert valve was used to introduce the LC eluent flow into the mass spectrometer over the period of 1.5–3.0 min, with the other eluent flows diverted to waste.
2.9. Statistical analysis Food consumption, body weight, hematological parameters and clinical chemistry data were analyzed by parametric one-way analysis using the F-test (ANOVA, two-sided). If the resulting p-value was <0.05, a comparison of each group with the control group using the Dunnett’s test (twosided) was performed for the hypothesis of equal means. When the data cannot be assumed to follow a normal distribution or homogeneity variance test even if converted, data will be analyzed by non-parametric one-way analysis using the Kruskal–Wallis test (twosided). If the resulting p-value was <0.05, a pairwise comparison of each dose group with the control group was performed using the Mann–Whitney U-test (two-sided for the equal medians).
3. Results 3.1. Test article The test article stored in the original container at 2–8 C was found to be stable for at least 13 weeks. 3.2. Clinical observation All animals survived the scheduled treatment period. Some clinic sign were observed at dose of 300 mg/kg/day of LZB. Transient vomiting and diarrhea were found for some animals at the beginning of administration. Anorexia was observed in both male and female dogs in the last month of administration. CNS (central nervous system) effect of lethargy was found at individual dog from day 72. In addition, high-dose dogs for both sex showed an evident tendency to decrease in body weight gain (Fig. 2) from about 9th week compared with the control dogs and to decrease in food intake (Tables 1 and 2). During the recovery period, the body weight of high-dose group increased, but was still less than controls at the time of sacrifice. No treatment-related ophthalmic changes were observed in any dog of either sex. The positive control shared almost the same symptoms as high-dose group with more severe degree and frequency of vomiting, diarrhea and anorexia across the whole test period. Most of positive control dogs (6/8) were extremely asthenia and bony at the end of administration. No CNS effect was observed at positive control in this study. 3.3. Hematology After 13-week of treatment, female dogs of high-dose had slightly decreased RBC, HB, Hct (Table 3, only results with significant difference were listed). One of the high-dose male dogs had a transient increase in WBC, which considered of incidental and there was no clear dose–response relationship. For positive control, lower RBC, HB and
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Fig. 2. Mean body weight (kg) for male and female dogs after 13 weeks of treatment with LZB. Dogs (4/sex/group) were treated orally with LZB for 13 weeks and then half sacrificed. Table 1 Food intakes of male dogs treated with LZB for 13 weeksa Period
Dose (mg/kg/day) 0
30
100
300
60 (positive)
Day Day Day Day Day Day Day Day Day
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 291 ± 13.3 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 396 ± 69.3 309 ± 57.8 250 ± 80.9 271 ± 90.2 153 ± 83.5 103 ± 77.7
500 ± 0.0 500 ± 0.0 500 ± 0.0 321 ± 73.6 386 ± 81.7 261 ± 76.8 221 ± 70.5 96 ± 84.7 74 ± 88.3
0 1 15 29 43 57 71 85 89
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. a Values are presented as means ± SD.
Hct were observed at male dogs, lower Hct and MCV were observed at female dogs after 13-week of treatment. All parameters come back at the end of the recovery period. 3.4. Clinical chemistry A few changes were observed in clinical chemistry parameters in dogs of high dose (Table 4, only results with
significant difference were listed). After 6-week and 13week administration, increased ALAT, ASAT, Tch, TG and gastrin were observed in males, increased Tch, TG and gastrin were seen in females. All other changes that attained statistical significance were considered not of toxicological significance. For positive control, lower Ca, Prot, Alb was found at male dogs, lower Ca, Prot, Alb and higher Tch, TG were found at female dogs after 13-week
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Table 2 Food intakes of female dogs treated with LZB for 13 weeksa Period
Dose(mg/kg/day) 0
30
100
300
60 (positive)
Day Day Day Day Day Day Day Day Day
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 291 ± 13.3 350 ± 0.0 350 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 450 ± 49.1 380 ± 58.4 352 ± 80.6 250 ± 75.9 221 ± 131.1
500 ± 0.0 500 ± 0.0 500 ± 0.0 306 ± 35.9 278 ± 70.4 189 ± 86.2 153 ± 67.3 96 ± 26.8 77 ± 61.1
0 1 15 29 43 57 71 85 89
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. a Values are presented as means ± SD. Table 3 Hematologic parameters in dogs after 13 weeks of treatment with LZB and 4 weeks recovery Dose (mg/kg/day)
Males
Females
WBC (109/L)
RBC (1012/L)
HCT (1/1 · 100)
RBC (1012/L)
Pretest 0 30 100 300 60 (positive)
13.1 13.2 12.8 14.3 12.6
6.07 6.08 5.69 6.00 5.63
154 145 149 158 145
38.4 37.2 36.4 38.0 0.36
5.56 5.24 5.98 5.77 5.82
6-week 0 30 100 300 60 (positive)
11.4 12.1 10.5 11.9 14.0
6.50 6.46 5.97 5.43 6.86
165 162 148 138 170
41.0 41.1 36.8 35.1 43.1
13-week 0 30 100 300 60 (positive)
13.0 11.8 11.5 14.6* 10.8
6.41 5.92 5.56 5.38 4.24*
160 158 149 137 112*
Recovery 0 30 100 300 60 (positive)
7.4 9.9 8.2 11.4 12.4
8.01 6.72 6.34 6.56 6.10
175 173 160 142 151
HB (g/L)
HCT (1/1 · 100)
MCV (fl)
148 141 144 153 146
35.9 34.3 37.6 38.1 38.0
64.6 65.5 63.1 66.1 65.0
5.94 6.45 5.72 6.43 6.22
154 169 146 153 156
39.3 42.4 36.0 39.9 41.2
66.6 65.7 63.0 62.2 65.2
41.8 40.3 35.0 33.1 28.6*
5.65 6.09 5.96 4.62* 5.25
159 143 135 120* 136
39.1 36.3 37.0 29.1* 29.0*
69.1 59.6 62.5 63.1 55.1*
51.3 44.6 39.2 40.1 39.2
6.18 6.58 6.02 7.08 6.65
175 160 153 178 186
42.7 40.6 38.4 45.0 46.1
69.1 61.7 63.8 63.6 68.4
HB (g/L)
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. * p < 0.05. Data represent the mean values.
of treatment. All of these changes reversed after 4-week recovery period.
0.75 KU/L at day 72, 1.25 KU/L versus 0.83 KU/L at day 90), while there was no difference between two groups after 4-week recovery.
3.5. Erythrocyte acetylcholinesterase (AChE) 3.6. Urinanalysis A kinetic analysis applied on automated biochemical analyzer for Erythrocyte AChE was programmed at day 72 (CNS effect of lethargy was found in one high-dose dog) and day 90 (end of administration period). The results showed that Erythrocyte AChE of high dose was statistically higher than control group (1.08 KU/L versus
No treatment-related changes were observed in urinanalysis parameters during the course of the study. All changes that attained statistical significance were considered to be incidental and there were no clear dose–response relationship (data not shown).
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Table 4 Clinical chemistry parameters in dogs after 13 weeks of treatment with LZB and 4 weeks recovery Dose (mg/kg/day)
ALAT (nmol/s/L)
ASAT (nmol/s/L)
Prot (g/L)
Alb (g/L)
Cholest (mmol/L)
Triglyceride (mmol/L)
Gastrin (ng/L)
Ca (mmol/L)
Males Pretest 0 30 100 300 60 (positive)
328 368 376 399 368
569 564 648 616 646
56.9 55.9 54.2 54.6 56.5
30.3 29.3 29.1 29.1 29.4
3.30 2.81 3.44 3.28 3.01
0.46 0.39 0.49 0.45 0.42
29.2 22.4 29.1 26.7 31.6
2.74 2.67 2.61 2.73 2.80
6-week 0 30 100 300 60 (positive)
335 409 449 517 435
511 616 692 779* 700
56.6 56.4 58.8 56.2 53.6
29.2 29.7 29.1 29.7 27.0
2.86 2.94 3.75 5.56* 3.09
0.48 0.47 0.60 0.80* 0.51
50.4 41.9 43.6 119.9* 121.8*
2.55 2.76 2.69 2.66 2.68
13-week 0 30 100 300 60 (positive)
405 538 455 684* 473
543 694 662 830* 669
64.0 58.8 61.6 56.3 48.6*
31.3 29.4 29.7 30.8 25.3*
3.02 2.83 4.03 5.78* 4.51
0.50 0.42 0.69 0.79* 0.59
26.6 32.1 49.7 90.8* 102.5*
2.61 2.63 2.70 2.72 1.75*
Recovery 0 30 100 300 60 (positive)
360 502 521 377 559
570 631 561 546 674
67.3 61.1 61.4 59.9 63.1
28.5 30.4 30.7 30.1 31.4
3.0 1.96 2.81 4.61 3.11
0.39 0.46 0.47 0.55 0.37
26.1 34.5 40.1 36.4 46.8
2.64 2.54 2.63 2.61 2.61
Cholest (mmol/L)
Triglyceride (mmol/L)
Prot (g/L)
Alb (g/L)
Gastrin (ng/L)
Ca (mmol/L)
Females Pretest 0 30 100 300 60 (positive)
59.1 55.7 55.0 58.4 57.6
31.0 30.4 30.0 30.0 29.4
3.10 3.56 2.83 3.69 3.62
0.53 0.44 0.44 0.55 0.57
31.6 30.4 28.3 34.2 27.6
2.61 2.76 2.90 2.73 2.71
6-week 0 30 100 300 60 (positive)
54.7 55.0 50.7 57.2 53.3
30.2 29.2 27.9 30.0 28.8
2.44 2.87 2.59 4.41* 4.04
0.47 0.51 0.49 0.76* 0.49
34.5 36.5 42.9 121.7* 135.6*
2.61 2.65 2.53 2.66 2.72
13-week 0 30 100 300 60 (positive)
60.4 59.1 55.5 60.2 44.0*
29.7 29.1 27.1 28.1 23.4*
2.64 2.79 2.93 5.81* 4.34*
0.61 0.57 0.60 0.88* 1.08*
30.4 38.1 39.7 85.3* 113.7*
2.54 2.61 2.59 2.63 1.87*
Recovery 0 30 100 300 60 (positive)
65.6 55.0 52.0 63.6 62.8
33.5 29.2 30.8 31.2 32.0
2.71 2.65 3.51 3.33 3.39
0.61 0.45 0.35 0.46 0.53
28.6 31.4 43.1 42.4 51.2
2.64 2.55 2.56 2.58 2.55
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. * p < 0.05. Data represent the mean values.
Y. Mao et al. / Regulatory Toxicology and Pharmacology 50 (2008) 75–86
3.7. Necropsy and organ weights
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day group was seen macroscopically. A few other isolated findings were observed, which were within the range of background lesions or did not show a dose relationship. These changes included congested spleen, thickened
At necropsy after 13-week of treatment, thickened mucus membrane in corpus ventriculi at the 300 mg/kg/
Table 5 Selected histopathological findings in dogs after 13 weeks of treatment with LZB Organ/finding
Dose (mg/kg/d) 0
30
100
300
60 (positive)
M
F
M
F
M
F
M
F
M
F
Lung Focal hemorrhage Chronic bronchitis
0/2 0/2
0/2 0/2
0/2 0/2
0/2 1/2
0/2 0/2
1/2 0/2
0/2 0/2
0/2 0/2
0/2 1/2
0/2 0/2
Liver Fatty change Hydropic degeneration Focal inflammatory cell infiltration Interstitial inflammation
0/2 0/2 0/2 0/2
0/2 0/2 1/2 0/2
0/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
1/2 0/2 0/2 1/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 1/2 0/2
0/2 1/2 0/2 0/2
0/2 0/2 1/2 0/2
Kidney Hydropic degeneration of epithelial cell of proximal convoluted tubule Cortical mineralization Interstitial nephritis
0/2 0/2 0/2
0/2 1/2 0/2
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 0/2
0/2 0/2 1/2
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 1/2
1/2 0/2 0/2
Stomach Karyopycnosis and isolated chief cell necrosis Acidophilic change of chief cell Slight inflammation in lamina propria Proliferation of argyrophilic cells (Grimelius method)
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 1/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 1/2
1/2 1/2 0/2 0/2
1/2 2/2 0/2 1/2
1/2 1/2 0/2 2/2
1/2 1/2 0/2 2/2
2/2 2/2 0/2 2/2
Thymus Atrophy
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
2/2
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. Data represent the number examined (bold) and incidence. Table 6 Selected histopathological findings in dogs after 4-week recovery Organ/finding
Dose mg/kg/d 0
30
100
300
60 (positive)
M
F
M
F
M
F
M
F
M
F
Lung Focal hemorrhage Chronic bronchitis
1/2 0/2
0/2 0/2
1/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
1/2 0/2
Liver Fatty change Hydropic degeneration Focal inflammatory cell infiltration Interstitial inflammation
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
0/2 1/2 0/2 0/2
0/2 0/2 0/2 0/2
Kidney Hydropic degeneration of epithelial cell of proximal convoluted tubule Cortical mineralization Interstitial nephritis
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 0/2
0/2 0/2 0/2
0/2 0/2 0/2
0/2 1/2 0/2
0/2 0/2 1/2
0/2 0/2 0/2
0/2 0/2 1/2
1/2 0/2 0/2
Stomach Karyopycnosis and isolated chief cell necrosis Acidophilic change of chief cell Slight inflammation in lamina propria Proliferation of argyrophilic cells (Grimelius method)
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 0/2 1/2
0/2 1/2 0/2 1/2
1/2 1/2 0/2 1/2
Thymus Atrophy
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
1/2
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. Data represent the number examined (bold) and incidence.
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oviducts, and discoloration of various organs (not listed in Table 5). After 4-week recovery, there were no macroscopic findings that were considered related to previous treatment with LZB. For male dogs treated with 300 mg/kg/day, higher relative weight of liver (35.6 versus 27.4) was observed. For female dogs treated with 300 mg/kg/day compared to controls, higher relative weight of liver (39.0 versus 29.4), kidney (7.0 versus 5.1) and brain (13.8 versus 7.6) were observed. Apparent decrease of both absolute and relative weight of thymus was found at all positive controls (grand mean, absolute weight: 2.3 g, relative weight: 0.04) compared to controls (grand mean, absolute weight: 5.1 g, relative weight: 0.07). No other treatment-related effects on organ weight were found.
• For liver, at 300 mg/kg/day, one each case of hydropic degeneration and focal inflammatory cell infiltrate were found; at 30 mg/kg/day, one case of hydropic degeneration was found; at control, one case of focal inflammatory cell infiltrate was seen; at positive control, one case of focal inflammatory cell infiltrate was seen. • For kidney, at 300 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at 100 mg/kg/day, one case of inter-
3.8. Histopathologic findings After 13-week treatment, histological changes were seen mainly in liver, kidney, lung and stomach (Table 5, only organs or tissues with histological changes were listed). These changes were reversed or showed a tendency to reverse during 4-week drug-free rest period, indicating that the effects of LZB were reversible (Table 6). In male dogs • For lung, one case of chronic bronchitis at positive control was found. • For liver, at 300 mg/kg/day, one case of fatty change was found; at 100 mg/kg/day, one each case of fatty change and interstitial inflammation were found; at positive control, one case of hydropic degeneration was seen. • For kidney, at 100 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at positive control, one each case of interstitial nephritis and hydropic degeneration of epithelial cell of proximal convoluted tubule were observed. • For stomach, at 300 mg/kg/day, one case of karyopycnosis and isolated necrosis of chief cell, one case of proliferation of argyrophilic cells and two case of acidophilic change of chief cell were found; at 100 mg/ kg/day, one each case of karyopycnosis and isolated necrosis of chief cell and proliferation of argyrophilic cells (Grimelius method) were found; at 30 mg/kg/day, one case of slight inflammation in lamina propria was found; at positive control, one case of karyopycnosis and isolated necrosis of chief cell, one case of acidophilic change of chief cell and two case of proliferation of argyrophilic cells were seen. • For thymus, one case of atrophy at positive control was found. In female dogs • For lung, one case of focal hemorrhage at 100 mg/kg/day, one case of chronic bronchitis at 30 mg/kg/day were found.
Fig. 3. SEM photomicrographs for the fundus area of the gastric mucosa from high-dose group (#24, A) and negative control group ($18, B). Note the irregular ‘honeycomb’ structure of the mucosa surface for #24.
Y. Mao et al. / Regulatory Toxicology and Pharmacology 50 (2008) 75–86
These changes were reversed or showed a tendency to reverse during 4-week drug-free rest period, indicating that the effects of LZB were reversible (Table 6). In the bone marrow, hyperplasia of myelocyte or metamyelocyte and hypoplasia of intermediate or late erythroblast were observed in dogs of high dose and positive control (more significant) after 13-week administration. Proliferation of erythrocyte series apparently becomes active 4-week drug-free period later.
3.9. SEM findings The appearance of the mucosa of high-dose dog and control was illustrated in Fig. 3. Apparently for high-dose dog (Fig. 3A), part of the surface epithelium built with mucous cell had been necrosis and some mucous cell ablated. This region can be typically characterized by ‘honeycomb’ pattern. This ‘honeycomb’ pattern covered the fundus area. For control dog (Fig. 3B), the columnar mucous cell was well-regulated and lines the gastric pit, and no vacuolation or necrosis were observed.
20.000 30mg/kg 100mg/kg
15.000
AUC/Dose
stitial nephritis was found; at 30 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at control, one case of cortical mineralization was seen; at positive control, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was seen. • For stomach, at 300 mg/kg/day, one case of karyopycnosis and isolated necrosis of chief cell, one case of acidophilic change of chief cell and two case of proliferation of argyrophilic cells were found; at 100 mg/kg/day, one each case of karyopycnosis and isolated necrosis of chief cell and acidophilic change of chief cell were found; at positive control, two case of karyopycnosis and isolated necrosis of chief cell and two case of proliferation of argyrophilic cells were seen. • For thymus, two case of atrophy at positive control were also found.
83
300mg/kg 10.000
5.000
0.000 single
6-week
13-week
Fig. 5. Dose normalized plots for AUC.
Fig. 4. Plasma level-time profiles of LZB in beagle dogs with dose of 30, 100, 300 mg/kg. Blood samples were obtained in the 1st day, 45th day and 89th day of administration at 0 (1st day), 0.5, 1, 2, 4, 8 and 24 h after dosing.
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Table 7 Mean (±SD) toxicokinetic parameters of LZB after oral administration in doses of 30, 100, and 300 mg/kg/day to beagle dogs (n = 4/sex/group) Dose (mg/kg/day) Grand mean 30 (n = 8)
Duration of administration
AUC0fi24 h (lg · h/ml)
Cmax (lg/ml)
Tmax (h)
Single 6-week 13-week
179.7 ± 7.03 207.7 ± 37.2 225.2 ± 52.5
44.3 ± 10.5 29.7 ± 10.1 38.7 ± 39.6
2±0 4±0 5±2
100 (n = 8)
Single 6-week 13-week
740.9 ± 341.1 758.9 ± 273.9 558.5 ± 294.0
54.1 ± 28.2 64.4 ± 66.4 128.4 ± 30.9
4±0 2±1 2±1
300 (n = 8)
Single 6-week 13-week
1504.8 ± 184.9 1069.5 ± 492.5 1062.3 ± 240.5
181.5 ± 17.4 214.6 ± 82.9 93.8 ± 35.3
4±0 3±1 4±2
3.10. Toxicokinetics Fig. 4 shows the plasma level-time profiles of LZB in the beagle dogs. Dose normalized plots was shown in Fig. 5. Relevant pharmacokinetic parameters are listed in Table 7. There were no significant differences in pharmacokinetic parameters of LZB between male and female dogs during 13-week oral administration for three different doses. Therefore, we combined the pharmacokinetic parameters from male and female dogs together. The plasma concentration of LZB on the 1st, 45th and 89th day showed a dose dependent, linear increase with maximum plasma values observed at about 2–4 h after treatment. There were considerable inter-individual differences in the plasma concentrations. At the 100 and 300 mg/kg/day dose level, lower exposures were noted on Day 45 and Day 89 as compared to Day 1 (Table 7). 4. Discussion The daily administration of LZB orally by enteric capsule at dose levels of 0, 30, 100 or 300 mg/kg/day to beagle dogs in a 13-week study was well tolerated and no mortality was observed. Most of the toxic effects were found at high dose and positive control group. Gastro intestinal disturbances always associated with PPIs (Marchetti et al., 2003; Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b) were not notable ever for high-dose group in this study. Some intrinsically associated symptoms for high-dose group were observed, such as decreased body weight gain and food intake, decreased RBC, Hb and HCT, hypoplasia of erythrocyte series in bone marrow, histopathlogical findings of acidophilic change and karyopycnosis or isolated necrosis of chief cell, surface epithelium necrosis and some mucous cells ablate. Theoretically, gastric acid can activate pepsinogen, and provide proper environment for absorption of nutriments. Long terms of acid suppression with high dose might injure the gastric mucosa and disturb the secreting function of chief cell (Kakei et al., 1993), accordingly inducing disorder of nutriments absorption (Koop, 1992; Stewart et al., 1995), hypothrepsia or even hemophthisis, and histopath-
logical change of cells in gastric glands. These changes came back at the end of the recovery period, suggesting that these by-effects of LZB were reversible. CNS effect of lethargy and astasia were found at individual dog of high-dose group after 72 days administration. While there were no histopathlogical findings associated with skeletal muscle and sciatic nerve. The result of Erythrocyte AChE showed that there was significant difference between high-dose and control group and recovered after 4-week rest period. So the effect of LZB on nerves system may be related to cholinergic nerve. CNS by-effect can be found in some cases from preclinical and clinical test (Arnold, 1994), or in dispensatory of some commercially available PPIs. What could be the molecular action mechanism and if LZB will exhibit CNS effect on human remain questions for further research and clinical trial. Increased ALAT, ASAT and higher relative liver weight were observed at high-dose dogs. It cannot be excluded to be related to the treatment. However, these changes did not accompanied by changes in hepatic histopathology. Higher relative kidney weight was also found at high-dose dogs with no changes in renal histopathology and in urinary or clinical chemistry parameters indicative of renal toxicity. Although there was case report implicating PPI in acute interstitial nephritis (AIN) and progression to acute renal failure (ARF) (Nimeshan et al., 2006), in this study the changes in relative kidney weights were considered of no toxicological significance. Increased cholesterol and triglyceride were observed at high dose, which may relate to abnormal fat metabolism due to acid suppression. Serum gastrin concentration of both sexes at high dose was significantly higher than control after 13-week treatment, which induce proliferation of argyrophilic cells in gastric fundus (seen by Grimelius stain) due to the trophic action of high gastrin level. PPIs, like H2-receptor antagonists, are associated with gastric carcinoid tumors in rats (Ekman et al., 1985), raising questions about the safety of these drugs. Both omeprazole and lansoprazole cause reversible hypergastrinemia. Serum gastrin levels increased to two to four times baseline levels during omeprazole therapy (Tielemans et al., 1989) and 1.6- to 2-fold
Y. Mao et al. / Regulatory Toxicology and Pharmacology 50 (2008) 75–86
increase was seen with lansoprazole (Misawa et al., 1991; Ogoshi et al., 1991). Enterochromaffin cells, the precursors of carcinoids, are hypertrophied in humans receiving PPIs. However, no carcinoids have been observed during PPI therapy. Gastrin levels and cellular hypertrophy are reversed when PPI therapy is stopped (Larsson et al., 1988). In this study, no carcinoid tumor was found among all groups, and gastrin levels and cellular hypertrophy were also reversible. Refer to the positive control, more severe vomiting and diarrhea compared to high-dose group was observed during the administration period. Dogs of both sex were anorexia and extremely feeble and emaciation at the last month of administration; the similar symptoms including body weight, food intake, hematological changes, high serum gastrin level and histopathological changes of gastric mucosa also can be found in positive controls with more severe degree than high-dose group; clinical chemistry parameters like protein, Alb and Ca were significantly lower than control, which had not been seen in LZB groups. It can be contributed to nutriments deficiency due to extremely severe anorexia and frequent vomit or diarrhea; in bone marrow, the hypoplasia of erythrocyte series was more significant than high-dose group; beside the liver, kidney and brain weight change, apparent atrophy of thymus could be observed. Considering the dose difference of LZB and rabeprazole (see Section 1), in this study oral administration of LZB appeared more toleration than that of rabeprazole. Systemic exposure to LZB was indicated by the plasma AUC0–24 h and Cmax over the 24 h dosing interval, which were proportional to the dose and no saturation was observed. After 6-week and 13-week of oral administration, the AUC0–24 h values at all doses were not significantly greater than those on the day 1, which suggests that LZB is not accumulated (Fig. 5). No non-linear kinetics of LZB was observed. From the result of this work and literature report, we can find there are both difference and similarity between toxicity of LZB and other PPIs. In a 13-week beagle dog study of tenatoprazol (TU-199), GS disturbance like vomit was seen in high-dose group. High urea nitrogen and serum gastrin concentration were found. Histological changes were seen in the stomach (hypertrophy and fibrosis of the mucous membrane; increase in parietal cell; partial epithelial necrosis in the fundic glands) and thyroid (not observed in this work) (Okamoto et al., 1998a,b). The reason why LZB could be different from the others are still remaining unknown. It need further research concerning structure–effect relationship and mechanism of action. Based on the absence of treat-related toxicologically changes, the no-observed-adverse-effect level (NOAEL) was considered to be at least 30 mg/kg/day and the toxic dose to be 100 mg/kg/day, which was much higher than other PPIs (Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b). The pharmacodynamic action of LZB for rats was proved to be
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substantially equivalent to PPIs like omeprazole, rabeprazole or tenatoprazole (unpublished data). From the safety vs. efficacy point of view, LZB could be a potential new PPI for treatment of GI disorder. References Abe, T., Nishida, N., Orita, S., Miyajima, H., 1990. Thirteen-week oral toxicity study of lansoprazole (AG-1749) in beagle dogs. Jpn. Pharmacol. Ther. 18 (suppl. 10), 25. ACIPHEX (rabeprazole sodium), Delayed-Release Tablets, NDA20973, March 5, 1999. Arnold, R., 1994. Safety of proton pump inhibitors an over view. Aliment. Pharmacol. Ther. 8, 65–70. Bardhan, K.D., Bishop, A.E., Polak, J.M., et al., 2005. Pantoprazole in severe acid-peptic disease: the effectiveness and safety of 5 years’ continuous treatment. Dig. Liver Dis. 37 (1), 10–22. Byrne, M.F., Murray, F.E., 1999. Formulary management of proton pump inhibitors. Pharmacoeconomics 16, 225–246. Ekman, L., Hansson, E., et al., 1985. Toxicologic studies on omeprazole. Scand. J. Gastroenterol. 20, 53–69. Feret, B., Quercia, R.A., Cappa, J., 1999. Rabeprazole: a proton pump inhibitor for the treatment of acid-related disorders. Formulary 34, 313–323. Fitton, A., Wiseman, L., 1996. Pantoprazole. A review of its pharmacological properties and therapeutic use in acid-related disorders. Drugs 51, 460–482. Freston, J.W., 1997. Long-term acid control and proton pump inhibitors: interactions and safety issues in perspective. Am. J. Gastroenterol. 92, 51S–57S. Garnett, W.R., 1998. Considerations for long-term use of proton-pump inhibitors. Am. J. Health Syst. Pharm. 55, 2268–2279. Gerald, M.L., Henry, W.S., Phillip, R., 1986. Omeprazole-induced hypergastrinemia: role of gastric acidity. J. Surg. Res. 40 (5), 504– 509. Jai, M.S., Michel, H., Florence, D., Herve, F., George, S., 2006. Characterization of the inhibitory activity of tenatoprazole on the gastric H+,K+-ATPase in vitro and in vivo. Biochem. Pharmacol. 71, 837–849. Kakei, N., Ichinose, M., Tsukada, S., et al., 1993. Omeprazole, a proton pump inhibitor, reduces the secretion, synthesis and gene expression of pepsinogen in the rat stomach. Biochem. Biophys. Res. Commun. 195 (2), 997–1004. Koop, H., 1992. Review article: metabolic consequences of long-term inhibition of acid secretion by omeprazole. Aliment. Pharmacol. Ther. 6, 399. Larsson, H., Carlsson, E., Hakanson, R., et al., 1988. Time-course of development and reversal of gastric endocrine cell hyperplasia after inhibition of acid secretion. Gastroenterology 95, 1477– 1486. Marchetti, F., Gerarduzzi, T., Ventura, A., 2003. Proton pump inhibitors in children: a review. Dig. Liver Dis. 35 (10), 738–746. Misawa, T., Chijiiwa, Y., Imazono, Y., et al., 1991. Effects of lansoprazole, a gastric proton pump inhibitor, on endocrine function and healing in patients with peptic ulcer. Ther. Res. 122, 175–189. National Research Council (NRC), 1996. Guide for the Care and Use of Laboratory Animals. National Research Council, National Academy of Sciences, Washington, DC. Nimeshan, G., Patrick, L.C., Angela, C.W., Simon, D.R., 2006. Proton pump inhibitors and acute interstitial nephritis. Clin. Gastroenterol. Hepatol. 4 (5), 597–604. Ogoshi, K., Kato, T., Saito, S., et al., 1991. Clinical study of AG-1749 (lansoprazole): effects serum gastrin levels and gastric mucosal ECL cell density. Yakuri Chiryo 19, 933–946. Okamoto, M. et al., 1998a. Thirteen-week oral toxicity study followed by five-week recovery study of TU-199 in beagle dogs. Jpn. Pharmacol. Ther. 26 (12), 1923–1949.
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Okamoto, M. et al., 1998b. Thirteen-week oral toxicity study followed by five-week recovery study of TU-199 in rats. Jpn. Pharmacol. Ther. 26 (12), 1897–1921. Stewart, C.A., Termanini, B., Gibril, F., et al., 1995. Prospective study of the effect of long-term gastric acid antisecretory treatment on serum vitamin B12 levels in patients with Zollinger–Ellison syndrome. Gastroenterology 108 (Suppl. 4), A226. Tielemans, Y., Hakanson, R., Sundler, F., Willems, G., 1989. Proliferation of enterochromaffin-like cells in omeprazole-treated hypergastrinemic rats. Gastroenterology 96, 723–729.
Valuck, R.J., Ruscin, J.M., 2004. A case-control study on adverse effects: H2 blocker or proton pump inhibitor use and risk of vitamin B12 deficiency in older adults. J. Clin. Epidemiol. 57 (4), 422–428. Watson, W.A., Litovitz, T.L., Rodgers, G.C., Klein-Schwartz, W., Reid, N., Youniss, J., Flanagan, A., Wruk, K.M., 2005. 2004 Annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am. J. Emerg. Med. 23, 589–666. Whittaker, M., 1985. Cholinesterase. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. IV, third ed. Verlag Chemie, WeinheimDeerfield Beach, Florida-Basel, pp. 57–62.
Regulatory Toxicology and Pharmacology 50 (2008) 75–86 www.elsevier.com/locate/yrtph
Subchronic toxicity and toxicokinetics of LZB, a new proton pump inhibitor, after 13-week repeated oral administration in dogs Yu Mao a, Xiaodong Zhang b
a,1
, Li Li b, Bojun Yuan
a,*
, Guocai Lu
a,*
a Center of Evaluation for Drug Safety, Second Military Medical University, Shanghai 200433, China Drug Metabolism Pharmacokinetics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Received 18 June 2007 Available online 25 October 2007
Abstract The subchronic toxicity and toxicokinetics of a novel proton pump inhibitor, pymeprazole (LZB), were investigated in beagle dogs by daily oral administration for 13 consecutive weeks. Three test groups received doses of 30, 100 and 300 mg/kg/day of LZB. Rabeprazole of 60 mg/kg/day was used as positive control. The 13-week repeated oral doses of LZB resulted in objective signs of mild gastrointestinal disturbance for high-dose group animals. One individual dog of high-dose group was found to be lethargy and astasia at the last month of administration; for hematology, mild anemia was observed at high-dose females; for clinical chemistry, higher cholest, trigly and gastrin were observed at high-dose females, higher ASAT, ALAT, cholesterol, triglyceride and gastrin at high-dose males were also observed; for histopathology, the primary effects of LZB were related to gastric mucosa of high-dose group seen by H and E or Grimelius stain. Impairment of surface epithelium was observed by SEM. The treat-related effects basically were reversible for a 4-week drug-free period. As for positive control group, 13-week oral administration of rabeprazole resulted in more severe toxicity than high-dose group of LZB although much lower dose was employed. The accumulation of LZB after 13-week oral administration was not notable at the toxic dose of 300 mg/kg/day. The toxic dose was considered to be 100 mg/kg/day and the no-observed-adverse-effect level (NOAEL) to be 30 mg/kg/day, which is much higher than other PPIs. The toxicological target could be stomach, liver, hematological system and nervous system. 2007 Elsevier Inc. All rights reserved. Keywords: LZB; Subchronic toxicity; Toxicokinetics; 13-week oral administration; Beagle dogs
1. Introduction Proton pump inhibitors (PPIs) are now the mainstay of treatment of peptic ulcers, reflux esophagitis and the Zollinger–Ellison syndrome, and have overtaken histamine-2receptor antagonists due to their superior effectiveness and faster onset of action. Current PPIs are mainly substituted pyridyl methyl sulfinyl benzimidazole or imidazopyridine prodrug that selectively inhibits the gastric acid secretion. After absorption into the circulation, PPIs readily permeate the basolateral parietal cell membrane. When the parietal cell is activated and H+/K+-ATPase is exposed *
1
Corresponding authors. Fax: +86 21 25074386. E-mail address: [email protected] (B. Yuan). Co-first author.
0273-2300/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2007.10.006
in the canaliculi, PPIs are activated by the acid environment. The activated form of the PPIs, sulfenamides, has a reactive sulfhydryl group that bonds with the cysteine residues of H+/K+-ATPase. The covalent bond permanently deactivate the proton pump-associated ATPase. Acid secretion can only occur when new pumps are synthesized (Jai et al., 2006). From the result of toxicity study in animals (Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b), the main toxicity of PPIs were vomit, some clinical parameters changes and stomach lesions. The most common clinical adverse effects associated with PPIs are gastrointestinal disturbances, upper respiratory infection, dizziness, asthenia, back pain, reversible hypergastrinemia, enterochromaffin-like (ECL) cell proliferation, hindrance of nutrition absorption, atrophic gastri-
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Fig. 1. Chemical structure of LZB.
The food and water used in the study was assayed for chemical as well as for microbiological contaminants and the levels were found to be under acceptable limits. Water was supplied ad libitum. Beagle dogs (4/sex/group) with LZB administered orally at dose levels of 0 (control), 30, 100, 300 mg/kg/day and rabeprazole at 60 mg/kg/day (positive control). Half animals were sacrificed after 13-week of treatment, the remained half (2/sex/group) were kept for a 4-week recovery period and then killed.
2.3. Selection of doses
tis, interstitial inflammation of liver, and so on (Valuck and Ruscin, 2004; Marchetti et al., 2003; Bardhan et al., 2005; Gerald et al., 1986; Byrne and Murray, 1999; Feret et al., 1999; Garnett, 1998; Freston, 1997; Fitton and Wiseman, 1996). Deaths have been reported with proton pump inhibitor use (Watson et al., 2005). LZB (recently developed by Research laboratory of Haoseng Pharmaceutical Manufacturing Company, Ltd., Shanghai, China) is a novel reversible proton pump inhibitor. The structure was shown in Fig. 1. It has demonstrated low acute toxicity for dogs and rats and is absorbed quickly following oral administration for rats (unpublished data). LZB is acid labile and white to offwhite powder with a purity of 99.5%, which is made to enteric capsule, and is now being considered for evaluation in phase I clinical trial. The purpose of this study is to determine the subchronic toxicity and toxicokinetics of LZB during 13-week oral administration at doses of 0 (to serve as a control), 30, 100 and 300 mg/kg/day. The toxic effects of LZB and rabeprazole (serve as positive control drug) were also compared. 2. Materials and methods 2.1. Test substance LZB and rabeprazole enteric capsule were supplied by Research Laboratory of Haoseng Pharmaceutical Manufacturing Company, Ltd. Other chemicals were of reagent grade or high-performance liquid chromatographic (HPLC) grade and therefore were used without further purification. LZB and rabeprazole were stored refrigerated at 2–8 C, protected from light and wet. The appropriate amount of LZB enteric capsules required for daily administration was calculated and was adjusted every two weeks based on the most recently recorded body weight. The control animals received empty enteric capsules of the same size and number as those given to the mid-dose group animals. Dogs were dosed daily (6 days per week), at approximately 8:00 AM.
2.2. Animal Twenty male (weighing 6.0–7.5 kg) and 20 female (weighing 5.5– 7.0 kg) purebred beagle dogs of 5.5–6.5 months of age were purchased from ZhongKe experimental animal Co. Ltd. (Suzhou, China). After quarantine and acclimation periods of 30 days, oral studies were performed (when male dogs weighed 7.0–8.5 kg and female dogs weighed 6.0–8.0 kg). Each animal was identified by an ear tattoo as well as kennel number and housed in individual kennel. Room temperature and humidity were monitored continually, with target ranges for room temperature of 20 ± 3 C, relative humidity 30–70% and a photocycle of 12 h. Diet (500 g of dry feed) (Japan Oriental Yeast Company, Tokyo, Japan) was provided to the animals at 10:00 each morning and removed at 15:00.
In the previous acute safety study for LZB in rat and dogs, all animals survived the 2-week observation period following a single oral administration of 5000 mg/kg and 2000 mg/kg in rats and dogs, respectively. No abnormal signs, clinical test parameters, or necropsy findings were observed except that vomit was observed at the dog of 2000 mg/kg at the first day after administration. It can be concluded that LZB has a low order of acute toxicity and that the oral MTD (maximum tolerated dose) >2000 mg/kg for dogs. Otherwise, in a 21-day repeated oral dose pretest (data not shown), slight vomiting were observed in dogs at doses of 300 mg/kg of LZB in both sexes, while severe anorexia, vomiting and diarrhea were observed in positive controls at dose of 100 mg/kg of rabeprazole in both sexes. According to the results of acute toxicity study and pretests, a dose of 300 mg/kg/day of LZB (900 times as rabeprazole’s clinical dose, rabeprazole enteric tablet, 20 mg/day) was selected for the highest dose in this study. Dose of 100 and 30 mg/kg/day (300 and 90 times as rabeprazole’s clinic dose) were selected as middle and low dose, using a common ratio of 3. Dose of 60 mg/kg/day (180 times as rabeprazole’s clinic dose, two times as low-dose of LZB) of rabeprazole was selected as positive control.
2.4. Experimental design The purpose of this study was to determine the subchronic toxicity, and the potential for recovery from any adverse effects, associated with repeated exposure to LZB when administered orally to beagle dogs 6 days per week for a period of 13-week, following an additional 4 weeks to determine the reversibility, persistence, or delayed occurrence of toxic effects. This nonclinical laboratory study was carried out in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification [H] GPT2-1 issued by China Food and Drug Administration on March 2005) and the Organization for Economic Cooperation and Development under the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. This experiment was conducted in facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAA LAC), and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996).
2.5. Clinical observations Observations for mortality and viability were recorded from the beginning of the quarantine period. Each animal was examined at least twice daily for any change in behavior, reaction to treatment or ill. Observations included, but were not limited to, changes in skin and fur; eyes and mucous membranes; respiratory, circulatory, autonomic, and central nervous systems; somatic motor system; and behavior patterns. Ophthalmoscopic examinations were performed on each animal every two weeks from the quarantine period to the end of recovery for abnormalities of the eyes, at least 20 min after the instillation of 0.5% tropicamide solution, using a binocular indirect ophthalmoscope. The observation area included the cornea, conjunctiva, sclera, iris, lens and fundus.
2.6. Body weights, weight gain, and food consumption Food consumption and body weight were measured every two weeks. Dose administration was based on the most recent individual body weights.
Y. Mao et al. / Regulatory Toxicology and Pharmacology 50 (2008) 75–86
2.7. Clinical test parameters In the pre-administration (twice, d01 and d02), after 6-week (d45), 13week (d90) oral administration and 4-week recovery (d120), the followings were checked and measured carefully. (1) Urinanalysis was carried out for items including glucose, pH, specific gravity, protein, bilirubin, ketone, occult blood and sediment, nitrite, and urobilinogen. (2) Hematological examination was performed for the items such as erythrocyte count (RBC), hemoglobin concentration (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (Plat), total leukocyte count (WBC), differential leukocyte count, reticulocyte count (Ret%), and prothrombin time (PT). (3) Biochemical examination was carried out for the items including alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), glucose (Glu), total protein (Prot), albumin (Alb), urea nitrogen (BUN), creatinine (Cr), triglyceride (TG), total cholesterol (Tch), total bilirubin (Tbil), gamma-glutamyl transferase (GGT), calcium (Ca), phosphorus (P), creatine phosphokinase (CPK), lactate dehydrogenase (LDH), chloride (Cl), sodium (Na), potassium (K) and gastrin. (4) Erythrocyte acetylcholinesterase (AChE) was determined according to Whittaker’s method (Whittaker, 1985) with slight modification. (5) Necropsy findings were performed after 13-week treatment and 4-week recovery. (6) Organ weights of brain, hypophysis, heart, liver, spleen, kidneys, adrenal glands, prostate, testes and ovaries were measured. (7) Histopathologic findings were recorded including brain, pituitary gland, thyroid (including the parathyroid), trachea, heart, pancreas, spleen, adrenal glands, testes, prostate, epididymides, ovaries, uterus, esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, mesenteric lymph nodes, submaxillary lymph nodes, aorta, eyes, skeletal muscle, sciatic nerve, mammary gland, sternum, salivary glands, abnormal lesion, spinal cord, urinary bladder, lung (including the bronchi), liver, kidneys, stomach, thymus and bone marrow. All results were compared with those of the control group. (8) SEM for gastric mucosa. The scanning electronic microscopy (SEM) analyses were carried out using an accelerating voltage of 20 kV after they were gold sputtered.
2.8. Toxicokinetics Blood samples were obtained in the 1st day, 45th day (6-week) and 89th day (13-week) of administration for plasma LZB level determinations at 0 (1st day), 0.5, 1, 2, 4, 8 and 24 h after dosing. On each occasion, approximately 2.0 ml of blood was drawn from the jugular vein and collected into tubes containing heparin. Blood samples were centrifuged immediately and a 50-ll aliquot of plasma sample was stored in a 80 C freezer until analysis. Each plasma sample (50 ll) was mixed with 10 ll IS (internal standard) (rabeprazole 100 ng/ml) spiking solution, and then extracted with 1000 ll EtOAc (ethyl acetate). After centrifugation at 16,060g, the supernatant (850 ll) was evaporated to dryness at 40 C under a stream of N2. The residue was reconstituted in 250 ll CH3CN/H2O (296:204, v/v, containing 0.02% HCOONH4) and centrifuged, and 60 ll of the resulting supernatant were subjected to LC/MS/MS (liquid chromatography/mass spectrometry) analysis. LZB and IS were separated on a Zorbax Eclipse SB C18 column (50 mm · 2.1 mm, maintained at 25 C; Chadds Ford, PA, USA) with a 0.2 mm pre-column filter (Upchurch Scientific, Oak Harbor, WA, USA). Samples were separated under isocratic conditions, with CH3CN/H2O (296:204, v/v, containing a mass fraction of 0.02% HCOONH4) used as the mobile phase at a flow rate of 0.2 ml/min. A ThermoFinnigan TSQ Quantum triple–quadrupole mass spectrometer (San Jose, CA, USA) was interfaced via an electrospray ionization (ESI) probe with an Agilent 1100 series liquid chromatograph. The Agilent Chemstation and Finnigan Xcalibur software packages were used to control the LC/MS/MS system, as well as for data acquisition and processing. The mass spectrometer was operated in the positive ESI mode with selected reaction monitoring for all the analytes. The instrumental parameters were tuned to maximize generation of the protonated analyte molecules ([M+H]+) and characteristic
77
fragment ions. The precursor-to-product ion transitions were monitored at m/z 391 fi 242 for LZB and m/z 360 fi 242 for IS. An on-line motorized six-port divert valve was used to introduce the LC eluent flow into the mass spectrometer over the period of 1.5–3.0 min, with the other eluent flows diverted to waste.
2.9. Statistical analysis Food consumption, body weight, hematological parameters and clinical chemistry data were analyzed by parametric one-way analysis using the F-test (ANOVA, two-sided). If the resulting p-value was <0.05, a comparison of each group with the control group using the Dunnett’s test (twosided) was performed for the hypothesis of equal means. When the data cannot be assumed to follow a normal distribution or homogeneity variance test even if converted, data will be analyzed by non-parametric one-way analysis using the Kruskal–Wallis test (twosided). If the resulting p-value was <0.05, a pairwise comparison of each dose group with the control group was performed using the Mann–Whitney U-test (two-sided for the equal medians).
3. Results 3.1. Test article The test article stored in the original container at 2–8 C was found to be stable for at least 13 weeks. 3.2. Clinical observation All animals survived the scheduled treatment period. Some clinic sign were observed at dose of 300 mg/kg/day of LZB. Transient vomiting and diarrhea were found for some animals at the beginning of administration. Anorexia was observed in both male and female dogs in the last month of administration. CNS (central nervous system) effect of lethargy was found at individual dog from day 72. In addition, high-dose dogs for both sex showed an evident tendency to decrease in body weight gain (Fig. 2) from about 9th week compared with the control dogs and to decrease in food intake (Tables 1 and 2). During the recovery period, the body weight of high-dose group increased, but was still less than controls at the time of sacrifice. No treatment-related ophthalmic changes were observed in any dog of either sex. The positive control shared almost the same symptoms as high-dose group with more severe degree and frequency of vomiting, diarrhea and anorexia across the whole test period. Most of positive control dogs (6/8) were extremely asthenia and bony at the end of administration. No CNS effect was observed at positive control in this study. 3.3. Hematology After 13-week of treatment, female dogs of high-dose had slightly decreased RBC, HB, Hct (Table 3, only results with significant difference were listed). One of the high-dose male dogs had a transient increase in WBC, which considered of incidental and there was no clear dose–response relationship. For positive control, lower RBC, HB and
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Fig. 2. Mean body weight (kg) for male and female dogs after 13 weeks of treatment with LZB. Dogs (4/sex/group) were treated orally with LZB for 13 weeks and then half sacrificed. Table 1 Food intakes of male dogs treated with LZB for 13 weeksa Period
Dose (mg/kg/day) 0
30
100
300
60 (positive)
Day Day Day Day Day Day Day Day Day
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 291 ± 13.3 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 396 ± 69.3 309 ± 57.8 250 ± 80.9 271 ± 90.2 153 ± 83.5 103 ± 77.7
500 ± 0.0 500 ± 0.0 500 ± 0.0 321 ± 73.6 386 ± 81.7 261 ± 76.8 221 ± 70.5 96 ± 84.7 74 ± 88.3
0 1 15 29 43 57 71 85 89
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. a Values are presented as means ± SD.
Hct were observed at male dogs, lower Hct and MCV were observed at female dogs after 13-week of treatment. All parameters come back at the end of the recovery period. 3.4. Clinical chemistry A few changes were observed in clinical chemistry parameters in dogs of high dose (Table 4, only results with
significant difference were listed). After 6-week and 13week administration, increased ALAT, ASAT, Tch, TG and gastrin were observed in males, increased Tch, TG and gastrin were seen in females. All other changes that attained statistical significance were considered not of toxicological significance. For positive control, lower Ca, Prot, Alb was found at male dogs, lower Ca, Prot, Alb and higher Tch, TG were found at female dogs after 13-week
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Table 2 Food intakes of female dogs treated with LZB for 13 weeksa Period
Dose(mg/kg/day) 0
30
100
300
60 (positive)
Day Day Day Day Day Day Day Day Day
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 291 ± 13.3 350 ± 0.0 350 ± 0.0
500 ± 0.0 500 ± 0.0 500 ± 0.0 500 ± 0.0 450 ± 49.1 380 ± 58.4 352 ± 80.6 250 ± 75.9 221 ± 131.1
500 ± 0.0 500 ± 0.0 500 ± 0.0 306 ± 35.9 278 ± 70.4 189 ± 86.2 153 ± 67.3 96 ± 26.8 77 ± 61.1
0 1 15 29 43 57 71 85 89
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. a Values are presented as means ± SD. Table 3 Hematologic parameters in dogs after 13 weeks of treatment with LZB and 4 weeks recovery Dose (mg/kg/day)
Males
Females
WBC (109/L)
RBC (1012/L)
HCT (1/1 · 100)
RBC (1012/L)
Pretest 0 30 100 300 60 (positive)
13.1 13.2 12.8 14.3 12.6
6.07 6.08 5.69 6.00 5.63
154 145 149 158 145
38.4 37.2 36.4 38.0 0.36
5.56 5.24 5.98 5.77 5.82
6-week 0 30 100 300 60 (positive)
11.4 12.1 10.5 11.9 14.0
6.50 6.46 5.97 5.43 6.86
165 162 148 138 170
41.0 41.1 36.8 35.1 43.1
13-week 0 30 100 300 60 (positive)
13.0 11.8 11.5 14.6* 10.8
6.41 5.92 5.56 5.38 4.24*
160 158 149 137 112*
Recovery 0 30 100 300 60 (positive)
7.4 9.9 8.2 11.4 12.4
8.01 6.72 6.34 6.56 6.10
175 173 160 142 151
HB (g/L)
HCT (1/1 · 100)
MCV (fl)
148 141 144 153 146
35.9 34.3 37.6 38.1 38.0
64.6 65.5 63.1 66.1 65.0
5.94 6.45 5.72 6.43 6.22
154 169 146 153 156
39.3 42.4 36.0 39.9 41.2
66.6 65.7 63.0 62.2 65.2
41.8 40.3 35.0 33.1 28.6*
5.65 6.09 5.96 4.62* 5.25
159 143 135 120* 136
39.1 36.3 37.0 29.1* 29.0*
69.1 59.6 62.5 63.1 55.1*
51.3 44.6 39.2 40.1 39.2
6.18 6.58 6.02 7.08 6.65
175 160 153 178 186
42.7 40.6 38.4 45.0 46.1
69.1 61.7 63.8 63.6 68.4
HB (g/L)
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. * p < 0.05. Data represent the mean values.
of treatment. All of these changes reversed after 4-week recovery period.
0.75 KU/L at day 72, 1.25 KU/L versus 0.83 KU/L at day 90), while there was no difference between two groups after 4-week recovery.
3.5. Erythrocyte acetylcholinesterase (AChE) 3.6. Urinanalysis A kinetic analysis applied on automated biochemical analyzer for Erythrocyte AChE was programmed at day 72 (CNS effect of lethargy was found in one high-dose dog) and day 90 (end of administration period). The results showed that Erythrocyte AChE of high dose was statistically higher than control group (1.08 KU/L versus
No treatment-related changes were observed in urinanalysis parameters during the course of the study. All changes that attained statistical significance were considered to be incidental and there were no clear dose–response relationship (data not shown).
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Table 4 Clinical chemistry parameters in dogs after 13 weeks of treatment with LZB and 4 weeks recovery Dose (mg/kg/day)
ALAT (nmol/s/L)
ASAT (nmol/s/L)
Prot (g/L)
Alb (g/L)
Cholest (mmol/L)
Triglyceride (mmol/L)
Gastrin (ng/L)
Ca (mmol/L)
Males Pretest 0 30 100 300 60 (positive)
328 368 376 399 368
569 564 648 616 646
56.9 55.9 54.2 54.6 56.5
30.3 29.3 29.1 29.1 29.4
3.30 2.81 3.44 3.28 3.01
0.46 0.39 0.49 0.45 0.42
29.2 22.4 29.1 26.7 31.6
2.74 2.67 2.61 2.73 2.80
6-week 0 30 100 300 60 (positive)
335 409 449 517 435
511 616 692 779* 700
56.6 56.4 58.8 56.2 53.6
29.2 29.7 29.1 29.7 27.0
2.86 2.94 3.75 5.56* 3.09
0.48 0.47 0.60 0.80* 0.51
50.4 41.9 43.6 119.9* 121.8*
2.55 2.76 2.69 2.66 2.68
13-week 0 30 100 300 60 (positive)
405 538 455 684* 473
543 694 662 830* 669
64.0 58.8 61.6 56.3 48.6*
31.3 29.4 29.7 30.8 25.3*
3.02 2.83 4.03 5.78* 4.51
0.50 0.42 0.69 0.79* 0.59
26.6 32.1 49.7 90.8* 102.5*
2.61 2.63 2.70 2.72 1.75*
Recovery 0 30 100 300 60 (positive)
360 502 521 377 559
570 631 561 546 674
67.3 61.1 61.4 59.9 63.1
28.5 30.4 30.7 30.1 31.4
3.0 1.96 2.81 4.61 3.11
0.39 0.46 0.47 0.55 0.37
26.1 34.5 40.1 36.4 46.8
2.64 2.54 2.63 2.61 2.61
Cholest (mmol/L)
Triglyceride (mmol/L)
Prot (g/L)
Alb (g/L)
Gastrin (ng/L)
Ca (mmol/L)
Females Pretest 0 30 100 300 60 (positive)
59.1 55.7 55.0 58.4 57.6
31.0 30.4 30.0 30.0 29.4
3.10 3.56 2.83 3.69 3.62
0.53 0.44 0.44 0.55 0.57
31.6 30.4 28.3 34.2 27.6
2.61 2.76 2.90 2.73 2.71
6-week 0 30 100 300 60 (positive)
54.7 55.0 50.7 57.2 53.3
30.2 29.2 27.9 30.0 28.8
2.44 2.87 2.59 4.41* 4.04
0.47 0.51 0.49 0.76* 0.49
34.5 36.5 42.9 121.7* 135.6*
2.61 2.65 2.53 2.66 2.72
13-week 0 30 100 300 60 (positive)
60.4 59.1 55.5 60.2 44.0*
29.7 29.1 27.1 28.1 23.4*
2.64 2.79 2.93 5.81* 4.34*
0.61 0.57 0.60 0.88* 1.08*
30.4 38.1 39.7 85.3* 113.7*
2.54 2.61 2.59 2.63 1.87*
Recovery 0 30 100 300 60 (positive)
65.6 55.0 52.0 63.6 62.8
33.5 29.2 30.8 31.2 32.0
2.71 2.65 3.51 3.33 3.39
0.61 0.45 0.35 0.46 0.53
28.6 31.4 43.1 42.4 51.2
2.64 2.55 2.56 2.58 2.55
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. * p < 0.05. Data represent the mean values.
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3.7. Necropsy and organ weights
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day group was seen macroscopically. A few other isolated findings were observed, which were within the range of background lesions or did not show a dose relationship. These changes included congested spleen, thickened
At necropsy after 13-week of treatment, thickened mucus membrane in corpus ventriculi at the 300 mg/kg/
Table 5 Selected histopathological findings in dogs after 13 weeks of treatment with LZB Organ/finding
Dose (mg/kg/d) 0
30
100
300
60 (positive)
M
F
M
F
M
F
M
F
M
F
Lung Focal hemorrhage Chronic bronchitis
0/2 0/2
0/2 0/2
0/2 0/2
0/2 1/2
0/2 0/2
1/2 0/2
0/2 0/2
0/2 0/2
0/2 1/2
0/2 0/2
Liver Fatty change Hydropic degeneration Focal inflammatory cell infiltration Interstitial inflammation
0/2 0/2 0/2 0/2
0/2 0/2 1/2 0/2
0/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
1/2 0/2 0/2 1/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 1/2 0/2
0/2 1/2 0/2 0/2
0/2 0/2 1/2 0/2
Kidney Hydropic degeneration of epithelial cell of proximal convoluted tubule Cortical mineralization Interstitial nephritis
0/2 0/2 0/2
0/2 1/2 0/2
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 0/2
0/2 0/2 1/2
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 1/2
1/2 0/2 0/2
Stomach Karyopycnosis and isolated chief cell necrosis Acidophilic change of chief cell Slight inflammation in lamina propria Proliferation of argyrophilic cells (Grimelius method)
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 1/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 1/2
1/2 1/2 0/2 0/2
1/2 2/2 0/2 1/2
1/2 1/2 0/2 2/2
1/2 1/2 0/2 2/2
2/2 2/2 0/2 2/2
Thymus Atrophy
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
2/2
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. Data represent the number examined (bold) and incidence. Table 6 Selected histopathological findings in dogs after 4-week recovery Organ/finding
Dose mg/kg/d 0
30
100
300
60 (positive)
M
F
M
F
M
F
M
F
M
F
Lung Focal hemorrhage Chronic bronchitis
1/2 0/2
0/2 0/2
1/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
0/2 0/2
1/2 0/2
Liver Fatty change Hydropic degeneration Focal inflammatory cell infiltration Interstitial inflammation
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
0/2 1/2 0/2 0/2
0/2 0/2 0/2 0/2
Kidney Hydropic degeneration of epithelial cell of proximal convoluted tubule Cortical mineralization Interstitial nephritis
0/2 0/2 0/2
1/2 0/2 0/2
1/2 0/2 0/2
0/2 0/2 0/2
0/2 0/2 0/2
0/2 1/2 0/2
0/2 0/2 1/2
0/2 0/2 0/2
0/2 0/2 1/2
1/2 0/2 0/2
Stomach Karyopycnosis and isolated chief cell necrosis Acidophilic change of chief cell Slight inflammation in lamina propria Proliferation of argyrophilic cells (Grimelius method)
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 0/2 0/2 0/2
0/2 1/2 0/2 0/2
1/2 0/2 0/2 0/2
0/2 1/2 0/2 1/2
0/2 1/2 0/2 1/2
1/2 1/2 0/2 1/2
Thymus Atrophy
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
1/2
Dogs (4/sex/group) were treated orally with LZB for 13 weeks, and one-half of the animals were sacrificed, the remaining half was sacrificed after 4-week recovery. Data represent the number examined (bold) and incidence.
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oviducts, and discoloration of various organs (not listed in Table 5). After 4-week recovery, there were no macroscopic findings that were considered related to previous treatment with LZB. For male dogs treated with 300 mg/kg/day, higher relative weight of liver (35.6 versus 27.4) was observed. For female dogs treated with 300 mg/kg/day compared to controls, higher relative weight of liver (39.0 versus 29.4), kidney (7.0 versus 5.1) and brain (13.8 versus 7.6) were observed. Apparent decrease of both absolute and relative weight of thymus was found at all positive controls (grand mean, absolute weight: 2.3 g, relative weight: 0.04) compared to controls (grand mean, absolute weight: 5.1 g, relative weight: 0.07). No other treatment-related effects on organ weight were found.
• For liver, at 300 mg/kg/day, one each case of hydropic degeneration and focal inflammatory cell infiltrate were found; at 30 mg/kg/day, one case of hydropic degeneration was found; at control, one case of focal inflammatory cell infiltrate was seen; at positive control, one case of focal inflammatory cell infiltrate was seen. • For kidney, at 300 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at 100 mg/kg/day, one case of inter-
3.8. Histopathologic findings After 13-week treatment, histological changes were seen mainly in liver, kidney, lung and stomach (Table 5, only organs or tissues with histological changes were listed). These changes were reversed or showed a tendency to reverse during 4-week drug-free rest period, indicating that the effects of LZB were reversible (Table 6). In male dogs • For lung, one case of chronic bronchitis at positive control was found. • For liver, at 300 mg/kg/day, one case of fatty change was found; at 100 mg/kg/day, one each case of fatty change and interstitial inflammation were found; at positive control, one case of hydropic degeneration was seen. • For kidney, at 100 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at positive control, one each case of interstitial nephritis and hydropic degeneration of epithelial cell of proximal convoluted tubule were observed. • For stomach, at 300 mg/kg/day, one case of karyopycnosis and isolated necrosis of chief cell, one case of proliferation of argyrophilic cells and two case of acidophilic change of chief cell were found; at 100 mg/ kg/day, one each case of karyopycnosis and isolated necrosis of chief cell and proliferation of argyrophilic cells (Grimelius method) were found; at 30 mg/kg/day, one case of slight inflammation in lamina propria was found; at positive control, one case of karyopycnosis and isolated necrosis of chief cell, one case of acidophilic change of chief cell and two case of proliferation of argyrophilic cells were seen. • For thymus, one case of atrophy at positive control was found. In female dogs • For lung, one case of focal hemorrhage at 100 mg/kg/day, one case of chronic bronchitis at 30 mg/kg/day were found.
Fig. 3. SEM photomicrographs for the fundus area of the gastric mucosa from high-dose group (#24, A) and negative control group ($18, B). Note the irregular ‘honeycomb’ structure of the mucosa surface for #24.
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These changes were reversed or showed a tendency to reverse during 4-week drug-free rest period, indicating that the effects of LZB were reversible (Table 6). In the bone marrow, hyperplasia of myelocyte or metamyelocyte and hypoplasia of intermediate or late erythroblast were observed in dogs of high dose and positive control (more significant) after 13-week administration. Proliferation of erythrocyte series apparently becomes active 4-week drug-free period later.
3.9. SEM findings The appearance of the mucosa of high-dose dog and control was illustrated in Fig. 3. Apparently for high-dose dog (Fig. 3A), part of the surface epithelium built with mucous cell had been necrosis and some mucous cell ablated. This region can be typically characterized by ‘honeycomb’ pattern. This ‘honeycomb’ pattern covered the fundus area. For control dog (Fig. 3B), the columnar mucous cell was well-regulated and lines the gastric pit, and no vacuolation or necrosis were observed.
20.000 30mg/kg 100mg/kg
15.000
AUC/Dose
stitial nephritis was found; at 30 mg/kg/day, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was found; at control, one case of cortical mineralization was seen; at positive control, one case of hydropic degeneration of epithelial cell of proximal convoluted tubule was seen. • For stomach, at 300 mg/kg/day, one case of karyopycnosis and isolated necrosis of chief cell, one case of acidophilic change of chief cell and two case of proliferation of argyrophilic cells were found; at 100 mg/kg/day, one each case of karyopycnosis and isolated necrosis of chief cell and acidophilic change of chief cell were found; at positive control, two case of karyopycnosis and isolated necrosis of chief cell and two case of proliferation of argyrophilic cells were seen. • For thymus, two case of atrophy at positive control were also found.
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300mg/kg 10.000
5.000
0.000 single
6-week
13-week
Fig. 5. Dose normalized plots for AUC.
Fig. 4. Plasma level-time profiles of LZB in beagle dogs with dose of 30, 100, 300 mg/kg. Blood samples were obtained in the 1st day, 45th day and 89th day of administration at 0 (1st day), 0.5, 1, 2, 4, 8 and 24 h after dosing.
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Table 7 Mean (±SD) toxicokinetic parameters of LZB after oral administration in doses of 30, 100, and 300 mg/kg/day to beagle dogs (n = 4/sex/group) Dose (mg/kg/day) Grand mean 30 (n = 8)
Duration of administration
AUC0fi24 h (lg · h/ml)
Cmax (lg/ml)
Tmax (h)
Single 6-week 13-week
179.7 ± 7.03 207.7 ± 37.2 225.2 ± 52.5
44.3 ± 10.5 29.7 ± 10.1 38.7 ± 39.6
2±0 4±0 5±2
100 (n = 8)
Single 6-week 13-week
740.9 ± 341.1 758.9 ± 273.9 558.5 ± 294.0
54.1 ± 28.2 64.4 ± 66.4 128.4 ± 30.9
4±0 2±1 2±1
300 (n = 8)
Single 6-week 13-week
1504.8 ± 184.9 1069.5 ± 492.5 1062.3 ± 240.5
181.5 ± 17.4 214.6 ± 82.9 93.8 ± 35.3
4±0 3±1 4±2
3.10. Toxicokinetics Fig. 4 shows the plasma level-time profiles of LZB in the beagle dogs. Dose normalized plots was shown in Fig. 5. Relevant pharmacokinetic parameters are listed in Table 7. There were no significant differences in pharmacokinetic parameters of LZB between male and female dogs during 13-week oral administration for three different doses. Therefore, we combined the pharmacokinetic parameters from male and female dogs together. The plasma concentration of LZB on the 1st, 45th and 89th day showed a dose dependent, linear increase with maximum plasma values observed at about 2–4 h after treatment. There were considerable inter-individual differences in the plasma concentrations. At the 100 and 300 mg/kg/day dose level, lower exposures were noted on Day 45 and Day 89 as compared to Day 1 (Table 7). 4. Discussion The daily administration of LZB orally by enteric capsule at dose levels of 0, 30, 100 or 300 mg/kg/day to beagle dogs in a 13-week study was well tolerated and no mortality was observed. Most of the toxic effects were found at high dose and positive control group. Gastro intestinal disturbances always associated with PPIs (Marchetti et al., 2003; Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b) were not notable ever for high-dose group in this study. Some intrinsically associated symptoms for high-dose group were observed, such as decreased body weight gain and food intake, decreased RBC, Hb and HCT, hypoplasia of erythrocyte series in bone marrow, histopathlogical findings of acidophilic change and karyopycnosis or isolated necrosis of chief cell, surface epithelium necrosis and some mucous cells ablate. Theoretically, gastric acid can activate pepsinogen, and provide proper environment for absorption of nutriments. Long terms of acid suppression with high dose might injure the gastric mucosa and disturb the secreting function of chief cell (Kakei et al., 1993), accordingly inducing disorder of nutriments absorption (Koop, 1992; Stewart et al., 1995), hypothrepsia or even hemophthisis, and histopath-
logical change of cells in gastric glands. These changes came back at the end of the recovery period, suggesting that these by-effects of LZB were reversible. CNS effect of lethargy and astasia were found at individual dog of high-dose group after 72 days administration. While there were no histopathlogical findings associated with skeletal muscle and sciatic nerve. The result of Erythrocyte AChE showed that there was significant difference between high-dose and control group and recovered after 4-week rest period. So the effect of LZB on nerves system may be related to cholinergic nerve. CNS by-effect can be found in some cases from preclinical and clinical test (Arnold, 1994), or in dispensatory of some commercially available PPIs. What could be the molecular action mechanism and if LZB will exhibit CNS effect on human remain questions for further research and clinical trial. Increased ALAT, ASAT and higher relative liver weight were observed at high-dose dogs. It cannot be excluded to be related to the treatment. However, these changes did not accompanied by changes in hepatic histopathology. Higher relative kidney weight was also found at high-dose dogs with no changes in renal histopathology and in urinary or clinical chemistry parameters indicative of renal toxicity. Although there was case report implicating PPI in acute interstitial nephritis (AIN) and progression to acute renal failure (ARF) (Nimeshan et al., 2006), in this study the changes in relative kidney weights were considered of no toxicological significance. Increased cholesterol and triglyceride were observed at high dose, which may relate to abnormal fat metabolism due to acid suppression. Serum gastrin concentration of both sexes at high dose was significantly higher than control after 13-week treatment, which induce proliferation of argyrophilic cells in gastric fundus (seen by Grimelius stain) due to the trophic action of high gastrin level. PPIs, like H2-receptor antagonists, are associated with gastric carcinoid tumors in rats (Ekman et al., 1985), raising questions about the safety of these drugs. Both omeprazole and lansoprazole cause reversible hypergastrinemia. Serum gastrin levels increased to two to four times baseline levels during omeprazole therapy (Tielemans et al., 1989) and 1.6- to 2-fold
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increase was seen with lansoprazole (Misawa et al., 1991; Ogoshi et al., 1991). Enterochromaffin cells, the precursors of carcinoids, are hypertrophied in humans receiving PPIs. However, no carcinoids have been observed during PPI therapy. Gastrin levels and cellular hypertrophy are reversed when PPI therapy is stopped (Larsson et al., 1988). In this study, no carcinoid tumor was found among all groups, and gastrin levels and cellular hypertrophy were also reversible. Refer to the positive control, more severe vomiting and diarrhea compared to high-dose group was observed during the administration period. Dogs of both sex were anorexia and extremely feeble and emaciation at the last month of administration; the similar symptoms including body weight, food intake, hematological changes, high serum gastrin level and histopathological changes of gastric mucosa also can be found in positive controls with more severe degree than high-dose group; clinical chemistry parameters like protein, Alb and Ca were significantly lower than control, which had not been seen in LZB groups. It can be contributed to nutriments deficiency due to extremely severe anorexia and frequent vomit or diarrhea; in bone marrow, the hypoplasia of erythrocyte series was more significant than high-dose group; beside the liver, kidney and brain weight change, apparent atrophy of thymus could be observed. Considering the dose difference of LZB and rabeprazole (see Section 1), in this study oral administration of LZB appeared more toleration than that of rabeprazole. Systemic exposure to LZB was indicated by the plasma AUC0–24 h and Cmax over the 24 h dosing interval, which were proportional to the dose and no saturation was observed. After 6-week and 13-week of oral administration, the AUC0–24 h values at all doses were not significantly greater than those on the day 1, which suggests that LZB is not accumulated (Fig. 5). No non-linear kinetics of LZB was observed. From the result of this work and literature report, we can find there are both difference and similarity between toxicity of LZB and other PPIs. In a 13-week beagle dog study of tenatoprazol (TU-199), GS disturbance like vomit was seen in high-dose group. High urea nitrogen and serum gastrin concentration were found. Histological changes were seen in the stomach (hypertrophy and fibrosis of the mucous membrane; increase in parietal cell; partial epithelial necrosis in the fundic glands) and thyroid (not observed in this work) (Okamoto et al., 1998a,b). The reason why LZB could be different from the others are still remaining unknown. It need further research concerning structure–effect relationship and mechanism of action. Based on the absence of treat-related toxicologically changes, the no-observed-adverse-effect level (NOAEL) was considered to be at least 30 mg/kg/day and the toxic dose to be 100 mg/kg/day, which was much higher than other PPIs (Ekman et al., 1985; Abe et al., 1990; ACIPHEX, 1999; Okamoto et al., 1998a,b). The pharmacodynamic action of LZB for rats was proved to be
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