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Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on April 14, 2008; DOI: 10.1124/jpet.108.137299

0022-3565/08/3261-163-170$20.00
JPET 326:163-170, 2008
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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

The Effect of Subchronic Administration of 7-(4-Fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526), a Novel Acid Pump Antagonist, on Gastric Acid Secretion and Gastrin Levels in Rats

Keiichi Ito, Kazuya Kinoshita, Atsuyuki Tomizawa, Yuka Morikawa-Inomata, Fumi Inaba, Yoshifumi Fujita, Keiichi Tabata, and Nobuhiko Shibakawa

Pharmacology Research Laboratories (K.I., K.K.), Biological Research Laboratories II (A.T., Y.M.-I.), and Biological Research Laboratories IV (F.I.), Daiichi Sankyo Co., Ltd., Shinagawa-ku, Tokyo, Japan; Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd., Edogawa-ku, Tokyo, Japan (Y.F.); Licensing Department, Daiichi Sankyo Co., Ltd., Chuo-ku, Tokyo, Japan (K.T.); and Pharmaceutical Research Department, Ube Laboratory, Corporate Research and Development, Ube Industries, Ltd., Ube City, Yamaguchi, Japan (N.S.)

Received for publication January 31, 2008
Accepted April 9, 2008.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
In the present report, we evaluated the effect of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) and 2-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-ylmethanesulfinyl]-1H-benzimidazole (lansoprazole) on rebound gastric acid secretion, using an intragastric dialysis membrane perfusion model and on the serum and antral gastrin level after a 14-day treatment in rats. The effect of CS-526 on gastric acid secretion was almost constant during the 14 days of treatment. After the 14-day treatment, gastric acid secretion had returned to pretreatment levels. However, CS-526 slightly increased and lansoprazole potently increased gastric acid secretion thereafter. In the posttreatment period, the influence on rebound gastric acid secretion by lansoprazole treatment was significant, but that by CS-526 was not. The serum gastrin concentration after the 14-day treatment with CS-526 did not increase significantly, even at 100 mg/kg/day. On the other hand, lansoprazole at 100 mg/kg/day significantly elevated the serum gastrin concentration. After the 14-day treatment with CS-526 at 100 mg/kg/day, the antral gastrin content significantly increased. Lansoprazole at the doses of 30 and 100 mg/kg/day also significantly increased the antral gastrin content after the 14-day treatment. The elevation of the serum gastrin level after the lansoprazole treatment was suppressed by the concomitant administration of CS-526. In conclusion, CS-526 has a potent antisecretory effect on gastric acid secretion without rebound gastric hypersecretion. Moreover, CS-526 had minimal effects on the serum and antral gastrin elevation. It is suggested that these effects on gastric acid secretion and serum gastrin after subchronic treatment with CS-526 would be beneficial in clinical use.

Proton pump inhibitors (PPIs) are now widely used for the treatment of gastric acid-related diseases such as peptic ulcer and gastroesophageal reflux disease (Robinson, 2001Go, 2005Go). Gastric H+,K+-ATPase is a proton pump located at the apical membrane of the parietal cells that transport H+ into the canaliculus of parietal cells in exchange for K+. Substituted benzimidazoles, such as omeprazole, lansoprazole, and rabeprazole, inactivate the H+,K+-ATPase by covalent binding to the sulfhydryl group of H+,K+-ATPase, resulting in the long-lasting inhibition of gastric acid secretion (Sachs, 1988Go). Although these derivatives exert superior healing of gastric acid-related diseases, long-term treatment with omeprazole causes bacterial overgrowth in the upper gut due to long-lasting gastric acid suppression (Larner and Lendrum, 1992Go). In addition, a recent report shows that the risk of community-acquired pneumonia is increased after chronic use of acid-suppressive drugs (Laheij et al., 2004Go).

The long-lasting inhibition of gastric acid secretion also induces hypergastrinemia, resulting in the induction of gastric enterochromaffin-like cell carcinoids in rats and hyperplasia of enterochromaffin cells in humans (Lamberts et al., 1988Go, 1993Go; Lindberg et al., 1990Go; Herling and Weidmann, 1994Go; Freston et al., 1995Go). It is well known that rebound acid hypersecretion (Fullarton et al., 1989Go; Nwokolo et al., 1991Go; Kummer et al., 1992Go) or tachyphylaxis (Wilder-Smith et al., 1990Go) occurs after or during histamine H2-receptor antagonist treatment, respectively. Moreover, in some reports, gastric acid hypersecretion has been observed after omeprazole treatment (Waldum et al., 1996Go; Gillen et al., 1999aGo) as well, and the degree of gastric acid hypersecretion is associated with fasting plasma gastrin levels (Gillen et al., 1999aGo).

Reversible acid pump antagonists (APAs), the other class of proton pump inhibitors, act by K+-competitive and reversible binding to the gastric proton pump (Pope and Parsons, 1993Go). The major classes of APAs are imidazopyridine (Wallmark et al., 1987Go; Kromer et al., 2000Go; Gedda et al., 2007Go) and acyl quinoline derivatives (Keeling et al., 1991Go; Pope et al., 1995Go; Cheon et al., 2001Go), and most of these compounds have been shown to inhibit H+,K+-ATPase in a K+-competitive mechanism without any covalent binding, thereby initiating reversible inhibition of H+,K+-ATPase activities.

CS-526 is a novel acid suppressant that is different from PPIs and the aforementioned APAs in that its chemical structure includes a pyrrolopyridazine structure (Ito et al., 2007Go). We have shown the potent and reversible inhibitory properties of CS-526 against porcine H+,K+-ATPase without a covalent binding formation. The potent gastric acid inhibitory effects and antiulcer efficacies of CS-526 have also been shown, and the efficacies of CS-526 are comparable with those of PPIs such as omeprazole, lansoprazole, and rabeprazole (Ito et al., 2007Go).

In the present study, we evaluated the effect of subchronic (14-day) and consecutive administration of CS-526 on gastric acid secretion and whether the serum and antral gastrin levels were elevated, as well as whether rebound gastric acid hypersecretion occurred in rats.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. CS-526 was synthesized in the Ube Research Laboratory of Ube Industries, Ltd. (Yamaguchi, Japan). Lansoprazole was extracted from Takepron (Takeda Pharmaceutical Co., Ltd., Osaka, Japan).

Animals. All animal experimental procedures were performed in accordance with the Animal Experimentation Guidelines of Sankyo, Co., Ltd.

Male SD rats (Charles River Laboratories Japan, Inc., Kanagawa, Japan) weighing 179 to 315 g were used.

Experimental Design. Three separate experiments were performed. Experiment 1 was an evaluation of the antigastric acidic effects during a 14-day treatment of CS-526 or lansoprazole using an intragastric dialysis membrane perfusion system in rats. In addition, the rebound gastric acid hypersecretion thereafter was also evaluated. CS-526 or lansoprazole at the dose of 30 mg/kg/day was administered orally for 14 days at approximately 9:00 AM. The gastric acid secretion was measured on the day before the first administration, on the 1st, 7th, and 14th days of administration, and from 1 to 4 days after the last administration.

In experiment 2, we measured the serum and the antral gastrin levels in rats. CS-526 or lansoprazole at doses of 30 and 100 mg/kg/day was administered orally for 14 days in the morning from 9:00 to 11:00 AM. On the day after the last administration, the animals were anesthetized by ether, blood was drawn from the abdominal aorta, and the gastric antral mucosa was collected in the morning (from 9:00 AM to 12:00 PM).

In experiment 3, we evaluated the effect of CS-526 on the gastrin elevation caused by lansoprazole treatment. CS-526 and lansoprazole at the dose of 30 mg/kg/day were administered separately or concomitantly. The compounds were administered for 14 days in the morning, from 9:00 to 11:00 AM. The serum and the gastric mucosa were collected on the day after the last administration in the morning (from 9:00 AM to 12:00 PM). In this experiment, in addition to the serum and antral gastrin content, the mRNA levels of gastrin and somatostatin in the gastric antral and corpus mucosa were measured by reverse transcription-polymerase chain reaction (RT-PCR).

Intragastric Dialysis Membrane Perfusion System in Rats. The animals were fasted for 16 to 20 h with free access to water, and then they were anesthetized with an i.p. administration of pentobarbital sodium (30–50 mg/kg) (Nembutal; Dainippon Pharma, Co., Ltd., Osaka, Japan). The midline of the abdomen was opened with an incision, and the stomach was exposed. A probe made from a dialysis tube (Spectra/Pore MWCO 6000–8000, 6.4-mm diameter, 5-cm length; Funakoshi Corporation, Tokyo, Japan) was inserted into the stomach via the small incision in the forestomach. The probe was ligated at the incision, and then the stomach was returned to its original position. The tubing (outer diameter of 1 mm) connected to the probe was exteriorized from the left abdominal wall and introduced s.c. to the nape of the neck. The animals were used for the experiment starting at least 1 week after the operation. The tubing from the probe was connected to a cannula swivel (TCS2-21; Tsumura and Co., Tokyo, Japan). The cannula swivel was mounted to the apparatus of a free-moving cannulation system (Sugiyama-gen iriki Co., Ltd., Tokyo, Japan). Saline was infused at a rate of 5 ml/h with a peristaltic pump (DECARF-N; Advantec Toyo Kaisha Ltd., Tokyo, Japan) from the tubing connected to the probe for at least 14 h before the experiment for equilibration. The perfusate from the other tubing connected to the probe was collected every 60 min with a fraction collector (222XL; M&S Instruments Trading Inc., Tokyo, Japan) during the experimental period. A schematic illustration is presented in Fig. 1.


Figure 1
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  		Fig. 1. Schematic representation of the intragastric dialysis membrane perfusion system. A probe made from a dialysis tube was inserted into the stomach by a forestomach incision. The tubing connected to the probe was exteriorized from the left abdominal wall and introduced s.c. to an incision made in the nape of the neck. At the nape incision, a dacron mesh button tether (Bio Research Center Co., Ltd., Aichi, Japan) was sutured and the tubes were passed through it. The tubing in the tether was connected to a two-channel cannula swivel using a pair of extension tubes passing through a stainless steel spring that was connected to the tether and the cannula swivel. The cannula swivel was mounted to the apparatus of a free-moving cannulation system. From one of the tubing, saline was infused at a rate of 5 ml/h with a peristaltic pump, and the perfusate from the other tubing was collected every 60 min with a fraction collector.

 
During the experimental period, the animals were able to move almost freely and were allowed free access to food and water. They were housed one per cage in a 12-h light/dark cycle (lights were on from 7:00 AM to 7:00 PM).

The acidity of the perfusate was determined by titration to be pH 7.0 using 0.05 N NaOH (COMTITE-980; Hiranuma Sangyo Co., Ltd., Ibaraki, Japan). The titratable acid output was expressed as microequivalents per hour and is indicated at the middle point of the collecting period in Figs. 2 and 3.


Figure 2
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  		Fig. 2. Circadian rhythm of gastric acid secretion in an intragastric dialysis membrane perfusion model. The rats could move freely in their cages and were allowed food and water ad libitum. Perfusate was collected every 60 min for 24 h on the day before the first administration.

 

Figure 3
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  		Fig. 3. Effect of CS-526 and lansoprazole on gastric acid secretion in an intragastric dialysis membrane perfusion model. Perfusate was collected every 60 min for sequential 24 h on day 0 (a day before the first administration), day 1 (a day of the first administration), day 7, day 14, day 15, day 16, day 17, and day 18. Vehicle or compounds were orally administered from day 1 to day 14 at 9:00 AM. A, from day 0 to day 14; B, from day 15 to day 18.

 
Measurement of Serum and Antral Gastrin Levels. The compounds were orally administered once a day for 14 days. The day after the last administration, under ether anesthesia, blood was collected from the abdominal artery into tubes containing a coagulator and a separator (Shino-Test Corporation, Tokyo, Japan) to isolate the serum from the clots. The serum was stored at –80°C until the gastrin measurement. The stomachs were removed, cut open along the greater curvature, and washed with ice-cold saline. The antral mucosa was wrapped in aluminum foil, frozen in liquid nitrogen, and stored at –80°C until the sample extraction. The extraction of gastrin from the antral tissues was performed as follows: the antral tissue was homogenized in lysate buffer; the lysate buffer was prepared by dissolving one vial of Protease Inhibitor Cocktail Set I (Merck Biosciences GmbH, Frankfurt, Germany) in 100 ml of Dulbecco's phosphate-buffered saline (Invitrogen, Carlsbad, CA); and then the tissue was boiled at 100°C for 30 min and centrifuged. The supernatant was collected into sample tubes and stored at –80°C until gastrin measurement. The serum and antral gastrin levels were measured by a radioimmunoassay (Gastrin RIA Kit II; Dainabot Radioisotope Laboratory, Tokyo, Japan). The radioactivity was counted with a gamma counter (Riastar; PerkinElmer Life and Analytical Sciences, Waltham, MA). The B/B0 ratio was calculated using the following equation: B/B0 = (mean dpm of each sample – mean dpm of nonspecific binding)/(mean dpm of nonspecific binding). The B/B0 ratio was converted to the logit form using the following equation: Logit (B/B0) = Loge ((B/B0)/(1-B/B0)). The logit (B/B0) of the standard solution against the log concentration was plotted, and the best-fit line based on the least-squares method was drawn. The concentration of the samples was calculated by the equation of the best-fit line.

Determination of mRNA Levels of Gastrin and Somatostatin by the Quantitative Real-Time RT-PCR Method. The compounds were orally administered for 14 days, and tissue samples were taken from the gastric corpus and the antrum. The total RNA was isolated from the tissues (0.05–0.2 g) using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) and purified using a S.N.A.P. total RNA isolation kit (Invitrogen Japan K.K., Tokyo, Japan). The yield of the RNA extract was determined by spectrophotometry, and 1 µg of RNA from each sample was reverse transcribed in 40 µl of TaqMan RT buffer containing 5.5 mM MgCl2, 0.5 mM each dNTP, 2.5 µM oligo(dT)16, 0.4 U/µl RNase inhibitor, and 1.25 U/µl MultiScribe Reverse Transcriptase (TaqMan Reverse Transcription kit; Applied Biosystems, Foster City, CA). The reverse transcription cycle conditions were as follows: 10 min at 25°C, 30 min at 48°C, 5 min at 95°C, and 5 min at 5°C.

The amount of gastrin and somatostatin cDNA was measured with the TaqMan assay system. The primer sequences (Oligo Express PCR; Amersham Pharmacia Biotech, Tokyo, Japan) were as follows: gastrin forward primer, 5'-TCTCACCATCGGAGACAGCTG-3'; gastrin reverse primer, 5'-GGTGGCCTCTGTTTCTTGGA-3'; somatostatin forward primer, 5'-CAGACAGAGAACGATGCCCTG-3'; and somatostatin reverse primer, 5'-CTCATCTCGTCGTGCTCAGC-3'. The 6-carboxyfluorescein-labeled probe sequences were as follows: gastrin, 5'-CCCAAGGTCCGCAACACTTCATAGCA-3'; and somatostatin, 5'-CTGAGGATTTGCCCCAGGCAGCT-3'. Each PCR solution of 50 µl was made up of TaqMan Universal PCR Mastermix (Applied Biosystems) containing 0.1 µM of each primer, 50 µM 6-carboxyfluorescein-labeled probe, and 10 µl of diluted cDNA solution. The TaqMan PCR conditions were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. During the assay, a linear increase in the fluorescence signals from the reporter dye was observed, as determined using a Prism 7700 (Applied Biosystems), and the quantification of mRNA was analyzed using a Sequence Detection System (version 1.6.3; Applied Biosystems). To calculate the quantities of each amplicon accurately, standard curves for each amplicon were plotted from five different concentrations of the standards. A glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (rodent housekeeping gene) gene was used as the internal control. Each RT solution containing 1 ng of GAPDH was used in the quantification of the gastrin mRNA. Likewise, an RT solution containing 5 ng of GAPDH was used as a template in the quantification of the somatostatin mRNA. The quantity of gastrin or somatostatin mRNA was expressed as a ratio against the mean of the corresponding vehicle treatment.

Statistical Analysis. Microsoft Excel (Microsoft, Redmond, WA) and SAS System for Windows (SAS Institute, Cary, NC) were used to calculate the mean ± S.E. of the values for each treatment.

For the evaluation of the daily value of the gastric acid secretion, the average of the hourly titratable acid output of perfusate (mean gastric acid secretion) was calculated each day for each animal. The effect of the compounds on gastric acid secretion was compared with the corresponding pretreatment (day 0) by the pairwise analysis of a Dunnett's test of longitudinal data. In the 14-day treatment period, repeated measures of a two-way analysis of variance (ANOVA) in which the day 0 values were used as a covariate, with "day" as the repeated factor and "compound" as another factor, were performed to compare the least-square mean of the mean gastric acid secretion of CS-526 or lansoprazole with that of the vehicle, and that of CS-526 with that of lansoprazole. In this ANOVA, an adjustment of the multiple comparisons by Dunnett-Hsu's method was performed. In the post-treatment period, a repeated measures ANOVA was also performed with the data of day 0 as a covariate.

The serum and antral gastrin concentration in the groups treated with the test compounds was compared with that of the control group, which received the vehicle, by a Dunnett's multiple comparison test. The effect of CS-526 on the serum or antral gastrin was compared with that of lansoprazole by a two-way ANOVA. The effects of the vehicle, CS-526, lansoprazole, and their combination (CS-526 + lansoprazole) on the mRNA of gastrin and somatostatin were compared with each other by a Tukey's multiple comparison test.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Circadian Rhythm of Gastric Acid Secretion in Rats Using Intragastric Dialysis Membrane Perfusion System. In advance of the evaluation of the gastric antisecretory effect of CS-526, we investigated the circadian rhythm of gastric acid secretion in rats (Fig. 2). The circadian rhythm of gastric acid secretion in rats revealed that the pattern of gastric acid secretion had two peaks: the top of the peaks occurred at approximately 9:30 AM and 11:30 PM, and the bottom of the peaks occurred at approximately 7:30 PM and 6:30 AM. The circadian rhythm pattern was varied in terms of the days and the animals. However, in general, gastric acid secretion decreased gradually during the light period, which was not the active period, and would increase again during the dark period. We determined to administer CS-526 at 9:00 in the morning, during the most acidic state of the circadian rhythm.

Effects of CS-526 and Lansoprazole on Gastric Acid Secretion in the Intragastric Dialysis Membrane Perfusion System. We examined the effect of 30 mg/kg/day of CS-526 and lansoprazole on gastric acid secretion in rats. The effect of CS-526 on gastric acid secretion was almost constant during the treatment for 14 days (Fig. 3A). The inhibitory effect of CS-526 on rat gastric acid secretion was revealed to be significant (P = 0.0027 versus vehicle) by a repeated measures ANOVA. In addition, a significant difference from day 0 was observed at days 1 (P = 0.0209) and 7 (P = 0.0385; Figs. 3A and 4). Lansoprazole also inhibited gastric acid secretion (P = 0.0009 versus vehicle by a repeated measures ANOVA) in rats. However, the effect of lansoprazole on gastric acid secretion on the 7th day of administration seemed to be weaker than that on the first day (Figs. 3A and 4). The effect of lansoprazole on the 14th day of administration was similar to that of the first administration (Figs. 3A and 4). During the 14-day treatment period, the effects of CS-526 and lansoprazole were not significantly different from each other (P = 0.4865 by a repeated measures ANOVA).


Figure 4
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  		Fig. 4. Effect of CS-526 and lansoprazole on gastric acid secretion in an intragastric dialysis membrane perfusion model. The average of 24 time points of hourly gastric acid secretion was calculated on each measurement day. Each point and bar represents the mean ± S.E. of the average gastric acid secretion obtained from 4 to 6 rats in each group. The statistical significance from day 0 was analyzed by a pairwise analysis of a Dunnett's test with longitudinal data. *, P < 0.05.

 

Then, we investigated the rebound on gastric acid secretion in rats after treatment with CS-526 or lansoprazole for 14 days. Vehicle-treated animals had constant gastric acid secretion after treatment (Figs. 3B and 4). In contrast, a slight and potent increase of gastric acid secretion after the 14-day treatment of CS-526 and lansoprazole was observed, respectively (Figs. 3B and 4). In the posttreatment period, gastric acid secretion in the CS-526-treated and lansoprazole-treated groups was not significantly different from that in the vehicle-treated group (P = 0.9867 and P = 0.2360, respectively), and there was no significant difference between the CS-526- and lansoprazole-treated groups (P = 0.2002) by a repeated measures ANOVA. However, a pairwise comparison with the day 0 value revealed that the gastric acid secretion from 2 to 4 days (day 16, day 17, and day 18) after the lansoprazole treatment was significantly increased (P = 0.0453, P = 0.0405, and P = 0.0328, respectively; Fig. 4). In contrast, the mean gastric acid secretion at any day in the CS-526-treated group was not significantly different from that of day 0 (Fig. 4).

Effect of CS-526 and Lansoprazole on Serum and Antral Gastrin. We examined the effect of CS-526 and lansoprazole on the serum and antral gastrin levels in rats (Fig. 5). The administration of CS-526 at the dose of 30 mg/kg/day for 14 days did not elevate the serum gastrin level (vehicle: 383 ± 68 pg/ml, n = 7 versus CS-526 30 mg/kg/day: 261 ± 31 pg/ml, n = 8, P = 0.8611). However, at the dose of 100 mg/kg/day, CS-526 slightly affected the serum gastrin level (695 ± 304 pg/ml, n = 8, P = 0.4136; Fig. 5A), although no statistical significance was observed. The effect of lansoprazole at the dose of 30 mg/kg/day (672 ± 131 pg/ml, n = 7, P = 0.1620; Fig. 5A) was almost the same as that of 100 mg/kg/day of CS-526. Increasing the dose to 100 mg/kg/day, lansoprazole significantly elevated the serum gastrin levels (1195 ± 135 pg/ml, n = 7, P = 0.0002; Fig. 5A). The administration of CS-526 at the dose of 100 mg/kg for 14 days significantly elevated the antral gastrin content (vehicle: 2.9 ± 0.3 ng/mg tissue, n = 7 versus 100 mg/kg/day: 6.0 ± 0.5 ng/mg tissue, n = 8, P = 0.0001; Fig. 5B). However, 30 mg/kg/day of CS-526 did not increase the antral gastrin content (4.1 ± 0.4 ng/mg tissue, n = 8; Fig. 5B). Lansoprazole (30 and 100 mg/kg/day) significantly increased the antral gastrin content (30 mg/kg/day: 7.9 ± 1.2 ng/mg tissue, n = 7, P = 0.0007; 100 mg/kg/day: 6.6 ± 0.7 ng/mg tissue, n = 7, P = 0.0088; Fig. 5B). The effect of CS-526 on the serum and antral gastrin levels was significantly weaker than that of lansoprazole (P = 0.0153 and P = 0.0092, respectively, two-way ANOVA).


Figure 5
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  		Fig. 5. Effect of CS-526 and lansoprazole on serum (A) and antral (B) gastrin level after a 14-day treatment. CS-526 and lansoprazole were orally administered once daily. Each column and bar represents the mean ± S.E. of the serum or antral gastrin level obtained from 6 to 8 rats in each group. The statistical significance was analyzed by a Dunnett's test. **, P < 0.01; ***, P < 0.001 versus vehicle.

 
Effect of CS-526 on Elevated Gastrin Level Caused by Lansoprazole Treatment. CS-526 did not elevate the serum gastrin (329 ± 32 pg/ml, n = 9, P = 0.8617; Fig. 6A) at the dose of 30 mg/kg/day. In contrast, lansoprazole significantly elevated the serum gastrin (924 ± 75 pg/ml, n = 10, P < 0.0001; Fig. 6A) in comparison with the vehicle control (273 ± 29 pg/ml, n = 11; Fig. 6A). Although no statistical significance was observed, the antral gastrin content was slightly increased by either CS-526 (3.0 ± 0.4 ng/mg tissue, n = 5, P = 0.5750; Fig. 6B) or lansoprazole treatment (2.9 ± 0.5 ng/mg tissue, n = 5, P = 0.7284; Fig. 6B) in comparison with the vehicle control (2.3 ± 0.5 ng/mg tissue, n = 5; Fig. 6B). However, the gastrin and somatostatin mRNA levels of the antral mucosa were elevated to 4.0 ± 1.0-fold (n = 5, P = 0.0324; Fig. 7A) and decreased to 0.4 ± 0.1-fold (n = 5, P = 0.0346; Fig. 7B) from that of the vehicle control, respectively, after the treatment of lansoprazole. Concomitant administration of lansoprazole and CS-526 lowered the elevation of the serum gastrin caused by lansoprazole treatment (474 ± 50 pg/ml, n = 11, P = 0.0001 versus lansoprazole; Fig. 6A). Likewise, antral gastrin mRNA was slightly decreased by the concomitant use of CS-526 and lansoprazole (3.3 ± 0.5-fold from vehicle, n = 5, P = 0.1192 versus vehicle; Fig. 7A), although no statistical significance was observed (P = 0.8978 versus lansoprazole). However, antral somatostatin mRNA was still significantly decreased in comparison with the vehicle (0.4 ± 0.1-fold from vehicle, n = 5, P = 0.0298; Fig. 7B). Somatostatin mRNA in the corpus showed an almost similar change (Fig. 7C) to that in the antrum, although no statistical significance by any treatment was observed.


Figure 6
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  		Fig. 6. Effects of CS-526 (30 mg/kg/day) and lansoprazole (30 mg/kg/day) and their combination on the serum (A) and antral (B) gastrin level after a 14-day treatment. CS-526 and lansoprazole were orally administered once daily. Each column and bar represents the mean ± S.E. of the serum and antral gastrin level obtained from 9 to 11 rats for serum and 6 to 8 rats for antral gastrin in each group. The statistical significance was analyzed by Tukey's multiple comparison tests. *, P < 0.05 versus vehicle; ***, P < 0.001 versus vehicle or lansoprazole.

 

Figure 7
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  		Fig. 7. Effects of CS-526 (30 mg/kg/day) and lansoprazole (30 mg/kg/day) and their combination on antral gastrin (A), antral somatostatin (SS) (B), and corpus SS (C) mRNA after a 14-day treatment. CS-526 and lansoprazole were orally administered once daily. Each column and bar represents the mean ± S.E. of the mRNA level obtained from five rats in each group. The statistical significance was analyzed by Tukey's multiple comparison tests. *, P < 0.05 versus vehicle.

 


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Consecutive measurements of gastric acid secretion have been frequently performed in humans by intragastric pH-metry. However, in terms of experimental animals, there are only a few reports involving dogs (Postius et al., 1991Go; Kromer et al., 2000Go). We succeeded in measuring gastric acid secretion consecutively in conscious, nonrestrained, and non-fasted rats in an intragastric dialysis membrane perfusion model. In a previous report about the circadian rhythms of gastric acid secretion in fasting and anesthetized rats, the peak time of the circadian period occurred at approximately 4:45 AM (1 h and 15 min before lights on), near the end of the nocturnal activity period of rats, and the bottom of circadian period occurred at approximately 4:45 PM, with a curve generated by a cosinor analysis (Larsen et al., 1991Go). In the present study, we observed two peaks of circadian rhythm (Fig. 2): one large peak occurred at approximately 9:30 AM (2 h and 30 min after lights on), and the other occurred at approximately 11:30 PM. The bottom of acid secretion occurred at approximately 7:30 PM (30 min after lights off) and 6:30 AM. There seemed to be some discrepancies between these data. However, these discrepancies could be caused by differences in the measurement conditions. We observed the rats under various physiological conditions, including being conscious and unrestrained. Moreover, we did not limit any access to food and water. Therefore, the buffering action of the food in the stomach could not be excluded in our system. We think that the peak in the nocturnal activity period in the circadian rhythm that we observed could be formed by the balance of the acid secretion and the buffering of the food that was given during this period. Otherwise, the circadian rhythms that we observed were in agreement with the former report in terms of the regularity of the 24-h cycle with a negligible (2–4 h) shift of the peak and the bottom time on the basis of the light/dark cycle.

Several studies have revealed that long-term acid suppression therapy using proton pump inhibitors may lead to elevation of the serum gastrin concentration, and this elevation of serum gastrin may cause enterochromaffin-like cell carcinoma and mucosal hypertrophy in rodents (Lindberg et al., 1990Go; Herling and Weidmann, 1994Go; Freston et al., 1995Go). In some reports, a rebound of gastric acid secretion was observed after acid suppression therapy in clinical use (Gillen et al., 1999aGo,bGo; Gillen and McColl, 2001Go) and in experimental animals (Larsson et al., 1988Go). In the present study, we observed the rapid rebound of gastric acid secretion in lansoprazole-treated rats. Significant rebound hypersecretion was observed at 2 days after the completion of lansoprazole treatment. These results are consistent with a previous report which indicated that significant hypersecretion was observed within 3 days after cessation of an 88-day consecutive administration of 400 µmol/kg omeprazole in rats (Larsson et al., 1988Go). However, in the present study, intriguingly, significant rebound hypersecretion was observed after a rather short treatment period of PPI, in comparison with the other study.

In the present study, CS-526 at the dose of 100 mg/kg/day significantly increased the antral gastrin contents, and although no statistical significance was observed, the serum gastrin was also slightly increased. However, lansoprazole showed more profound effects on the serum and the antral gastrin levels. The effects of lansoprazole on the serum and antral gastrin levels were different between experiments 2 and 3. The antral gastrin level after 30 mg/kg/day of lansoprazole increased in experiment 2 but not in experiment 3. On the other hand, the serum gastrin concentration was somewhat higher in experiment 3 and was significantly different from the vehicle treatment. It is considered that the timing of sampling could have been slightly mismatched, i.e., antral gastrin could have been released more from G-cells to the blood stream in experiment 3.

It is commonly believed that a more potent antisecretory effect on gastric acid causes a more potent serum gastrin elevation as a compensation mechanism for acid loss. In a previous report (Ito et al., 2007Go), it is interesting to note that the gastric antisecretory effects of CS-526 have been shown to be comparable with those of lansoprazole in terms of potency and duration of activity. Accordingly, we evaluated whether CS-526 has direct effects on gastrin release or synthesis in compensation for acid loss. CS-526 reduced the serum gastrin elevation caused by lansoprazole treatment with slight changes in the antral gastrin content and mRNA levels. This suggested that CS-526 could affect the synthesis of gastrin in antral gastrin-producing cells. However, the mechanisms of the inhibition of gastrin synthesis were not clarified. A previous study revealed that pirenzepine (muscarinic M1 antagonist) attenuates the gastrin elevation caused by omeprazole treatment (Tari et al., 1996Go). In that report, pirenzepine attenuated the serum gastrin and antral gastrin mRNA elevation caused by omeprazole treatment and increased somatostatin-like immunoreactivity and somatostatin mRNA in antral tissue significantly, albeit in small increments. Somatostatin is known to be a paracrine hormone that inhibits gastrin synthesis. Therefore, Tari et al. (1996Go) concluded that pirenzepine would block the elevation of serum gastrin and antral gastrin mRNA at least partly by somatostatin synthesis through muscarinic receptors on D cells. We also evaluated the effects of CS-526 on the antral and corpus somatostatin mRNA levels. However, neither the antral nor corpus somatostatin mRNA was increased by CS-526 treatment, and the level remained low in concomitant treatment of CS-526 and lansoprazole. Therefore, we concluded that the lowering effect of CS-526 on serum gastrin would not be induced by a modification of somatostatin synthesis.

Gillen et al. (1999aGo) reported that basal acid output and maximal acid output (MAO) at 15 days after the treatment of 40 mg once a day of omeprazole for 8 weeks on Helicobacter pylori-negative patients were significantly increased compared with the output at pretreatment. In that study, the degree of increase in MAO at posttreatment was significantly associated with the fasting plasma gastrin levels. The subjects whose fasting serum gastrin was >25 ng/l increased MAO by 49.2%, whereas the subjects whose fasting serum gastrin was <25 ng/l increased MAO by only 19.8% (P < 0.006). It is hypothesized that the rebound acid hypersecretion would be induced by serum gastrin elevation after oxyntic mucosal hypertrophy. In the present study, we did not evaluate the thickness or number of parietal cells in the oxyntic mucosa and whether such changes occurred within the rather short treatment period. However, we speculate that lower serum gastrin would be one of the reasons that CS-526 did not induce significant rebound hypergastric acid secretion.

In conclusion, the antisecretory effect of CS-526 was not significantly different from that of lansoprazole. However, CS-526 did not show the same rebound hypergastric acid secretion as lansoprazole. Moreover, CS-526 had a weaker effect on the serum gastrin concentration and antral gastrin content than that of lansoprazole, although the mechanism(s) of the minimal effect on the serum and antral gastrin levels by CS-526 was not clarified. It is considered that these effects of CS-526 would be beneficial in clinical use.


   Footnotes
 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.137299.

ABBREVIATIONS: PPI, proton pump inhibitor; lansoprazole, 2-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-ylmethanesulfinyl]-1H-benzimidazole; APA, acid pump antagonist; CS-526, 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ANOVA, analysis of variance; MAO, maximal acid output.

Address correspondence to: Keiichi Ito, Pharmacology Research Laboratories, Daiichi Sankyo, Co., Ltd., Hiromachi 1-2-58, Shinagawa-ku, Tokyo 140-8710, Japan. E-mail: ito.keiichi.vz{at}daiichisankyo.co.jp


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