Effect of DA-9701 on gastric emptying in a mouse model: Assessment by 13C-octanoic acid breath test

Abstract

AIM: To evaluate the effects of DA-9701 on the gastric emptying of a solid meal using the ¹³C-octanoic acid breath test in a mouse model.

METHODS: Male C57BL/6 mice aged > 8 wk and with body weights of 20-25 g were used in this study. The solid test meal consisted of 200 mg of egg yolk labeled with 1.5 L/g ¹³C-octanoic acid. The mice were placed in a 130 mL chamber flushed with air at a flow speed of 200 mL/min. Breath samples were collected for 6 h. The half-emptying time and lag phase were calculated using a modified power exponential model. To assess the reproducibility of the ¹³C-octanoic acid breath test, the breath test was performed two times at intervals of one week in ten mice without drug treatment. To assess the gastrokinetic effects of DA-9701, the breath test was performed three times in another twelve mice, with a randomized crossover sequence of three drug treatments: DA-9701 3 mg/kg, erythromycin 6 mg/kg, or saline. Each breath test was performed at an interval of one week.

RESULTS: Repeatedly measured half gastric emptying time of ten mice without drug treatment showed 0.856 of the intraclass correlation coefficient for the half gastric emptying time (P = 0.004). The mean cumulative excretion curve for the ¹³C-octanoic acid breath test showed accelerated gastric emptying after DA-9701 treatment compared with the saline control (P = 0.028). The median half gastric emptying time after the DA-9701 treatment was significantly shorter than after the saline treatment [122.4 min (109.0-137.9 min) vs 134.5 min (128.4-167.0 min), respectively; P = 0.028] and similar to that after the erythromycin treatment [123.3 min (112.9-138.2 min)]. The lag phase, which was defined as the period taken to empty 15% of a meal, was significantly shorter after the DA-9701 treatment than after the saline treatment [48.1 min (44.6-57.1 min) vs 52.6 min (49.45-57.4 min), respectively; P = 0.049].

CONCLUSION: The novel prokinetic agent DA-9701 accelerated gastric emptying, assessed with repeated measurements in the same mouse using the ¹³C-octanoic acid breath test. Our findings suggest that DA-9701 has therapeutic potential for the treatment of functional dyspepsia.

Key Words: DA-9701, Gastric emptying, Prokinetic agent, Breath test, Functional dyspepsia

Core tip: DA-9701 is a newly formulated prokinetic agent obtained from extracts of Pharbitis Semen and Corydalis Tuber. The ¹³C-octanoic acid breath test is a reliable and responsive method for measuring gastric emptying in small laboratory animal. This technique can be performed repeatedly in the same animal and reflect exact pharmacological effects without sacrifice of the animal. This study demonstrated the gastrokinetic effects of DA-9701, using repeated ¹³C-octanoic acid breath tests in the same animal. The gastrokinetic effect of DA-9701 could have therapeutic potential for the treatment of familial dysautonomia.

INTRODUCTION

DA-9701 is a newly formulated prokinetic agent obtained from extracts of Pharbitis Semen and Corydalis Tuber. Both the seed of Pharbitis Semen and the root of Corydalis Tuber have been used in traditional Oriental medicine for the treatment of gastrointestinal symptoms. DA-9701 and its components accelerated gastric emptying and improved gastric accommodation in animal model[1-4]. A previous study demonstrated the gastrokinetic effect of DA-9701 in a rat model[1], using the method of Ozaki et al[5]. With this model, the animal must be killed to assess gastric emptying, and consequently, gastric emptying can only be measured at that one point in time. The disadvantage of this method is that it is impossible to assess gastric emptying repeatedly in the same animal. Therefore, the utility of the model is limited for evaluating pharmacologically induced gastric emptying because there is great intersubject variability.

Stable isotope breath tests are indirect noninvasive tests for measuring gastric emptying. Their advantages include the absence of a radiation hazard, ease of handling, and no requirement for positioning, unlike scintigraphic tests[6]. Breath tests offer an attractive method of measuring gastric emptying in small laboratory animals because the animal need not be killed, thus allowing repeated measurements to be made in the same animal. With the ¹³C-octanoic acid breath test, gastric emptying can be assessed noninvasively, and the differences in gastric emptying induced in small laboratory animals by pharmacological agents can be quantified[7-11]. This test is a useful tool in the development of new prokinetic agents that modulate gastric emptying. The aim of this study was to evaluate the effects of DA-9701 on the gastric emptying of a solid meal using the ¹³C-octanoic acid breath test in a mouse model.

MATERIALS AND METHODS

Animals

Male C57BL/6 mice, aged > 8 wk and with body weights of 20-25 g, were used for the breath test. Ten mice were used to assess the reproducibility of the ¹³C-octanoic acid breath test and another 12 mice were used to evaluate the effects of DA-9701. Sample sizes were calculated from the equation below by referring to a previous study using C57BL/6 mice[8]: n = 2 + C (s/d)², where s is SD, d is expected difference between two means, and C is constant. A sample size of 12 mice was suitable to identify, with a power of 0.8, the expected difference in half gastric emptying time of 50 min with an alpha significance level of 0.05. All the mice were housed in a room maintained at 21 °C-23 °C on a 14/10 h light/dark cycle. The mice had continuous access to water and a standard commercial diet. All experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea, Seoul, South Korea.

Test meal

The gastric emptying rates were assessed for a solid egg yolk meal. ¹³C-Octanoic acid (Octanoic acid-1-¹³C, 99 atom % ¹³C, Sigma-Aldrich Co. LLC, St Louis, Missouri, United States) was added to raw yolk at a concentration of 1.5 μL/g. The yolk was homogenized and heated in water at 60 °C for 120 min. The test meal (200 mg) was given to each animal after an overnight fast with free access to water and was completely consumed by the mouse within 1 min in the breath test chamber.

Breath test protocol

To assess the gastric emptying rate using the ¹³C-octanoic acid breath test with measurements made up to 360 min, the mice were fasted overnight[7,8]. They were then placed in a 130 mL gas-tight rubber-sealed chamber with room air. The flow rate of air was 200 mL/min, which was selected because this rate was sufficient to flush any residual CO₂ from the chamber between the sampling time points. Three-way valves were connected to the chamber for air inlet and outlet to allow sampling. To collect a breath sample, the airflow through the chamber was stopped for 4 min. At the end of the breath-accumulation period, 10 mL of breath was syringed from the chamber, and the airflow through the chamber was restored. Each mouse was maintained in the chamber for the whole 360 min sampling procedure. A baseline breath sample was taken before the test meal was consumed. Further breath samples were collected at 5 min intervals for the first 30 min, and at 15 min intervals thereafter until 360 min after the consumption of the test meal.

Reproducibility of the breath test

To assess the reproducibility of the ¹³C-octanoic acid breath test, the breath test was performed two times at intervals of 1 wk in ten mice without drug treatment.

Drug treatment

This study was designed as a randomized crossover study. DA-9701 (3 mg/kg) was the test drug, erythromycin (6 mg/kg) the positive control, and normal saline the placebo. There were six possible permutations of the drug treatment sequences, and two animals were allocated to each permutation. The test drug and the positive control were suspended in distilled water for their oral administration. A 10 mL/kg volume of DA-9701, erythromycin, or saline was given orally to each mouse by gavage 1 h before the breath test. The breath test was performed three times in each animal at intervals of 1 wk.

Data analysis

The ¹³CO₂ content of each breath sample was analyzed using a HeliView isotope ratio mass spectrometer (Medichems, Seoul, South Korea). The [¹³C]/[¹²C] ratio (δ) was expressed as parts per thoUnited Statesnd relative to the Pee Dee Belemnite calcium carbonate international primary standard. The gastric emptying rates were calculated from the resultant ¹³CO₂ excretion curves. The CO₂ production rate of the mice was assumed to be 40 mL/kg per minite based on normal values for the resting metabolic parameters measured in C57BL/6 mice[12]. The percentage ¹³CO₂ cumulative values were fitted using a modified power exponential model[13]: y = m (1 - e^-kt)^β, where y is the cumulative percentage of ¹³CO₂ excretion in breath at time t (h), and m, k and β are estimated parameters. In this model, m is interpreted as the total cumulative ¹³C recovery when the time is infinite. The cumulative ¹³CO₂ excretion cannot reach 100% because a substantial amount of the orally administered dose is fixed in the bicarbonate pool in the body. Because the cumulative excretion reaches a steady state, sampling up to 360 min allows the calculation of m, the theoretical recovery after infinite time. The half gastric emptying time (T_1/2), the lag phase for 10% emptying (T_lag10), and the lag phase for 15% emptying (T_lag15) in the breath test were calculated from this model as T_1/2 = (-1/k) × ln[1 - (0.5^1/β)]; T_lag10 = (-1/k) × ln[1 - (0.1^1/β)]; T_lag15 = (-1/k) × ln [1 - (0.15^1/β)].

Statistical analysis

The results are expressed as medians and interquartile ranges. The reproducibility of breath test was evaluated using the intraclass correlation coefficient. A repeated measurements ANOVA followed by post hoc test at the point of every 60 min was performed for comparison of total cumulative excretion curve after drug treatment. Differences in the data were evaluated using the Wilcoxon signed-rank test for comparisons of the half gastric emptying times and lag phases after drug treatment. A difference was considered significant when the P value was less than 0.05.

RESULTS

A typical excretion curve for ¹³C-octanoic acid breath test of a mouse is presented in Figure 1. Repeatedly measured half gastric emptying time of ten mice is presented Figure 2. The half gastric emptying times of the first and second breath tests were 127.5 min (96.7-137.6 min) and 115.4 (79.9-176.4 min). The half gastric emptying times of the first and second breath tests showed good correlation (intraclass correlation coefficient = 0.856, P = 0.004). The effects of the drug treatments on gastric emptying are shown in Figure 3. The cumulative excretion curve for ¹³C-octanoic acid breath showed accelerated gastric emptying after treatment with DA-9701 compared with that after treatment with saline (P = 0.028). The effect of DA-9701 treatment on gastric emptying was similar to that of erythromycin.

Figure 1 Typical excretion curve of the ¹³C-octanoic acid breath test in the mouse model. m: The total cumulative ¹³C recovery when time is infinite; T_1/2: Half gastric emptying time; T_lag10: Lag phase of 10 % emptying; T_lag15: Lag phase for 15% emptying.

Figure 2 Reproducibility of the ¹³C-octanoic acid breath test in ten mice.

Figure 3 Mean excretion curve of the ¹³C-octanoic acid breath test in mice treated with DA-9701, erythromycin, and saline. m: The total cumulative ¹³C recovery when time is infinite. ^aP < 0.05 between DA-9701 and saline.

The half gastric emptying times and lag phases are presented in Table 1. Both DA-9701 and erythromycin treatments induced significantly shorter half gastric emptying times compared with that induced with saline (P = 0.028 and P = 0.049, respectively). There was no significant difference in the half gastric emptying times of the DA-9701- and erythromycin-treated mice. There was no significant difference in T_lag10 among the treatments. T_lag15 was significantly shorter in mice treated with DA-9701 than in mice treated with saline (P = 0.049). The half gastric emptying time and T_lag15 for each mouse treated with saline, DA-9701, or erythromycin is presented in Figure 4.

Table 1 Half gastric empting time and lag phase measured by the ¹³C-octanoic acid breath test in the mice treated with DA-9701, erythromycin, and saline.

	DA-9701 (n = 12)	Erythromycin (n = 12)	Saline (n = 12)
T1/2 (min)	122.4 (109.0-137.9)1	123.3 (112.9-138.2)2	134.5 (128.4-167.0)
Tlag10 (min)	38.5 (33.2-46.4)	37.9 (35.8-41.4)	39.5 (35.2-45.3)
Tlag15 (min)	48.1 (44.6-58.5)2	49.3 (46.5-53.3)	52.6 (48.9-57.6)

Data represent median with interquartile range in parentheses.

¹P = 0.028,

²P = 0.049 vs saline treatment (Wilcoxon's signed ranks test). T_1/2: Half gastric emptying time; T_lag10: Lag phase for 10 % emptying; T_lag15: Lag phase for 15% emptying.

Figure 4 Half gastric emptying time and lag phase for 15% emptying measured by the ¹³C-octanoic acid breath test in the each mouse treated with DA-9701, erythromycin, and saline. A: Half gastric emptying time (T_1/2); B: Lag phase for 15% emptying (T_log15). ^aP < 0.05 vs saline.

DISCUSSION

Dyspeptic symptoms are common problem in primary health care and gastroenterology practice. Although the precise pathophysiology of functional dyspepsia (FD) is not fully understood, disturbed gastric emptying is one of the important abnormalities in FD patients[14,15]. Current treatment approaches to FD include gastric acid suppression and gastroprokinetic drugs as primary treatment options[16]. However, no satisfactory therapeutic approach is currently available in clinical practice. There is an increasing need for the development of safe and effective prokinetic drugs without adverse effects[17-19].

We have demonstrated the effects of DA-9701 on gastric emptying, using repeated ¹³C-octanoic acid breath tests in the same animal. DA-9701 shortened both the half emptying time and the lag phase, and these effects were similar to those of erythromycin. The half gastric emptying times achieved with the placebo and erythromycin in our model were similar to those reported in previous studies that used the same breath test[8]. We selected a dose of 3 mg/kg DA-9701 based on a previous study of the appropriate DA-9701 doses in a rat model, which showed dose-dependent effects in the range from 0.03 to 3 mg/kg[1]. Another experiment in which DA-9701 was administered at 3 mg/kg also showed accelerated gastric emptying in a model of delayed gastric emptying induced with apomorphine and cisplatin. DA-9701 and its components (corydaline and tetrahydroberberine) accelerated gastric emptying in a rat model when a method that requires the animals to be killed was used, although comparisons within the same animal were impossible[1-3]. We performed the repeated breath tests in each mouse after treatment with either DA-9701, the positive control, or the placebo, according to all possible permutations of the drug treatment sequence, and identified the prokinetic effects of DA-9701.

DA-9701 is a newly formulated prokinetic agent obtained from extracts of Pharbitis Semen and Corydalis Tuber. Both the seed of Pharbitis Semen and the root of Corydalis Tuber have been used in traditional Oriental medicine for the treatment of gastrointestinal symptoms. As well as accelerating gastric emptying, DA-9701 and its components improved gastric accommodation in a dog model with a barostat[1-4]. DA-9701 is composed of several isoquinoline alkaloid compounds, including corydaline, berberine, protopine, and palmatine[20-23]. Although the exact mechanism of its action has not been identified, tetrahydroberberine from DA-9701 has the properties of a D₂ receptor antagonist and a 5-HT_1A receptor agonist, with micromolar affinities for the dopamine D₂ and 5-HT_1A receptors[2]. DA-9701 significantly improved the symptoms of patients with FD, with efficacy similar to that of itopride and a comparable safety profile[24].

In this study, we have demonstrated the gastrokinetic effect of DA-9701 using the ¹³C-octanoic acid breath test in mice. A variety of techniques have been used to assess gastric emptying in small laboratory animals. Most techniques require the animal to be killed and the contents of the stomach and intestine to be quantified by measuring their radioactivity, counting the glass beads remaining, or measuring the concentration of a marker. The ¹³C-octanoic acid breath test has been reported to be a reliable and responsive method for measuring gastric emptying in small laboratory animal[7,8,10,25], and has several advantages over other techniques. First, it allows repeated measurements to be made in the same animal, allowing exact pharmacological effects to be identified and minimizing the number of animals killed. Second, it does not require the animals to be handled or restrained, in contrast to scintigraphic tests. The results can also be expressed as rate curves and cumulative excretion curves. Therefore, the breath test can be used to determine the effects of physiological or pharmacological interventions on gastrointestinal motility, especially in developmental programs for new prokinetic drugs. However, to measure gastric emptying, the breath test requires a relatively long time and considerable effort to sample the animals’ breath.

In conclusion, DA-9701 accelerates gastric emptying, and its effect is similar to that of erythromycin. The ¹³C-octanoic acid breath test can be a useful tool to reflect physiological or pharmacological effects on gastric motility, especially in developmental programs for new prokinetic drugs. The gastrokinetic effect of DA-9701 could have therapeutic potential for the treatment of FD.

ACKNOWLEDGMENTS

We would like to thank Professor Gianrico Farrugia of Mayo Clinic for providing the breath test chambers. We also would like to thank Vet. Yong Sam Kwon of Dong-A Pharmaceutical Co., Ltd for providing technical supports in animal handling.

COMMENTS

Background

Dyspeptic symptoms are common problem in primary health care and gastroenterology practice. Disturbed gastric emptying is one of the important abnormalities in functional dyspepsia (FD) patients. Gastroprokinetic drugs in one of the primary treatment options. There is an increasing need for the development of safe and effective prokinetic drugs without adverse effects

Innovations and breakthroughs

A previous study demonstrated the gastrokinetic effect of DA-9701 in a rat model using the method of Ozaki and Sukamoto. With this model, the animal must be killed to assess gastric emptying, and consequently, gastric emptying can only be measured at that one point in time. The disadvantage of this method is that it is impossible to assess gastric emptying repeatedly in the same animal. This study demonstrated the gastrokinetic effect of DA-9701 using repeated the ¹³C-octanoic acid breath test in same animal with minimizing the number of animals killed.

Applications

The gastrokinetic effect of DA-9701 could have therapeutic potential for the treatment of FD. The ¹³C-octanoic acid breath test can be a useful tool to reflect physiological or pharmacological effects on gastric motility, especially in developmental programs for new prokinetic drugs.

Terminology

¹³C-octanoic acid breath test is one of stable isotope breath tests for measuring gastric emptying. The test meal containing ¹³C-labelled substrates is retained in the stomach and emptied into the duodenum. The ¹³C-labelled substrates are rapidly absorbed and transported to the liver. They are oxidized to ¹³CO₂ in the liver and excreted in the breath. Gastric emptying is the rate-limiting step that makes it possible to use the rate of ¹³CO₂ appearance in the breath approximate to the gastric emptying rate.

Peer review

This study examines the effects of DA-9701, a novel phyto-derived prokinetic agent, on gastric emptying of normal C57BL/6 mice. The authors used ¹³C-octanoic acid breath test to investigate gastric emptying of three treatments (DA-9701, erythromycin vs saline, the latter used as placebo) with a randomized crossover design. This study is interesting and original in its methodological part because it presents a novel method to assess gastric emptying over time rather than evaluating a single moment after sacrificing laboratory animals. The authors precisely describe their experiments using the breath test technique, including a power calculation. Compared to saline, DA-9701 evoked a more rapid emptying similarly to erythromycin. The finding of a prokinetic effect of DA-9701 on gastric emptying is very promising in view of potential therapeutic options in FD.