Over-the-counter medicine (Seirogan) containing wood creosote kills Anisakis larvae

Anisakis food posoning (anisakiasis) is characterized by the onset of severe abdominal pain. It is a parasitic disease caused by eating raw or undercooked seafoods that harbor larvae of the anisakid nematodes such as Anisakis simplex, A. pegreffi i and Pseudoterranova decipiens [1,2]. There has been a recent increase in the reported prevalence of anisakiasis worldwide [3,4]. In Japan, anisakiasis accounted for 35% of all cases of food poisoning in 2018 [5]. Once consumed, Anisakis larvae attempt to penetrate into the gastrointestinal wall, inducing severe gastrointestinal pain and serious allergic reactions such as anaphylactic shock against allergens (many proteins of unknown function) sereted from the nematodes or substances on the surface of nematodes [2,6-10]. Anisakis larvae are most frequently detected in the stomach (gastric anisakiasis), whereas bowel anisakiasis is not very frequent [1,11]. In gastric anisakiasis, symptoms occur 2-3 h after the consumption of raw fi sh or other seafoods [12,13]. Anisakis larvae are tolerant to gastric acid containing pepsin [14] and apparently many tested compounds including anti-nematodal agents such as ivermectin and albendazole [15]. In cases of gastric anisakiasis, the main effective treatment is an endoscopic removal of nematodes [16], while the effective treatment for intestinal anisakiasis has not yet been established.

Citation: Matsuoka  as a treatment for stomachache or diarrhea due to digestive disorders. Ten years ago, it was reported that the administration of Seirogan alleviates gastric anisakiasis symptoms (a report of two cases) [19]. In addition, in vitro experiment showed that Seirogan quickly suppressed the motility of Anisakis larvae, and resulted in the deformation of the nematodal head and disruption of sheath striation [19]. Based on these results, Seirogan was suggested to suppress the viability of nematodes [19]. However, it is still unclear whether the Seirogan-treated immobile nematodes are killed or temporarily paralysed. The allergens secreted by living nematodes may be responsible for an abrupt hypersensitivity reaction leading to the severe stomach pain or other allergic symptoms [2,9]. If the nematodes are killed by Seirogan administration, the production and secretion of allergens by living nematodes may be stopped, thereby resulting in alleviation of allergic symptom. The nematodes rarely penetrate through the gastrointestinal wall to reach the peritoneal cavity [20]. Such a high-risk case may be avoided if the nematodes are killed by Seirogan administration before they deeply penetrate into the gastrointestinal wall. Therefore, we conducted the present study to determine whether or not Seirogan kills Anisakis larvae.

Samples
Anisakis simplex L3 larvae (15-30mm) were isolated from internal organs of mackerel and pacifi c cod, and then maintained in 0.9% NaCl at room temperature (~25 o C). A. simplex larva was identifi ed by the shape of stomach and protrusion of posterior end.

Treatment with seirogan
One pill (containing 44 mg of wood creosote) of Seirogan (Taiko Pharmaceutical Co.) was dissolved in 5 mL or 10 mL of 0.01 M HCl (pH 2). Anisakis larvae kept in 0.9% NaCl were transferred into small petri dishes fi lled with a Seirogan solution or with 0.01 M HCl, and the petri dishes were then kept in an incubator for 30 min at 37 o C. After treatment, the nematodes were transferred into 0.9% NaCl and kept at room temperature (~25 o C) for 2 days of observation.

Trypan blue staining
To determine whether the Anisakis larvae that were treated with the Seirogan test solutions remained alive or not, the nematodes were transferred into 0.4% trypan blue solution (Fujifi lm Wako Pure Chemical Co., Osaka, Japan) and left them to be stained for 5 h at 37 o C.

Results and discussion
When Anisakis larvae were treated with either of the two different concentrations of Seirogan solutions, all nematodes stopped moving within 30 min. The treated nematodes were transferred into 0.9% NaCl and then observed at 0, 24 and 48.
The motility of the nematodes treated with a Seirogan solution (1 pill/5 mL) was not regained at all, whereas 13.3% of the nematodes treated with a diluted Seirogan solution (1 pill/10 mL) began to move 1 day later (Figure 1). In the 0.01 M HCl solution, all nematodes continued to move for ≥2 days ( Figure   1).
If motionless nematodes are killed, they will be stained blue by trypan blue staining, which has been widely used for the selective staining of dead tissues or cells [21]. To determine whether Seirogan-treated immobile nematodes are killed or simply temporarily paralyzed, Seirogan (1 pill/10 mL)- h, but some of the immobile nematodes may be alive.  Living Anisakis larvae are resistant to human gastric fl uid, which contains the digestive enzyme pepsin [14]. The question thus arises: Are the nematodes killed by Seirogan treatment digested by pepsin? The estimated concentration of pepsin contained in human gastric fl uid is 13.8 μg/mL [22]. When the living nematodes without Seirogan treatment were incubated in a pepsin solution for 24 h at 37 o C, they were still moving actively ( Figure 3A). On the other hand, the nematodes treated with Seirogan solution (1 pill/10 mL) were disrupted by pepsin digestion (Figure 3B, arrowheads). The results of the pepsin treatment were summarized in Table 2, showing that most (83.3%) of Seirogan-treated (1 pill/10 mL) immobile nematodes were digested by pepsin treatment. These results suggest that Anisakis larvae may begin to be digested within 24 h in the stomach cavity only if they are fi rst killed by oral administration of Seirogan.
When the 4/5 portion from the posterior end of the nematode body was dipped in Seirogan solution (1 pill/5 mL) at room temperature, the posterior 4/5 portion stopped moving soon. The anterior 1/5 portion continued to actively move during observation for 10 min (8 nematodes observed). This result suggests that Seirogan components that are effective for killing nematodes may be absorbed from the nematodes' body surface, and that Anisakis larvae can be killed by Seirogan administration even if they partially penetrated into the gastric wall.
The usual dose of Seirogan is three pills (providing 133 mg of wood creosote). The gastric fl uid volume in the fasted state of an adult is ~30 mL [23,24]. It is possible that Anisakis larvae attached to the stomach wall may be killed by this single dose if three pills of Seirogan are completely dissolved in 30 mL of gastric fl uid in the fasted state. If the nematodes are killed by Seirogan before they deeply penetrate into the gastrointestinal wall, a high-risk situation in which the nematodes penetrate through the gastrointestinal wall to reach the peritoneal cavity may be avoided. The secretion of allergens from killed nematodes may also be stopped. The results of the present in vitro experiments demonstrate that a widely available OTC intestinal medicine killed Anisakis larvae at its normal dose. As the next step, a clinical study should be conducted to elucidate whether the nematodes that have attached to the stomach wall can be actually killed by oral administration of Seirogan.

Conclusion
The present in vitro study suggested that Anisakis larvae might be killed by oral administration of widely available OTC intestinal medicine 'Seirogan' at its normal dose, and the killed nematodes are probably degested by pepsin contained in the gastric juice. Anisakis larvae can be killed by Seirogan even if they partially penetrate into the gastric wall, because Seirogan components may be absorbed from the nematodes' body surface. To our knowledge, this is the fi rst report of the medicine that possesses strong nematocidal activity against Anisakis larvae.