Abstract. The mouse embryonic stem cell test (mEST) is used to assess the embryotoxicity of drug candidates by evaluating the effects on the cardiac differentiation of stem cells. However, thalidomide embryotoxicity has not yet been reported using the mEST. To detect the effects of thalidomide, we used human induced pluripotent stem cells (hiPSCs) instead of mouse embryonic stem cells, and assessed three endpoints: the inhibition of cardiac differentiation, the cytotoxicity to hiPSCs, and the cytotoxicity to human dermal fibroblasts, according to the mEST. From these data (IC 50 values), the embryotoxicity was classified into one of three different classes based on the mEST and our criteria. Valproate was used as a positive control and ascorbic acid was used as a negative control, and their effects were assessed. Similar to valproate, thalidomide was classified as a Class 2 agent, with weak embryotoxicity, by the mEST criteria, and was classified as Category 3 embryotoxic based on our criteria. Ascorbic acid was classified as a Class 1 / Category 1, non-embryotoxic agent, based on both criteria. Thalidomide embryotoxicity was detected in the embryonic stem cell test based on hiPSCs. This test system is thus considered to have a much greater predictive ability than the mEST.

In vitro human induced pluripotent stem (iPS) cells testing (iPST) to assess developmental toxicity, e.g., the induction of malformation or dysfunction, was developed by modifying a mouse embryonic stem cell test (EST), a promising animal-free approach. The iPST evaluates the potential risks and types of drugs-induced developmental toxicity in humans by assessing three endpoints: the inhibitory effects of the drug on the cardiac differentiation of iPS cells and on the proliferation/survival of iPS cells and human fibroblasts. In the present study, the potential developmental toxicity of drugs was divided into three classes (1: non-developmentally toxic, 2: weakly developmentally toxic and 3: strongly developmentally toxic) according to the EST criteria. In addition, the type of developmental toxicity of drugs was grouped into three types (1: non-effective, 2: embryotoxic [inducing growth retardation/dysfunction]/ deadly or 3: teratogenic [inducing malformation]/deadly) by comparing the three endpoints. The present study was intended to validate the clinical predictability of the iPST. The traditionally developmentally toxic drugs of aminopterin, methotrexate, all-trans-retinoic acid, thalidomide, tetracycline, lithium, phenytoin, 5-fluorouracil, warfarin and valproate were designated as class 2 or 3 according to the EST criteria, and their developmental toxicity was type 3. The non-developmentally toxic drugs of ascorbic acid, saccharin, isoniazid and penicillin G were designated as class 1, and ascorbic acid, saccharin and isoniazid were grouped as type 1 while penicillin G was type 2 but not teratogenic. These results suggest that the iPST is useful for predicting the human developmental toxicity of drug candidates in a preclinical setting.

KW-5617 (zaldaride maleate), 1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin -4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one maleate, is a selective calmodulin inhibitor. We studied the effects of KW-5617 on secretory diarrhea and gastrointestinal propulsion in rats, as compared with those of loperamide, a conventional anti-diarrheal drug. Diarrhea was induced in rats either by 16,16-dimethyl prostaglandin E2 (500 microg/kg, i.p.) or by castor oil (1 ml/100 g body weight, p.o.). In the 16,16-dimethyl prostaglandin E2 model, KW-5617 at the doses of 3 mg/kg (p.o.) and higher ameliorated the diarrhea. Similarly, loperamide improved the diarrhea, the activity of loperamide being equivalent to that of KW-5617. In the castor oil model, KW-5617 significantly delayed the onset of diarrhea at the doses of 3 mg/kg (p.o.) and higher, while loperamide delayed the onset of diarrhea at the doses of 0.3 mg/kg (p.o.) and higher. KW-5617 only at the high doses of 30 and 100 mg/kg (p.o.) reduced gastric emptying, small intestinal propulsion, proximal colonic propulsion and distal colonic propulsion. In contrast, loperamide at its anti-diarrheal doses inhibited gastrointestinal propulsion. Our results show that KW-5617, unlike loperamide, at its anti-diarrheal doses does not exert anti-propulsive effects in rats. KW-5617 may be a useful drug for the treatment of diarrhea in terms of less side effects such as constipation.

The antidiarrheal action of zaldaride maleate (ZAL) after oral, intravenous and subcutaneous administration was examined to determine whether ZAL acts systemically or locally in the intestine of rats. Oral administration of ZAL inhibited castor oil- and 16,16-dimethyl prostaglandin E2-induced diarrhea; however, intravenous or subcutaneous administration of ZAL was ineffective. When ZAL was orally administered, the area under the plasma concentration time curve of the compound was lower than that of ZAL following intravenous or subcutaneous administration at the maximum doses studied. The antidiarrheal effect of ZAL was not dependent on its plasma concentration level. These results suggest that ZAL acts locally in the intestinal tract in rats.

The effect of zaldaride, a calmodulin inhibitor, on fecal pellet output in rats was compared with that of loperamide, an antidiarrheal drug. 5-Hydroxytryptamine (10 mg/kg, s.c.), neostigmine (0.3 mg/kg, s.c.) and nicotine (1.0 mg/kg, s.c.) increased fecal pellet output. Zaldaride (> or = 30 mg/kg, p.o.) reduced these increases in fecal pellet outputs. Loperamide (10 mg/kg, p.o.) inhibited fecal pellet output induced by 5-hydroxytryptamine and neostigmine but not nicotine. Under normal conditions, zaldaride and loperamide did not affect fecal pellet output at doses used in these studies. In conclusion, zaldaride may inhibit increases in fecal pellet output induced by hyperpropulsion of the gastrointestinal tract without causing constipation as a side effect.