Toxicity of novel anti-hepatitis drug bicyclol: A preclinical study

Table 1 Effect of bicyclol on blood picture and biochemical markers in rats (mean±SD).

Group (mg/kg)		Blood picture					Classification of leukocytes,RET and PT
		Sex	RBC(104/mm3)	WBC(/mm3)	Hb (g/dL)	HCT (%)	Leukocytes (%)			RET (%)	Platelet (1×109/L)
							Neutrophil	Lymphocyte	Reticulocyte
Control		M	713.8±95.8	14400±3969	17.3±1.4	37.5±5.2	39.3±7.05	58.6±4.32	4.5±2.06	1.55±0.99	900.75±136.07
Bicyclol	150		753.7±55.0	14102±3531	17.1±0.8	38.8±3.6	39.0±8.12	58.6±6.97	2.0±0.89	0.86±0.26	837.60±155.62
	300		609.1±109	15850±5813	15.4±5.3	31.7±11	29.6±9.22	68.4±9.07	2.0±0.63	1.14±0.91	905.60±106.10
	600		593.2±86.8a	11477±1919	14.7±1.5a	29.7±3.8	-	-	-	-	-
Control		F	681.9±104	13230±3671	16.8±1.99	35.1±5.0	29.4±13.47	67.8±13.9	2.4±2.5	1.04±0.62	763.20±144.50
Bicyclol	150		620.1±104	13220±2721	15.7±1.25	31.3±3.7	33.4±10.07	65.4±11.7	1.0±0.9	0.74±0.37	715.60±76.20
	300		626.9±70	12656±1461	15.3±1.52	34.6±3.9	45.0±8.7	53.4±8.7	1.6±1.6	0.92±0.55	831.6±82.20
	600		622.9±80	10777±4355	14.7±1.51	34.1±4.4	42.0±15.6	54.4±14.6	3.6±2.9	0.70±0.49	562.4±273.30

^aP<0.05 vs control group.

Table 2 Effect of bicyclol on organ weight in rats (n = 10, mean±SD).

Organ weightmg/100g BW	Sex	Control	Bicyclol (mg/kg)
			150	300	600
Heart	M	308±38	300±42	310±74	360±70
Liver		2960±310	3120±610	3200±810	3300±700
Lung		570±120	620±280	580±160	700±180
Kidney		670±70	660±120	700±180	580±270
Spleen		160±30	170±40	170±43	140±45
Thymus		60±20	60±20	60±17	45±18
Pituitary		3±4	2±1	2±1	2±1
Adrenal		20±10	24±30	14±3	16±4
Prostate		110±30	120±30	109±29	110±40
Seminal		250±80	240±80	250±55	260±66
Testis		1150±160	1120±180	1040±301	1130±296
Heart	F	350±40	360±38	380±40	370±41
Liver		3200±330	3300±220	3300±210	3300±290
Lung		970±320	890±280	700±80	680±291
Kidney		810±110	820±100	810±140	770±130
Spleen		220±44	210±36	240±34	320±54b
Thymus		102±36	67±23b	53±24b	120±88
Pituitary		5±2	3±1	5±2	12±15
Adrenal		29±4	26±4	29±3	51±8
Uterus		173±50	147±48	163±31	158±57
Ovary		39±19	34±24	37±3	47±25

^bP<0.01 vs control group.

Table 3 Effect of bicyclol on blood biochemical markers in rats (n = 10, mean± SD).

Parameter	Control	Bicyclol (mg/kg)
		150	300	600
Male rats
TP 10 g/L	6.68±0.31	6.30±0.63	7.30±0.61	6.47±0.41
ALB 10 g/L	3.82±0.22	3.55±0.29	3.79±0.42	3.68±0.21
GLB 10 g/L	2.86±0.22	2.75±0.43	3.51±0.38	2.80±0.32
A/G	1.34±0.13	1.31±0.17	1.09±0.15	1.33±0.17
SGOT IU/L	391±74	411±57	406±50	431±76
SGPT IU/L	63±10	50±8	46±8	62±10
ALP IU/L	293±42	255±53	303±67	285±68
LAP IU/L	63±5	62±7	69±8	65±5
T-CHO 10 mg/L	73.6±6.68	65.7±8.26	72.1±9.51	69.3±5.87
TG 10 mg/L	107.4±33.7	109.9±30.6	97.1±15.1	106.7±25.8
GLU 10 mg/L	4.2±5.33	2.2±1.87	8.5±12.7	8.2±8.8
D-BIL 10 mg/L	0.14±0.03	0.16±0.07	0.18±0.08	0.18±0.06
T-BIL 10 mg/L	1.33±0.22	1.32±0.71	1.58±0.67	1.68±0.53
BUN 10 mg/L	20.7±4.8	18.3±4.7	18.4±2.7	17.0±5.8
Creat 10 mg/L	1.14±0.19	1.13±0.16	1.13±0.23	0.97±2.2
Female rats
TP 10 g/L	6.53±0.78	6.58±0.75	6.93±0.64	6.73±0.44
ALB 10 g/L	3.55±0.57	3.58±0.46	3.97±0.36	4.06±0.36
GIB 10 g/L	2.99±0.53	2.99±0.45	2.96±0.45	2.68±0.40
A/G	1.22±0.26	1.22±0.19	1.37±0.21	1.55±0.30
sGOT IU/L	439±95	403±70	520±119	433±100
sGPT IU/L	59±31	38±11	49±16	46±10
ALP IU/L	227±170	203±156	189±46	151±26
LAP IU/L	59±18	59±5	69±7	69±6
T-CHO 10 mg/L	66.4±14.5	63.2±10.5	63.5±4.20	60.4±9.2
TG 10 mg/L	108.6±49.5	117.5±58.7	121.9±23.1	120.8±4.3
GLU 10 mg/L	7.4±4.8	6.1±5.5	8.2±4.0	16.3±12.0
D-BIL 10 mg/L	0.36±0.45	0.27±0.09	0.19±0.05	0.16±0.08
T-BIL 10 mg/L	2.84±3.03	2.35±0.65	1.79±0.54	1.57±0.77
BUN 10 mg/L	23.0±7.2	32.7±16.7	24.6±9.6	23.5±4.5
Creat 10 mg/L	0.90±0.14	1.06±0.25	0.98±0.18	0.81±0.28

Table 4 Blood test of female and male beagle dogs in control group before and after bicyclol treatment (mean±SD).

Parameter	Before treatment	After treatment (mo)
		1.5	3	4.5	6	7 (n = 1)
Female (n = 2)
RBC ×1012/L	9.2±0.5	9.2±0.5	7.3±2.2	8.2±0.9	9.8±2.0	8.4
Hb g/L	140±14	140±7	145±7	143±11	148±11	140
RET (%)	0.9±0.3	0.8±0.3	0.9±0.3	0.7±0.3	0.8±0.1	0.6
PLT ×109/L	200±14	205±21	215±35	370±14a	265±78	240
WBC ×109/L	14.6±2.2	13.7±2.0	15.2±3.7	13.6±2.6	16.2±7.2	14.5
Lymphocyte (%)	55±11	55±12	62±14	61±12	69±8	74
Polymorphonuclear leukocyte (%)	41±7	41±13	36±13	34±9	28±4	21
Monocyte (%)	5.5±2.1	5.0±1.4	2.5±0.7	4.0±1.4	3.5±0.7	5
Male (n = 3)
RBC ×1012/L	8.7±1.7	7.3±0.6	8.7±2.1	8.5±1.6	7.9±1.1	8.6
Hb g/L	135±10	140±5	135±9	145±7	150±7	140
RET (%)	0.9±0.4	0.6±0.1	0.6±0.2	0.9±0.4	0.8±0.4	0.8
PLT ×109/L	223±38	250±26	197±32	260±85	290±28	312
WBC ×109/L	12.1±3.1	12.7±0.8	10.6±2.3	11.9±2.1	12.2±1.6	20.1
Lymphocyte (%)	52±12	57±11	48±11	69±4	67±8	66
Polymorphonuclear leukocyte (%)	45±11	39±13	47±10	28±4	26±1	26
Monocyte (%)	3.7±0.6	3.3±1.5	2.7±0.6	3.5±0.7	7.0±1.4a	8

^aP<0.05 vs before dosing.

Table 5 Blood test of female and male beagle dogs before and after treatment with 225 mg/kg bicyclol (mean±SD).

Parameter	Before treatment	After treatment (mo)
		1.5	3	4.5	6	7 (n = 1)
Female (n = 4)
RBC ×1012/L	8.9±2.1	9.0±2.5	8.1±1.6	10.0±0.8	9.0±2.3	8.8
Hb g/L	141±9	151±9	141±6	153±12	165±13a	155
RET (%)	1.0±0.2	0.6±0.1	0.8±0.2	0.7±0.1	0.8±0.2	0.8
PLT ×109/L	195±55	230±37	217±29	237±15	313±25a	368
WBC ×109/L	10.6±2.2	10.2±0.7	12.2±3.2	10.5±2.4	12.2±3.0	10.9
Lymphocyte (%)	51±6	52±8	60±11	59±11	59±10	63
Polymorphonuclear
Leukocyte (%)	44±6	43±8	37±6	39±9	36±8	31
Monocyt (%)	3.8±1.3	4.5±0.6	3.0±1.2	2.0±2.0	5.3±3.1	6
Male (n = 2)
RBC ×1012/L	8.9±0.3	10.4±0.0a	9.9±1.6	7.4	7.9	7.5
Hb g/L	140±14	142±9	140±7	155	160	145
RET (%)	1.1±0.1	0.9±0.2	1.0±0.4	0.9	0.8	0.6
PLT ×109/L	215±7	290±0a	138±23a	210	260	248
WBC ×109/L	9.3±0.6	9.5±0.7	11.2±1.9	6.9	12.8	14.4
Lymphocyte (%)	55±4	51±11	52±6	48	55	67
Polymorphonuclear
Leukocyte (%)	41±3	46±11	46±6	48	40	26
Monocyt (%)	3.5±0.7	3.0±0.0	4.0±1.4	4	5	7

^aP<0.05 vs before dosing.

Table 6 Blood biochemical parameters of female and male beagle dogs 6 mo after bicyclol treatment (mean±SD).

Parameter	Control	Bicyclol (mg/kg)
		25	75	225
Female
Glu mmol/L	4.94±0.83	6.06±0.38	5.60±0.08	6.36±0.62
T-CHO mmol/L	2.56±1.08	2.82±0.60	2.97±1.02	3.43±1.39
TP g/L	71.3±0.2	74.3±4.2a	73.5±3.3	76.1±6.2
ALB g/L	37.0±0.9	39.9±1.7	41.0±3.7	44.0±4.1
Crea mmol/L	119±28	138±10	145±42	143±27
BUN mmol/L	4.25±0.05	3.51±0.24	4.22±0.29	3.60±0.78
T-BIL mmol/L	2.80±1.70	4.00±2.26	3.60±0.57	1.20±0.40
SGPT 10 U/L	26.2±1.3	28.3±15.0	12.8±8.9	25.2±8.1
SGOT 10 U/L	61.8±14.9	41.3±14.6	45.1±8.0	45.8±23.8
ALP 10 U/L	2.65±1.51	1.95±0.39	2.55±0.38	3.14±1.12
Male
Glu mmol/L	5.79±0.29	5.19±0.44	5.58±0.88	4.65
T-CHO mmol/L	2.37±0.35	2.82±0.60	2.39±0.06	2.65
TP g/L	74.2±5.2	71.6±9.8	79.1±12.2	70.6
ALB g/L	34.9±1.6	38.7±1.5	42.4±7.5	33.4
Crea mmol/L	136±10	136±9.8	165±39	105
BUN mmol/L	3.43±0.63	3.94±0.58	2.95±0.34	3.2
T-BIL mmol/L	4.40±0.57	4.00±0.00	3.20±0.80	2.8
SGPT 10 U/L	34.6±9.9	23.1±6.9	25.7±9.9	36
SGOT 10 U/L	57.9±11.6	37.9±5.8	54.5±7.3	53.3
ALP 10 U/L	2.51±0.46	5.41±4.59	2.23±1.16	1.94

^aP<0.05 vs control group.

Table 7 Mutagenicity of bicyclol in bacteria (revertants/plate) (mean±SD).

(mg/plate)		TA97		TA102
		0	0	0	0
Bicyclol	0	86±15	130±15	227±15	272±12
	10	68±8	111±1	237±13	230±10
	100	80±1	116±20	259±33	304±40
	500	57±14	120±6	236±16	279±5
	1000	88±8	140±4	237±35	344±18
	5000	80±4	88±1	259±23	153±2
9-aminoacridine	0.2	938±10
	2AF 10		1736±509
	DMC 20
	MMS 2 mL			1606±48
	Vp16 1 mg				1270±37

Table 8 Mutagenicity in mice after bicyclol treatment.

Group	Dosage (mg/kg)	n	MNPCE (‰)	P/N		P value
Control		6	2.2±0.04	100/56		1.8
Bicyclol	750	6	1.8±0.09	135/65	2.1	>0.05
	1500	6	2.5±0.14	126/74	1.7	>0.05
	3000	6	1.7±0.08	130/70	1.9	>0.05
Cyclophosphamide	50	6	38.2±3.0	100/250	0.4	<0.001

Table 9 Chromosome aberrations in mice after bicyclol treatment.

Dosage (mg/kg)		-S9			+S9
		Aberration (%)	P		Aberration (%)	P
Control (DMSO)	100	2		100	3
Bicyclol 50	100	2		100	2	>0.05
			>0.05
100	100	3	>0.05	100	3	>0.05
200	100	2	>0.05	100	3	>0.05
Carboplatin 100	50	84	<0.001
CY 100
				50	66	<0.001

Table 10 Effect of bicyclol on pregnant rats (mean±SD).

Group	n	Number of pregnancies	Death	Body weight (g)
				D 0	D 9	D 18
Control	20	15	0	251.9±6.8	271.0±6.8	337.7±8.2
Bicyclol 200 mg/kg	20	15	1	253.5±5.4	295.4±5.5	373.3±6.7
Bicyclol 1000 mg/kg	20	17	0	258.2±5.7	277.4±5.64	352.9±7.5

Table 11 Effect of bicyclol on embryonic development.

Dosage (mg/kg)	n	Corpus luteum	Living fetus(%)	Dead fetus (%)	Resorption (%)
Control	15	177	171 (96.6)	0	6 (3.4)
Bicyclol 200	15	181	155 (93.3)	6 (3.6)	5 (3.0)
Bicyclol 1000	17	225	203 (97.1)	1 (0.5)	5 (2.4)

Table 12 Effect of bicyclol on fetus (mean±SD).

Dosage (mg/kg)	Brood number (X)	BW (g)	Height (cm)	Tail length (cm)	Sex ratio (F/M)
Control	39.9±4.1	3.5±0.01	3.4±0.02	1.28±0.02	89/82
Bicyclol 200	43.3±3.1	4.3±0.08	3.4±0.02	1.26±0.01	81/74
Bicyclol 1000	47.7±2.2	4.0±0.09	3.3±0.02	1.17±0.01	97/106

Table 13 Effect of bicyclol on ossification of fetus, n (%).

Dosage (mg/kg)	n	Ossification of sternum
		Defect of the 5th and 6th sternums	Defect of the 6th sternum	Number of sternum defects
Control	87	0	8 (9.2)	79 (91.0)
Bicyclol 200	77	1 (1.3)	6 (7.8)	70 (90.9)
Bicyclol 1000	108	9 (8.3)	1 (0.9)	94 (87.0)

Table 14 Effect of skeleton development of fetus.

Dosage (mg/kg)	n	Cervical -rib	Defect of the 13th rib	Defect of the 14th rib
Control	87	0	0	0
Bicyclol 200	77	0	0	0
Bicyclol 1000	108	0	0	4 (3.7%)