Acute and repeated dose toxicity studies of recombinant saxatilin, a disintegrin from the Korean snake (Gloydius saxatilis)

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Toxicon 51 (2008) 406–417 www.elsevier.com/locate/toxicon

Acute and repeated dose toxicity studies of recombinant saxatilin, a disintegrin from the Korean snake (Gloydius saxatilis)$ Young-Doug Sohna,1, Sung-Yu Hongb,1, Kil-Sang Choe, Won-Seok Choic, Si-Whan Songd, Jin-Sook Baed, Doo-Sik Kimc, Kwang-Hoe Chunge, a Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul 120-729, Republic of Korea National Research Lab for Cardiovascular Nanobiology, BioBud Inc., Geumchen-gu, Seoul 153-777, Republic of Korea c Department of Biochemistry, College of Science, Bioproducts Research Center, Yonsei University, Seoul 120-749, Republic of Korea d Chemon Preclinical Research Center, 334 Jeil-ri, Yangji-myeon, Chein-gu, Yongin-si, Kyunggi-do 449-826, Republic of Korea e Thrombosis and Vascular Biochemistry Laboratory, Department of Biochemistry, College of Medicine, Pochon CHA University, Bundang-gu, Sungnam 463-836, Republic of Korea b

Received 21 May 2007; received in revised form 27 October 2007; accepted 29 October 2007 Available online 17 November 2007

Abstract To examine the toxicological effect of saxatilin, a disintegrin isolated from the venom of a Korean snake (Gloydius saxatilis), recombinant saxatilin was highly expressed as a biologically active form in Pichia pastoris, and was successfully purified to homogeneity from the culture broth supernatant. The molecular and biological properties of the recombinant protein were the same as those of its natural form. Plasma half-life of the protein in rat was determined to 13.8 min. The maximum tolerated dose of the recombinant saxatilin was examined in ICR mice. The determined LD50 values were 400 and 600 mg/kg of the body weight of a male and female mouse, respectively. To investigate the repeated dose toxicity of saxatilin in mice, the test item was intravenously administered to groups of ICR mice every day for 4 weeks. We observed a decrease in locomotor activity, piloerection, and crouching in clinical findings, a decrease of red blood cells (RBCs) in hematology, and hyperplasia of the spleen in histology related to administration of the test item. These results suggest that the target organ of intravenous administration of the test item is the spleen. The no adverse effect level (NOAEL) in this test for both males and females is considered to be 3 mg/kg. Our results also indicate that recombinant saxatilin is nontoxic at an administration dose with an anti-platelet effect, and might be a potential anti-adhesion therapeutic agent for thrombosis, cancer, restenosis, cataract, and osteoporosis. r 2007 Elsevier Ltd. All rights reserved. Keywords: Disintegrin; Saxatilin; Mass production; Plasma half-life; Acute toxicity; Repeated dose toxicity

$

Ethical statement: This study was carried out in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification No. 1999-61 issued by the Korean Food and Drug Administration on 22 December 1999) and under the Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. This experiment was conducted in facilities of the Chemon Preclinical Research Center approved by the Korean Government, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996). Corresponding author. Tel.: +82 31 725 8379; fax: +82 31 725 8350. E-mail address: [email protected] (K.-H. Chung). 1 These authors equally contributed to this paper. 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.10.019

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1. Introduction Disintegrins are a family of cysteine-rich, low molecular weight proteins (PROs) that are found in various snake venoms (Niewiarowski et al., 1994). Most disintegrins contain the Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD) sequence, which are the structural motifs recognized by various kinds of integrins (Pfaff et al., 1994). These PROs selectively bind to integrins GP IIb–IIIa, avb3, and a5b1, which are expressed in platelets (PLTs), vascular endothelial cells, and some tumor cells (Gould et al., 1990; Scarborough et al., 1993). Disintegrins inhibit fibrinogen-dependent PLT aggregation by binding to integrin GP IIb–IIIa (Kang et al., 1998). They also inhibit angiogenesis induced by basic fibroblast growth factor (bFGF) and suppress tumor growth and metastasis through selective blockade of avb3 on endothelial and tumor cells (Kang et al., 1999, 2000). The PROs suppress the migration of smooth muscle cells (SMCs), which represents an important process in restenosis induced by angioplasty (Jones et al., 1996; Hong et al., 2002a, b). An animal study has demonstrated that blockade of avb3 integrin by disintegrin after balloon angioplasty results in the reduction of neointimal formation (Sheu et al., 2001). We recently purified and cloned a novel disintegrin, saxatilin, from the venom of a Korean snake, Gloydius saxatilis (Hong et al., 2002a, b). Saxatilin is a single-chain polypeptide that is composed of 73 amino acids, including 12 cysteines, as well as the tripeptide sequence Arg-Gly-Asp, a proposed recognition site of adhesive PROs. Like other disintegrins, this PRO strongly inhibits human PLT aggregation, bFGF-induced proliferation of HUVEC, and vitronectin-induced migration of SMCs. It also regulates PLT activation associated with HUVEC migration and invasion (Jang et al., 2007), suppresses TNF-alpha-induced ovarian cancer cell proliferation and invasion (Kim et al., 2007a, b), and inhibits angiogenesis and tumor progression (Kim et al., 2006). However, despite the various biological effects of the disintegrins and their clinical implications in many diseases, including thrombosis, restenosis, cancer metastasis, and osteoporosis, toxicological data from laboratory animals have not been available. In this study, we developed an efficient, largescale production system through fermentation of Pichia pastoris and simple purification steps to obtain enough saxatilin (about 5 g) for in vivo toxicity

407

testing. Here, we newly report single and repeated dose toxicity as well as plasma half-life data of recombinant saxatilin in vivo. These investigations will be highly informative to the therapeutic application of disintegrins. 2. Materials and methods 2.1. Expression and mass production of recombinant saxatilin in Pichia pastoris The cDNA cloning and expression of saxatilin has been described previously (Hong et al., 2002a, b). Briefly, full-length cDNA encoding saxatilin was cloned from the venom gland cDNA library of a Korean snake, G. saxatilis. This saxatilin cDNA was inserted into P. pastoris expression vector pPIC9 and transformed into GS115 cells. Fermentation was performed with a modified protocol of Chen et al. (1996). The 2.5 ml frozen stock of transformant was inoculated into 250 ml of yeast nitrogen base-glycerol media, and was incubated in a shaker at 240 rpm for 24 h at 30%. The entire 250 ml volume of culture was transferred into a 5 l fermenter vessel containing 2.5 l of basal salt medium (H3PO4 27 ml/l, CaSO4  2H2O 0.9 g/l, K2SO4 18 g/l, MgSO4  7H2O 15 g/l, KOH 4.13 g/l) plus a PTMI trace metal solution and glycerol (40 g/l). The temperature and dissolved oxygen (DO) setting point were controlled at 30% and 30%, respectively. The set point of pH 5.0 was automatically adjusted by the addition of ammonium hydroxide (30%) solution. After 18 h of batch culture, the optical density (at 600 nm) reached 40, the point at which the fed-batch processing of glycerol was initiated. The feeding medium consisted of 50% glycerol and 12 ml/l of the trace elements mixture. Thereafter, the growth phase, a half-hour carbon source starvation period, was established before the culture was switched to the production phase. The production phase started after 48 h of growth. The feed medium consisted of 100% methanol and 12 ml/l trace metal solution. The recombinant saxatilin was purified from the culture supernatant of P. pastoris. The culture supernatant was collected by centrifugation and treated with ammonium sulfate to a final concentration of 2.0 M. The ammonium sulfate mixture was loaded to a phenyl-Sepharose column (Amersham Pharmacia Biotech., NJ, USA), and the PRO fraction was eluted with a 1 M ammonium sulfate solution. From this fraction, we purified

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recombinant saxatilin with an acetonitrile gradient in a Source 30 reverse-phase column (Amersham Pharmacia Biotech., NJ, USA). Fractions that showed PLT aggregation inhibition activity were collected and lyophilized for further use. 2.2. Mass spectrometry and N-terminal amino acid sequencing of recombinant saxatilin Matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS) for the purified recombinant saxatilin was obtained by a Kratos Kompact model II mass spectrometer. The N-terminal amino acid sequence of the recombinant PRO was determined using the Applied Biosystem Procise Protein Sequencing system (Perkin-Elmer, Wellesley, MA, USA). 2.3. PLT aggregation assay A PLT aggregation assay was performed in human platelet-rich plasma (PRP). The PLT concentrate was diluted to 300,000PLTs per microliter of PLTml-poor plasma. Ten microliters of sample was added to 450 ml of plasma and incubated for 3 min in an incubation well of the Chrono-Log Aggregometer (Chrono-Log Co., Havertown, PA, USA) at 37%. The impedance was recorded, and ADP (20 mM) was added in order to initiate PLT aggregation. The inhibition of PLT aggregation was measured at the maximum aggregation response. 2.4. Inhibition assay of fibrinogen– GP IIb– IIIa complex formation Fibrinogen/GP IIb–IIIa ELISA was performed using a modified form of the method described by Nachman and Leung (1982). In brief, 96-well plates were coated with purified human fibrinogen (10 mg/ml). After blocking with 1% bovine serum albumin, the plate was washed and each PRO sample to be tested was added, followed immediately by the addition of purified GP IIb–IIIa (40 mg/ml) (Calbiochem, La Jolla, CA, USA). After incubation, the plate was washed and mouse anti-human GP IIIa antibody (Chemicon, Temecula, CA, USA) was added. Following one additional hour of incubation and washing, goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) was added. A final wash was performed and a developing substrate (1-Step ABTS, Pierce, Rock-

ford, IL, USA) solution was added. The plate was then incubated for about 10 min until color developed. The reaction was stopped with 3 M HCl, followed by absorbance measurement at 492 nm. 2.5. Preparation of recombinant saxatilin for in vivo toxicity test The purity of saxatilin was 499% confirmed by high-performance liquid chromatography (HPLC). For the intravenous toxicity test, saxatilin was solved in pyrogen-free sterile saline and filtered using a 0.2 mg pore syringe membrane (Adventec MFS Inc., CA, USA). All dosage formulations were prepared on the day of administration. 2.6. Animal care and maintenance Four-week-old ICR mice were used for the toxicological study. The test animals were identified by cage labeling with the test number, sex, dosage level, and the period of the test. The room temperature was maintained at 2372 1C with a relative humidity of 50710%. All experiments were carried out in compliance with the Testing Guidelines for Safety Evaluation of Drugs (Notification No. 1999-61 issued by the Korean Food and Drug Administration on 22 December 1999) and under the Korean Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. This experiment was conducted in facilities of the Chemon Preclinical Research Center approved by the Korean Government, and animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, 1996). 2.7. Plasma half-life determination of recombinant saxatilin in rat Male Spraque Dawley rats (8 weeks of age, 23676.7 g) were administered a single i.v. dose of rhodamin-labeled saxatilin in isotonic phosphatebuffered vehicle. Rhodamin-labeled saxatilin was injected via tail vein (volume 200 ml) of animals (n ¼ 5) at a dose of 5 mg/kg. Whole blood was collected to time course and processed for plasma. Rhodamin-labeled peptide in plasma was detected using Fluorescence Spectrometer LS 50B (Perkin-Elmer, USA). Plasma clearance parameters were calculated from the fluorescence intensity of residual peptide in plasma versus time data curves.

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2.8. LD50 and single dose toxicity of recombinant saxatilin From preliminary tests, lethal doses were observed at 1000 and 1500 mg/kg in male and female mice, respectively, but all mice survived injection at 250 mg/kg. Using a table of random numbers, 20 males and 20 females were randomized by sex into dose groups (n ¼ 5 per sex per group). Saxatilin was administered once by a bolus of intravenous injection into the lateral tail vein, with a dose ranging from a maximum of 600 mg/kg, an intermediate of 400 mg/kg, and a minimum of 200 mg/kg. The injection volume was prepared at 10 ml/kg of body weight prior to administration. 2.9. Repeated dose toxicity study of recombinant saxatilin From the preliminary challenge of repeated administration of saxatilin, drug-related deaths of both males and females were observed on day 2 at 300 mg/kg/day and on day 7 at 100 mg/kg/day, respectively. The general signs in animals before death were a reduction of locomotor activity, piloerection, crouching, and difficulty in breathing. The necropsy findings indicated organ enlargement and weight gain of the spleen at an overdose of 33 mg/kg/day, but a dose of 11 mg/kg/day showed no organ abnormalities. Based on these findings, the toxic dose was estimated to range from 33 to 100 mg/kg/day, and the non-effective dose was determined to be 11 mg/kg/day. The highest dose of repeated toxicity was estimated to be 48 mg/kg/day, although drug-related death occurred on day 1 in male mice at 48 mg/kg/day. Therefore, the highest doses for males and females were 24 and 48 mg/kg/day, respectively. For the multiple dose study, 40 males and 40 females were randomized by sex into dose groups (n ¼ 10 per sex per group) on the basis of body weight. Recombinant saxatilin was administered once daily for 4 consecutive weeks (starting on day 1) by intravenous bolus injection into the lateral tail vein at 0 (vehicle control), 3, 12, and 24 mg/kg/day for males and at 3, 12, and 48 mg/kg/day for females. To examine recovery, five males of the 24 mg/kg/day group, five females of the 48 mg/kg/day group, and five each of the male and female controls were added to experiment as a satellite group. The dose volume was 1.0 ml/kg/day. Body weights were determined at random, on days 1.5, once weekly

409

thereafter, and at termination. Animals were observed for clinical signs, and physical examinations were conducted as below. Blood was collected on the day of test termination. 2.10. Analysis of hematological and clinical chemistry parameters The clinical symptoms of mice were observed shortly after the injection, from 30 min to 4 h, and at least three times daily (8 h interval) thereafter for 14 days. After the test, all animals were sacrificed and autopsied. In addition to the regular observations described above, animals underwent a physical examination once a week. Body weights were determined at random, on days 1, 3, 7, and once weekly thereafter (including termination). Hematological measurements included white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, PLT count, and WBC differential count. Clinical chemistry parameters included aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase (ALP), creatine phosphokinase (CPK), blood urea nitrogen, total bilirubin (TBIL), albumin/globulin ratio, glucose (GLU), cholesterol (CHO), creatinine (CRE), total PRO, and triglycerides. 2.11. Necropsy Five mice per sex per group were necropsied on the day of the test termination, 14 days after the final injection. The necropsy procedure consisted of a thorough examination of the viscera and carcass, and the collection and fixation of approximately 40 tissues. In addition, the adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, and testes/ epididymides were weighed. All collected tissues were microscopically examined. 2.12. Statistical analysis During the experimental period, Levene’s test was performed to compare the equality of variance between the results of body weight measurement and the weight gain ratio. When the variance had equality, data were analyzed by one-way analysis of variance using SPSS10K and SAS6.12 software.

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3. Results

a

3.1. Mass production of recombinant saxatilin in P. pastoris

Recombinant saxatilin 7712.28

100

1097.

90

Induction time (h) kDa

0

12

24

36

48

60

72 r-saxatilin

97.4 66.3 55.4

36.5 31.0 21.5 14.4 6.0 3.5

Fig. 1. Time-course analysis for protein expression. Coomassiestained SDS-PAGE (4–12% gradient) of culture: lane 1, molecular markers; lane 2, 24 h of fermentation corresponding to the uninduced glycerol-fed stage. Lanes 3–7, and 8 are 100 ml of culture from the induction stage at 12, 24, 36, 48, 60, and 72 h, respectively. Lane 9 is 5 mg of purified recombinant saxatilin. Arrow indicates purified recombinant saxatilin.

% Intensity

80

The expression clone obtained from a selection process of His4 complementation and methanol utilization was used for high cell density fermentation. During the fermentation, culture broth was analyzed for PRO expression by SDS-PAGE analysis (Fig. 1). The initial growth phase ended after 24 h of glycerol feeding, and induction of expression started with the methanol-adaptation and -utilization phases. By adding methanol for 12 h, the secreted saxatilin of 7.7 kDa was clearly detected by SDS-PAGE. The accumulated amount of secreted saxatilin in media was highest at 72 h after induction, and was estimated to be approximately 300 mg/l by relative staining of the PRO on SDS analysis, in comparison with the purified saxatilin standard. Recombinant saxatilin was purified to homogeneity from the P. pastoris culture supernatant using the combination of phenyl-Sepharose and Source30 reverse-phase chromatography. MALDI–MS analysis of purified recombinant saxatilin determined that it had a molecular mass of 7712 (Fig. 2a), the same as that of native saxatilin. The N-terminal sequencing analysis showed that recombinant saxatilin was correctly processed by yeast

70 60 50 40 30 20

7639.87 7935.04

10 0 3,000

4,000

5,800

7,200

8,600

0 10,000

Mass (m/z)

b Fig. 2. Characterization of the purified saxatilin from P. pastoris: (a) the molecular mass of recombinant saxatilin. (b) The Nterminal amino acid sequence of the purified recombinant saxatilin from P. pastoris.

KEX2 endo-peptidase during the secretion event (Fig. 2b). 3.2. Inhibition of PLT aggregation and fibrinogen– GP IIb– IIIa complex formation To confirm that recombinant saxatilin has the same biological activity as native saxatilin, we performed the following two in vitro activity assays. Dose-dependent inhibitions of human PLT aggregation by purified recombinant and native saxatilin were also measured in an ADP-induced PLT aggregation assay. The IC50 value of recombinant saxatilin is the same as the native form, which was approximately 130 nM, and complete inhibition was observed at a concentration of 500 nM (Fig. 3a). Inhibition of the fibrinogen–GP IIb–IIIa complex by recombinant saxatilin was determined through the solid-phase fibrinogen–GP IIb–IIIa binding assay. The IC50 values of native and recombinant saxatilin for the inhibition of fibrinogen–GP IIb–IIIa binding were in the range of 2.2–2.7 nM (Fig. 3b). These results indicated that recombinant saxatilin was expressed and purified with the fully active form. 3.3. Plasma half-life of recombinant saxatilin in rat To measure the clearance time of saxatilin in the plasma, we labeled saxatilin with a fluorescence dye,

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Fig. 4. Plasma half-life of rhodamin-labeled recombinant saxatilin in rat. The calculated half-life of saxatilin was 13.8 min.

Fig. 3. Comparison of biological activities of recombinant and natural saxatilin: (a) inhibition of ADP-induced platelet aggregation was performed in PRP as described in Section 2. The IC50 values were 131 nM (native saxatilin), 140 nM (recombinant saxatilin), and 270 mM (GRGDSP). (b) Inhibition of GP IIb–IIIa binding to immobilized fibrinogen was measured by solid-phase ELISA assay as described in Section 2. The concentration is plotted on a log scale. The IC50 values determined were 2.2 nM (native saxatilin), 2.7 nM (recombinant saxatilin), and 4.2 mM (GRGDS).

rhodamin, and administrated the rhodamin-labeled saxatilin to rats via the tail vain. The calculated half-life in plasma of saxatilin was 13.8 min (Fig. 4). 3.4. Single-dose (intravenous) toxicity of recombinant saxatilin in mice At 400 and 600 mg/kg, two of five mouse deaths occurred within 30 and 20 min, respectively, whereas a 200 mg/kg dose level caused no deaths, thus indicating that the latent period of the toxic effect to lethality was dose dependent (data not

shown). The LD50 value of recombinant saxatilin was determined to be approximately 400 mg/kg body weight in male mice and 600 mg/kg in female mice, respectively. The body weight of mice was not significantly changed during the period of the test (data not shown). Other clinical symptoms observed in test mice included that, immediately after the injection, all mice showed a decline in behavioral activity and some surviving female mice had diarrhea (Table 1). However, all of these symptoms disappeared within 24 h, and the activity of mice was fully recovered. From the autopsies of test mice, we found hepatic congestion in dead mice, but the organs of surviving mice lacked any other abnormal indications (data not shown). 3.5. Repeated dose (intravenous) toxicity of recombinant saxatilin in mice 3.5.1. General signs and death rate Recombinant saxatilin-related animal death occurred in one case in a male mouse injected with a dosage of 24 mg/kg at the day of injection (Table 2). In the mean time, one test drug-unassociated death was observed at day 21 in a male mouse treated with a dosage of 12 mg/kg due to suffocation by the animal holder. In terms of general symptoms, male mice showed decreased locomotor activity, piloerection, and crouching within 2–3 h after injection on the day of administration. These symptoms were reduced on the day after injection, except in the dead animals. The animals receiving a low dose of 3 mg/kg and in animals in the recovery period had no abnormal symptoms related to the administration of the test drug in the experiment.

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Table 1 Observation of clinical symptoms of ICR mice administered with single-dose injection of recombinant saxatilin Group

Dose (mg/kg)

Sex

N

Hours (day 0) after treatment Signs

0.5

1

2

3

4

5

6

7

8

C

0

Male Female

5 5

NAD NAD

5 5

5 5

5 5

5 5

5 5

5 5

5 5

5 5

5 5

T1

200

Male Female

5 5

Hypoactivity Hypoactivity

5 5

5 5

5 5

5 5

5 5

5 5

5 5

5 5

5 5

T2

400

Male

5

Female

5

Death Polypnea Hypoactivity Hypoactivity Diarrhea

2 2 5 5 3

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

Male

5

Female

5

Death Polypnea Hypoactivity Hypoactivity Diarrhea

2 2 5 5 3

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

0 0 3 5 0

T3

600

N: animal numbers. NAD: no abnormalities detected.

Table 2 Mortality and clinical signs in mice treated intravenously with recombinant saxatilin for 4 weeks Sex

Dose (mg/kg)

Final mortality

Clinical signs

Male

0 4 12 24

0/15a 0/10 1/10 1/15

Rough fur Normal Decrease in locomotor activity and crouching, piloerection Decrease in locomotor activity, crouching, piloerection, paleness, soiled fur, loss of fur

Female

0 4 12 48

0/15 0/10 0/10 0/15

Loss of fur Normal Decrease in locomotor activity, and piloerection Decrease in locomotor activity, piloerection, and, loss of fur

a

Number of dead animal/total number of animals

3.5.2. Body weight changes and ophthalmological signs Drug-related weight loss was statistically significantly observed in the fourth week of administration in males at 24 mg/kg and females at 48 mg/kg, respectively (Fig. 5). There was no significant change in food or water consumption (data not shown). Drug-related abnormalities in the eye such as congestion were not observed during the entire period of testing. 3.5.3. Urine analysis The pH values of female urine at 3 and 12 mg/kg were statistically (po0.05) higher than that of the

control group. The ketones of male mice in the recovery period were statistically significantly increased (po0.05) compared with the control group (Table 3). 3.5.4. Hematological findings All drug-treated male mice showed a statistically significant (po0.05) reduction in HGB count. An increased mean platelet volume (MPV) was also observed in the 12 mg/kg-injected group. In female mice, the 12 mg/kg- (po0.05) and 48 mg/kg-injected (po0.01) groups showed low HGB counts; the PLT count was decreased in the 48 mg/kg group (po0.05), and the MPV was statistically

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a

413

Table 3 Urinalysis of mice, which were treated intravenously with recombinant saxatilin for 4 weeks

40

Weight (Gram)

Main group 30

20 0 mg/kg 3 mg/kg 12 mg/kg 24 mg/kg

10 P < 0.05 0 0

b

1

2

3 Weeks

4

5

6

7

Female 40

Weight (Gram)

30

20

0 mg/kg 3 mg/kg 12 mg/kg 48 mg/kg

10 P < 0.05 0 0

1

2

3

4

5

6

7

Weeks

Fig. 5. Change of body weight (gram) in mice which were intravenously treated with recombinant saxatilin for 6 weeks. The number of animals was 10–15 and P value was lower than 0.05.

significantly increased in the 48 mg/kg group (po0.05). There were no toxicologically significant changes in the recovery periods of the tested animals (Table 4). 3.5.5. Blood biochemical findings The levels of blood biochemical parameters in each group of mice are shown in Table 5. The level of ALP was reduced in male mice administered 24 mg/kg. The 48 mg/kg-injected female group showed statistically significant reductions of ALP (po0.05) and total CHO (po0.05). In all cases of drug administration, females showed low levels of TBIL compared with the control group values. In the recovery period, serum GLU was increased in males (po0.05), while statistically significant (po0.05) reductions of PRO and CPK were observed in females. No variation was observed among control group animals.

Sex

Male

Female

Dose (mg/kg)

0

3

12

24

0

3

12

48

No. of animals 5 (1) Specific gravity p1.005 0 1.010 1 1.015 0 1.020 3 1.025 1 X1.030 0

5

5

5

5

5

5

5

0 0 0 5 0 0

0 0 2 2 1 0

0 0 1 2 1 1

0 0 0 4 1 0

0 0 0 1 6 1

0 0 2 1 2 0

0 0 4 1 0 0

(2) pH 6 7 7.5 8

0 1 2 1

0 0 2 2

0 0 2 3

0 1 2 2

0 0 2 0

0 1 2 2*

0 0 2 3*

1 2 1 1

(3) Ketone  7 +

0 5 0

0 3 2

1 4 0

0 4 1

2 3 0

1 4 0

3 2 0

3 2 0

(4) Protein – 7 1+ 2+ 3+

0 0 2 3 0

0 0 1 4 0

0 1 4 0 0

0 0 2 3 0

1 4 0 0 0

0 3 2 0 0

0 3 2 0 0

2 3 0 0 0

(5) Occult blood – 7 1+ 2+

5 0 0 0

5 0 0 0

5 0 0 0

5 0 0 0

4 0 0 1

5 0 0 0

4 0 1 0

4 0 1 0

*Significantly different from control value (Po0.05).

3.5.6. Organ weight determination Both male and female drug-administered groups showed increased spleen weight following treatment. The spleen weight was increased in a dosedependent manner in both male and female mice (Fig. 6). Absolute organ weights of the spleen, liver, and right adrenal gland were statistically significantly (po0.05) increased in males treated at 24 mg/kg. The absolute organ weight increment of the adrenal gland was also observed in males treated at 12 mg/kg. In female mice treated at 48 mg/kg, statistically significant variations of absolute organ weight were observed in the increment of the spleen (po0.01) and the reduction of the brain (po0.05). The variations of relative organ weight were statistically significantly increased in the liver

414

Table 4 Hematological changes of mice which were treated intravenously with recombinant saxatilin for 4 weeks Sex

Dose (mg/kg)

WBC (K/ml)

RBC (M/ml)

HGB (g/dl)

MCV (fl)

MCHC (g/dl)

PLT (k/mL)

MPV (fL)

Male (main)

0 3 12 24

3.8571.887 3.6271.734 3.8970.712 3.9571.484

9.26 70.368 8.9370.423 8.8370.505 8.6870.568

15.5170.479 14.8470.611* 14.7070.490* 14.8470.617*

55.80 71.755 55.8771.813 55.6871.418 57.3872.173

30.0771.007 29.7470.966 29.9470.604 29.8070.642

1202.407128.625 1138.80776.530 1156.337179.368 1122.11784.606

6.66 70.336 6.7970.413 7.1270.284* 7.3270.767

Female (main)

0 3 12 48

6.0771.248 4.5271.873 4.6071.569 5.8572.216

9.5070.520 8.9170.502 8.9970.322 7.9272.715

16.0970.451 15.3970.912 15.3970.479* 14.6671.405**

56.1372.248 58.1471.787 57.4871.666 58.2872.305

30.2270.993 29.7571.244 29.9271.083 29.2471.324

Male (satellite)

0 24

5.2871.097 3.7970.816

9.1670.371 9.3770.440

14.5870.497 15.0870.834

56.1471.780 57.4073.905

28.3470.974 28.1370.874

1235.807176.579 1323.33728.919

6.9470.414 6.6270.336

Female (satellite)

0 48

4.5772.498 4.6771.289

9.4870.218 9.1270.485

15.7470.921 14.9670.764

56.3070.606 56.7572.346

28.7870.340 28.6370.714

1051.757100.570 1066.757113.523

6.8470.381 6.8270.219

990.78777.701 992.207141.139 939.337124.515 777.347264.718*

6.8570.573 7.0270.559 7.2970.691 7.8270.714**

Sex

Dose AST (mg/kg) (IU/l)

ALT (IU/l)

Male (main)

0 3 12 24

85.95727.60 78.06717.68 73.08724.44 71.99720.40

33.44715.99 27.7175.16 22.1178.40 30.46722.22

Female (main)

0 3 12 48

Male (satellite)

Female (satellite)

ALP (IU/l)

BUN (mg/dl)

CRE (mg/dl)

62.07718.04 54.02718.95 51.28717.12 41.26713.06

27.4174.66 27.1773.90 31.4078.13 32.68711.85

0.3770.04 0.3470.03 0.3470.02 0.3770.03

121.22748.62 94.93726.46 101.76729.82 141.67724.22

73.01713.33 73.24720.56 76.00719.67 94.07753.65

21.8975.10 78.50716.46 26.4075.99 72.30714.42 23.82713.26 74.50720.99 27.01710.81 63.69*714.48

23.7073.72 28.01712.20 27.1478.30 24.7174.30

0.3970.03 0.4070.08 0.3970.03 0.3970.04

130.98714.54 105.49714.20 118.88721.50 99.23714.87 121.00730.69 104.92721.44 126.14718.73 91.64*718.14

0 24

80.24719.58 60.95710.77

31.61712.35 26.1479.01

26.2072.68 26.7373.09

0.3670.02 92.80711.75 136.20713.45 0.3570.04 112.94*78.15 140.58728.22

0 48

68.12710.67 22.5473.67 63.3476.62 18.7037.57

46.6279.99 43.5671.49

64.98713.11 23.68711.94 0.3970.03 59.83718.91 20.3474.41 0.3770.03

*Significantly different from control value (Po0.05).

GLU (mg/dl)

CHO (mg/dl) 139.93732.65 160.59725.43 132.99731.17 127.89719.03

PRO (g/dl) 5.2370.28 5.1970.28 5.1570.19 5.1770.18

CPK (IU/l)

ALB (g/dl)

1720.837975.31 1706.107721.53 1515.4971041.13 1133.797772.26

3.0970.19 3.0470.08 3.0970.13 3.0370.11

5.3270.29 559.667385.62 5.2370.25 736.827945.60 5.3170.34 574.757428.62 5.2270.08 1060.997901.80

3.2570.19 3.2270.16

TG (mg/dl)

0.0770.05 105.87724.80 0.0970.05 104.38717.23 0.0970.05 92.69728.89 0.0970.05 111.26735.05

3.3170.14 0.0970.21 3.2570.13 0.0470.04 3.2970.23 0.0370.03 3.1970.09 0.02270.02

5.1170.22 1567.0571018.47 3.0770.14 5.4570.28 674.147312.57 3.0970.09

145.67722.44 103.01716.61 5.3070.32 895.197426.01 119.70730.40 92.4174.85 5.19*70.26 307.30*7226.95

TBIL (mg/dl)

81.57724.62 66.94723.28 68.79714.28 64.19712.25

A/G (mg/dl) 1.4470.07 1.4270.12 1.4970.09 1.4370.07 1.6670.07 1.8070.44 1.6370.06 1.8970.66

0.0770.06 0.0770.06

65.03711.40 1.5070.07 88.89734.63 1.32*70.11

0.0670.06 0.0570.04

59.1179.77 55.14714.47

1.6070.19 1.6470.09

ARTICLE IN PRESS

Table 5 Serological changes of mice which were treated intravenously with recombinant saxatilin for 4 weeks

Y.-D. Sohn et al. / Toxicon 51 (2008) 406–417

*Significantly different from control value (Po0.05). **Significantly different from control value (Po0.01).

ARTICLE IN PRESS Y.-D. Sohn et al. / Toxicon 51 (2008) 406–417

b

1.8 0 mg/kg 12 mg/kg 24 mg/kg

1.6 1.4 Weight (Gram)

1.4

1.2 P< 0.05

1.0 0.8 0.6

0 mg/kg 12 mg/kg 48 mg/kg

1.2 Weight (Gram)

a

415

1.0

P< 0.05

0.8 0.6 0.4

0.4 0.2

0.2

Male

ey va ry U te us O

en

Ki

dn

r

le

Sp

rt

ve

ng

ea

Li

H

Lu

ey Te st Pr i s ot eu s

en

Ki

dn

r

le

Sp

rt

ve Li

ng

ea H

Lu

Br a Th in ym us

Br ai Th n ym us

0.0

0.0

Female

Fig. 6. Absolute organ weight of mice which were intravenously treated with recombinant saxatilin for 4 weeks. The number of animals was 10–15 and P value was lower than 0.05.

(po0.05), spleen (po0.01), and left adrenal gland (po0.01) in male mice at 24 mg/kg (data not shown). In female mice, groups given doses of 48 mg/kg (po0.05) and 12 mg/kg (po0.05) showed significantly increases in the organ weight of the spleen (data not shown). 3.5.7. Necropsy features Necropsies revealed slight enlargements of the spleen in one male treated at 24 mg/kg and in three females in the 12 mg/kg and nine females in the 48 mg/kg-injected groups. The pale and dark color variations were sporadically observed in lung tissue, but did not appear to be dose dependent. 3.5.8. Histopathological findings In the main group of male mice, cases of chronic progressive nephropathy, cyst formation in the kidney, mineralization in the heart, microgranuloma in the liver, hyperplasia in the spleen, and submandibular lymph node hyperplasia were observed in the saline controls. Spleen hyperplasia were observed in all main groups due to the recombinant saxatilin treatment. Chronic progressive nephropathy in the kidney, microgranuloma in the liver, myopathy of the muscle, two cases of chronic inflammation of the lung, two cases each of submandibular lymph node and mesenteric lymph node hyperplasia, and five cases of spleen hyperplasia were detected following treatment with 24 mg/kg. In the main group of female mice, spleen hyperplasia and a vacuolation of the adrenal gland were observed in saline controls. The 3 and 12 mg/kg

groups each experienced three cases of spleen hyperplasia. Two cases each of cyst formation in the kidney, chronic inflammation of the lung, and submandibular lymph node hyperplasia were observed at 48 mg/kg, along with three cases of microgranuloma in the liver, six cases of hyperplasia of the spleen, and a case of uterine and vaginal atrophy. In the male satellite (recovery) group, a microgranuloma of the liver was observed in the control group, and one case of chronic progressive nephropathy of the kidney and one case of submandibular lymph node occurred at 24 mg/kg. In the female satellite (recovery) group, one case of chronic progressive nephropathy, one case of microgranuloma of the liver, and two cases of adrenal gland vacuolation were observed in the control group. Additionally, one case of liver microgranuloma, one case of submandibular lymph node hyperplasia, and two cases of adrenal gland vacuolation were observed at 48 mg/kg (Table 6). 4. Discussion Recombinant saxatilin was purified to homogeneity by the combination of phenyl-Sepharose column chromatography and reverse-phase HPLC from yeast culture broth. This simple two-step purification yields highly purified and fully active saxatilin. The overall yield was approximately 150 mg per 1 l culture broth, and molecular and biological properties of the purified saxatilin were almost the same as those of its natural form. We previously reported that salmosin, which has 95% homology to saxatilin in terms of its amino

ARTICLE IN PRESS Y.-D. Sohn et al. / Toxicon 51 (2008) 406–417

416

Table 6 Histopathological findings of mice which were treated intravenously with recombinant saxatilin for 4 weeks Group

Main group

Satellite group

Sex

Male

Dose (mg/kg)

0

3

12

24

0

3

12

48

0

24

0

48

Organ Kidney Chronic progressive nephropathy Cyst formation

1 1

0 0

0 0

1 0

0 0

0 0

0 0

0 2

0 0

1 0

1 0

0 0

Heart Mineralization

1

0

0

0

0

0

0

0

0

0

0

0

Liver Microgranuloma Granuloma

1 0

0 0

0 0

1 0

0 0

0 0

0 0

3 0

1 0

1 0

1 0

1 1

Lung Chronic inflammation

0

0

0

2

0

0

0

2

0

0

0

0

Muscle Myopathy

0

0

0

1

0

0

0

0

0

0

0

0

Spleen Hyperplasia

1

3

5

5

1

3

3

6

0

0

0

0

Mesenteric lymph node Hyperplasia

0

0

0

2

0

0

0

0

0

0

0

0

Submandibular lymph node Hyperplasia

1

0

0

2

0

0

0

2

0

1

0

1

Adrenal gland Vacuolation

0

0

0

0

1

0

0

0

0

0

2

2

Uterus Atrophy

0

0

0

0

0

0

0

1

0

0

0

0

Vagina Atrophy

0

0

0

0

0

0

0

1

0

0

0

0

Female

acid sequence, suppressed the tumor metastasis caused by B16 melanoma injection in a dosedependent manner (Kang et al., 2000). At a concentration of 1.5 mg/kg, salmosin completely inhibited B16F10 melanoma-induced metastasis (Kang et al., 2000). Saxatilin also inhibited 99% of the tumor metastasis caused by B16 melanoma at the same concentration (unpublished data). In vivo clearance study of saxatilin in rat plasma revealed that intravenous administration of saxatilin is rapidly cleared from blood circulation with 13.8 min of halflife. This plasma half-life was shorter than 20–30 min of an other GPIIb–IIIa inhibitor, abciximab, which is an antibody fragment (Schror and Weber, 2003). The LD50 value of saxatilin was determined to be approximately 400 mg/kg body

Male

Female

weight in male mice and 600 mg/kg body weight in female mice. An intravenous toxicity study determined that the LD50 value of saxatilin in ICR mice was extremely high compared with the complete inhibition concentration (1.5 mg/kg) for B16F melanoma-induced tumor metastasis in the same animal. To investigate the repeated dose toxicity of saxatilin in mice, the test item was administered daily to groups containing 10 male and 10 female ICR mice at a dose level of 0, 3, 12, and 24 mg/kg (48 mg/kg at start) in males and at 0, 3, 12, and 48 mg/kg in females for 4 weeks. Parameters evaluated in this study included the mortalities, clinical findings, body and organ weight measurement, food and water consumption measurement,

ARTICLE IN PRESS Y.-D. Sohn et al. / Toxicon 51 (2008) 406–417

ophthalmologic examination, urinalysis, hematological examination, clinical chemistry, gross pathology, and histopathology. Five additional males and females were assigned to the vehicle control group, and males treated with 24 mg/kg and females treated with 48 mg/kg were maintained for a 2-week withdrawal period on completion of dosing. Parameters showed decreases in locomotor activity, piloerection, and crouching in clinical findings, and a decrease of RBC in hematology and hyperplasia of the spleen in histology related to saxatilin administration. These results suggest that the target organ of intravenous administration of saxatilin is the spleen, and the toxicity to the spleen is temporal and reversible. The cause of spleen toxicity induced by saxatilin is unknown, and is currently under further study. The NOAEL in this test for both males and females is considered to be 3 mg/kg. Saxatilin appears to be a safe drug candidate for use in further clinical applications, but particular caution must be taken, as it can cause hepatic congestion by increasing the blood pressure when an overdose is given. These results will be useful for the further study of disintegrin in vivo to evaluate its potential as a therapeutic drug for thrombosisrelated cardiovascular diseases, cancers, restenosis, and osteoporosis. Acknowledgments This study was supported by a grant from the National Research Laboratory program funded by the Ministry of Commerce, Industry, and Energy (no. 10020214), and by a grant from the National Cancer R&D program (Project no. 0420210-3) from the Korean Ministry of Health and Welfare.

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