{'fraction_non_alphanumeric': 0.03970553684705145, 'fraction_numerical': 0.037512726133604825, 'mean_word_length': 4.485180412371134, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Anti-metastatic effect of 131I-labeled Buthus martensii Karsch chlorotoxin in gliomas

The present study investigated the underlying molecular mechanism by which Buthus martensii Karsch chlorotoxin (BmK CT) inhibits the invasion and metastasis of glioma cells and the possibility of 131I-labeled BmK CT (131I-BmK CT) as a novel targeted agent for the treatment of glioma. The impact of BmK CT with and without 131I radiolabeling on the invasion and metastasis of glioma cells in vitro was studied. Cell viability was assessed using Cell Counting Kit-8 and plate colony formation assays in order to confirm the cytotoxicity of BmK CT and 131I-BmK CT at different concentrations. Transwell invasion and wound-healing assays were conducted in order to investigate the inhibitory effects BmK CT and 131I-BmK CT on cell migration and invasion. Furthermore, western blotting, ELISA immunofluorescence and a gelatin zymography assay were performed to evaluate changes in the protein expression levels of glioma cells following treatment with BmK CT or 131I-BmK CT. The results indicated that BmK CT inhibits the invasion and metastasis of glioma cells via regulation of tissue inhibitor of metalloproteinase-2 expression and that 131I-BmK CT has the potential to be a novel targeted therapeutic drug for glioma.


Introduction
Glioma accounts for 80% of malignant brain tumors and has one of the highest mortality rates among all types of cancer (1). The current standard glioma treatment comprises surgical excision followed by radiotherapy and chemotherapy (2). However, as glioma cells infiltrate into the brain parenchyma, combination therapy can only minimally prolong survival time and almost all patients relapse (3)(4)(5). The high risk of recurrence following combination therapy is a major therapeutic challenge. Thus, it is necessary to investigate novel therapeutic approaches to glioma and the underlying mechanism of the invasion and metastasis of glioma cells.
Tumor metastasis is a multi-step process that comprises neovascularization and cell adhesion, invasion, migration and proliferation, in which the crucial step is degradation of the extracellular matrix (EcM) and basement membrane (6,7). A growing body of evidence supports the perspective that tumor progression is associated with the overexpression of matrix metalloproteinases (MMPs) in tumor cells, particularly that of MMP-2 and -9, which are able to modulate the tumor microenvironment (8). Apart from their role in the degradation of the ECM and cancer metastasis, MMPs also regulate inflammatory processes, cell proliferation and angiogenesis (9). These functions of MMPs provide an alternative method for targeting cancer therapy. There are multiple methods to regulate the activities of MMPs, including regulation of the following: MMP mRNA expression, conversion from the zymogen to the active enzyme, and tissue inhibitors of metalloproteinases (TIMPs) (9). A number of studies have demonstrated that overexpression of TIMPs can reduce tumor metastasis (10)(11)(12). In the TIMP family, TIMP-1 and -2 are able to suppress the activities of most known MMPs (11).
Peptide toxins have offered a novel approach for targeted treatment of glioma. chlorotoxin (cTX), a 36-amino-acid peptide that is isolated from the venom of Leiurus quinquestriatus (13), has been considered to be a novel blocker of small conductance chloride ion channels (14) and MMP-2 (15). It has an anti-invasive effect that is due to inhibition of the enzymatic activity of MMP-2 and reduced expression of MMP-2 via its interactions with MMP-2 (15). Furthermore, 131 I-labeled cTX is considered to be a promising agent for imaging or therapy of glioma (16)(17)(18) and has been used in clinical trials (19,20). Similarly, a cTX-like peptide purified from Buthus martensii Karsch (BmK cT), which possesses similar biological features to cTX, has been demonstrated to inhibit glioma cell proliferation, migration and invasion (21). Our previous studies have revealed that BmK CT can specifically bind to C6 glioma cells and suppress cell invasion through downregulation of MMP-2 expression (22)(23)(24), while not affecting mRNA expression of MMP-2 at the genetic level (24). However, these studies only investigated the MMP-2 protein. Thus, the aim of the present study was to further investigate the potential anti-metastasis mechanism of BmK cT on glioma cells and the possibility of 131 I-labeled BmK cT ( 131 I-BmK cT) as a novel targeted therapeutic agent for glioma.
In the present study study, cell counting Kit-8 (ccK-8) and plate colony formation assays demonstrated that BmK cT had no influence on cell proliferation at concentrations of 0-1 mg/ml, while a significant reduction in proliferation was observed in the glioma cells treated with 131 I-BmK cT. Furthermore, the results of the cell cycle assay demonstrated that only 131 I-BmK cT could induce evident changes in the cell cycle and arrest glioma cells at the S and G 2 /M phases. Transwell invasion and wound healing assays demonstrated that 131 I-BmK cT possessed a stronger effect than BmK cT in terms of inhibiting cell migration and invasion. Furthermore, western blotting, ELISA, immunofluorescence and a gelatin zymography assay demonstrated that both 131 I-BmK cT and BmK cT were able to downregulate the expression of MMP-2 and -9 proteins, and upregulate that of TIMP-2 protein. Notably, 131 I-BmK cT was more effective than BmK cT at inhibiting cell migration and invasion and the enzyme activity of MMP-2 and -9, but not at inhibiting the protein expression of MMP-2, MMP-9 and TIMP-2. The results indicate that BmK cT inhibits the metastasis of U87MG cells via regulation of TIMP-2 expression and that 131 I-BmK cT has the potential to be a novel targeted drug for glioma therapy.
Synthesis of 131 I-BmK CT. For the 131 I radiolabeling, a tyrosine was modified in the N-terminus of BmK CT that could be labeled with 131 I easily using the chloramine-T method. Briefly, sterile Na 131 I solution (370-740 MBq; 0.1-0.2 ml) was mixed with 0.2 ml PBS containing BmK cT (200 µg) and chloramine-T (100 µg). The mixture was continuously stirred for 5 min at room temperature and then 100 µg Na 2 S 2 O 5 was added to stop the reaction. Subsequently, 131 I-BmK cT was purified from the reaction mixture using disposable PD-10 desalting columns. In the follow-up experiments, the specific activity was 10 mci/mg.
Plate colony formation assay. In order to evaluate colony formation, 400 log-phase U87MG cells were seeded in 6-well plates and then treated with BmK cT (0, 0.05, 0.5, 5, 10 and 20 µg/ml) or 131 I-BmK cT (0, 0.5, 5, 50, 100 and 200 µci/ml) for 24 h. Subsequently, the culture medium was replaced with fresh culture medium. Following incubation at 37˚C for 10 days, the cells were fixed in methyl alcohol and stained with crystal violet for 15 min at room temperature. The colonies were then counted and images of the plates were captured.
Cell cycle analysis. U87MG cells were seeded at a density of 2x10 5 cells/ml in 6-well culture plates and then were treated with different concentrations of BmK cT (0, 5, 10 and 20 µg/ml) or 131 I-BmK cT (0, 50, 100 and 200 µci/ml). Following incubation for 72 h at 37˚C, the cells were trypsinized, washed twice with PBS and fixed with 70% ethanol overnight at 4˚C. The cells were then resuspended in PI/RNase staining buffer for 30 min at 37˚C and analyzed using flow cytometry (BD Accuri c6 Software; version 1. 0. 264. 21; Bd Biosciences, San Jose, cA, USA).
Transwell invasion assay. Transwell inserts (corning Incorporated, corning, NY, USA) were coated with Matrigel (Bd Biosciences) to form a thin layer of gel and incubated overnight at 37˚C. U87MG cells were treated with PBS, Na 131 I (0.5, 5 and 50 µci/ml), BmK cT (0.05, 0.5 and 5 µg/ml) or 131 I-BmK cT (0.5, 5 and 50 µci/ml) for 24 h. A total of 5x10 4 cells were seeded in the upper chamber with serum-free dMEM medium and then the chambers were placed into a 24-well plate containing complete medium. Following incubation for 24 h at 37˚C, cells on the upper surface of the chambers were removed with a cotton swab and the chambers were washed 3 times with PBS. Subsequently, the cells on the lower surface of the chambers were fixed with methanol for 15 min and stained with crystal violet solution for 30 min at 37˚C.
The number of invaded cells was counted under an inverted microscope (magnification, x200).
Wound healing assay. U87MG cells were seeded at a density of 4 x10 5 cells/ml in a 6-well plate to form a confluent monolayer. A wound was made with a pipette tip and then fresh medium containing the treatment (PBS, 50 µci/ml Na 131 I, 5 µg/ml BmK cT or 50 µci/ml 131 I-BmK cT) was added to the wells. Then the cells were incubated at 37˚C. Images of the cells were captured at 0, 12 and 24 h with an inverted phase contrast microscope, and cell migration was assessed by measuring the size of the gap in multiple fields. The microscope images were quantified by a distance measurement. The magnification of the microscope was set as x100.
Western blotting assays. The primary antibodies used were rabbit anti-MMP-2, -MMP-9 and -TIMP-2 (1:1,000). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (cat. no. 7076 and 7074; 1:10,000; cell Signaling Technology, Inc.) were used to detect the primary antibodies on the western blots. Briefly, cells were incubated with PBS, BmK cT (5 µg/ml), Na 131 I or 131 I-BmK CT (50 µCi/ml) for 72 h at 37˚C. Total protein from the U87MG cells was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). A total of 20 µg total protein was separated by 10% SdS-PAGE and transferred onto a polyvinylidene difluoride (PVdF) membrane. The PVdF membranes weres then blocked in PBS containing 5% skimmed milk and 0.1% Tween-20 for 2 h at room temperature. Subsequently, the membranes were incubated at 4˚C overnight with the primary antibodies, followed by incubation with the corresponding secondary antibody for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology). The expression of the proteins was quantified using ImageJ software (version 1.52a; National Institutes of Health, Bethesda, Md, USA) and compared with that of the control (β-actin).
Confocal microscopy. U87MG cells were seeded at a density of 5x10 4 cells/ml in glass bottom cell culture dishes. The cells were initially incubated in medium containing the treatment for 48 h and the incubation conditions were the same as those of the western blotting assay. Subsequently, cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature. The cells were then washed 3 times with PBS, permeabilized with 0.2% Triton X-100 for 15 min at 37˚C and blocked with 5% bovine serum albumin (cat. no. P0260; Shanghai doBio Biotech co., Ltd.) in PBS for 30 min at room temperature. Following blocking, cells were incubated with the appropriate primary antibody (MMP-2, MMP-9 and TIMP-2; 1:250) overnight at 4˚C. The cells were then washed 3 times with PBS and stained with fluorescein isothiocyanate-labelled goat anti-rabbit immunoglobulin G (cat. no. SAB3700873; 1:300; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at 37˚C. Subsequently, the cells were washed 3 times with PBS and were then stained with dAPI for 2 min at room temperature. Images were captured using a confocal microscope (FV300; Olympus corporation, Tokyo, Japan).
ELISA. Rat MMP-2, MMP-9 and TIMP-2 ELISA kits were purchased from Sigma-Aldrich (cat. nos. RAB1916, RAB1112 and RAB1156, respectively; Merck KGaA). U87MG cells were treated with BmK cT (0.05, 0.5 and 5 µg/ml), Na 131 I or 131 I-BmK CT (0.5, 5 and 50 µCi/ml) for 72 h at 37˚C. The secreted proteins MMP-2, MMP-9 and TIMP-2, which are produced by U87MG cells, in the supernatant from each well were evaluated via ELISA. Briefly, 96-well ELISA plates were coated with the appropriate antibodies (MMP-2, MMP-9 and TIMP-2) at 4˚C overnight. Subsequently, the supernatants of the cell cultures (100 µl) collected by centrifugation (3,661 x g; 15 min; 4˚C) were added to the plates and the plates were incubated for 1 h at 37˚C. The plates were washed 5 times with PBS and then incubated with the horseradish peroxidase-conjugated detection antibodies provided with each kit for 1 h at 37˚C. The plates were washed a further 5 times and were then incubated for 15 min at 37˚C with tetramethyl benzidine dilution. The reaction was stopped with 2N H 2 SO 4 and the absorbance at 450 nm was determined using a microplate reader (BioTek Instruments, Inc.).
Gelatin zymography assay. The activities of MMP-2 and -9 were examined using a gelatin zymography assay. U87MG cells were treated with PBS, BmK cT (0.05, 0.5 and 5 µg/ml) or 131 I-BmK CT (0.5, 5 and 50 µCi/ml) for 72 h at 37˚C. Protein was isolated and the concentration determined as in the western blotting protocol. Total protein (10 µg) was separated using 10% SdS-PAGE that included 1% gelatin (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) without prior heating or reduction. The gel was washed with 2.5% Triton-X 100 to strip the SdS and incubated with developing buffer (50 mM Tris-Hcl, 150 mM Nacl and 5 mM cacl 2 ) for 16 h at 37˚C. Following staining with 0.5% Coomassie Brilliant Blue R-250 (Sigma-Aldrich; Merck KGaA) for 3 h at room temperature, the gel was destained and photographed. Signal intensities were measured by densitometry using Bio-1d software, version 15.01 (Vilber Lourmat deutschland GmbH, Eberhardzell, Germany).
Statistical analysis. data are presented as the mean ± standard deviation. All results were analyzed using SPSS software, version 20.0 (IBM corp., Armonk, NY, USA). Student's t-test was used to analyze the variances between two groups. Multi-group comparisons of the means were carried out by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

I-BmK CT inhibits U87MG cell proliferation.
In order to investigate the proliferation of U87MG cells treated with BmK cT or 131 I-BmK cT, ccK-8 and plate colony formation assays were performed. The viability of the U87MG cells did not change significantly following treatment with BmK CT at concentrations of 0-1,000 µg/ml (Fig. 1Aa-c). However, there was significant inhibition in the growth of the cells in the 131 I-BmK cT group. This effect was time-(24-72 h) and dose-dependent (0-200 µci/ml; Fig. 1Ad-f). The half maximal inhibitory concentration of the 131 I-BmK cT cells at the 72-h time point was 50 µci/ml. In addition, the colony formation assay demonstrated that 131 I-BmK cT reduced colony formation compared with the blank control (Fig. 1B). These results demonstrated that 131 I-BmK cT could inhibit U87MG cell proliferation, whereas BmK CT had no significant influence on cell viability at a concentration of ≤1,000 µg/ml. 131

I-BmK CT induces changes in the cell cycle of U87MG cells.
As presented in Fig. 2A, BmK cT (0, 5, 10 and 20 µg/ml) did not significantly affect the cell cycle progression of U87MG cells. In contrast to blank control, 131 I-BmK cT (50, 100 and 200 µCi/ml) significantly increased the proportion of U87MG cells in the S and G 2 /M phases and reduced the proportion of cells in the G 1 phase (Fig. 2B). These results suggest that 131 I-BmK cT inhibits cell proliferation by inducing cell cycle arrest at the S and G 2 /M phases.

I-BmK CT inhibits U87MG cell invasion and migration.
Tumor metastasis depends on the capacity of tumor cells to invade and migrate to distant sites. Transwell invasion assays demonstrate the invasive capacity of tumor cells. The results in the present study demonstrated that the number of cells that passed through the membrane was reduced following treatment with BmK cT and 131 I-BmK cT. As the treatment concentration increased, a significant reduction in the cell number was observed (Fig. 3A). As presented in Fig. 3B, the results of wound healing assay in vitro demonstrated that 24 h following scraping, the cells in the PBS group had migrated across the wound. The percentage of wound closure in the Na 131 I, BmK cT and 131 I-BmK cT groups was 78.3±19.6,   56.6±10.0 and 45.0±8.2%, respectively, and these results were statistically significant compared with the blank control. The Transwell and wound healing assays demonstrated that BmK cT can inhibit the invasion and migration of U87MG cells, and that 131 I-BmK cT has a more marked effect than BmK cT at the same concentration.  (Fig. 4). These results indicate that BmK cT upregulates TIMP-2 and downregulates MMP-2 and -9. Furthermore, 131 I-BmK cT was not significantly more effective than BmK cT. The upregulation of TIMP-2 and downregulation of MMP-2 and -9 were also visualized by confocal imaging of the U87MG cells (Fig. 5A). 131 I-BmK CT stimulates the secretion of TIMP-2 and suppresses the expression of MMP-2 and -9. ELISA was used  The results demonstrated that BmK cT and 131 I-BmK cT inhibited the secretion of MMP-2 and -9, and stimulated the secretion of TIMP-2 in glioma cells (Fig. 5B). These results were in accordance with those of the western blotting. 131 I-BmK CT reduces the enzymatic activities of MMP-2 and -9 that are secreted by U87MG cells. The enzymatic activities of MMP-2 and -9 in the treated groups were analyzed and compared with the blank control using gelatin zymography. The activities of MMP-2 and -9 were reduced in the treated groups in a dose-dependent manner (Fig. 6), which may partially explain the inferior ability of cells to migrate and invade following treatment with BmK cT or 131 I-BmK cT.

Discussion
As malignant glioma is difficult to treat and recurs easily, novel therapies are urgently required. BmK CT has been identified as a small peptide that can specifically bind to glioma cells and inhibit their metastasis (21,27). certain previous studies also demonstrated that BmK cT can inhibit proliferation glioma cells (21,24), but similar results were not obtained in the present study. The proliferation of U87MG cells treated with BmK cT (0-1,000 µg/ml) was assessed. Although the results do not demonstrate outcomes such as its anti-proliferative effect, the data demonstrated that BmK cT did not affect cell survival following 24, 48 and 72 h of treatment. These results suggest that BmK cT may be non-toxic at concentrations of ≤1,000 µg/ml. The results of the plate colony formation assay and flow cytometric analysis also supported this. The invasion and migration of U87MG cells was subsequently investigated. The Transwell invasion assay indicated that BmK cT exposure (0.05-5 µg/ml) for 24 h inhibited the invasiveness of U87MG cells in a dose-dependent manner. The wound-healing assay demonstrated that BmK cT exposure significantly inhibited U87MG cell migration. Notably, the data demonstrated that the anti-invasive effect of BmK cT on U87MG cells was not due to its cytotoxicity and that 131 I-BmK cT was more effective than BmK cT at inhibiting the invasion and migration of U87MG cells.
A previous study demonstrated that BmK cT displayed anti-metastatic ability and inhibited glioma cell invasion via MMP-2 (28). Invasion and metastasis are the later stages of tumor progression and are the major cause of treatment failure in patients with cancer due to the spreading of tumor cells from the localized stage to distant sites in order to form a secondary tumor (29). EcM degradation is a fundamental step in the process of tumor metastasis (30). MMPs that can degrade and remodel the EcM serve a major role in tumor growth, angiogenesis and metastasis. Without MMPs, tumor cells would be unable to cross through the EcM barriers that restrict their migration (31). MMP-2 and -9 are the key enzymes in the MMP family due to their critical role in EcM degradation (32). The enzymatic activity of MMPs is fundamental to their physiological role in tumor progression. BmK cT has been demonstrated to downregulate MMP-2 expression (28). In the present study, the western blotting results, laser scanning confocal images and ELISA results demonstrated that the levels of MMP-2 and -9 in or secreted by U87MG cells were reduced. The enzyme activities of MMP-2 and -9 were measured by gelatin zymography and the results indicated that the enzyme ability of MMP-2 and -9 to degrade gelatin was reduced.
MMPs can be regulated by TIMPs. TIMPs are the natural endogenous inhibitors of MMP proteins and the family contains four members (TIMP-1, -2, -3 and -4) (33). Among the TIMP family, TIMP-2 serves an essential role in regulating the activities of MMP-2 and -9. Furthermore, increasing the expression of TIMP-2 has been demonstrated to suppress cell invasion (34,35). Therefore, an aim of the present study was to investigate whether BmK cT could reduce the expression of MMP-2 and -9 through TIMP-2. The western blotting results demonstrated that TIMP-2 was increased in the U87MG cells compared with the control group, which was further supported by the laser scanning confocal images and the ELISA results, suggesting that TIMP-2 has a significant influence on the expression of MMP-2 and -9 and the invasiveness of U87MG cells. These results indicate that the BmK cT may downregulate MMP-2 and -9 by upregulating TIMP-2. 131 I has been widely used for the therapy of patients with cancer (36)(37)(38). Its therapeutic effect is due to its β radiation, whereas the γ rays can be used for SPEcT imaging. The β emission results in the production of free radicals and toxic substances that can damage crucial macromolecules, including dNA, cell membranes and enzymes, and can cause cell death (39). These functions allow the combination of targeted tumor-specific agents to this radionuclide. Due to the high affinity to thyroid, 131 I has been routinely used in radionuclide therapy and imaging of thyroid diseases, especially thyroid cancer (37,40), and 131 I whole-body SPEcT imaging has been developed as a diagnostic method in detecting recurrences or metastases from thyroid carcinoma (41). clinical trails have demonstrated the safety of 131 I-labeled cTX used in the treatment of glioma patients (19,20). As a cTX-like peptide, 131 I-BmK cT will likely not affect normal tissues. However, some relative experiments should be performed to elucidate the safety and radiation dose in vivo which will be reported in our further work. In the present study, it was demonstrated that not only can 131 I-BmK cT inhibit metastasis, but it can also induce cell apoptosis. Although there was no obvious advantage in the effectiveness of 131 I-BmK cT at downregulating MMP-2 and -9 by upregulating TIMP-2 compared with BmK cT, it was more effective in reducing the enzyme abilities of MMP-2 and -9. The ccK-8 and plate colony formation assays demonstrated that 131 I-BmK cT had a marked effect on U87MG cell proliferation. The effect of 131 I-BmK cT on the Figure 6. The activity of MMP-2 and -9 was reduced in culture supernatants secreted by U87MG cells following treatment with BmK cT or 131 I-BmK cT. MMP, matrix metalloproteinase; BmK cT, Buthus martensii Karsch chlorotoxin; 131 I-BmK cT, 131 I-labeled BmK cT. cell cycle was analyzed and the cells were demonstrated to be arrested in the S phase. The main reason that 131 I-BmK cT demonstrated a stronger effect than BmK cT was due to the presence of 131 I emitting the β minus that may induce cell apoptosis and inhibit the invasion and migration of U87 cells. This finding is consistent with a previous study, which demonstrated that 131 I-BmK cT significantly induced cell apoptosis (22). Induction of cell apoptosis by 131 I-BmK cT is a good explanation for its superior inhibitory effect on glioma cell invasion. In the present study, the effectiveness of BmK cT and 131 I-BmK cT at regulating MMP-2, MMP-9 and TIMP-2 were similar due to the presence of BmK cT. Following 131 I radiolabeling, 131 I-BmK cT induced apoptosis and fewer cells passing through the membrane in the Transwell invasion assay were observed, which was in accordance with the results of the wound healing assay.
Tumor-specific radionuclide therapy ensures the preferential accumulation of radiopharmaceuticals in tumor cells and not in normal tissue. As a monotherapy, BmK cT could inhibit metastasis and 131 I-BmK cT could be used in imaging and therapy of malignant glioma. Following BmK cT binding to glioma cells (15), the β radiation from 131 I induces cell death. This therapeutic method could increase the radiation reaching tumor tissues, decrease the radioactive damage to surrounding tissues and reduce the chance of relapse. However, the effect of 131 I-BmK cT in glioma therapy in vivo requires investigation in future studies.
In conclusion, the present study demonstrated that BmK cT inhibits U87MG cell metastasis, invasion and migration by downregulating MMP-2 and -9 expression and upregulating TIMP-2 expression. Additionally, BmK cT and 131 I-BmK cT are able to suppress the activities of MMP-2 and -9. Therefore, 131 I-BmK cT may be a promising therapeutic treatment for malignant glioma. 131 I is suitable for imaging and dose determination purposes and is simple to conjugate. In order to further determine the mechanism of the effect of BmK cT on glioma cell invasion, in vivo studies are required.