Synthesis and biological evaluation of technetium-labeled d-glucose-MAG3 derivative as agent for tumor diagnosis

A d-glucose-MAG₃ derivative was successfully synthesized and radiolabeled in high labeling yield. Biodistribution studies in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle ratio and moderate tumor-to-blood ratio. Thus, d-glucose-MAG₃ is a potential agent for tumor diagnosis.     

Preparation and biodistribution of a 99mTc tricarbonyl complex with deoxyglucose dithiocarbamate as a tumor imaging agent for SPECT

The deoxyglucose dithiocarbamate (DGDTC) was successfully labeled with the ^99mTc(CO)₃ core to provide the corresponding ^99mTc(CO)₃–DGDTC complex in good yields. The radiochemical purity of the ^99mTc(CO)₃–DGDTC complex was over 90%, as measured by high performance liquid chromatography (HPLC). The complex possessed good stability in saline at room temperature and in mouse plasma at 37 °C. Its partition coefficient result indicated that it was a hydrophilic complex. The electrophoresis results showed the complex was neutral. The biodistribution of ^99mTc(CO)₃–DGDTC in mice bearing S 180 tumor showed that the complex clearly accumulated in tumor, exhibiting high tumor/blood and tumor/muscle ratios and good tumor retention. Single photon emission computed tomography (SPECT) image studies showed there was a visible uptake in tumor sites, suggesting ^99mTc(CO)₃–DGDTC could be considered as a potential tumor imaging agent.     

Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging

One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y₂O₃). Cyclic RGD peptide binds specifically to integrin α_vβ₃ which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y₂O₃ was observed for U87MG cancer cell (high integrin α_vβ₃ expression), but not for MCF-7 cancer cell (low integrin α_vβ₃ expression), this UC imaging is considered to be integrin α_vβ₃ specific. The non-invasive imaging of integrin α_vβ₃ expression using UCP-based probes will have great potential in cancer imaging in general in living subjects.     

Synthesis and biological evaluation of 99mTc(CO)3(His–CB) as a tumor imaging agent

The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [^99mTc(CO)₃]⁺ core to form the ^99mTc(CO)₃(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention, fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images highlighted the potential of ^99mTc(CO)₃(His–CB) as a tumor imaging agent.     

Investigation of a MMP-2 Activity-Dependent Anchoring Probe for Nuclear Imaging of Cancer

Purpose

Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.

Methods

We designed and synthesized MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀, which consist of 4 independent moieties: RI unit (¹¹¹In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.

Results

MDAP₁₀₀₀, MDAP₃₀₀₀, and MDAP₅₀₀₀ were cleaved by MMP-2 in a concentration-dependent manner. MDAP₃₀₀₀ pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP₃₀₀₀ exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP₃₀₀₀ exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP₃₀₀₀ existed in tumor xenografts in vivo.

Conclusions

The results indicate the possible usefulness of our MDAP strategy for tumor imaging.     

Activation of Integrin αVβ3 Regulates Cell Adhesion and Migration to Bone Sialoprotein

α_Vβ₃, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of α_Vβ₃, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for α_Vβ₃ in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of α_Vβ₃ activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn²⁺ markedly enhanced α_Vβ₃-dependent adhesion to BSP. α_Vβ₃-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that α_Vβ₃ activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by α_Vβ₃-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of α_Vβ₃ can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of α_Vβ₃ on neoplastic cells may contribute to tumor growth and metastatic potential.     

Tumor regression with Q fever rickettsiae and a mycobacterial glycolipid

Summary Regression of established tumors by Coxiella burneti, the rickettsial agent which causes Q fever, was studied using the transplantable line-10 tumor in strain 2 guinea pigs. Suspensions of formalin-killed, purified rickettsiae induced an average of 42% tumor regression after intratumor injection. The activity of C. burneti was enhanced by incorporation of the rickettsiae into oil droplet vaccines containing the mycobacterial glycolipid, P₃. Such vaccines induced 64% tumor regression. The activity of C. burneti that was enhanced by P₃ was found in phenol-water extracts of the rickettsiae. These extracts contained lipopolysaccharides which were less toxic than bacterial endotoxins, and they induced 63% tumor regression when combined with P₃. These lipopolysaccharides may provide agents of high therapeutic activity but relatively low toxicity for cancer immunotherapy.     

Identification of the chemokine CX3CL1 as a new regulator of malignant cell proliferation in epithelial ovarian cancer

Background

Little is known about the molecules that contribute to the growth of epithelial ovarian carcinomas (EOC), which remain the most lethal gynecological cancer in women. The chemokine Fractalkine/CX₃CL1 has been widely reported to play a biologically relevant role in tumor growth and spread. We report here the first investigation of the expression and role of CX₃CL1 in EOC.

Results

Epithelial cells from the surface of the ovary and the Fallopian tubes and from benign, borderline and malignant tumors all stained positive for CX₃CL1. In tumor specimens from 54 women who underwent surgical treatment for EOC diagnosis, CX₃CL1 immunoreactivity was unevenly distributed in epithelial tumor cells, and ranged from strong (33%) to absent (17%). This uneven distribution of CX₃CL1 did not reflect the morphological heterogeneity of EOC. It was positively correlated with the proliferation index Ki-67 and with GILZ (glucocorticoid-induced leucine zipper), previously identified as an activator of the proliferation of malignant EOC cells. Hierarchical clustering analysis, including age at diagnosis, tumor grade, FIGO stage, Ki-67 index, CX₃CL1, SDF-1/CXCL12 and GILZ immunostaining scores, distinguished two major clusters corresponding to low and high levels of proliferation and differing in terms of GILZ and CX₃CL1 expression. GILZ overexpression in the carcinoma-derived BG1 cell line resulted in parallel changes in CX₃CL1 products. Conversely, CX₃CL1 promoted through its binding to CX₃CR1 AKT activation and proliferation in BG1 cells. In a mouse subcutaneous xenograft model, the overexpression of GILZ was associated with higher expression of CX₃CL1 and faster tumor growth.

Conclusion

Our findings highlight the previously unappreciated constitutive expression of CX₃CL1 preceding tumorigenesis in ovarian epithelial cells. Together with GILZ, this chemokine emerges as a regulator of cell proliferation, which may be of potential clinical relevance for the selection of the most appropriate treatment for EOC patients.     

Synthesis of 99mTc-labeled 2-Mercaptobenzimidazole as a novel radiotracer to diagnose tumor hypoxia

Discovery of ^99mTc-labeled imidazole derivatives as a potential radiotracer for hypoxic tumor imaging is considered to be of great interest because of non-invasive detection capabilities. 2-Mercaptobenzimidazole (2-MBI) was successfully synthesized, characterized and radiolabeled with [^99mTc (CO)₃(H₂O)₃]⁺ intermediate to form ^99mTc-2-MBI complex with radiochemical purity of ≥95% yield as observed by instant-thin layer chromatography (ITLC) and radio-high performance liquid chromatography (radio-HPLC). The ^99mTc-2-MBI complex was observed to be stable in saline and serum with no noticeable decomposition at room temperature and 37 °C, respectively, over a time period of 24 h. Biodistribution results in Balb/c mice bearing S180 tumor show that ^99mTc-2-MBI highly internalized in tumor tissue, also possess preferably high tumor/muscle and tumor/blood ratios 4.14 ± 0.77 and 3.91 ± 0.63, respectively at 24 h incubation. Scintigraphic imaging study shows ^99mTc-2-MBI is visibly accumulated in hypoxic tumor tissue, suggesting it would be a promising radiotracer for early stage diagnosis of tumor hypoxia.     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer

Introduction

Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO₂T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease.

Methods

We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO₂T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging.

Results

KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO₂T alone did not influence cancer progression, combining the KD with HBO₂T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls.

Conclusions

KD and HBO₂T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.     

A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer

Background

Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).

Results

The cc49scFv-FasL_ext is highly effective in in vitro killing of human TAG-72⁺ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasL_ext only increased cytotoxicity 500-fold as compared with sFasL against TAL6⁺ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasL_ext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.

Conclusion

Our study demonstrated that scFv-FasL_ext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.     

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

Background

α-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for α-particle emitting isotopes facilitating selective tumor therapies.

Principal Findings

A dimer of the vascular tumor homing peptide F3 was chemically coupled to the α-emitter ²¹³Bi (²¹³Bi-DTPA-[F3]₂). We found ²¹³Bi-DTPA-[F3]₂ to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of ²¹³Bi-DTPA-[F3]₂ we treated mice bearing intraperitoneally growing xenograft tumors with ²¹³Bi-DTPA-[F3]₂. In a tumor prevention study between the days 4–14 after inoculation of tumor cells 6×1.85 MBq (50 µCi) of ²¹³Bi-DTPA-[F3]₂ were injected. In a tumor reduction study between the days 16–26 after inoculation of tumor cells 6×1.85 MBq of ²¹³Bi-DTPA-[F3]₂ were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found ²¹³Bi-DTPA-[F3]₂ to accumulate in tumors but only low activities were found in control organs except for the kidneys, where ²¹³Bi-DTPA-[F3]₂ is found due to renal excretion.

Conclusions/Significance

In conclusion we report that ²¹³Bi-DTPA-[F3]₂ is a novel tool for the targeted delivery of α-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography Can Contribute to Discriminate Patients with Poor Prognosis in Hormone Receptor-Positive Breast Cancer

Background

Patients with hormone receptor-positive breast cancer typically show favorable survival. However, identifying individuals at high risk of recurrence among these patients is a crucial issue. We tested the hypothesis that [¹⁸F]-fluorodeoxyglucose positron emission tomography (FDG-PET) scans can help predict prognosis in patients with hormone receptor-positive breast cancer.

Methods

Between April 2004 and December 2008, 305 patients with hormone receptor-positive breast cancer who underwent FGD-PET were enrolled. Patients with luminal B subtype were identified by positivity for human epidermal growth factor receptor-2 (HER2) or high Ki67 (≥14%) according to criteria recently recommended by the St. Gallen panelists. The cut-off value of SUV_max was defined using the time-dependent receiver operator characteristic curve for recurrence-free survival (RFS).

Results

At a median follow up of 6.23 years, continuous SUV_max was a significant prognostic factor with a hazard ratio (HR) of 1.21 (p = 0.021). The cut-off value of SUV_max was defined as 4. Patients with luminal B subtype (n = 82) or high SUV_max (n = 107) showed a reduced RFS (p = 0.031 and 0.002, respectively). In multivariate analysis for RFS, SUV_max carried independent prognostic significance (p = 0.012) whereas classification with immunohistochemical markers did not (p = 0.274). The Harell c-index was 0.729. High SUV_max was significantly associated with larger tumor size, positive nodes, HER2 positivity, high Ki67 (≥14%), high tumor grade, and luminal B subtype.

Conclusions

Among patients with hormone receptor-positive breast cancer, FDG-PET can help discriminate patients at high risk of tumor relapse.     

Synthesis and antitumor activity of 2,5-bis(3′-indolyl)-furans and 3,5-bis(3′-indolyl)-isoxazoles,nortopsentin analogues

A series of novel 2,5-bis(3′-indolyl)furans and 3,5-bis(3′-indolyl)isoxazoles were synthesized as antitumor agents. The antiproliferative activity was evaluated in vitro toward diverse human tumor cell lines. Initially 5 isoxazoles and 3 furan derivatives were tested against a panel of 10 human tumor cell lines and the most active derivatives 3c and 4a were selected to be evaluated in an extended panel of 29 cell lines. By exhibiting mean IC₅₀ values of 17.4 μg/mL (3a) and 20.5 μg/mL (4c), in particular 4c showed a high level of tumor selectivity toward the 29 cell lines.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Molecular Magnetic Resonance Imaging of Tumors with a PTPµ Targeted Contrast Agent

Molecular magnetic resonance imaging (MRI) of tumors improves the specificity of MRI by using targeted probes conjugated to contrast-generating metals. The limitation of this approach is in the identification of a target molecule present in sufficient concentration for visualization and the development of a labeling reagent that can penetrate tumor tissue with the fast kinetics required for use in a clinical setting. The receptor protein tyrosine phosphatase PTPµ is a transmembrane protein that is continuously proteolyzed in the tumor microenvironment to generate a high concentration of extracellular fragment that can be recognized by the SBK2 probe. We conjugated the SBK2 peptide to a gadolinium chelate [SBK2-Tris-(Gd-DOTA)₃] to test whether the SBK2 probe could be developed as an MR molecular imaging probe. When intravenously injected into mice bearing flank tumors of human glioma cells, SBK2-Tris-(Gd-DOTA)₃ labeled the tumors within 5 minutes with a high level of contrast for up to 2 hours post-injection. The contrast enhancement of SBK2-Tris-(Gd-DOTA)₃ was significantly higher than that observed with a current MRI macrocyclic gadolinium chelate (Gadoteridol, ProHance) alone or a scrambled control. These results demonstrate that SBK2-Tris-(Gd-DOTA)₃ labeling of the PTPµ extracellular fragment is a more specific MR molecular imaging probe than ProHance or a scrambled control. Consequently, the SBK2 probe may be more useful than the current gold standard reagent for MRI to identify tumors and to co-register tumor borders during surgical resection.     

Synthesis and biological evaluation of new [1,2,4]triazolo[4,3-a]pyridine derivatives as potential c-Met inhibitors

A series of [1,2,4]triazolo[4,3-a]pyrazine derivatives (4a–4i) were designed, synthesized and evaluated for their c-Met kinase inhibition and antitumor activity against SNU5 gastric cell line in vitro. Among these compounds, 4d was found to show the highest activity against c-Met and high selectivity against the tumor cells which are believed to be dependent on the c-Met oncogene amplification, because 4d selectively inhibited c-Met while had no effect on other 59 kinases. In vivo efficacy study on human gastric (MKN-45) and human non-small cell lung (NCI-H1993) tumor xenograft in nude mouse demonstrated that 4d·CH₃SO₃H had a better inhibiting activity than SGX-523 in a dose-dependent manner. When tested in mice, compound 4d·CH₃SO₃H was found to have biological half-lives and plasma exposure values higher than those of JNJ-38877605, and its long-term toxicity and acute toxicity turned out to be acceptable, all of which indicates that 4d·CH₃SO₃H is a desirable drug candidate.