GMI-1359 is a novel and potent dual CXCR4/E-selectin antagonist with anticancer activities
Physicochemical Properties
| Appearance | Typically exists as solid at room temperature |
| Synonyms | GMI-1359; GMI1359 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | CXCR4; E-Selectin |
| ln Vitro |
GMI-1359 Potentiates the Cytotoxicity of DTX in Drug Sensitive Cells and Sensitizes to DTX in Drug-Resistant Cells In Vitro: Comparison with CTCE-9908 and GMI1272. [1] Researchers evaluated if the increased expression of CXCR4 and reduced DTX sensitivity may support the use of a combinatory treatment between a dual CXCR4/E-selectin antagonist and DTX. So, GMI-1359, CTCE-9908 and GMI-1271 were co-administered at respective IC20 values with different doses of DTX in manner to calculate the IC50 values for this chemotherapeutic agent both in DTX sensitive and resistant cells. Here, we demonstrated that these combinatory strategies may reduce IC50 values for DTX both sensitive or resistant cells. As shown in panels C and D of Figure 3, GMI-1359 sensitizes to DTX administration (Figure 3C) and, at the same time, reverts (Figure 3D) the DTX resistance of PCa cell lines. Similarly, CTCE-9908 and DTX combination results also effective in these cell models. Nevertheless, the DTX sensitization was higher in presence of a co-treatment with GMI-1359 (4.8-fold on the CTRL) when compared to the single E-selectin (GMI-1271, 1.6 times on CTRL) or CXCR4 (CTCE-9908, 2.7 times vs. CTRL) in PC3 DTX sensitive cells with an increment of 3 and 1.78 times on GMI-1271 and CTCE-9908, respectively. We observed also that these differences were less pronounced in other PCa cell lines with values of 1.43 (C4-2B), 1.58 (DU145) and 1.65 (22rv1) times. GMI-1271 showed always lower sensitizing effects. When we have compared the effectiveness of different combinations in DTX resistant cell lines, we showed that GMI-1359 was still more active versus untreated animals in all cell models (with values of 2.47 [DU145DTXR], 2.78 [PC3DTXR], 3.00 [C4-2BDTXR] and 3.21 [22rv1DTXR] times) when compared to CTCE-9908 (with values of 1.76 [DU145DTXR], 2.31 [22rv1DTXR], 2.57 [PC3DTXR] and to 2.64 [C4-2BDTXR] times) and GMI-1271 (with values starting 1.2 up to 1.7 times vs. CTRL). The increment of sensitivity was of 1.08 [PC3DTXR], 1.14 [C4-2B], 1.39 [C4-2BDTXR and 22rv1DTXR] times versus CTCE-9908. These data indicate that GMI-1359 shows better in vitro effects as DTX sensitizing agent when compared to CTCE-9908 and comparable effects to CTCE-9908 as DTX reverting agent. |
| ln Vivo |
GM1359 impacted bone marrow colonization and growth in intraventricular and intratibial cell injection models. The anti-proliferative effects of GMI-1359 and DTX correlated with decreased size, osteolysis and serum levels of both mTRAP and type I collagen fragment (CTX) in intra-osseous tumours suggesting that the dual CXCR4/E-selectin antagonist was a docetaxel-sensitizing agent for bone metastatic growth. Single agent CXCR4 (CTCE-9908) and E-selectin (GMI-1271) antagonists resulted in lower sensitizing effects compared to GMI-1359. These data provide a biologic rationale for the use of a dual E-selectin/CXCR4 inhibitor as an adjuvant to taxane-based chemotherapy in men with mCRPC to prevent and reduce bone metastases.
Compared effects of GMI-1359, CTCE-9908 and GMI-1271 Single Treatments in Locally Aggressive/Non Metastatic 22rv1 Xenografts [1] Next researchers compared the sensitization effects in vivo treating 22rv1 DTX sensitive subcutaneous xenografts with GMI1259, CTCE-9908 and GMI-1271 administered as single therapies in agreement with the protocol of treatments indicated in Figure 4A. We showed that GMI-1359 administration resulted in an approximate 30% reduction in 22rv1 tumour weight (0.725 g ± 0.071 vs. 1.069 ± 0.220- means ± SD, p < 0.05, Figure 4B). CTCE-9908 administration resulted in a decrement of tumour weight by about 20% (0.845 g ± 0.090, p < 0.05) and GMI-1271 did not significantly impact tumour weight in this primary setting. The comparisons between GMI-1359 and CTCE-9908 shows a significant difference with p < 0.05 as indicated in the Supplementary Materials, Figure S1A. These data suggest that dual antagonism of CXCR4 and E-selectin afforded by GMI-1359 was greater of about 14.2% than the individual CXCR4 activity alone. Then we compared the time to progression (defined as the time necessary to have a doubling of tumour volume, TTP) on the basis of the growth curves plotted from single tumors. Control tumors showed a TTP of 9.0 ± 1.9 days (mean ± SE). This value reached 14.6 ± 2.0 days with GMI-1359 and 12.2 ± 2.2 days with CTCE-9908. The differences with untreated animals were statistically significant (p < 0.05) as indicated in Figure 4C and Supplementary Materials Figure S1B. GMI-1271 showed a non-significant value of 10.4 ± 0.5 days. The comparison between TTP values shown in GMI-1359 and those shown in CTCE-9908-treated animals was not statistically significant. GMI-1359 administration show statistically significant values of TTP when compared to GMI-1271 (p < 0.05) whereas these values were not statistically different in the comparison between GMI-1271 and CTCE-9908. Kaplan Meyer curves were generated (Figure 4D) and hazard ratio values calculated by the evaluation of log-rank test (Figure 4E). Kaplan Meyer curves demonstrate that GMI-1359 was more effective in slowing the progression of the tumour as compared to CTCE-9908 and GMI-1271 (Figure 4D) with a HR = 2.8 (p < 0.05). In order to verify if GMI-1359 and CTCE-9908 are playing on CXCR4 and GMI-1359 and GMI-1271 are playing on E-selectin ligands we blotted 8 tissue extracts on nitrocellulose paper analysis by dot blot and serum from treated animals by ELISA. researchers demonstrated that CXCR4 dots were similar to untreated animals when we considered extracts from GMI-1271, whereas this seems to be reduced in tumors treated with GMI-1359 or CTCE-9908, suggesting that GMI-1359 and CTCE-9908 are actually inhibiting CXCR4 since was reported that CXCR4 antagonists reduce CXCR4 expression in vivo due to internalization and degradation. In Figure 4G we added a graphical representation of densitometric units collected for the single immune-spot grouped for each therapeutic arm. Regarding GMI 1271 (Figure 4), we observed that effectively GMI-1271 and GMI-1359 reduced the levels of HECA-452 when compared to control: vehicle 10.4 ± 1.8 pg/mL; GMI-1359 6.2 ± 1.3 pg/mL (p = 0.040) and GMI-1271 5.0 ± 0.7 (p = 0.016) whereas the comparison with CTCE-9908 showed similar results: 10.4 ± 1.8 (NS). No differences were observed between GMI-1271 and GMI-1359. In addition, no drug seemed to affect toxicity in vivo, as attested by evidence that mice did not significantly decrease their weight during treatment. Compared Effects of GMI-1359, CTCE-9908 and GMI-1271 as DTX Chemo-Sensitizing Agents in 22rv1 Xenografts [1] Next we evaluated if GMI-1359, CTCE-9908 or GMI-1271 increased the in vivo efficacy of DTX (7.5 mg/kg/week, ip). We used the administration schedule shown in Figure 5A. We found that DTX showed a tumour weight reduction of about 50% (0.515 g ± 0.129, p < 0.05) versus untreated animals as indicated in Figure 5B and Supplementary Materials, Figure S1A. Intraventricular Tumour Cell Injection: Reduction of Bone Marrow Colonization (Anti-Bone Metastatic Activities) from GMI-1359, GMI-1271 and CTCE-9908 [1] Next researchers verified if GMI-1359, GMI-1271 or CTCE-9908 influenced bone colonization of PCa cells. For this reason we used the bone metastatic PC3 cell derivatives, PCb2 cells, which were inoculated by an intra-ventricular (IV) route according to our previous reports. X-ray determinations were performed weekly. We know that the overall rate of bone colonization by using this cell line results to be up to 75%. The colonization of tibiae (80%), femurs (20%) or both (75%) was maximal whereas the localization in the other sites (anterior legs, vertebrae, skull or mandible) ranged between 2 and 5% (Figure S2B). The overall percentage of bone metastases was 85%. In Figure S2C we show that the PC3b2 cells colonize the bone marrow of tibiae or femurs. The staining with an epithelial markers expressed from PC3 and its cell derivatives as cytokeratin 18, CXCR4 and E selectin ligands allow us to detect epithelial tumor cells growing as dispersed or organized in sheets of cytokeratin (K18), CXCR4 and E-selectin positive cells (Figure S1C). E-selectin expression was present both in the bone marrow and tumor cells. The expression of CXCR4 and the HECA-452 immunoreactivity were also evaluated in human tissues derived from bone metastases. CXCR4 expression was increased in bone metastases in agreement with our previous report. |
| Cell Assay | Cells were seeded at a density of 2 × 104 cells/mL in 24 well plates. Cells were left to attach and grow in 5% FCS DMEM for 24 h. After this time, cells were maintained in the control or experimental culture conditions for the considered time. Morphological controls were assessed daily with an inverted phase-contrast photomicroscope, before cell trypsinization and counting. Cells trypsinized and resuspended in 1.0 mL of saline were counted using the NucleoCounterTM NC-100. The effect on cell proliferation was measured by taking the mean cell number with respect to controls over time for the different treatment groups as described. Results were represented as data from three independent experiments performed in triplicate. IC50 values were calculated by using the GraFit plotting the percentage of inhibition versus drug concentration in a semi-logarithmic way [1]. |
| Animal Protocol |
Subcutaneous Xenograft Model [1] Male CD1 nude mice were anesthetized with a mixture of ketamine (100 mg/kg)/xylazine (5 mg/kg) in saline and subsequently received S.C. flank injections of 1 × 106 PC3 and 22v1 cells and DTX resistant strains. Tumour growth was assessed by bi-weekly measurement of tumour diameters with a Vernier calliper (length × width). Randomization was performed when subcutaneous tumors reached volumes ranged between 80 and 100 mm3. This was commonly obtained 7–10 days after cell injection. Tumour weight was calculated according to the formula: TW (mg) = tumour volume (mm3) = d2 × D/2, where d and D are the shortest and longest diameters, respectively. The effects of the treatments were examined as previously described. Evaluation of Treatment Response In Vivo [1] In order to get closer to the parameters used to analyze thee pharmacological efficacy assessments in the man, we quantified the antitumor effects of different treatments as previously described [15,44,45,50]. Briefly: (1) tumor volume, measured throughout the experiment, (2) tumor weight, measured at the end of experiment; (3) complete response (CR) defined as the disappearance of the tumor; (4) partial response (PR) defined as a reduction of greater than 50% of tumor volume with respect to baseline; (5) stable disease (SD) defined as a reduction of less than 50% or an increase of less than 100% of tumor volume with respect to baseline; (6) tumor progression (TP) defined as an increase of greater than 50% of tumor volume with respect to baseline; (7) time to progression (TTP) defined as the time necessary to have progression. These modalities of analysis reduced both the differences of single tumor volume measurements in the time linked to differences of engraftment efficacy of the tumor cells as well as the individual variability of the response (even though the mice For the in vivo analysis, the synergy/additivity index (combination index, CI) may be calculated in different manner i.e., by using relative risk (RR), odds ratios (OR), or hazard ratios (HR). We considered the HR values through the formula: CI = [HR(a) + HR(b)]: HR (a + b) as described where a and b represent the drug 1 and 2 when two compounds are combined. CI > 1.3 indicates antagonism, CI = 1.1 to 1.3 moderate antagonism, CI = 0.9 to 1.1 additive effects, CI ≤ 0.9 synergism. Intracardiac (IC) Tumour Model [1] Briefly, anesthetized animals received cardiac injection of 1 × 105 PCb2 cells in 0.1 mL of PBS was performed as previously described [39,50]. In the intracardiac tumour model, treatments were started 2 days before tumour cell injection and stopped after 50 days. Animals were sacrificed by carbon dioxide inhalation 70 days after heart injections, or earlier if there were early signs of serious distress. All animals were subjected to an accurate necroscopy and portions of various organs were processed for routine histological examination. Intratibial (IT) Tumour Model [1] Intratibial tumour injection was performed as previously described. Briefly, anesthetized animals received 1 × 105 luciferase transfected PC3 cells/10 µL PBS which were inoculated in the tibiae. Treatments were started 2 days after tumour injection and stopped after 28 days the end of drug administration. Assessment of Treatment Response in Bone Tumour Models [1] Tumour and bone treatment response was determined using in vivo and ex vivo evaluations. The development of metastases was monitored by radiography using a Faxitron cabinet x-ray system and lytic units were quantified by Image J analysis performed on lytic lesions as arbitrary densitometric units. Mice were administered 150 mg/kg body weight luciferin subcutaneously and imaged 15 min later in a bioluminescence imager to identify intra-tibial implants similar to the method described by Kemper et al. The mice were photographed while placed on their front. Imaging data were normalised to the acquisition conditions and expressed as radiance (photons/second/cm2/steradian (p/s/cm2/sr)), and the colour scale was adjusted according to the strength of signal detected by using wasabi software, a Hamamatsu dedicated software. This semi-quantitative analyses allows to us to determine the bioluminescence intensity (BLI) which was measured in the region of interest. The BLI values were used to calculate the BLI increment for each individual animal. Mice were euthanized when they displayed distress signs (eg, altered gait, tremors/seizures, lethargy) or weight loss of 20% or greater of pre-surgical weight. All animals were subjected to an accurate necroscopy to document secondary sites of metastases. Treatments for In Vivo Experiments [1] Before the start of treatment, xenografts were randomised into eight groups as follows: Group 1: mice (10 animals) receiving intraperitoneal (i.p.) injections of 100 µL PBS; Group 2: mice (10 animals) receiving GMI-1359 twice a day i.p. at 40 mg/kg for consecutive 14 days; Group 3: mice (10 animals) receiving CTCE-9908 at 25 mg/kg/day IP. Group 4: mice (10 animals) received GMI-1271 25 mg/Kg/day for 28 days; Group 5: mice (10 animals) receiving docetaxel alone (DTX i.p. injection of 7.5 mg/kg per week); Group 6: mice (10 animals) received CTCE-9908 plus DXT at doses above mentioned for single treatments; Group 7: mice (10 animals) receiving GMI-1359 plus DTX at dose mentioned above for single treatments and Group 8: mice (10 animals) receiving GMI-1271 plus DTX at doses mentioned above for single treatments. The choice of these doses derived from the literature data. Groups 1–4 and groups 1–8 were considered for the IC and IT experiment, respectively. Measurements of serum mouse cross linked C-telopeptide of type I collagen and mouse TRAP-5b (mTRAP) were performed according to the manufacturer’s protocol on blood plasma. Cachexia was also considered and analyzed. This is a complex syndrome associated with an underlying illness causing ongoing muscle loss that is not entirely reversed with nutritional supplementation. A body weight loss of 15–20% is indicative for cachecsia. This is associated to anorexia, curved spine and absence or scarcity of responses to external solicitations. All these aspects were evaluated during the experiments and accounted at the end of analyses. A chi square test analyses for trend followed by Bonferroni correction. |
| References | [1]. Dual CXCR4 and E-Selectin Inhibitor, GMI-1359, Shows Anti-Bone Metastatic Effects and Synergizes with Docetaxel in Prostate Cancer Cell Intraosseous Growth. Cells logo Cells. 2019 Dec 20;9(1):32. |
| Additional Infomation | In order to evaluate the anti-tumour effects in bone microenvironment bypassing the extravasation phenomenon, the intratibial tumour model was used. This model allowed us to inject a substantial higher number of tumour cells within bone marrow with respect to intracardiac tumour model. In addition, in order to evaluate possible effects of the compounds in analysis on intra-bone tumor growth, we injected into the tibiae luciferase transfected PC3 cells (PC3luc). Luciferase activity (BLI) was analyzed as indicated in the Material and Methods section. Effectively, we demonstrated that GMI-1359 affected intra-osseous tumour growth and increases the efficacy of docetaxel. Osteolysis and tumor growth were, indeed, significantly reduced after co-treatment of GMI-1359, CTCE-9908 and GMI-1271 with DTX. In addition these anti-tumour effects were stronger when compared to subcutaneous 22rv1 tumour suggesting a major role of CXCR4 in the bone microenvironment dependent tumour growth and drug resistance respect to a primary lesion. GMI-1359-mediated DTX sensitizing effects were more marked when compared to those observed for GMI-1271 and very similar, instead, when compared to those observed for CTCE-9908. Our report provides the rationale to use GMI-1359 in adjuvation to taxane-based chemotherapy in men with high-risk prostate cancer. Although the combination between GMI-1359 and DTX seems to be, indeed, low when compared to the combination between CTCE-9908 and DTX, we can be satisfied by a similar increment of activity. A low increase of effectiveness is welcome for the mCRPC treatments. It is necessary to consider, indeed, that the mCRPC is a lethal and almost incurable disease and the pharmacological therapies (ie. use of bisphosphonates) are aimed at improving the quality of life of metastatic patients through the reduction of osteolysis and bone fractures. In addition, we must also take into account that the use of targeted clinical trials is necessary to evaluate the feasibility in humans of the use of GMI-1359 and DTX combinations. Among other things, the inhibition of CXCR4 and E-selectin signaling pathways also modulates immune responses by regulating the recruitment of inflammatory cells in the tumor site and in bone metastases. To get answers on the clinical use of GMI-1359, it would also be necessary to use non-immuno-compromised animal models to evaluate efficacy in a native immune system. [1] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |