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| Targets |
MMP-2 MMP-9
CTTHWGFTLC, CYCLIC TFA targets matrix metalloproteinase-2 (MMP-2, gelatinase A) and MMP-9 (gelatinase B). These enzymes degrade type IV collagen, a major component of the basement membrane, and are involved in extracellular matrix remodeling, tumor invasion, and metastasis. The cyclic peptide inhibits MMP-9 with an IC₅0 value of approximately 8 microM. The binding stabilizes the enzyme-inhibitor complex, preventing substrate access and subsequent catalytic action. |
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| ln Vitro |
In vitro, CTTHWGFTLC, CYCLIC TFA inhibits the migration of tumor cells and endothelial cells[1].
In vitro, CTTHWGFTLC, CYCLIC TFA inhibits endothelial and tumor cell migration. The cyclic peptide reduces the invasive capacity of tumor cells by blocking MMP-2 and MMP-9 activity, thereby preventing degradation of the extracellular matrix and basement membrane. At concentrations around 10-50 microM, the peptide inhibits cell migration in Boyden chamber assays. It does not show direct cytotoxicity at these concentrations; instead, it inhibits invasion and migration. |
| ln Vivo |
In vivo tumor progression is inhibited by CTTHWGFTLC, CYCLIC TFA in mice models[1].
In vivo, CTTHWGFTLC, CYCLIC TFA is used in research models to study the role of MMP-2/9 in tumor angiogenesis, invasion, and metastasis. The cyclic peptide has been incorporated into liposomes for targeting to tumor cells. Peptide-mediated targeting of MMP-2 and MMP-9 inhibitors has been studied for anti-angiogenic and anti-metastatic effects in animal models, although detailed efficacy data are limited. The cyclic structure improves stability and resistance to proteolytic degradation compared to linear peptides, making it suitable for in vivo applications. Binding to phospholipid membranes enables use in liposome targeting to tumor cells in vitro and potentially in vivo. |
| Enzyme Assay |
For in vitro enzyme inhibition assays (non-cell-based), recombinant human MMP-2 or MMP-9 enzyme (catalytic domain) is activated with 1 mM APMA (p-aminophenylmercuric acetate) for 1-2 hours at 37degC in activation buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35). After activation, the enzyme (2-10 nM) is incubated with varying concentrations of CTTHWGFTLC, CYCLIC TFA (0-100 microM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35) for 30 minutes at 25degC. A fluorogenic substrate (e.g., Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, which is cleaved by MMP-2 and MMP-9) is added to a final concentration of 5-10 microM. The reaction is monitored continuously for 30-60 minutes at 37degC using a fluorescence plate reader with excitation at 320-340 nm and emission at 390-420 nm. The IC₅0 value is calculated from a dose-response curve by fitting the inhibition of initial reaction velocities. For MMP-9, the reported IC₅0 is approximately 8 microM. Alternatively, a quenched fluorescent substrate (DQ-gelatin) can be used, which upon cleavage yields fluorescent fragments. A zymography assay can be performed for semi-quantitative analysis of MMP activity: cell culture supernatants or tissue lysates are run on SDS-PAGE gels containing gelatin (1 mg/mL) under non-reducing conditions. Gels are washed with 2.5% Triton X-100 (to remove SDS), incubated in activation buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35) with or without CTTHWGFTLC (0-50 microM) for 24-48 hours at 37degC, and then stained with Coomassie Blue. Clear zones (digested gelatin) indicate MMP activity; inhibition is assessed by reduced clearing zones in the presence of the peptide.
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| Cell Assay |
For cell-based assays, human umbilical vein endothelial cells (HUVECs) or tumor cell lines (e.g., HT1080 fibrosarcoma cells, highly invasive due to high MMP-9 expression) are used. For migration/invasion assays: Transwell inserts (8 microm pore size) are coated with Matrigel (50 microg/insert) for invasion assays. Cells (1-5×10⁵ cells per insert) are resuspended in serum-free medium containing CTTHWGFTLC, CYCLIC TFA (0-100 microM) and placed into the upper chamber. The lower chamber contains medium with 10% fetal bovine serum (FBS) as a chemoattractant. The cells are incubated at 37degC for 16-24 hours. Non-migrated cells on the upper side of the membrane are removed with a cotton swab. Migrated/invaded cells on the lower side are fixed with 4% paraformaldehyde, stained with 0.1% crystal violet or Giemsa, and counted under a microscope in 5-10 random fields. The percent inhibition of migration/invasion is calculated relative to vehicle control. For viability control, cells are treated with the same concentration range of the peptide in 96-well plates for 24-48 hours, and cell viability is measured using the MTT or CellTiter-Glo assay to ensure that the observed reduction in migration is not due to cytotoxicity. Additional functional assays: (1) Gelatin zymography of cell culture supernatants to assess MMP-2/9 activity: cells are treated with the peptide for 24 hours, and conditioned media are collected, concentrated if necessary, and run on gelatin zymography gels as described above; (2) Western blotting for MMP-2/9 protein expression to distinguish between inhibition of activity and downregulation of expression (the peptide is an activity inhibitor, not a protein synthesis inhibitor).
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| Animal Protocol |
For in vivo animal studies, CTTHWGFTLC, CYCLIC TFA is typically administered to immunocompromised mice bearing subcutaneous tumor xenografts (e.g., HT1080 fibrosarcoma, MDA-MB-231 breast carcinoma, or B16-F10 melanoma) to study anti-metastatic or anti-angiogenic effects. For intravenous administration, the cyclic peptide (10-50 mg/kg) is dissolved in sterile PBS (with 0.1-0.5% DMSO if needed) and administered via tail vein injection daily or every other day for 2-4 weeks. Tumor volume is measured twice weekly using calipers. At the end of the study, primary tumors are excised and weighed. For metastasis models (e.g., intravenous injection of tumor cells for lung colonization), the number of metastatic nodules in the lungs is counted after euthanasia. Tumors and tissues are processed for immunohistochemistry (IHC) using anti-MMP-2 and anti-MMP-9 antibodies, and for gelatin zymography to assess MMP activity. Alternative administration routes: subcutaneous injection near the tumor site, or intraperitoneal injection. Due to the cyclic structure and the presence of a disulfide bond, CTTHWGFTLC has improved stability compared to linear peptides, but its half-life in vivo is still limited; for sustained effects, the peptide may be conjugated to PEG or encapsulated in nanoparticles/liposomes. Liposome targeting to tumor cells using this peptide has been described in the literature.
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| ADME/Pharmacokinetics |
CTTHWGFTLC, CYCLIC TFA has a molecular weight of approximately 1166.33 Da (for the free base; the TFA salt increases the mass slightly). The peptide sequence: CTTHWGFTLC with a disulfide bridge between Cys1 and Cys10 (cyclic structure). CAS number: 244082-19-7 (for the cyclic peptide free base). The peptide is supplied as a lyophilized powder (white to off-white). The TFA salt form enhances water solubility. Storage: store at -20degC or -80degC, protect from light. Avoid repeated freeze-thaw cycles. For stock solutions, dissolve in sterile DMSO (10-20 mM) or in water (if solubility permits). For cell-based assays, dilute the DMSO stock in culture medium; the final DMSO concentration should be ≤0.5% to avoid cytotoxicity. For in vivo administration, dissolve in PBS or saline (pH 7.4); sonication may be required to achieve full dissolution. Peptide purity is typically ≥95% by HPLC. The cyclic structure provides resistance to exopeptidases but may still be susceptible to endoproteases; for extended in vivo studies, consider using mini-pumps or repeated dosing.
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| Toxicity/Toxicokinetics |
CTTHWGFTLC, CYCLIC TFA is a cyclic peptide inhibitor of MMP-2 and MMP-9, intended for research use only, not for human diagnostic or therapeutic applications. At typical research concentrations (10-50 microM), the peptide is considered non-toxic; however, cytotoxicity may occur at higher concentrations (>100 microM) due to non-specific effects. MMP-2 and MMP-9 play important roles in normal physiological processes (e.g., wound healing, angiogenesis), so prolonged inhibition could have undesirable effects. Standard safety practices for handling peptides should be followed: use of gloves, lab coat, and eye protection; avoid inhalation of dust. For in vivo studies, standard safety monitoring (body weight, behavior, signs of inflammation) should be performed.
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| References | |
| Additional Infomation |
CTTHWGFTLC, CYCLIC TFA (also known as CTT cyclic peptide or gelatinase inhibitor peptide) is a cyclic peptide inhibitor of type IV collagenases MMP-2 and MMP-9. Key features: (1) Cyclic structure via disulfide bridge (Cys1-Cys10) confers stability and protease resistance; (2) IC₅0 for MMP-9 is approximately 8 microM; (3) Inhibits endothelial and tumor cell migration in vitro; (4) Can be used for targeting MMP-overexpressing tumors when conjugated to liposomes or nanoparticles; (5) TFA salt enhances solubility. Common applications: anti-metastasis research, anti-angiogenesis studies, extracellular matrix (ECM) remodeling, tumor invasion assays, and development of MMP-targeted drug delivery systems. The peptide was described in the literature: Koivunen E, et al. Nat Biotechnol. 1999;17(8):768-774, and binding to phospholipid membranes was further characterized by Medina OP, et al. (2001). This product is not approved for clinical use.
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| Molecular Formula |
C54H72F3N13O16S2
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| Molecular Weight |
1280.35
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| Related CAS # |
CTTHWGFTLC, CYCLIC;244082-19-7
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :≥ 50 mg/mL (~39.05 mM)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.7810 mL | 3.9052 mL | 7.8104 mL | |
| 5 mM | 0.1562 mL | 0.7810 mL | 1.5621 mL | |
| 10 mM | 0.0781 mL | 0.3905 mL | 0.7810 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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