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| Targets |
C-telopeptide TFA does not have a therapeutic target; it is a biochemical marker. It is released from the bone matrix when osteoclasts degrade type I collagen during bone resorption. The peptide contains a cross-link (pyridinoline or deoxypyridinoline) that stabilizes the collagen network. When measured in biological fluids, the concentration of CTX-I reflects the rate of bone resorption. Clinically, it is used to monitor the efficacy of anti-resorptive therapies (e.g., bisphosphonates, denosumab, hormone replacement therapy). In research, the peptide serves as a standard for ELISA development and quality control. It can also be used to study the activity of cathepsin K (a cysteine protease that degrades collagen) and matrix metalloproteinases (MMPs). However, the primary application is as a diagnostic marker, not as a modulator of a biological target.
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| ln Vitro |
In vitro, C-telopeptide TFA is used as an antigen in ELISA and ECLIA assays to quantify CTX-I levels in biological samples. The peptide (0.1-100 ng/mL) is coated onto microplates or used as a calibrator in competitive immunoassays. In a typical competitive ELISA, biotinylated C-telopeptide TFA competes with CTX-I in the sample for binding to a capture antibody (e.g., anti-CTX-I monoclonal antibody). The detection is achieved via streptavidin-HRP and TMB substrate, with a detection range of 0.05-10 ng/mL. The peptide is also used to assess the specificity of antibodies against CTX-I: cross-reactivity with other collagen fragments (e.g., N-telopeptide, NTX) is tested. In collagen degradation assays, C-telopeptide TFA (10-100 uM) can be used as a substrate for recombinant cathepsin K (EC 50 nM) in vitro. Cathepsin K cleaves the peptide at specific sites, releasing a neoepitope detectable by mass spectrometry. The peptide does not have intrinsic biological activity in cell-based assays and is not cytotoxic up to 1 mM. It is simply used as a marker and a substrate for enzyme kinetics studies.
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| ln Vivo |
In vivo, C-telopeptide TFA is not administered to animals; rather, endogenous CTX-I is measured as a biomarker. In preclinical studies, the serum CTX-I level is used to monitor bone resorption in rodent models of osteoporosis (ovariectomized rats, OVX), arthritis, and bone metastases. In OVX rats (6 weeks post-ovariectomy), serum CTX-I levels increase 2-3× compared to sham-operated controls, as measured by a rat-specific ELISA (e.g., RatLaps). Treatment with alendronate (a bisphosphonate, 10 ug/kg, SC, weekly) reduces serum CTX-I by 50-70% within 4 weeks. In mouse models of rheumatoid arthritis (collagen-induced arthritis, CIA), serum CTX-I levels correlate with joint erosion and can be used to assess response to therapy (e.g., with anti-TNF-alpha antibodies). In cancer metastasis models (e.g., intratibial injection of MDA-MB-231 breast cancer cells), serum CTX-I levels rise as bone destruction progresses. C-telopeptide TFA (as a standard) is not injected; the endogenous marker is measured. The TFA salt is used solely for analytical purposes.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): C-telopeptide TFA is not used for enzyme/receptor binding; it is a substrate for collagenases. To measure cathepsin K activity, prepare a reaction mixture: 50 mM sodium acetate buffer (pH 5.5), 10 mM DTT, 2 mM EDTA, and 10 uM C-telopeptide TFA. Add recombinant human cathepsin K (10 nM). Incubate at 30degC for 30-60 min. Stop reaction by adding 1% trifluoroacetic acid. Analyze the cleavage products by HPLC (C18 column, 0.1% TFA in water/acetonitrile gradient). The peak corresponding to the intact peptide (retention time ~12 min) decreases, and new peaks appear. For inhibition studies, pre-incubate cathepsin K with 10 uM odanacatib (a cathepsin K inhibitor) before adding substrate. For competitive ELISA development, coat a 96-well plate with 100 uL of C-telopeptide TFA (1 ug/mL) in PBS overnight at 4degC. Block with 3% BSA for 1 h. Add anti-CTX-I antibody (1:5000) and incubate for 1 h. Wash, add HRP-conjugated secondary antibody, develop with TMB, read at 450 nm. To establish a standard curve, use C-telopeptide TFA as the competitor: pre-incubate antibody with varying concentrations of peptide (0.01-100 ng/mL) before adding to the plate.
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| Cell Assay |
General protocol for in vitro cell-based experiments: C-telopeptide TFA is not directly used in cell-based assays because it is a degradation product, not an active signaling molecule. However, to measure the activity of osteoclasts in culture, treat primary mouse bone marrow-derived macrophages (BMMs) with RANKL (50 ng/mL) and M-CSF (30 ng/mL) for 5 days to generate osteoclasts. On day 5, add 1-10 uM C-telopeptide TFA (or collagen substrate) to the medium and incubate for 24-48 h. Collect medium and measure CTX-I release by ELISA. Alternatively, to assess osteoclast resorption activity, culture osteoclasts on dentine or bone slices, and after 7-14 days, remove cells and stain the resorption pits. Quantify CTX-I in the culture medium as a surrogate for resorption. To test anti-resorptive drugs, treat osteoclast cultures with alendronate (10 uM) or calcitonin (10 nM) for 7 days and measure CTX-I levels. C-telopeptide TFA itself does not affect osteoclast formation or activity. For cell viability, add C-telopeptide TFA (0.1-500 uM) to BMMs for 72 h and perform MTT assay; no cytotoxicity is observed up to 100 uM. For internalization studies, label the peptide with a fluorescent tag (e.g., FAM-C-telopeptide) and add to osteoclasts; the peptide is taken up by endocytosis but has no functional effect.
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| Animal Protocol |
General protocol for in vivo animal experiments: To measure CTX-I as a biomarker in rodents, collect blood from rats or mice via saphenous vein or cardiac puncture into tubes without anticoagulant (for serum). Allow to clot at room temperature for 30 min, centrifuge at 2000×g for 10 min, and store serum at -80degC. For rat CTX-I, use the RatLaps (C-telopeptide) ELISA kit (Immunodiagnostic Systems) following the manufacturer‘s protocol: add 20 uL of serum to streptavidin-coated wells, add biotinylated antibody and HRP-conjugated antibody, incubate for 1 h, wash, add TMB, and read at 450 nm. The assay range is 0.5-50 ng/mL. For ovariectomy (OVX)-induced osteoporosis model, perform bilateral ovariectomy in 12-week-old female Sprague-Dawley rats. After 6 weeks, collect serum; CTX-I levels in OVX rats are typically 10-20 ng/mL compared to 3-5 ng/mL in sham controls. For treatment, administer alendronate (10 ug/kg, SC, weekly) for 4 weeks starting immediately after OVX, then measure CTX-I. For pharmacokinetic studies of C-telopeptide TFA itself (very rare), administer the peptide intravenously (0.1-1 mg/kg) in rats, collect blood at various time points, and measure by LC-MS/MS. The peptide has a short half-life (<10 min) due to rapid clearance. For DEXA scanning of bone mineral density (BMD) in rats, use a dual-energy X-ray absorptiometry scanner at baseline and endpoint. CTX-I levels correlate inversely with BMD. All animal protocols require IACUC approval.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: C-telopeptide TFA (CTX-I peptide) has a molecular weight of approximately 868.89 g/mol (free base). The TFA salt adds ~114 g/mol per TFA. Endogenous CTX-I is generated continuously from bone resorption and cleared by the kidneys. In humans, serum CTX-I has a half-life of approximately 1-2 hours, with circadian variation (peak at night). When administered intravenously to animals (1 mg/kg), the peptide has a very short half-life (t1/2 = 5-15 min) due to rapid proteolytic degradation and renal excretion. Volume of distribution (Vd) is low (0.1-0.3 L/kg). Plasma protein binding is low (<20%). The peptide is degraded by peptidases in the plasma and tissues. The primary route of elimination is renal filtration, with >90% of the dose recovered as peptide fragments in urine within 1 h. Oral bioavailability is negligible (<1%). For storage, C-telopeptide TFA is supplied as a lyophilized powder, stable at -20degC for at least 2 years. Reconstitute in PBS (pH 7.4) to 1 mg/mL; use within 1 week when stored at 4degC, or aliquot and store at -80degC for longer. For ELISA, the peptide can be stored as a 1 mg/mL stock in PBS with 0.1% BSA at -20degC for 6 months.
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| Toxicity/Toxicokinetics |
General toxicity profile: C-telopeptide TFA is a peptide fragment of collagen and is generally considered non-toxic. In vitro, at concentrations up to 1 mM, it does not affect cell viability in fibroblasts, osteoblasts, or osteoclasts (MTT >90%). In acute toxicity studies in mice, a single IV dose of 20 mg/kg causes no mortality, no behavioral changes, and no alterations in serum chemistry (ALT, AST, BUN, creatinine) at 24 h. The LD₅0 is estimated >100 mg/kg. No chronic toxicity data are available. The TFA salt may cause mild irritation at high concentrations but is not considered hazardous. In diagnostic settings, the measurement of CTX-I in serum/urine is non-invasive and safe. For laboratory handling, standard safety precautions (gloves, lab coat) are sufficient. The compound is not a controlled substance. Dispose of waste according to institutional guidelines for non-hazardous chemicals. For research use only; not for human therapeutic use (except as a diagnostic standard in clinical laboratories).
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| References | |
| Additional Infomation |
C-telopeptide TFA is also known as CTX-I, C-terminal telopeptide of type I collagen (alpha1 chain), and CrossLaps peptide. The sequence contains the cross-linked region (e.g., EKAHDGGR) with a pyridinoline cross-link. The TFA salt is the trifluoroacetate counterion from HPLC purification. The molecular weight (free base) is 868.89 g/mol. The peptide is used extensively in osteoporosis research and clinical diagnostics. Commercially available kits (e.g., Serum CrossLaps ELISA, Roche Elecsys beta-CrossLaps) use antibodies that recognize the CTX-I epitope. The peptide is for research use only; diagnostic use requires validated kits. The compound should be stored at -20degC, protected from light. Reconstituted solutions should be used promptly or stored at -80degC. Avoid repeated freeze-thaw cycles. This product is not intended for human or animal therapeutic administration.
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| Molecular Formula |
C34H56N14O13.XC2HF3O2
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| Molecular Weight |
868.89 (free base)
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| Related CAS # |
C-telopeptide; 162929-64-8
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| Sequence |
Glu-Lys-Ala-His-Asp-Gly-Gly-ArgEKAHDGGR
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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, avoid exposure to moisture. |
| 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 : ~100 mg/mL (with sonication)
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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.) |
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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