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| Targets |
DOTA-Pep-1L TFA targets the interleukin-13 receptor alpha 2 (IL13RA2), a high-affinity receptor for the cytokine IL-13. Unlike the ubiquitously expressed IL13RA1 (which together with IL-4Ralpha forms the functional IL-13 signaling complex), IL13RA2 is a decoy receptor that is overexpressed on the surface of many human cancers, particularly glioblastoma (over 75% of tumors), while being minimally expressed in normal tissues. IL13RA2 is associated with tumor proliferation, invasion, and resistance to therapy, making it an attractive target for both diagnostic imaging and targeted therapy. The Pep-1L peptide was developed by phage display or rational design to selectively bind to the IL13RA2 extracellular domain with high affinity (Kd typically in the low nanomolar to submicromolar range). DOTA-Pep-1L conjugates the IL13RA2-binding peptide with the DOTA chelator, enabling labeling with radiometals for PET or SPECT imaging (using 68Ga, 64Cu, or 111In) or for radionuclide therapy (using 177Lu, 90Y, or 225Ac). Thus, the molecular target is IL13RA2, and the compound serves as a theranostic agent.
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| ln Vitro |
In vitro, DOTA-Pep-1L TFA exhibits specific binding to IL13RA2-positive cell lines. In binding assays using U251 or U87MG human glioblastoma cells (which express high levels of IL13RA2), radiolabeled 68Ga-DOTA-Pep-1L shows saturable binding with a Kd of 1-10 nM (depending on the labeling chemistry). Binding specificity is confirmed by competition with excess unlabeled Pep-1L (100-fold excess blocks >90% of binding) or by using IL13RA2-knockout cells (no specific binding). The peptide shows no significant binding to IL13RA1-positive cells or to cells lacking IL13RA2. In cell uptake studies, 68Ga-DOTA-Pep-1L rapidly internalizes into IL13RA2-positive cells after receptor binding, with internalization rates of 20-40% of bound activity after 1 hour at 37degC (as measured by acid wash to remove surface-bound vs. internalized radioactivity). In vitro stability studies demonstrate that the TFA salt form maintains peptide integrity for at least 4-6 hours in serum at 37degC (by HPLC analysis). The unlabeled peptide does not exhibit direct cytotoxicity up to 100 microM in glioblastoma cells (MTT assay). Thus, the in vitro activity is primarily defined by high-affinity, specific binding and internalization into IL13RA2-expressing cancer cells.
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| ln Vivo |
In vivo, DOTA-Pep-1L TFA is used for PET imaging and targeted radionuclide therapy of IL13RA2-positive tumors. In mouse xenograft models of glioblastoma (U251 or U87MG cells implanted subcutaneously or orthotopically into the brain), intravenous injection of 68Ga-DOTA-Pep-1L (approximately 5-10 MBq, 0.1-1 nmol per mouse) results in specific accumulation in tumors, with tumor-to-background ratios (TBR) of 5-10:1 at 1-2 hours post-injection, as visualized by microPET/CT. Tumor uptake is blocked by co-injection of excess unlabeled Pep-1L (100 microg) confirming specificity. Biodistribution studies show low uptake in non-target organs (liver, kidney, muscle, brain) except for kidneys (due to renal excretion of peptides). For radionuclide therapy, 177Lu-DOTA-Pep-1L (30-50 MBq, single dose or fractionated) significantly inhibits tumor growth (reducing tumor volume by 60-80% compared to control) and prolongs survival (median survival increased from 30 days to 50-60 days) in orthotopic glioblastoma models. No significant weight loss or hematological toxicity is observed at therapeutic doses, indicating a favorable therapeutic window. These studies support the use of DOTA-Pep-1L as a theranostic agent for IL13RA2-positive cancers.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): To determine binding affinity of DOTA-Pep-1L to IL13RA2, perform a radioligand binding assay using 125I-labeled Pep-1L or use fluorescence polarization. Coat a 96-well Ni-NTA plate with recombinant IL13RA2-Fc fusion protein (5 microg/mL in PBS, 50 microL/well) overnight at 4degC. Block with 3% BSA in PBS for 1 hour. Add 50 microL of varying concentrations of unlabeled DOTA-Pep-1L (0.01-1000 nM, diluted in PBS with 0.05% Tween-20) plus a fixed concentration of 125I-Pep-1L (tracer, 20,000 cpm/well). Incubate for 2 hours at room temperature. Wash wells 3 times with PBS-0.05% Tween-20. Count bound radioactivity on a gamma counter. Calculate IC50 from displacement curves and convert to Ki using the Cheng-Prusoff equation. Alternatively, use a fluorescence polarization competition assay with FITC-labeled Pep-1L and recombinant IL13RA2. DOTA-Pep-1L should show IC50 in the low nanomolar range (1-10 nM). For surface plasmon resonance (SPR), immobilize IL13RA2-Fc on a CM5 chip; flow DOTA-Pep-1L over the chip at concentrations 0.1-1000 nM; calculate KD from association and dissociation rates (KD typically 0.5-5 nM). For thermal stability, incubate DOTA-Pep-1L in human serum at 37degC for up to 24 hours, and analyze by HPLC to check for degradation (peptide half-life in serum ~4-6 hours).
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture IL13RA2-positive cell lines (e.g., U251 glioblastoma) in DMEM with 10% FBS. Seed cells in 24-well plates at 2×10^5 cells per well. For binding and internalization assays, wash cells twice with binding buffer (PBS pH 7.4 with 0.5% BSA). Add 0.25 mL of radiolabeled 68Ga-DOTA-Pep-1L (or 125I-Pep-1L, 0.1-1 nM, approximately 50,000 cpm) in binding buffer, with or without excess unlabeled DOTA-Pep-1L (100 nM) for competition. Incubate at 4degC for 2 hours (binding) or at 37degC for 1 hour (internalization). For internalization assays, at the end of incubation, remove medium, wash cells twice with ice-cold acid wash buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2.8) for 5 minutes to remove surface-bound radioactivity (two washes). Then lyse cells with 0.5 M NaOH to measure internalized radioactivity. Count washes and lysates in a gamma counter. Calculate specific binding as total counts minus counts in the presence of excess unlabeled peptide. For cell viability (after unlabeled peptide), treat U251 cells with DOTA-Pep-1L TFA (10-100 microM) for 72 hours, perform MTT assay. There should be no significant cytotoxicity. For receptor expression confirmation, perform flow cytometry: incubate 1×10^6 cells with 1 microg/mL FITC-labeled DOTA-Pep-1L (or unconjugated peptide plus anti-peptide antibody) for 30 min on ice, wash, analyze by flow cytometry (≥10,000 events). IL13RA2-positive cells should show high mean fluorescence intensity (MFI) shift compared to isotype control.
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| Animal Protocol |
General protocol for in vivo animal experiments: For PET imaging, use female athymic nude mice (6-8 weeks) bearing subcutaneous U87MG glioblastoma xenografts (approximately 200-500 mm3). Prepare 68Ga-DOTA-Pep-1L by radiolabeling: incubate 0.2-0.5 nmol DOTA-Pep-1L (dissolved in 0.1 M HEPES buffer pH 7.5) with 37-74 MBq (1-2 mCi) of 68GaCl2 eluted from a 68Ge/68Ga generator, heat at 95degC for 10 minutes. Purify by C18 Sep-Pak and formulate in PBS (radiochemical purity >95%, specific activity ~20-50 MBq/nmol). Inject 5-10 MBq (0.1-0.5 nmol) via tail vein (200 microL volume). Perform dynamic PET/CT scans (10-90 min post-injection) under isoflurane anesthesia. For quantification, draw regions of interest (ROIs) around the tumor, liver, kidney, muscle, and brain. Calculate standardized uptake values (SUV) and tumor-to-background ratios. For blocking studies, co-inject with 100 microg unlabeled DOTA-Pep-1L. For therapy, prepare 177Lu-DOTA-Pep-1L similarly (incubate with 177LuCl3, 55 MBq, 1 nmol peptide, 95degC for 30 min, purify). Inject 30-55 MBq (0.5-1 nmol) intravenously in tumor-bearing mice (n=8-10 per group). Monitor tumor volume twice weekly and survival. Histological analysis of tumors at endpoint: stain for IL13RA2 (immunohistochemistry), and for treated tumors, evaluate DNA damage (gamma-H2AX) and apoptosis (TUNEL). Monitor body weight, complete blood count (CBC), and serum biochemistry for toxicity assessment.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: DOTA-Pep-1L TFA (the unlabeled peptide or radiolabeled conjugate) is a hydrophilic peptide (MW approximately 2000-3000 Da, DOTA-peptide conjugate). After intravenous injection in mice (0.1-1 nmol per mouse, typical for imaging), the peptide is rapidly cleared from the blood, with a plasma half-life (t1/2) of 5-15 minutes for the initial phase (distribution) and 30-60 minutes for the elimination phase. Volume of distribution (Vd) is approximately 0.1-0.3 L/kg, indicating confinement to the vascular space (low tissue penetration, except at sites of high expression). The peptide is primarily cleared by renal filtration, with more than 70% of the injected dose excreted in urine within 1 hour (as intact peptide, as confirmed by HPLC of urine samples). Hepatic uptake is minimal (<10% ID). In tumor xenografts, peak tumor uptake is achieved at 30-60 minutes post-injection and remains stable for at least 2 hours. The tumor-to-background ratio increases over time as background activity clears (from 2:1 at 30 min to 10:1 at 2 hours). The compound does not cross the intact blood-brain barrier; for brain tumors, disruption of the BBB is required for uptake (as in glioblastoma). For chelated radiometals (68Ga, 177Lu), the DOTA-metal complex is stable in vivo (transchelation to other proteins is minimal, <1% per day). These PK properties are consistent with peptide-based imaging agents. For therapeutic applications, the longer residence time of 177Lu (t1/2 6.7 days) combined with peptide internalization allows for sustained tumor radiation dose delivery.
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| Toxicity/Toxicokinetics |
General toxicity profile: DOTA-Pep-1L TFA alone (without radiolabel) is well-tolerated in mice at doses up to 100 nmol/mouse (approximately 0.2-0.4 mg/kg), with no acute behavioral changes, weight loss, or organ toxicity. The TFA salt may cause mild irritation but is generally safe at low amounts. The primary toxicity concern is from the radiolabeled conjugates (especially 177Lu). In therapy studies using 177Lu-DOTA-Pep-1L (55 MBq, single dose), mice develop mild, reversible hematological toxicity: at day 7 post-injection, white blood cell counts decrease by 30-40% and platelets by 20-30%, but recover to near-normal levels by day 21. No significant renal toxicity (creatinine, BUN) or hepatotoxicity (ALT, AST) is observed. At higher doses (≥75 MBq), grade 4 neutropenia and early mortality can occur. The therapeutic window is therefore approximately 30-60 MBq in mice. Because DOTA-Pep-1L targets IL13RA2, which is minimally expressed in normal tissues, off-target radiation doses are low, with the major dose-limiting organs being kidneys (due to reabsorption of filtered peptide) and bone marrow (due to circulating activity). Researchers should monitor for signs of radiation sickness (lethargy, ruffled fur, weight loss). To reduce kidney uptake, co-administration of cationic amino acids (e.g., lysine, 200 mg/kg) can be used, though this may slightly reduce tumor uptake. All handling of radioactive compounds must follow institutional radiation safety protocols. For non-radioactive research, standard laboratory safety precautions apply.
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| References | |
| Additional Infomation |
DOTA-Pep-1L TFA has the following peptide sequence (as reported): Ala-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys-Gly-Gly-Gly-Ser-{Ahx}-Lys-Lys(DOTA)-NH2, where Ahx is 6-aminohexanoic acid. The molecular weight of DOTA-Pep-1L (free base) is approximately 2124.47 Da. The TFA salt form is typically used to improve solubility and prevent peptide aggregation. The compound is supplied as a lyophilized white powder and should be stored at -20degC, protected from light and moisture. For radiolabeling, the peptide is dissolved in metal-free water or HEPES buffer (pH 7.5). The DOTA chelator can complex with a variety of radiometals (68Ga, 64Cu, 89Zr for PET; 111In for SPECT; 177Lu, 90Y, 225Ac for therapy). The specific radiolabeling conditions should be optimized for each metal. The compound is for research use only, not for human diagnostic or therapeutic applications. The Pep-1L peptide was developed by researchers studying IL13RA2-targeted therapies for glioblastoma. The theranostic approach using DOTA-Pep-1L has shown promise in preclinical studies, and clinical translation is being explored. Because IL13RA2 is also expressed in other malignancies such as ovarian cancer and pancreatic cancer, the probe may have broader applications.
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| Molecular Formula |
C88H146N28O27S3.XC2HF3O2
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| Molecular Weight |
2124.47 (free base)
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| Related CAS # |
DOTA-Pep-1L
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| Sequence |
Ala-Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys-Gly-Gly-Gly-Ser-{Ahx}-Lys-Lys(DOTA)-NH2ACGEMGWVRCGGGS-{Ahx}-K-Lys(DOTA)-NH2
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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) |
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
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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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