μ Opioid Receptor/MOR 3.5 nM (IC50) δ Opioid Receptor/DOR 4500 nM (IC50)
CTAP TFA targets the micro-opioid receptor (MOR) with an IC₅0 of 3.5 nM. It displays over 1,200-fold selectivity over the delta-opioid receptor (IC₅0 = 4500 nM) and also over somatostatin receptors. CTAP is a cyclic octapeptide with the sequence D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, where Pen is penicillamine, forming a disulfide bridge with Cys. It acts as a silent antagonist, blocking the effects of micro-opioid agonists without producing intrinsic opioid activity.

In vitro, CTAP TFA exhibits high affinity binding to micro-opioid receptors in radioligand binding assays. It potently antagonizes micro-opioid agonist-induced responses, such as inhibition of cAMP accumulation and activation of G protein-coupled inwardly rectifying potassium channels (GIRKs), in cells expressing micro-opioid receptors. The IC₅0 for micro-opioid receptor antagonism is 3.5 nM, and it has minimal activity at delta-opioid and kappa-opioid receptors. The cyclic structure ensures high selectivity and stability against proteolytic degradation. In guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays, CTAP antagonizes micro-opioid receptor-mediated inhibition of electrically evoked contractions.

The analgesic effects of morphine are inhibited by CTAP TFA (0–1 mg/kg, IP, single dose) [1]. When it comes to abnormal involuntary movements of the limbs, axis, oral tongue, or motor produced by L-DOPA, CTAP TFA (10 mg/kg; IP, single dosage) has no effect. [1]. In rat blood and serum, CTAP TFA is stable (T1/2 > 500 minutes), suggesting that the peptide's structure is resistant to enzymes [2]. In perfusion mediums, CTAP TFA binds substantially to albumin (68.2%) and to proteins (84.2%) in rat serum [2].

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In vivo, CTAP TFA is a brain-penetrant micro-opioid antagonist. It has been used in rodent models to study the role of micro-opioid receptors in L-DOPA-induced dyskinesia (LID), a common side effect of dopamine replacement therapy for Parkinson's disease. CTAP does not block L-DOPA-induced dyskinesia in a rodent model, as demonstrated in behavioral studies using highly selective micro-opioid receptor antagonism. CTAP is also used to study micro-opioid receptor involvement in pain modulation, reward, and addiction. Its blood-brain barrier (BBB) permeability makes it suitable for systemic administration.

For in vitro micro-opioid receptor binding assays (non-cell-based), membranes from HEK293 cells expressing recombinant human micro-opioid receptor or from rat brain (cortex, thalamus) are prepared. For radioligand displacement: membranes (50-100 microg protein) are incubated with 1 nM [3H]DAMGO ([D-Ala2, N-MePhe⁴, Gly-ol⁵]-enkephalin) or 0.5 nM [3H]naloxone in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.1% BSA). Varying concentrations of CTAP TFA (0.1-1000 nM) are added, and the mixture is incubated at 25degC for 60-90 minutes. Non-specific binding is determined with 10 microM naloxone or 1 microM unlabeled DAMGO. Bound radioligand is separated by rapid vacuum filtration through GF/B glass fiber filters pre-soaked in 0.3% polyethyleneimine. Filters are washed 3× with ice-cold buffer, dried, and counted by liquid scintillation. The IC₅0 is calculated from displacement curves, and the Ki is calculated using the Cheng-Prusoff equation. For functional GTPgammaS binding assay: membranes are incubated with [3⁵S]GTPgammaS (50-100 pM) in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 0.1% BSA, 30 microM GDP) with 0.5-1 microM DAMGO (micro-agonist) and varying concentrations of CTAP TFA (0.1-1000 nM). Incubate at 25degC for 60 minutes, then filter and count. The percent inhibition of DAMGO-stimulated [3⁵S]GTPgammaS binding is used to calculate the antagonistic IC₅0.

For cell-based assays, CHO or HEK293 cells stably expressing human micro-opioid receptors are used. For cAMP accumulation assay (a functional readout of micro-opioid receptor activation, which is inhibitory): cells are seeded in 96-well plates (50,000 cells/well) and incubated overnight. The next day, cells are pre-incubated with 500 microM IBMX for 10 minutes at 37degC. Then, CTAP TFA (0.1-1000 nM) is added in the presence or absence of a micro-opioid agonist (e.g., DAMGO 1-100 nM) and 10 microM forskolin (to stimulate cAMP production). After 20-30 minutes, cells are lysed, and cAMP levels are measured using a competitive immunoassay (e.g., HTRF or ELISA). The EC₅0 for antagonism of the agonist-induced inhibition of cAMP accumulation is calculated. For GIRK channel activation assay (G protein-coupled inwardly rectifying potassium channels): cells co-expressing micro-opioid receptor and GIRK1/2 channels are used for whole-cell patch-clamp recording. Cells are held at -60 mV, and the GIRK current (inward rectifying) is measured as a decrease in holding current (or as current amplitude in the presence of high extracellular K+ solution). Application of DAMGO (0.1-1 microM) induces a GIRK current. Co-application of increasing concentrations of CTAP TFA (0.1-1000 nM) causes a concentration-dependent blockade of the DAMGO-induced GIRK current. The IC₅0 for antagonism is determined from the dose-response curve. CTAP should have no agonist activity (i.e., no current in the absence of DAMGO) at the concentrations tested. Cell viability is checked using trypan blue exclusion or an MTT assay to rule out cytotoxic effects.

Animal/Disease Models: Male SD (Sprague-Dawley) rats[1]
Doses: 0, 0.1, 0.5, 1 mg/kg
Route of Administration: IP, single
Experimental Results: Completely blocked morphine's antinociceptive effect at 0.5 or 1 mg/kg.

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For in vivo animal studies, CTAP TFA can be administered to mice or rats via subcutaneous (s.c.), intraperitoneal (i.p.), or intravenous (i.v.) routes due to its brain-penetrant properties. For micro-opioid receptor antagonism studies, CTAP TFA is dissolved in sterile saline or PBS (pH 7.4) at concentrations of 1-10 mg/mL. A typical dose is 1-10 mg/kg in a volume of 100-200 microL per mouse (for i.p. injection). For studies on L-DOPA-induced dyskinesia (LID) in a rodent model of Parkinson's disease: Male Sprague-Dawley rats (or C57BL/6 mice) are lesioned unilaterally with 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle to deplete dopamine. After lesion confirmation (amphetamine-induced rotation test), animals receive chronic L-DOPA treatment (e.g., 6 mg/kg L-DOPA + 15 mg/kg benserazide, i.p., once or twice daily for 14-21 days) to induce dyskinesia. CTAP TFA (e.g., 1-10 mg/kg, i.p.) is administered 30 minutes prior to L-DOPA injection on test days. Dyskinesia severity is rated using the Abnormal Involuntary Movements (AIMs) scale (axial, limb, orolingual, and locomotive). Rotational behavior and forepaw adjusting steps are also measured. The micro-opioid antagonist does not block L-DOPA-induced dyskinesia, indicating the non-involvement of micro-opioid receptors in this model (as demonstrated in published literature: Bartlett MJ, et al. BMC Res Notes). For pain studies (e.g., tail-flick test), CTAP is administered either systemically or intrathecally (via lumbar puncture) to block the antinociceptive effects of micro-opioid agonists (e.g., morphine). Baseline tail-flick latency is measured, followed by administration of the micro-opioid agonist (e.g., 5 mg/kg morphine, s.c.), and tail-flick latency is measured again at various time points (15, 30, 60, 90, 120 minutes). If CTAP is given 10-30 minutes before the agonist, it should abolish the antinociceptive effect. For brain penetration studies: after systemic administration of CTAP TFA, mice are euthanized at various time points, and brain tissue is collected, homogenized, and analyzed by LC-MS/MS to quantify the concentration of CTAP (requires a validated bioanalytical method). CTAP is known to cross the blood-brain barrier, but quantitative distribution data may not be available for the TFA salt specifically.

CTAP TFA has a molecular weight of 1218.33 Da for the TFA salt (free base: approximately 1105.3 Da). CAS number: 103429-32-9 (free base). The TFA salt form is the trifluoroacetate salt, which is commonly used for peptides to enhance water solubility and improve handling. The peptide sequence: H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (Pen = penicillamine, forming a disulfide bridge with Cys). The cyclic structure is essential for high affinity and selectivity. Storage: protect from light and moisture; store at -20degC for up to 3 years; store in solution at -80degC for up to 1 year. Solubility: water (soluble, due to TFA salt; typically >1 mg/mL), DMSO (soluble). For in vivo administration, dissolve CTAP TFA in sterile 0.9% saline or PBS; the TFA salt may give an acidic pH (pH 4-5); adjust pH to 6.5-7.5 with 0.1 M NaOH if necessary for injection to avoid tissue irritation. Peptide purity is typically ≥95-98% by HPLC. Stock solutions (1-10 mM in DMSO) should be stored at -80degC and protected from light; avoid repeated freeze-thaw cycles.

CTAP TFA is a research-grade compound for laboratory use only, not for human diagnostic or therapeutic applications. At typical research doses (mg/kg range in animals, or microM range in vitro), CTAP TFA is considered non-toxic. However, as a micro-opioid receptor antagonist, it may block endogenous opioid signaling, leading to changes in pain perception, stress response, and reward behavior in animal models. No significant acute toxicity has been reported. Standard safety practices for handling peptides and chemical compounds should be followed: use of gloves, lab coat, and eye protection; avoid inhalation of dust; work in a well-ventilated area. For in vivo studies, monitor animals for any behavioral changes (e.g., increased pain sensitivity or reduced locomotion) and for signs of injection site reactions (the TFA salt may be mildly irritating). This product is for research use only.

[1]. Highly-selective µ-opioid Receptor Antagonism Does Not Block L-DOPA-induced Dyskinesia in a Rodent Model.BMC Res Notes.

[2]. Blood-brain barrier permeability and bioavailability of a highly potent and mu-selective opioid receptor antagonist, CTAP: comparison with morphine. J Pharmacol Exp Ther. 1997 Jan;280(1):402-9.

CTAP TFA (H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, disulfide bridge between Cys and Pen) is a cyclic somatostatin analog and a highly selective, potent micro-opioid receptor antagonist. Key features: (1) IC₅0 for micro-opioid receptor = 3.5 nM; (2) >1,200-fold selectivity over delta-opioid receptor (IC₅0 = 4500 nM) and somatostatin receptors; (3) Brain penetrant (crosses the blood-brain barrier); (4) No intrinsic agonist activity; (5) Used as a tool to study micro-opioid receptor function in pain, dyskinesia, addiction, and mood disorders. CTAP is structurally similar to CTOP (which contains Orn at position 5 instead of Arg). CTAP is the arginine-containing analog, which may have slightly different selectivity and stability profiles. Common applications: opioid receptor pharmacology, L-DOPA-induced dyskinesia (LID) research, pain research, studies of endogenous opioid systems. This product is not approved for human use.

C53H70F3N13O12S2

1218.32

CTAP;103429-32-9

White to off-white solid powder

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~82.08 mM)

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)