μ Opioid Receptor/MOR
CTOP TFA targets the micro-opioid receptor (MOR) with high affinity and selectivity. It is a somatostatin analogue but lacks significant activity at somatostatin receptors. The Ki for micro-opioid receptor is 0.96 nM, while the Ki for delta-opioid receptor is >10,000 nM, indicating very high selectivity. CTOP acts as an antagonist, blocking the effects of micro-opioid agonists such as morphine and enkephalins. The structure is cyclic: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (Pen = penicillamine, which forms a disulfide bridge with Cys). CTOP increases potassium currents in rat locus coeruleus neurons in vitro via a pertussis toxin-sensitive G protein-coupled pathway.

In vitro, CTOP TFA is a highly potent and selective micro-opioid receptor antagonist. In rat locus coeruleus neurons, CTOP increases K+ currents through G protein-coupled inwardly rectifying potassium channels (GIRKs) via a pertussis toxin-sensitive mechanism. CTOP dose-dependently blocks micro-opioid agonist-induced inhibition of adenylyl cyclase and stimulation of potassium channel activity. Its high selectivity (>10,000-fold over delta-opioid receptors) makes it a valuable pharmacological tool for dissecting the roles of micro-opioid receptors in various tissue preparations and in the brain. In opioid receptor binding assays, CTOP has a Ki of 0.96 nM for micro-opioid receptors and over 10,000 nM for delta-opioid receptors. The cyclic structure stabilizes the peptide conformation and enhances receptor selectivity.

In a dose-dependent manner, CTOP TFA (0-0.5 nmol, ICV, once) counteracts the analgesic effect[1]. Animals exposed to CTOP TFA (0–2 nmol, ICV, once) experience withdrawal hypothermia and body weight loss[ 1]. In the nucleus accumbens, CTOP TFA (0–1.5 nmol per side, Intra-VTA injection) raises extracellular dopamine levels and dose-dependently increases locomotor activity[2].

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In vivo, CTOP TFA antagonizes acute analgesic effects and hypermotility induced by micro-opioid agonists such as morphine. It dose-dependently increases locomotor activity in mice. Central administration (intracerebroventricular or intrathecal) of CTOP causes behavioral effects, including an increase in locomotor activity and the blockade of morphine-induced antinociception in pain assays (e.g., tail-flick test). CTOP enhances extracellular dopamine levels in the nucleus accumbens, indicating that endogenous micro-opioid receptor tone tonically inhibits dopamine release in this reward-related brain region. CTOP is brain-penetrant, making it suitable for systemic administration in studies of micro-opioid receptor function in the central nervous system. However, the TFA salt form is commonly used for in vitro and ex vivo studies, and the peptide is usually administered via central injection for CNS studies.

For in vitro micro-opioid receptor binding assays (non-cell-based), membranes from rat brain (cortex or striatum, rich in micro-opioid receptors) or from HEK293 cells expressing recombinant human micro-opioid receptor are prepared. For radioligand binding displacement assays: membranes (50-100 microg protein) are incubated with 0.5-1 nM of a micro-selective radioligand such as [3H]DAMGO ([tyrosyl-3,5-3H] [D-Ala2, N-MePhe⁴, Gly-ol⁵]-enkephalin) or [3H]naloxone in assay buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1% BSA, protease inhibitors) in the presence of varying concentrations of CTOP TFA (0.1-1000 nM). Incubate at 25degC or 37degC for 60-90 minutes. Non-specific binding is defined using 10 microM naloxone. Bound radioactivity is separated by rapid filtration through glass fiber filters pre-soaked in 0.3% polyethyleneimine. Filters are washed 3× with ice-cold buffer, dried, and counted in a scintillation counter. IC₅0 values are converted to Ki using the Cheng-Prusoff equation. For functional antagonism assays (GTPgammaS binding): membranes are incubated with [3⁵S]GTPgammaS (0.05-0.1 nM) 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 CTOP TFA (0.1-1000 nM). Incubate at 25degC for 60 minutes. The increase in [3⁵S]GTPgammaS binding stimulated by DAMGO is measured, and the antagonism (IC₅0 for inhibiting the DAMGO-stimulated signal) is calculated.

For cell-based assays, cells expressing micro-opioid receptors (e.g., CHO or HEK293 cells stably transfected with human MOR) are seeded in 96-well plates (50,000 cells/well). For cAMP accumulation assays: cells are pre-incubated with 500 microM IBMX (phosphodiesterase inhibitor) for 10 minutes at 37degC. Cells are then treated with CTOP TFA (0.1-1000 nM) in the presence or absence of a micro-opioid agonist (e.g., DAMGO 0.1-1 microM) and 10 microM forskolin (to stimulate cAMP) for 20-30 minutes at 37degC. cAMP accumulation is measured using a homogeneous time-resolved fluorescence (HTRF) cAMP kit or a chemiluminescence-based immunoassay. The EC₅0 for antagonism is calculated. For GIRK channel activation (electrophysiology): cells co-expressing micro-opioid receptor and GIRK1/2 channels are used for whole-cell patch-clamp recording. Cells are superfused with extracellular solution containing 0.5-1 microM DAMGO, and then increasing concentrations of CTOP (0.1-1000 nM) are co-applied. The percentage reversal of the DAMGO-induced GIRK current by CTOP is plotted to determine the IC₅0. In both assays, CTOP acts as a silent antagonist, blocking agonist-induced responses without producing agonist effects on its own (no intrinsic activity).

Animal/Disease Models: Male CFLP mice (25-30 g)[ 1]
Doses: 0, 0.001, 0.05, 0.075, 0.1, and 0.5 nmol (made up in artificial cerebrospinal fluid (CSF) and kept in plastic tubes at -25℃ until use)
Route of Administration: Intracerebroventricular (icv) administration, once
Experimental Results: Antagonized the analgesic effect in a dose-dependent manner, antagonized the induced hypermotility in a dose-dependent manner.

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Animal/Disease Models: Male CFLP mice (25-30 g, Acute dependence to morphine was induced by a single dependence-inducing (100 mg/ kg) dose of morphine-HC1)[1]
Doses: 0, 0.001, 0.05, 0.2, and 2 nmol
Route of Administration: Intracerebroventricular (icv) administration, once
Experimental Results: diminished the body temperature in a dose-dependent manner, and caused withdrawal hypothermia and a loss of body weight in animals.

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Animal/Disease Models: Long-Evans hooded rats (12, male, 350-450 g)[2]
Doses: 0, 0.015, 0.15, and 1.5 nmol per side
Route of Administration: Intra-VTA (ventral tegmental area) injection
Experimental Results: Enhanced extracellular dopamine levels in the nucleus accumbens, dose-dependently increased activity, whereas had no effect on feeding and drinking behavior.

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For in vivo animal studies, CTOP TFA is typically administered via intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection to rodents (mice or rats). For i.c.v. injection: male C57BL/6 mice (6-8 weeks) are anesthetized with isoflurane and stereotaxically implanted with a guide cannula into the lateral ventricle (coordinates: AP -0.5 mm, ML +/-1.0 mm, DV -2.0 mm relative to bregma). After 5-7 days of recovery, CTOP TFA is dissolved in sterile artificial cerebrospinal fluid (aCSF) at concentrations of 0.1-10 microg/microL. A volume of 2-5 microL (0.1-5 microg/mouse) is injected via a microsyringe over 1-2 minutes, and the injector is left in place for an additional minute to allow diffusion. Behavioral testing (e.g., tail-flick or hot-plate test for analgesia, open-field test for locomotor activity) is performed 10-30 minutes post-injection. For systemic administration (subcutaneous or intraperitoneal) to test brain penetration, CTOP TFA is administered at doses of 1-10 mg/kg in 100-200 microL saline. However, due to its peptide nature, systemic bioavailability is limited; central administration is more reliable. For studies on dopamine release, microdialysis probes are implanted into the nucleus accumbens, and CTOP is administered systemically or centrally while dialysate is collected for HPLC analysis of dopamine and metabolites. For studies on L-DOPA-induced dyskinesia, CTOP TFA has been used to examine the role of micro-opioid receptors in rodent models (see literature references).

CTOP TFA is a cyclic peptide with a disulfide bridge. The sequence: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (Pen = penicillamine, which forms the disulfide bond with Cys). The TFA salt form enhances water solubility and purity. Molecular weight: Approximately 1218.33 Da (for CTAP TFA, see CTAP details; CTOP TFA molecular weight is similar). The compound is supplied as a lyophilized powder (white to off-white). Storage: protect from light; store at -20degC for up to 3 years; in solution at -80degC for up to 1 year. Solubility: water (soluble, 1 mg/mL or higher with sonication); DMSO (soluble). For in vivo administration, dissolve in aCSF or sterile saline; the TFA salt may be slightly acidic (pH 4-5); if needed, adjust pH to 7.0-7.4 with 0.1 M NaOH for central injections to avoid tissue irritation. Stock solutions (1-10 mM in DMSO) are stable at -80degC for 6 months. Avoid repeated freeze-thaw cycles. Peptide purity is typically ≥95-98% by HPLC.

CTOP TFA is a research-grade peptide for laboratory use only, not for human diagnostic or therapeutic applications. At typical research doses (microg range for central administration, or mg/kg for systemic), no significant toxicity has been reported. However, as a micro-opioid receptor antagonist, it may alter pain perception, stress responses, and reward behavior in animal models. 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, monitor for any behavioral changes (e.g., increased locomotor activity or altered pain responses). The TFA counterion is generally considered safe at the concentrations used, but careful handling is advised. This product is for research use only.

[1]. Central effects of the potent and highly selective μ opioid antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) in mice. Eur J Pharmacol. 1988 Jun 10;150(3):355-60.

[2]. Intra-VTA injections of the mu-opioid antagonist CTOP enhance locomotor activity. Brain Res. 1995 Aug 28;690(1):112-6.

CTOP TFA (cyclic octapeptide somatostatin analog) is a potent, highly selective, brain-penetrant micro-opioid receptor antagonist. Key features: (1) Cyclic structure (disulfide bridge) confers stability and receptor selectivity; (2) Ki for micro-opioid receptor = 0.96 nM, with >10,000-fold selectivity over delta-opioid receptors; (3) Antagonizes micro-opioid agonist-induced analgesia and hypermotility; (4) Increases extracellular dopamine in the nucleus accumbens, suggesting tonic inhibition of dopamine release by endogenous micro-opioid activity; (5) Increases K+ currents in locus coeruleus neurons via a G protein-coupled mechanism. Common applications: studies of micro-opioid receptor function in pain, addiction, reward, and mood disorders. CTOP is structurally related to CTAP (another micro-antagonist). CTOP is the ornithine (Orn)-containing analog, whereas CTAP contains arginine (Arg) at position 5. The peptide is not approved for clinical use.

C52H68F3N11O13S2

1176.28

CTOP;103429-31-8

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 :~50 mg/mL (~42.51 mM)

Solubility in Formulation 1: 100 mg/mL (85.01 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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