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| Targets |
DALDA acetate specifically targets the micro-opioid receptor (MOR), a G protein-coupled receptor (GPCR) that is the primary molecular target of endogenous opioid peptides (e.g., endorphins, enkephalins, dynorphins) and classical opioid analgesic drugs (e.g., morphine, fentanyl). DALDA acts as a full agonist at the micro-opioid receptor. Binding of DALDA to MOR activates the Gi/o protein signaling pathway, leading to inhibition of adenylyl cyclase, decreased cAMP levels, activation of G protein-coupled inwardly rectifying potassium channels (GIRKs), and inhibition of voltage-gated calcium channels. This results in neuronal hyperpolarization and reduced neurotransmitter release, producing antinociception (analgesia) and, at higher doses, respiratory depression. The Ki of DALDA for the micro-opioid receptor is 1.69 nM. It has weak affinity for delta- and kappa-opioid receptors, showing high selectivity (over 1000-fold).
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| ln Vitro |
DALDA acetate is more polar and hydrophilic than morphine because it has a net positive charge (3+) at physiological pH [1].
DALDA acetate is a potent micro-opioid receptor agonist in vitro. In receptor binding assays, it shows high affinity for the micro-opioid receptor (Ki = 1.69 nM) with excellent selectivity over delta (Ki > 1000 nM) and kappa (Ki > 1000 nM) receptors. In functional assays (e.g., [35S]GTPgammaS binding to measure G protein activation) using CHO cells expressing the micro-opioid receptor, DALDA acts as a full agonist, stimulating [35S]GTPgammaS binding with an EC50 in the low nanomolar range. The compound is more potent than morphine in these assays. DALDA acetate carries a net positive charge (3+) at physiological pH due to the presence of three basic amino acid residues (D-Arg and Lys, and the N-terminal Tyr). Consequently, it is highly hydrophilic and more polar than morphine, which has a net charge of +1. This increased polarity reduces passive diffusion across cell membranes and the blood-brain barrier, making it a useful tool for studying peripheral opioid receptors. |
| ln Vivo |
Rats treated with DALDA acetate (0–7 nmol/rat; once) show analgesic and respiratory effects [1]. Rats' horizontal locomotion, uprightness, and stereotyped timing are all biphasic, with initial inhibition, intermediate substantial inhibition, and following activation when exposed to DALDA acetate (0.1 and 1.0 μg/side; ICV; once) [2].
In vivo, DALDA acetate produces antinociception (analgesia) when administered by routes that bypass the blood-brain barrier, such as intrathecal (i.t., into the spinal cord) or intracerebroventricular (i.c.v., into the brain ventricles) injection. It is much less effective after systemic (i.v., i.p., s.c.) administration because it does not readily cross the BBB. When administered intrathecally to rats (0.1-10 nmol/rat), DALDA produces dose-dependent antinociception in the tail-flick and hot-plate tests, with a potency significantly higher than morphine. The antinociceptive effect is mediated by micro-opioid receptors, as it is blocked by the selective micro-antagonist naloxone. However, compared to morphine, DALDA produces less respiratory depression at equianalgesic doses in animal models. The reduced respiratory depression is attributed to its poor central nervous system (CNS) penetration after systemic administration. In rats, intrathecal DALDA (0-7 nmol/rat) also shows dose-dependent respiratory effects (decreased respiratory rate) and modifies locomotor activity, causing biphasic effects (initial inhibition, then activation). DALDA also produces physical dependence in animal models, but its abuse liability is lower than that of morphine due to its poor oral bioavailability and BBB penetration. |
| Enzyme Assay |
DALDA acetate is a potent and highly selective micro-opioid receptor agonist. The in vitro binding affinity (Ki) for the micro-opioid receptor is 1.69 nM. Receptor binding assays are performed using rat or human micro-opioid receptor (MOR) expressed in CHO cells or HEK-293 cells, or using rat brain membranes. For radioligand competition binding, membranes (50-100 microg protein) are incubated with 0.5-1 nM of a radiolabeled micro-selective antagonist such as [3H]-DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin) or [3H]-naloxone. DALDA acetate is dissolved in DMSO (10 mM stock) and serially diluted in binding buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 5 mM MgCl2, 0.1% BSA). Non-specific binding is determined with 10 microM naloxone. The reaction is incubated for 60-90 min at 25degC, and bound radioactivity is separated by filtration through GF/B filters presoaked in 0.3% polyethyleneimine (PEI). Filters are washed three times with ice-cold buffer, and radioactivity is counted by liquid scintillation. Ki is calculated from the IC50 using the Cheng-Prusoff equation. For functional activity (agonist), [35S]GTPgammaS binding assays are performed. Membranes (50 microg protein) are incubated in assay buffer (50 mM Tris-HCl pH 7.7, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA, 50 microM GDP, 0.1 nM [35S]GTPgammaS) and various concentrations of DALDA (0.01-10 microM). After 60 min at 30degC, the reaction is terminated by rapid filtration. Non-specific binding is defined with 10 microM unlabeled GTPgammaS. EC50 values are calculated. The maximal stimulation (Emax) is typically >80% relative to the reference agonist DAMGO. DALDA is a full agonist with an EC50 of ~5-20 nM in these assays.
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| Cell Assay |
Cellular functional assays (e.g., cAMP inhibition) are performed in CHO or HEK-293 cells stably expressing the human micro-opioid receptor. Cells are seeded in 96-well plates at 2 × 10⁴ cells/well and grown to 80% confluency. The medium is replaced with HBSS containing 0.5 mM IBMX (a phosphodiesterase inhibitor) and incubated for 10 min. Cells are treated with DALDA acetate (0.01 pM - 100 nM) for 5-10 min at 37degC. For cAMP inhibition assays, cells are then stimulated with forskolin (1-10 microM) for 10 min to elevate cAMP levels. The reaction is stopped by lysis, and intracellular cAMP is measured using a competitive ELISA or HTRF (homogeneous time-resolved fluorescence) cAMP detection kit (e.g., Cisbio). The EC50 for inhibition of cAMP accumulation is calculated. Typical EC50 values for DALDA are in the 1-10 nM range. Maximal inhibition of cAMP is 70-90% of the forskolin control. To demonstrate receptor selectivity, the same experiments are performed with cells expressing delta- or kappa-opioid receptors. DALDA shows negligible activity at these receptors at concentrations up to 10 microM. For proliferation assays (e.g., to assess cytotoxicity), an MTT assay is performed: cells (1 × 10⁴/well) are treated with DALDA (0.1-100 uM) for 24-72 h, and viability is compared to untreated controls. DALDA shows no significant cytotoxicity up to 100 uM.
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| Animal Protocol |
In vivo antinociception is evaluated in male Sprague-Dawley rats (200-250 g) or CD-1 mice (20-25 g). For central administration, an intrathecal (i.t.) cannula is implanted into the subarachnoid space of the spinal cord (L4-L6) under anesthesia. Animals are allowed to recover for 5-7 days. DALDA acetate is dissolved in sterile 0.9% saline (or artificial CSF). Doses of 0.1, 1, 3, 10, and 30 nmol (for rats; for mice, 0.01-3 nmol) are administered intrathecally in a volume of 5-20 uL (rats) or 5 uL (mice), followed by a 10 uL flush of saline. For intracerebroventricular (i.c.v.) administration, a guide cannula is implanted into the right lateral ventricle (coordinates: AP -0.8 mm, ML 1.5 mm, DV 3.5 mm from bregma). The tail-flick test is used to assess thermal nociception: the tail is immersed in a water bath at 52-55degC, and the latency to tail withdrawal is recorded (cut-off time: 10 s). Measurements are taken at baseline and at 5, 10, 15, 30, 45, 60, 90, and 120 min after i.t. injection. The maximum possible effect (%MPE) is calculated as [(test latency - baseline) / (cut-off - baseline)] × 100. The ED50 (dose producing 50% of MPE) is calculated by log-probit analysis. The ED50 for DALDA in the tail-flick test is approximately 0.4-0.8 nmol/rat, which is 10-50 fold more potent than morphine. The duration of action is 45-90 minutes. For respiratory studies, rats are placed in a whole-body plethysmograph, and respiratory rate, tidal volume, and minute ventilation are recorded. DALDA (0-7 nmol, i.t.) produces a dose-dependent decrease in respiratory rate (e.g., 20-30% decrease at 7 nmol). For locomotor studies, rats are placed in an open field apparatus, and horizontal locomotion, rearing, and stereotypy are recorded. DALDA (0.1-1.0 ug/side, i.c.v.) produces biphasic effects: initial inhibition (first 5-15 min), followed by substantial inhibition, and then activation (after 30-60 min).
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| ADME/Pharmacokinetics |
The pharmacokinetics of DALDA acetate have been characterized in rodents. After intravenous (i.v.) administration (e.g., 1 mg/kg in rats), DALDA has a very short plasma half-life (t½ < 5 minutes) due to rapid proteolytic degradation and renal clearance. The compound is not orally bioavailable because it is a tetrapeptide that is extensively degraded by digestive enzymes. The peptide is highly hydrophilic (logP < 0) due to its net positive charge at physiological pH (3+), which prevents passive diffusion across the blood-brain barrier (BBB). As a result, after systemic administration, DALDA has very limited penetration into the central nervous system (CNS). The CNS-to-plasma concentration ratio is less than 0.05 (i.e., <5% of the plasma concentration reaches the brain and spinal cord). This property is exploited to study peripheral micro-opioid receptors without central side effects (e.g., analgesia in inflamed tissue without sedation or addiction liability). After intrathecal (i.t.) administration, DALDA is cleared from the CSF with a half-life of approximately 15-30 minutes, which corresponds to the duration of antinociceptive action. The acetate salt form is used for research; the acetate counterion improves solubility in water and buffers. The compound is stable when stored as a lyophilized powder at -20degC. For in vivo use, it is dissolved in sterile saline or PBS immediately before use, as solutions are not stable for long periods at room temperature.
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| Toxicity/Toxicokinetics |
The toxicity of DALDA acetate has not been extensively studied because it is a research tool, not a clinical drug candidate. At antinociceptive doses (0.1-3 nmol/rat i.t.), no acute toxicity is observed. At higher intrathecal doses (≥30 nmol/rat), transient motor impairment (ataxia, hind limb weakness) may occur due to non-specific effects or diffusion to higher spinal segments. No long-term neurotoxicity (e.g., neurodegeneration) has been reported for intrathecal DALDA in rodents at therapeutic doses. In the respiratory studies, at 7 nmol i.t., rats experienced a 20-30% decrease in respiratory rate, which was not life-threatening. No studies on hepatotoxicity, nephrotoxicity, or genotoxicity have been reported. The compound is not considered cytotoxic in vitro (IC50 > 100 uM in normal cells). Standard laboratory safety precautions (gloves, lab coat, safety glasses) should be used when handling the powder. Avoid inhalation and skin contact. DALDA acetate is for research use only; it is not approved for human or veterinary therapeutic use. It is not a controlled substance in many jurisdictions, but as a micro-opioid agonist, it should be handled with appropriate care and security to prevent misuse.
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| References |
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| Additional Infomation |
DALDA acetate (H-Tyr-D-Arg-Phe-Lys-NH2·CH3COOH) is a tetrapeptide amide. The name DALDA stands for D-Arg2, Lys4-Dermorphin (1-4) amide. It is an analog of dermorphin, an endogenous heptapeptide (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) isolated from the skin of the South American frog Phyllomedusa sauvagei. Dermorphin is one of the most potent naturally occurring micro-opioid receptor agonists. DALDA was designed as a shorter, more hydrophilic analog with a D-Arg (instead of D-Ala) and a C-terminal amide, which confers resistance to carboxypeptidase degradation. The presence of D-Arg increases the net positive charge (from +2 in dermorphin to +3 in DALDA), which enhances solubility and reduces blood-brain barrier penetration while retaining high micro-opioid affinity. Because of its poor CNS penetration, DALDA is a valuable tool for studying peripheral micro-opioid receptor-mediated analgesia (e.g., in inflammatory pain models) without central side effects such as sedation, addiction, and respiratory depression. However, at high doses or when administered centrally, it still produces these effects. DALDA is not approved as a therapeutic drug, but it has been used as a lead compound for developing peripherally-restricted analgesics. The acetate salt is the common research form; the TFA salt is also available. DALDA should be stored as a lyophilized powder at -20degC, protected from light and moisture. It is soluble in water (up to 100 mg/mL) and in DMSO. For research use only.
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| Molecular Formula |
C32H49N9O7
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| Molecular Weight |
671.79
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| Related CAS # |
DALDA;118476-85-0;DALDA TFA
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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 :~100 mg/mL (~148.86 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 | 1.4886 mL | 7.4428 mL | 14.8856 mL | |
| 5 mM | 0.2977 mL | 1.4886 mL | 2.9771 mL | |
| 10 mM | 0.1489 mL | 0.7443 mL | 1.4886 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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