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| Targets |
Difelikefalin-d5 hydrochloride targets the kappa opioid receptor (KOR), a G protein-coupled receptor (GPCR) primarily expressed in the central and peripheral nervous systems. However, Difelikefalin is specifically designed to be peripherally restricted, meaning it does not readily cross the blood-brain barrier. As a result, it activates KORs located on peripheral sensory nerves and immune cells without causing central nervous system side effects (such as dysphoria, sedation, or hallucinations) typically associated with central KOR activation. By binding to and activating KORs on peripheral nerve endings, Difelikefalin reduces the transmission of itch signals to the spinal cord. KOR activation also produces anti-inflammatory effects by modulating cytokine release from immune cells. The mechanism is G protein-dependent, involving inhibition of adenylate cyclase, decreased cAMP production, and reduced neuronal excitability. Thus, the molecular target is the peripheral kappa opioid receptor.
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| ln Vitro |
Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools.
In vitro, Difelikefalin (the parent compound, and by extension its deuterated form) exhibits potent and selective agonism at the human kappa opioid receptor (KOR). In a transactivation assay using HEK293 cells expressing the human KOR, Difelikefalin activates the receptor with an EC50 of 0.16 nM. It also inhibits forskolin-stimulated cAMP production in R1.G1 mouse thymoma cells expressing KOR with an EC50 of 0.048 nM, indicating potent functional activity. The compound is highly selective for KOR over other opioid receptors: it shows >1000-fold selectivity over mu (u) and delta (delta) opioid receptors. In vitro, Difelikefalin does not cause significant activation of other GPCRs, ion channels, or enzymes at concentrations up to 10 uM. The deuterated version (Difelikefalin-d5) is not used in activity assays because it serves as an internal standard; however, it is expected to have identical biological activity to unlabeled Difelikefalin on a molar basis due to the minimal effect of deuterium substitution on receptor binding and signaling. |
| ln Vivo |
Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.
In vivo, Difelikefalin (the parent compound) has demonstrated efficacy in multiple animal models of itch and inflammation. In mouse models of acute and chronic pruritus (itch), subcutaneous administration of Difelikefalin (0.03-3 mg/kg) dose-dependently reduces scratching behavior induced by various pruritogens (including compound 48/80, chloroquine, and histamine). The anti-itch effect is blocked by the selective KOR antagonist nor-BNI, confirming target engagement. In models of inflammatory pain, Difelikefalin reduces hyperalgesia and allodynia without affecting motor coordination. In phase 3 clinical trials in hemodialysis patients with chronic kidney disease-associated pruritus (CKD-aP), Difelikefalin (0.5 ug/kg IV after each dialysis session) significantly reduced itch intensity scores (WI-NRS) and improved quality of life measures. The deuterated version is not administered in vivo; it is used as an internal standard for pharmacokinetic studies to quantify unlabeled Difelikefalin concentrations in plasma and tissues by LC-MS/MS. |
| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): To measure binding affinity of Difelikefalin to KOR, perform a radioligand competition binding assay using membrane preparations from HEK293 cells expressing human KOR. Prepare assay buffer: 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM EDTA, 0.1% BSA. Incubate 20 ug of membrane protein with 1 nM [3H]-U-69593 (selective KOR radioligand) and increasing concentrations of Difelikefalin-d5 hydrochloride (used as a competitor, 0.001-1000 nM, or use unlabeled Difelikefalin) in a total volume of 250 uL for 60 min at 25degC. Define non-specific binding using 10 uM nor-BNI. Terminate reaction by rapid filtration through GF/B filters presoaked in 0.5% polyethyleneimine. Wash filters 3× with ice-cold buffer. Count filters in a scintillation counter. Calculate IC₅0 values using non-linear regression and convert to Kᵢ using the Cheng-Prusoff equation. For functional activity (cAMP accumulation), use CHO-K1 cells expressing human KOR. Incubate cells with 1 uM forskolin to stimulate cAMP production, plus various concentrations of Difelikefalin-d5 hydrochloride (0.001-1000 nM) for 30 min at 37degC. Measure intracellular cAMP using a homogeneous time-resolved FRET (HTRF) cAMP kit. Calculate EC₅0 for inhibition of cAMP production.
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| Cell Assay |
General protocol for in vitro cell-based experiments: For KOR activation studies, use CHO-K1 cells stably expressing human kappa opioid receptor (KOR) and a CRE-luciferase reporter gene. Culture cells in F-12K medium with 10% FBS. Seed in 96-well white opaque plates at 2×10⁴ cells/well and incubate overnight. The next day, replace medium with serum-free medium and treat cells with Difelikefalin-d5 hydrochloride (as a surrogate for unlabeled Difelikefalin, 0, 0.001, 0.01, 0.1, 1, 10, 100, 1000 nM) for 5-6 hours at 37degC. Add luciferase detection reagent and measure luminescence using a plate reader. Normalize to DMSO control. The deuterated compound should produce the same concentration-response curve as unlabeled Difelikefalin. For selectivity testing, repeat the assay using CHO cells expressing mu (u) or delta (delta) opioid receptors; Difelikefalin should show minimal activation (>1000-fold higher EC₅0). For anti-inflammatory activity, culture primary mouse macrophages or RAW264.7 cells, treat with 0.1-1000 nM Difelikefalin-d5 for 1 hour, then stimulate with LPS (1 ug/mL) for 24 hours. Measure TNF-alpha and IL-6 in supernatant by ELISA. Difelikefalin should reduce cytokine production with EC₅0 around 1-10 nM.
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| Animal Protocol |
General protocol for in vivo animal experiments: Difelikefalin-d5 hydrochloride is not used in efficacy studies; it is a stable isotope-labeled internal standard for PK assays. For pharmacokinetic studies of unlabeled Difelikefalin, administer Difelikefalin (0.1-3 mg/kg) subcutaneously or intravenously to rats or mice. Collect blood samples at multiple time points (0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 h) into EDTA tubes containing a protease inhibitor (e.g., aprotonin). Centrifuge to obtain plasma. To 50 uL plasma, add 10 uL of internal standard solution containing Difelikefalin-d5 hydrochloride (e.g., 10-50 ng/mL in water/acetonitrile). Add 200 uL cold acetonitrile for protein precipitation, vortex, centrifuge at 12,000 rpm for 10 min. Inject supernatant onto LC-MS/MS system with C18 column (mobile phase: 0.1% formic acid in water / acetonitrile gradient). Monitor transitions: unlabeled Difelikefalin (parent mass to fragment) and Difelikefalin-d5 (mass +5). Construct calibration curve with known concentrations of unlabeled Difelikefalin and fixed concentration of internal standard. Calculate PK parameters using non-compartmental analysis. For efficacy studies in animal models of itch, administer Difelikefalin (unlabeled, 0.1-3 mg/kg, SC) to mice 30 min before pruritogen challenge (e.g., compound 48/80, 50 ug in 50 uL injected intradermally into the nape of the neck). Record scratching behavior for 30-60 min. Difelikefalin should reduce number of scratches by 50-80% compared to vehicle.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: Difelikefalin (and its deuterated form as an internal standard) exhibits favorable pharmacokinetic properties. After subcutaneous administration (0.5 ug/kg, the clinical dose in humans after hemodialysis), the compound reaches peak plasma concentration (Cmax) of approximately 1-2 ng/mL at 1-2 hours (Tmax). The terminal elimination half-life (t1/2) in humans is approximately 24-30 hours, allowing for dosing three times per week. Bioavailability after subcutaneous injection is nearly 100%. The compound is not metabolized extensively; less than 10% is metabolized, primarily via hydrolysis and oxidative processes. Protein binding is approximately 80-90%. Difelikefalin is cleared primarily by renal excretion as unchanged drug (approximately 80% of the dose recovered in urine). Due to its peripheral restriction (high polar surface area, poor brain penetration), the brain-to-plasma ratio is less than 0.1. In hemodialysis patients, the compound is efficiently removed by dialysis (extraction ratio ~0.6), so dosing is scheduled after dialysis sessions. The deuterated version is metabolically stable and does not undergo significant deuterium exchange under physiological conditions. For LC-MS/MS analysis, the lower limit of quantification (LLOQ) is 0.05 ng/mL using Difelikefalin-d5 as internal standard.
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| Toxicity/Toxicokinetics |
General toxicity profile: Difelikefalin has an established safety profile based on extensive clinical trials in hemodialysis patients. In phase 3 trials (KALM-1 and KALM-2), the most common adverse events reported were diarrhea (approximately 10%), nausea (8%), dizziness (5%), and headache (4%). These were generally mild to moderate in severity. Serious adverse events were rare and comparable to placebo. Difelikefalin does not cause respiratory depression, sedation, or euphoria, which distinguishes it from mu opioid agonists. No significant QT prolongation or cardiac toxicity was observed. In preclinical toxicology studies, Difelikefalin was well-tolerated in rats and dogs at doses up to 10 mg/kg/day for 28 days, with no target organ toxicity. The compound showed no genotoxicity in the Ames test, no reproductive toxicity in animal studies, and no carcinogenicity potential. The deuterated form is chemically stable and non-toxic at the low concentrations used as an internal standard (typically ng/mL levels). Standard laboratory safety precautions (gloves, lab coat) are sufficient when handling Difelikefalin-d5 hydrochloride.
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| References | |
| Additional Infomation |
Difelikefalin-d5 hydrochloride has the molecular formula C3₆H₅1D₅Cl3N₇O₆ and a molecular weight of approximately 794.26 g/mol. The deuterium atoms are incorporated at specific positions (typically five D atoms) and are stable under physiological conditions, with no back-exchange. The compound appears as a white to off-white powder and is soluble in water and DMSO. It is supplied as a hydrochloride salt to improve aqueous solubility. Storage: lyophilized powder at -20degC, protected from light, for up to 3 years; reconstituted solutions in water (1 mM) can be stored at -80degC for up to 6 months, avoid repeated freeze-thaw cycles. The parent drug Difelikefalin (CR-845) was approved by the FDA in 2021 (Korsuva®) and by the EMA in 2022 for the treatment of moderate-to-severe pruritus associated with chronic kidney disease in hemodialysis patients. The deuterated version is intended for research use only, as an analytical internal standard for pharmacokinetic and bioequivalence studies. It is not for human therapeutic administration. The compound is typically handled in a biosafety cabinet when preparing stock solutions for LC-MS/MS analysis.
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| Molecular Formula |
C36H51D5CL3N7O6
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| Molecular Weight |
794.26
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| Related CAS # |
Difelikefalin-d5
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| Appearance |
White to off-white solid powder
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| Synonyms |
CR-845-d5 hydrochloride; FE-202845-d5 hydrochloride
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2590 mL | 6.2952 mL | 12.5903 mL | |
| 5 mM | 0.2518 mL | 1.2590 mL | 2.5181 mL | |
| 10 mM | 0.1259 mL | 0.6295 mL | 1.2590 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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