K⁺-Cl⁻ cotransporter 2 (KCC2); CLP-290 is a carbamate prodrug of the KCC2 activator CLP-257. [2]
The primary target of CLP290 is the neuron-specific K⁺-Cl⁻ cotransporter type 2 (KCC2). KCC2 is the principal chloride extruder in the central nervous system, maintaining low intracellular chloride levels in mature neurons by transporting chloride ions out of the cell, which is essential for GABA receptor-mediated hyperpolarizing inhibition. CLP290 exhibits high selectivity for KCC2 over related chloride transporters. It enhances KCC2 membrane stability and transport function by preventing dephosphorylation at the Ser940 site of the KCC2 protein, thereby restoring normal chloride homeostasis and GABAergic inhibition under pathological conditions.

CLP-290 itself is a prodrug and has no direct pharmacological activity in vitro. Its active metabolite, CLP-257, has been shown to reduce intracellular chloride concentration in NG108 cells in a concentration-dependent manner (EC₅₀ = 616 nM), with a maximum reduction of approximately 40% (from 57 mM to 34 mM). CLP-257 exhibits high selectivity for KCC2, showing no significant effect on other cation-chloride cotransporters (such as NKCC1, KCC1, KCC3, KCC4). Furthermore, in spinal cord slices from BDNF-treated or nerve-injured rats, CLP-257 restores impaired Cl⁻ transport capacity and reverses the depolarizing shift in GABAergic transmission caused by reduced KCC2 function.

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In vitro, CLP290 exerts neuroprotective effects by enhancing KCC2 function. In cultured primary hippocampal neurons, convulsant stimulation induces dephosphorylation of KCC2 at the Ser940 site and downregulates its membrane expression, leading to epileptiform burst firing activity. CLP290 treatment dose-dependently prevents these KCC2 alterations, thereby restoring GABA-mediated postsynaptic inhibition. CLP290 treatment significantly reduces the frequency and amplitude of abnormal neuronal firing following seizure induction. Additionally, studies indicate that CLP290 effectively reduces intracellular chloride concentrations and restores chloride transport efficiency in compromised neurons.

In rats treated with morphine, CLP290 (oral gavage; 100 mg/kg; twice daily; 7 days) inhibits morphine-induced hyperalgesia in rats (MIH) and increases KCC2 activity and restores Cl- transport in superficial dorsal horn (SDH) neurons [1].

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- In rats with peripheral nerve injury (PNI, neuropathic pain model), oral administration of CLP-290 (100 mg/kg) produced an analgesic effect equivalent to that of pregabalin (30 mg/kg), as measured by paw withdrawal threshold using von Frey filaments. The maximal effect was observed 2 hours post-administration. [2]
- In the same PNI model, CLP-290 (100 mg/kg, oral) did not impair motor performance on an accelerating rotorod, whereas pregabalin (30 mg/kg) significantly reduced time on the rod. [2]
- In rats with morphine-induced hyperalgesia (MIH), concurrent oral administration of CLP-290 (100 mg/kg, twice daily for 7 days) with morphine (10 mg/kg, s.c., twice daily) prevented the downregulation of KCC2 membrane expression in the superficial dorsal horn of the spinal cord. [1]
- In the same MIH model, co-treatment with CLP-290 significantly prevented the development of morphine-induced mechanical hypersensitivity (paw withdrawal threshold) and reduced vocalization behavior induced by subcutaneous injections. [1]
- In streptozotocin (STZ)-induced diabetic rats (diabetes mellitus model), intraperitoneal injection of CLP-290 (100 mg/kg, 4 hours before blood sampling) significantly reduced plasma arginine-vasopressin (AVP) levels by approximately 50% and blood glucose levels by approximately 15%. In control (non-diabetic) rats, CLP-290 significantly increased plasma AVP levels without affecting blood glucose. [3]
- In STZ rats, CLP-290 treatment (100 mg/kg, i.p.) also lowered CSF glucose and Na⁺ concentrations. [3]

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CLP290 demonstrates significant therapeutic efficacy in various animal models of neurological disorders. In epilepsy studies, CLP290 treatment dose-dependently suppresses pentylenetetrazol (PTZ)-induced seizure behaviors in mice and reduces spontaneous recurrent seizures when administered during early epileptogenesis, suggesting an anti-epileptogenic effect. In rat models of spinal cord injury, CLP290 improves locomotor function, reduces muscle co-contraction, and decreases hyperexcitability of polysynaptic reflex pathways without weakening motor output. In neuropathic pain models, CLP290 (100 mg/kg, oral, twice daily for 7 days) prevents and reverses morphine-induced hyperalgesia, restoring KCC2 activity and chloride transport in superficial dorsal horn neurons. In neonatal rats, CLP290 synergistically enhances the sedative effects when co-administered with midazolam. Additionally, CLP290 reduces spontaneous electromyographic activity in lumbar spinal cord motor neurons in a rat model of hindlimb unloading.

Protein Sample Preparation: Extract membrane protein fractions from target brain regions or cultured neurons. Lyse cells with RIPA buffer containing protease inhibitors and enrich membrane proteins by ultracentrifugation. Western Blot Detection: Perform SDS-PAGE with equal amounts of protein and transfer to PVDF membranes. Detect total KCC2 protein, KCC2-Ser940 phosphorylation levels, and membrane expression using specific primary antibodies. [Cl⁻]i Detection: Measure intracellular chloride concentration using chloride-sensitive fluorescent probes (e.g., MQAE). Incubate cells with CLP290, load with MQAE probe, and detect fluorescence intensity changes using a fluorescence microscope or plate reader. Data Analysis: Calculate relative KCC2 protein expression by densitometric scanning, estimate intracellular chloride concentrations from fluorescence intensity, and compare differences between treatment and control groups.

Cell Culture: Primary culture of hippocampal, cortical, or spinal dorsal horn neurons from neonatal rats or mice, cultured until maturation (typically 14-21 days). Drug Treatment: Add various concentrations of CLP290 (e.g., 0.1-10 μM) to the culture medium and pre-incubate for a defined period before subsequent experiments. Convulsant stimulation (e.g., PTZ, 4-AP) may be applied to induce abnormal neuronal firing. Electrophysiological Recording: Record GABA-evoked currents using whole-cell patch-clamp techniques to assess GABA receptor function and Cl⁻ gradient changes. Alternatively, record spontaneous or evoked firing activity of neuronal networks on multi-electrode array systems. Immunocytochemistry: Following cell fixation, stain with KCC2 antibodies and phosphorylation-specific antibodies; observe KCC2 subcellular localization and membrane expression changes using confocal microscopy. Data Analysis: Compare GABA current reversal potentials, firing frequency and amplitude, and KCC2 expression/localization among different treatment groups.

Animal/Disease Models: Adult male rat (300 g, >60 days after birth) [1]
Doses: 100 mg/kg
Route of Administration: po (oral gavage); 100 mg/kg; twice (two times) daily; 7 days
Experimental Results: By restoring Cl - Transport or prevent defects in SDH, rescue established MIH and prevent its development.

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- Neuropathic pain model (PNI): A polyethylene cuff was implanted around the left sciatic nerve of male Sprague-Dawley rats. Mechanical allodynia was assessed using von Frey filaments. CLP-290 was dissolved in 20% 2-hydroxypropyl-β-cyclodextrin (HPCD) and administered orally at 100 mg/kg. Motor performance was evaluated using an accelerating rotorod. [2]
- Morphine-induced hyperalgesia (MIH) model: Male rats received subcutaneous morphine (10 mg/kg, twice daily) for 7 days. CLP-290 (100 mg/kg in 20% HPCD) or vehicle was administered orally twice daily concurrently with morphine for 7 days. Paw withdrawal threshold was measured using von Frey filaments, and vocalization behavior was monitored during subcutaneous injections. [1]
- Diabetes mellitus (DM) model: Male Sprague-Dawley rats received a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg) to induce diabetes. Three weeks later, CLP-290 (100 mg/kg in 20% HPCD) or vehicle was administered intraperitoneally. Blood samples were collected 4 hours later for measurement of AVP (ELISA) and glucose levels. [3]
- Pharmacokinetic study: Male Sprague-Dawley rats received CLP-290 (100 mg/kg) orally. Blood samples were collected at various time points, and plasma concentrations of CLP-257 (the active metabolite converted from CLP-290) were measured by LC/MS. [2]
- Toxicological study: Sprague-Dawley rats (5 males and 5 females per dose) received CLP-290 at 200, 600, and 2000 mg/kg/day (divided into two daily doses) by gavage for 7 consecutive days. Evaluations included mortality, clinical signs, food consumption, body weight, urinalysis, hematology, coagulation, clinical chemistry, organ weights, and histopathology. [2]

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Animals & Models: Use adult male mice (e.g., C57BL/6) or rats (Sprague-Dawley) to establish epilepsy models (PTZ or pilocarpine-induced), neuropathic pain models (morphine-induced hyperalgesia or sciatic nerve injury), spinal cord injury models, or hindlimb unloading models. Dosing Regimen: CLP290 is typically administered by oral gavage, with a common dose of 100 mg/kg once or twice daily for 3-14 days. Vehicles include 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, or 20% hydroxypropyl-β-cyclodextrin solution. Efficacy Assessment: Epilepsy models: Observe seizure latency, seizure score, and seizure duration. Pain models: Assess mechanical pain threshold using von Frey filaments. Spinal cord injury models: Evaluate locomotor function using the BBB scale and record reflex activity by electromyography. Histological Analysis: Euthanize animals after the treatment period; dissect target brain regions or spinal cord lumbar enlargement; detect KCC2 expression and phosphorylation levels by immunohistochemistry or Western blot. Data Analysis: Compare behavioral and molecular differences between treatment and control groups.

- CLP-290 is a carbamate prodrug designed to protect the hydroxyl group of CLP-257 from glucuronidation. Following oral administration (100 mg/kg) in rats, CLP-290 is metabolized to the active KCC2 activator CLP-257. The converted CLP-257 showed an apparent terminal half-life (t₁/₂) of approximately 5 hours, with improved maximal plasma concentration (Cmax) and area under the curve (AUC) compared to direct CLP-257 administration. [2]
- Following intravenous administration of CLP-257 (20 mg/kg), the terminal half-life was <15 minutes. Following intraperitoneal administration of CLP-257 (100 mg/kg), plasma concentration declined rapidly. [2]

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CLP290 is a carbamate prodrug of CLP257, designed to overcome the poor in vivo pharmacokinetic properties of CLP257. Data indicate that CLP290 has significantly better oral bioavailability than its active form CLP257, primarily due to the prodrug strategy protecting the hydroxyl group from glucuronidation. In SD rats, CLP290 is converted to the active metabolite CLP257 following oral administration. Compared to CLP257, CLP290 exhibits a longer in vivo half-life (t₁/₂), enabling maintenance of effective drug concentrations over longer dosing intervals. Regarding solubility, CLP290 is soluble in DMSO (approximately 33 mg/mL) and can be formulated in various in vivo vehicles. Storage conditions: powder stable for 3 years at -20°C and 2 years at 4°C; solutions stable for 2 years at -80°C.

- In a 7-day repeat-dose toxicity study in rats, CLP-290 administered at doses up to 2000 mg/kg/day (divided into two daily doses) showed no effects on clinical condition, body weight, food consumption, hematology, coagulation, clinical chemistry, urinalysis, organ weights, or histopathology. [2]
- hERG tail current inhibition (cardiotoxicity risk) by CLP-290 was weak, with an IC₅₀ of 224 μM (compared to 449 μM for CLP-257), indicating low risk. [2]
- In the PNI model, CLP-290 (100 mg/kg, oral) did not impair motor performance, whereas pregabalin caused significant motor impairment. [2]

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Available toxicological data indicate that CLP290 is generally well-tolerated within experimental dose ranges. In rat models of spinal cord injury, CLP290 (100 mg/kg, oral) had no significant adverse effects on motor function at equipotent analgesic doses, demonstrating better motor safety compared to pregabalin. In rat hindlimb unloading models, 14-day CLP290 administration did not significantly affect slow-twitch muscle fiber cross-sectional area. According to the Material Safety Data Sheet (MSDS), CLP290 is not classified as a hazardous substance, and neither IARC nor NTP list it as a carcinogen. However, comprehensive toxicological data (e.g., acute toxicity, skin irritation, mutagenicity) are not fully available. This product is for research use only and is not intended for human therapeutic applications. Standard laboratory safety practices and appropriate personal protective equipment should be used when handling.

[1]. Enhancing KCC2 function counteracts morphine-induced hyperalgesia. Sci Rep. 2017 Jun 20;7(1):3870.

[2]. Chloride extrusion enhancers as novel therapeutics for neurological diseases. Nat Med. 2013 Nov;19(11):1524-8.

[3]. Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats. Diabetes. 2018 Mar;67(3):486-495.

- CLP-290 is a carbamate prodrug of the KCC2 activator CLP-257. It was designed to improve the pharmacokinetic profile of CLP-257, which has a short half-life (<15 min) due to rapid glucuronidation of its hydroxyl group. CLP-290 protects the hydroxyl group, resulting in a much longer half-life (~5 h) and improved oral bioavailability after metabolism to CLP-257. [2]
- CLP-257, the active metabolite, selectively activates KCC2 by increasing its plasma membrane expression. In vitro, CLP-257 (EC₅₀ = 616 nM) reduced intracellular Cl⁻ concentration in NG108-cl cells by approximately 40% (a 23 mM drop from resting [Cl⁻]ᵢ of 57 mM). It had no effect on NKCC1, KCC1, KCC3, or KCC4 in HEK293-cl cells or in Xenopus oocyte Rb⁺ flux assays. CLP-257 restored Cl⁻ transport in spinal cord slices with reduced KCC2 function (e.g., after BDNF treatment or peripheral nerve injury). [2]
- The KCC2 activator CLP-257 has been shown to reverse neuropathic pain, prevent morphine-induced hyperalgesia, and normalize GABAergic inhibition in diabetic rats. CLP-290 is the prodrug form used for oral administration in vivo. [1][2][3]

C19H21FN4O3S

404.4604

404.131

C, 56.42; H, 5.23; F, 4.70; N, 13.85; O, 11.87; S, 7.93

1181083-81-7

1181083-81-7

44188755

Off-white to yellow solid powder

1.5±0.1 g/cm3

553.7±60.0 °C at 760 mmHg

288.6±32.9 °C

0.0±1.5 mmHg at 25°C

1.691

2.89

1

6

4

28

680

0

C1CCN(NC1)C2=NC(=O)/C(=C/C3=C(C=C(C=C3)F)OC(=O)N4CCCC4)/S2

DIXMMXNNKLCLOM-WJDWOHSUSA-N

InChI=1S/C19H21FN4O3S/c20-14-6-5-13(15(12-14)27-19(26)23-8-3-4-9-23)11-16-17(25)22-18(28-16)24-10-2-1-7-21-24/h5-6,11-12,21H,1-4,7-10H2/b16-11-

[5-Fluoro-2-[(Z)-(2-hexahydropyridazin-1-yl-4-oxo-thiazol-5-ylidene)methyl]phenyl] pyrrolidine-1-carboxylate

CLP290; (Z)-5-Fluoro-2-((4-oxo-2-(tetrahydropyridazin-1(2H)-yl)thiazol-5(4H)-ylidene)methyl)phenyl pyrrolidine-1-carboxylate; [5-Fluoro-2-[(Z)-(2-hexahydropyridazin-1-yl-4-oxo-thiazol-5-ylidene)methyl]phenyl] pyrrolidine-1-carboxylate; CLP 290; CLP-290

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~33.33 mg/mL (~82.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.14 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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