probenecid

Probenecid is a potent and selective agonist of transient receptor potential vanilloid 2 (TRPV2) channels.

Signaling Pathways >> Membrane Transporter/Ion Channel >> TRP Channel

Research Areas >> Metabolic Disease

TRPV2[1]

Probenecid efficiently inhibits ATP-dependent active vesicular N-ethylmaleimide glutathione (NEM-GS) uptake by both MRP1 and MRP2. A significant inhibition of the MRP1-ATPase is observed at higher organic anion concentrations. In contrast, the ATPase activity of MRP2 is strongly stimulated by both Probenecid (approximate KACT=250 μM), sulfinpyrazone (KACT=300 μM), and indomethacin (KACT=150 μM), and ATPase activation is even stronger than in the case of NEM-GS. The organic anion activation of the MRP2-ATPase followed bell-shaped curves, with maximum values obtained at about 2 mM for Probenecid, 800 μM for sulfinpyrazone, and 400 μM for indomethacin[2]. Probenecid is an inhibitor of the hTAS2R16, hTAS2R38, and hTAS2R43 bitter taste receptors. Probenecid acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. Probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism[3].

Administration of Probenecid to WT mice results in increased contractility as measured via ejection fraction (EF) relative to EF in control mice given saline. The increased contractility is noted within 5 minutes of the bolus injection with all doses at or above 75 mg/kg (peak change of 5.26±3.35, 8.40±2.80, 7.32±2.52 for 75mg/kg, 100mg/kg and 200mg/kg, respectively). The measured change in contractility as measured at 5 minute intervals (for 30 minutes total) revealed a dose dependent increase in contractility with an estimated EC50 of 49.33 mg/kg. The EF remained at an elevated state for at least 1 hour on subjects (n=5, dose of 200 mg/kg IV) that are evaluated for a longer period of time (average increase in EF over baseline of 8.9±2.57)[1].

HEK-293T cells are transfected with hTAS2R expression constructs using Lipofectamine 2000 in poly-lysine coated, black 384-well plates with clear bottoms and incubated for 22 hours at 37°C. Growth media is removed and cells are washed twice with HBSS containing 20 mM HEPES, then loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit) with or without 1 mM Probenecid. Cells are incubated at 37°C for 1 hour in the presence of both dye and Probenecid, then moved to a Flexstation II-384 set for 32°C. After a 15-minute temperature equilibration (without washout), indicated compounds are injected (at t=~25 seconds) and fluorescence is measured for 100 to 180 seconds, reading every 3 seconds. Data sets are analyzed and represented as % over baseline signal using Prism 5.0 software. For Schild plots, replicates of raw calcium flux values are expressed as % over baseline signal. The mean value at 36 seconds (corresponding to the maximum flux signal) for each concentration of TAS2R ligand in the presence of the indicated concentration of Probenecid is plotted against the log of ligand concentration. Data points are fit using non-linear regression in GraphPad Prism[1].

Mice[1] In order to obtain a dose response curve, male C57 WT (n=39) mice 12-16 weeks of age are anesthetized with isoflurane while intravenous jugular access (IV) is obtained under a microscope. Subsequently, an echocardiographic study with both M-mode and B-mode is obtained in parasternal long axis (PSLAX) as described below. Either saline or different doses of Probenecid (increasing from 2 to 200mg/kg) are injected (bolus IV) for the initial contractility studies in WT mice.

[1]. Koch SE, et al. Probenecid: novel use as a non-injurious positive inotrope acting via cardiac TRPV2 stimulation. J Mol Cell Cardiol. 2012 Jul;53(1):134-44.

[2]. Bakos E, et al. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol. 2000 Apr;57(4):760-8.

[3]. Greene TA, et al. Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin. PLoS One. 2011;6(5):e20123.

capsaicin | Capsazepine | EIPA | Diphenyleneiodonium chloride | HC-030031 | SKF-96365 | HC 067047 | AMG 9810 | GSK-1016790A | SAR7334 | PF-4840154 | RN-1734 | AMG-517 | SB-366791 | IcilinAG-3-5

DATA ITEMS CITED :

23

MOLECULAR FORMULA :

C13-H19-N-O4-S

MOLECULAR WEIGHT :

285.39

WISWESSER LINE NOTATION :

QVR DSWN3&3

HEALTH HAZARD DATA

ACUTE TOXICITY DATA

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Human - man

DOSE/DURATION :

50 mg/kg/1W-I

TOXIC EFFECTS :

Blood - other hemolysis with or without anemia

TYPE OF TEST :

LD50 - Lethal dose, 50 percent kill

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - rat

DOSE/DURATION :

1600 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Intraperitoneal

SPECIES OBSERVED :

Rodent - rat

DOSE/DURATION :

394 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

REFERENCE :

CLDND* Compilation of LD50 Values of New Drugs. (J.R. MacDougal, Dept. of National Health and Welfare, Food and Drug Divisions, 35 John St., Ottawa, Ont., Canada)

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Subcutaneous

SPECIES OBSERVED :

Rodent - rat

DOSE/DURATION :

611 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

REFERENCE :

CLDND* Compilation of LD50 Values of New Drugs. (J.R. MacDougal, Dept. of National Health and Welfare, Food and Drug Divisions, 35 John St., Ottawa, Ont., Canada)

TYPE OF TEST :

LD50 - Lethal dose, 50 percent kill

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

1666 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Intraperitoneal

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

1 gm/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Subcutaneous

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

1156 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

REFERENCE :

CLDND* Compilation of LD50 Values of New Drugs. (J.R. MacDougal, Dept. of National Health and Welfare, Food and Drug Divisions, 35 John St., Ottawa, Ont., Canada)

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Intravenous

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

458 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

REFERENCE :

CLDND* Compilation of LD50 Values of New Drugs. (J.R. MacDougal, Dept. of National Health and Welfare, Food and Drug Divisions, 35 John St., Ottawa, Ont., Canada)

TYPE OF TEST :

LDLo - Lowest published lethal dose

ROUTE OF EXPOSURE :

Intravenous

SPECIES OBSERVED :

Mammal - dog

DOSE/DURATION :

230 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

REFERENCE :

CLDND* Compilation of LD50 Values of New Drugs. (J.R. MacDougal, Dept. of National Health and Welfare, Food and Drug Divisions, 35 John St., Ottawa, Ont., Canada)

TYPE OF TEST :

LD50 - Lethal dose, 50 percent kill

ROUTE OF EXPOSURE :

Intravenous

SPECIES OBSERVED :

Rodent - rabbit

DOSE/DURATION :

304 mg/kg

TOXIC EFFECTS :

Details of toxic effects not reported other than lethal dose value

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - rat

DOSE/DURATION :

9600 mg/kg/14D-C

TOXIC EFFECTS :

Nutritional and Gross Metabolic - weight loss or decreased weight gain

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - rat

DOSE/DURATION :

52 gm/kg/13W-I

TOXIC EFFECTS :

Sense Organs and Special Senses (Eye) - effect, not otherwise specified Liver - other changes Nutritional and Gross Metabolic - weight loss or decreased weight gain

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

38400 mg/kg/14D-C

TOXIC EFFECTS :

Related to Chronic Data - death

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

52 gm/kg/13W-I

TOXIC EFFECTS :

Nutritional and Gross Metabolic - weight loss or decreased weight gain Related to Chronic Data - death

TYPE OF TEST :

TDLo - Lowest published toxic dose

ROUTE OF EXPOSURE :

Oral

SPECIES OBSERVED :

Rodent - mouse

DOSE/DURATION :

206 gm/kg/2Y-C

TOXIC EFFECTS :

Tumorigenic - neoplastic by RTECS criteria Liver - tumors

MUTATION DATA

TYPE OF TEST :

Sister chromatid exchange

TEST SYSTEM :

Rodent - hamster Ovary

DOSE/DURATION :

5 mg/L

REFERENCE :

NTPTR* National Toxicology Program Technical Report Series. (Research Triangle Park, NC 27709) No.206- Volume(issue)/page/year: NTP-TR-395,1991 *** NIOSH STANDARDS DEVELOPMENT AND SURVEILLANCE DATA *** NIOSH OCCUPATIONAL EXPOSURE SURVEY DATA : NOES - National Occupational Exposure Survey (1983) NOES Hazard Code - X4171 No. of Facilities: 66 (estimated) No. of Industries: 2 No. of Occupations: 9 No. of Employees: 4059 (estimated) No. of Female Employees: 1675 (estimated)