OAT1/OAT3; Pannexin-1 (Panx1) ; MRP1; transient receptor potential vanilloid 2 (TRPV2)

Probenecid potently inhibits the absorption of ATP-dependent active vesicles N-ethylmaleimide glutathione (NEM-GS) by MRP1 and MRP2. Significant inhibition of MRP1-ATPase was seen at increasing organic anion concentrations. The ATPase activity of MRP2 is influenced by probenecid (about KACT=250 μM), sulfinpyrazone (KACT=300 μM) and indomethacin (KACT=150 μM), and the ATPase activation is even stronger than that of NEM-GS. Activation of MRP2-ATPase by organic anions follows a bell-shaped curve, with maximum values at 2 mM for Probenecid, 800 μM for sulfinpyrazone, and 400 μM for indomethacin [2]. Probenecid is an inhibitor of hTAS2R16, hTAS2R38 and hTAS2R43 bitter taste receptors. Probenecid operates on a subset of TAS2R and suppresses through a new allosteric mechanism of action. Probenecid is also often utilized to improve cell signaling in GPCR calcium mobilization experiments. Probenecid specifically suppresses cellular responses mediated by the bitter taste receptor hTAS2R16 and provides molecular and pharmacological evidence for direct contact with this GPCR using a non-competitive (allosteric) mechanism [3].

When compared to control mice fed saline, probenecid increased the contractility of WT mice as indicated by their ejection fraction (EF). At all doses of 75 mg/kg and higher, increased contractility was observed within 5 minutes of the bolus (peak changes at 75 mg/kg, 100 mg/kg, and 200 mg/kg were 5.26±3.35, 8.40±2.80, and 7.32± 2.52, respectively). With an estimated EC50 of 49.33 mg/kg, changes in contractility assessed at 5-minute intervals for a total of 30 minutes showed a dose-dependent increase in contractility. EF stayed raised for at least an hour in patients examined over a longer duration (n=5 at 200 mg/kg IV) (mean increase in EF from baseline 8.9±2.57) [1].

HEK-293T cells were plated in poly-lysine-coated, black-walled, clear-bottom 384-well plates and transfected with hTAS2R expression constructs using Lipofectamine 2000, followed by a 22-hour incubation at 37°C. After transfection, the growth medium was aspirated, and the cells were washed twice with HBSS buffer containing 20 mM HEPES. The cells were then loaded with a calcium indicator dye (from the Calcium 4 Assay kit) prepared in the same HBSS/HEPES buffer, either in the presence or absence of 1 mM Probenecid. Dye loading was performed during a 1-hour incubation at 37°C. Subsequently, the plate was transferred to a Flexstation II-384 microplate reader pre-equilibrated to 32°C. Following a 15-minute temperature equilibration period (without removing the dye solution), the test compounds were automatically injected at approximately 25 seconds. Fluorescence was monitored immediately after injection for a duration of 100 to 180 seconds, with readings taken at 3-second intervals. For data analysis, fluorescence signals were processed using Prism 5.0 software (GraphPad) and are presented as the percentage change relative to the baseline signal (% over baseline). Specifically for the construction of Schild plots, replicate measurements of raw calcium flux were first converted to this percentage format. The mean response value at the 36-second time point—which corresponds to the peak calcium flux signal—for each concentration of TAS2R ligand, tested alongside a given concentration of Probenecid, was plotted against the logarithm of the corresponding ligand concentration. The resulting data points were fitted using a non-linear regression model in GraphPad Prism.

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.

Absorption, Distribution and Excretion
Probenefit is primarily excreted in the urine as monoacyl glucuronide and unchanged drug. Alkalinization of the urine increases renal excretion of probenecid. Probenecid is completely absorbed after oral administration. Peak plasma concentrations are reached within 2–4 hours. The plasma half-life of the drug is dose-dependent, ranging from less than 5 hours to more than 8 hours. 85% to 95% of the drug is bound to plasma albumin, with the majority bound to albumin. A small amount of free drug enters the glomerular filtrate; much larger amounts are actively secreted by the proximal tubules. Despite its low PKA value (3.4), the high lipid solubility of the undissociated form results in almost complete absorption via retrodiffusion unless the urine is significantly alkaline. Small amounts of probenecid glucuronide may be present in the urine. ... /Organic acid drugs (such as probenecid)/are not easily absorbed/by parenchymal tissues or reticuloendothelial tissues/, and have high plasma concentrations… For more complete data on the absorption, distribution, and excretion of probenecid (8 metabolites), please visit the HSDB record page. Metabolites/Metabolites: p-Dipropylsulfonamidobenzoyl-β-D-glucuronic acid; p-(2-hydroxypropylN-propylsulfonamido)benzoic acid; p-(3-hydroxypropylN-propylsulfonamido)benzoic acid; and p-propylsulfonamidobenzoic acid in humans. /Excerpt from tables/ The structures of all probenecid metabolites in rat bile and human urine have been elucidated. Propionic acid has been identified as another metabolite of probenecid. The main metabolic pathways involve side-chain oxidation and glucuronide conjugation… β-glucuronides of the 2- and 3-hydroxylated metabolites, as well as acyl glucuronides of probenecid itself, have been identified. Significant differences exist in metabolism across species. In mice and monkeys, oxidation is dominant. ...In dogs, the binding pathway is the primary pathway, while in humans, the oxidation pathway is just as important as the glucuronidation pathway. Long-term drug use not only stimulates the metabolism of other metabolites, but in some cases, the pharmacological or toxic effects of a drug may be weakened by long-term use because it stimulates its own metabolism. For example, a drug that produces this effect in dogs is... probenecid. For more complete metabolite/metabolite data on probenecid (6 metabolites), please visit the HSDB record page.

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Biological Half-Life
6-12 hours
The plasma half-life of probenecid is dose-related, ranging from less than 5 hours to more than 8 hours...
After oral administration of 2 grams of probenecid, the plasma half-life of the drug is 4-17 hours; as the dose decreases from 2 grams to 500 mg, the half-life also decreases.

Hepatotoxicity
Currently, there are no reports on the frequency of liver function abnormalities during probenecid treatment. However, since the drug is primarily excreted unchanged in the urine, liver function abnormalities are likely rare. A case of severe hypersensitivity to probenecid was reported over 50 years ago, with rapid and severe recurrence of jaundice after re-administration. Similar to typical hypersensitivity reactions, this case developed within days of starting probenecid administration, accompanied by fever and rash. Probability score: D (likely a rare cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of use during lactation Limited information suggests that low concentrations of probenecid in breast milk, even when the mother takes no more than 2 grams daily, are not expected to have any adverse effects on breastfed infants, especially those older than 2 months. Animal studies have shown that probenecid can increase the excretion of cimetidine in breast milk, possibly through interaction with active transport mechanisms in the mammary glands. The effects of probenecid enhancing drug excretion on breastfeeding mothers and their infants have not been studied; however, only a few drugs are known to enter breast milk via active transport.
◉ Effects on breastfed infants
A woman with mastitis received 3 days of intravenous cefotaxime treatment, followed by 16 days of oral probenecid 500 mg and cephalexin 500 mg four times daily. Her infant developed green, loose stools, severe diarrhea, discomfort, and crying. The authors believe these effects are likely related to cefotaxime and cephalexin in breast milk, rather than probenecid.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
75-95%

[1]. Cell Calcium.1989 Apr;10(3):171-80;
[2]. Cardiovasc Toxicol.2012 Mar;12(1):1-9.

Probenecid appears is an odorless white or off-white crystalline powder. It has a slightly bitter taste with a pleasant aftertaste. (NTP, 1992)
Probenecid is a sulfonamide drug in which the nitrogen atom of 4-aminosulfonylbenzoic acid is replaced by two propyl groups. It is a uricosuric drug. It belongs to the sulfonamide class and is also a benzoic acid class of drugs.
It is a typical uricosuric drug. It inhibits the excretion of organic anions by the kidneys and reduces the reabsorption of urate by the renal tubules. Probenecid has also been used to treat patients with renal insufficiency and, because it reduces the renal tubular excretion of other drugs, it is also used as an adjunct to antibacterial therapy.
Probenecid is a uricosuric drug and is often used in combination with other drugs to treat gout. Probenecid is associated with mild elevation of serum transaminases, and in rare cases, it can cause hypersensitivity reactions, and even rarer cases, hypersensitivity reactions may be accompanied by acute liver injury.
Probenecid is a benzoic acid derivative with uric acid-lowering effects. Probenecid competitively inhibits the active reabsorption of uric acid in the proximal tubules of the kidneys, thereby increasing uric acid excretion and lowering serum uric acid concentration. This prevents uric acid deposition and promotes the regression of existing uric acid deposits. Furthermore, probenecid can regulate the transport of organic acids and acidic drugs in the proximal and distal tubules, thereby increasing serum drug concentrations. It is a typical uricosuric agent. It inhibits the renal excretion of organic anions and reduces the reabsorption of uric acid in the renal tubules. Probenecid has also been used to treat patients with renal insufficiency and, because it reduces the renal tubular excretion of other drugs, it is also used as an adjunct to antibacterial therapy. See also: colchicine; probenecid (ingredient); ampicillin/ampicillin trihydrate; probenecid (ingredient). Indications: For reducing serum uric acid concentrations in patients with chronic gouty arthritis and tophaceous gout, especially those with frequent and disabling gout attacks. It can also effectively promote uric acid excretion in patients with secondary hyperuricemia caused by thiazide diuretics and related diuretics.

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Mechanism of Action
Probenecid inhibits renal tubular reabsorption of uric acid, thereby increasing uric acid excretion and lowering serum uric acid levels. At subtherapeutic concentrations, probenecid may also reduce plasma urate binding and inhibit renal secretion of uric acid. The mechanism by which probenecid inhibits renal tubular transport is not fully understood, but the drug may inhibit transport enzymes requiring high-energy phosphate bonds and/or nonspecifically interfere with substrate binding to renal tubular protein receptors.
When the dose of probenecid is higher than that required to produce a uricosuric effect, it also inhibits organic acid transport in other sites, such as the transport system that clears organic acids from cerebrospinal fluid.
It inhibits renal tubular reabsorption of urate, thereby increasing uric acid excretion and lowering serum uric acid levels.
Probenecid is a renal tubular blocker. This drug competitively inhibits the active reabsorption of uric acid in the proximal tubules, thereby promoting urinary excretion of uric acid and reducing serum uric acid concentration. Probenecid reduces the plasma protein binding rate of uric acid and inhibits renal secretion of uric acid at subtherapeutic doses. In healthy individuals, probenecid has no effect on glomerular filtration rate or renal tubular reabsorption of normal urinary components (such as glucose, arginine, urea, sodium, potassium, chloride, or phosphate). In the proximal and distal tubules, probenecid competitively inhibits the secretion of a variety of weak organic acids, including penicillin, most cephalosporins, and some other β-lactam antibiotics. Generally, the net effect of probenecid on the plasma concentration of weak acids depends on the ratio of the amount of organic acids secreted by the kidneys to the amount of organic acids filtered by the glomerulus. Therefore, probenecid can significantly increase the plasma concentration of acidic drugs that are primarily excreted through renal secretion, while for drugs that are primarily excreted through filtration, the plasma concentration is only slightly increased. Probenecid typically more than doubles the plasma concentration of penicillin; it also increases the concentration of penicillin in cerebrospinal fluid (CSF). Probenecid can also significantly increase the plasma concentrations of most cephalosporins and some other β-lactam antibiotics. Furthermore, probenecid can prolong the half-life of penicillins and cephalosporins and may reduce their volume of distribution. …The cellular mechanism by which probenecid inhibits renal tubular transport is unclear. The drug may inhibit transport enzymes that require high-energy phosphate bonds and/or nonspecifically interfere with substrate binding to renal tubular protein receptor sites. Following probenecid administration, the concentrations of 5-hydroxyindoleacetic acid, homovanillic acid, cyclic adenosine monophosphate (cAMP), and 4-hydroxy-3-methoxyphenylethylene glycol (4-HMA) in CSF increase. Some studies suggest that probenecid blocks the active transport of these organic acids from CSF to blood. The probenecid-induced increase in homovanillic acid (a dopamine metabolite) in the cerebrospinal fluid of patients with Parkinson's syndrome and 5-hydroxyindoleacetic acid (a serotonin metabolite) in the cerebrospinal fluid of patients with depression was significantly lower than that in healthy patients.

C13H19NO4S

285.36

285.103

C, 54.72; H, 6.71; N, 4.91; O, 22.43; S, 11.24

57-66-9

Probenecid;57-66-9

4911

White to off-white solid powder

1.2±0.1 g/cm3

438.0±47.0 °C at 760 mmHg

194-196°C

218.7±29.3 °C

0.0±1.1 mmHg at 25°C

1.542

3.3

1

5

7

19

374

0

CCCN(CCC)S(=O)(=O)C1=CC=C(C=C1)C(=O)O

DBABZHXKTCFAPX-UHFFFAOYSA-N

InChI=1S/C13H19NO4S/c1-3-9-14(10-4-2)19(17,18)12-7-5-11(6-8-12)13(15)16/h5-8H,3-4,9-10H2,1-2H3,(H,15,16)

4-(dipropylsulfamoyl)benzoic acid

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)

Solubility in Formulation 1: 2.5 mg/mL (8.76 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (8.76 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 25.0 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.)