Diuretic; anti-hypertensive

Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)-approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, researchers screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of hPXR. The data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in hPXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates hPXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates hPXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy.[3]

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Under the brand name Zytanix, Zydus Cadila, Zaroxolyn, and Mykrox distribute the thiazide diuretic metolazone (SR-720-22). It is mainly used to treat hypertension and congestive heart failure. Metolazone causes a decrease in blood volume and an increase in urine production by indirectly lowering the quantity of water that the kidneys reabsorb into the blood. As a result, blood pressure is lowered and excessive fluid accumulation in heart failure is avoided. Metolazone is occasionally used with highly effective loop diuretics like bumetanide or furosemide, however doing so might cause electrolyte imbalances and dehydration. The sodium-chloride symporter is inhibited by metolazone and other thiazide diuretics, which keeps water and salt from entering the renal tubular cells from the lumen. As a result, instead of being reabsorbed into the bloodstream, water stays in the lumen and is expelled as urine. When the filtrate reaches the distal convoluted tubule, the majority of the sodium in the lumen has already been reabsorbed, therefore thiazide diuretics have minimal effects on water balance and electrolyte levels. On the other hand, negative consequences like hypovolemia, hypotension, and low sodium levels might be linked to them.

Preeclampsia is a disorder that continues to exact a significant toll with respect to maternal morbidity and mortality as well as fetal wastage. Furthermore, the treatment of this disorder has not changed significantly in 50 years and is unsatisfactory. The use of diuretics in this syndrome is controversial because there is a concern related to potential baleful effects of volume contraction leading to a possible further decrement in the perfusion of the maternal-fetal unit. Metolazone is a diuretic/antihypertensive agent, which has a therapeutic effect on blood pressure (BP) in human essential hypertension without causing a natriuresis. We administered the drug in nondiuretic doses in a rat model of preeclampsia previously developed in this laboratory. The drug reduced BP without an accompanying natriuresis. Although there was a trend toward an improvement in intrauterine growth restriction, as determined by litter size and the number of pups demonstrating malformations, the values did not reach statistical significance. We conclude that metolazone, in low dosage, is an effective antihypertensive in this rat model. These studies have implications for the treatment of the human disorder [4].

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) assays [3]
Total RNA was isolated from LS180 cells, LS174T cells, and human primary hepatocytes by using Maxwell 16 LEV simplyRNA purification kits. Then, qRT-PCR was performed by using Taqman gene expression assays specific for CYP3A4, MDR1, and β-actin (ACTB), which was used as the reference gene according to the manufacturer’s protocol in an ABI 7900HT system. The comparative Ct method was used for relative quantification for gene expression with the following formula: ΔCt = Ct (test gene) — Ct (ACTB); ΔΔCt (test gene) = ΔCt (test gene in treatment group) — ΔCt (test gene in vehicle or siPXR control group); the fold changes of mRNA = 2−ΔΔCt, which indicated the relative mRNA level of the corresponding transcript to that of the control samples. DMSO (solvent control) or siPXR and rifampicin were used as negative and positive control, respectively.

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Small interfering RNA transfection [3]
We knocked down hPXR expression by transiently transfecting small interfering RNA (siRNA) into cells by using the previously described protocol. Briefly, LS180 cells stably expressing hPXR and CYP3A4-luc were cultured in 6-well plates and treated with a final concentration of 25 nM ON-Targetplus SMARTpool SiRNA targeting PXR or Nontargeting Pool by using Lipofectamine RNAiMAX. After 72 h, the cells were washed, treated with indicated compounds for 48 h and then collected for qRT-PCR analysis.

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Competitive ligand-binding assay A LanthaScreen TR-FRET PXR competitive binding assay was conducted according to the manufacturer’s protocol as described previously. Briefly, assays were performed in a volume of 20 μL in 384-well solid black plates with 5 nM GST-hPXR ligand-binding domain, 40 nM fluorescent-labeled hPXR agonist, 5 nM terbium-labeled anti-GST antibody, and test compound at different concentrations. The reactions were incubated at 25°C for 60 min before the fluorescent emission of each well at 495 and 520 nm was measured by using a 340-nm excitation filter, 100-μs delay time, and 200-μs integration time on a PHERAStar plate reader (BMG Labtech, Durham, NC). The curve-fitting software GraphPad Prism 4.0 was used to generate the plot.

Small-molecule screening in HEK293T cell–based luciferase assay [3]
HEK293T cells (5000 cells/well; 25 μL/well) transiently transfected with FLAG-hPXR and CYP3A4-luciferase (CYP3A4-luc) were grown in 384-well, tissue culture–treated solid white plates for 24 h prior to treatment with the drug compounds to be tested (70 nl; 10 mM in DMSO). Pintools were used to add compound to each well (final compound concentration: 28 μM; final DMSO concentration: 0.28%), and the plates were incubated for 24 h before the Dual-Glo luciferase assay was performed and read on a EnVision microplate reader.

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Transient transfection and luciferase reporter gene assays [3]
The methods for these procedures were described previously. Briefly, the cells were transfected with Flag-hPXR, CMV-Renilla, and CYP3A4-luc plasmids by using FuGENE 6 (Roche Diagnostics). After 24 h, cells were seeded in 384-well plates (5000 cells/well) in phenol red–free medium containing 5% charcoal/dextran-treated FBS and incubated for another 24 h before treatment with compounds to be tested. Compounds were transferred by using pintools and incubated with the cells for 24 h before Dual-Glo Luciferase Assays were performed. Renilla luciferase activity was used to normalize the firefly luciferase activity. CYP3A4 promoter activity (percentage of activation; a.u.) was determined as described previously. Rifampicin (7 μM) and DMSO were used as positive (100%, or a.u. = 100) and negative (0%, or a.u. = 0) controls, respectively in the dose-responsive evaluation of compound activity. Curve-fitting software was used to generate the curves and to determine the EC50.

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Cell viability assay [3]
HepG2, LS180, and LS174T cells were transiently transfected with FLAG-hPXR and treated with compounds as described in Section 2.4 before the CellTiter-Glo assay was used to measure cell viability. Briefly, CellTiter-Glo reagent was added to the wells and incubated at room temperature for 10 min in the dark. Luminescence was measured and recorded by using an Envision plate reader. DMSO was used as a negative control for cell viability. Values of the viability of compound-treated cells were expressed as a percentage of that of DMSO-treated cells.

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Mammalian two-hybrid assay [3]
The CheckMate mammalian two-hybrid system (Promega) was performed as described previously and consists of VP16-hPXR, Gal4-SRC-1, and a luciferase reporter (pG5-luc) co-transfected into HepG2 cells. The Gal4 vector (pBIND) also constitutively expresses Renilla luciferase, which was used as an internal transfection control. The Dual-Glo Luciferase Assay was used to measure luciferase activity as an indicator of protein–protein interactions. The relative luciferase activity for pG5-luc was determined by normalizing firefly luciferase activity with Renilla luciferase activity.

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Western blot analysis [3]
All cell extracts were harvested in 1X RIPA buffer, and samples were centrifuged at 12,000g at 4°C for 25 min. The samples were then boiled in sample loading buffer containing SDS, and equal amounts of samples were resolved on a 4–12% SDS-PAGE gradient gel and then transferred onto a nitrocellulose membrane. Unbound sites on the membrane were blocked, and the membrane was incubated with the indicated antibodies overnight at 4°C. We used anti-CYP3A4, anti-MDR1, anti-PXR, anti-FLAG M2 (1:1000 dilutions) and anti-β-actin (1:5000 dilution) antibodies to detect CYP3A4, MDR1, PXR, FLAG-PXR and β-actin, respectively. All Western blot analyses were performed on the Odyssey Infrared Imaging system. The intensity of each protein band was quantified using ImageJ 1.48 software. The intensity of each protein band was normalized to that of actin to generate the relative intensity, with the relative intensity of the DMSO treated sample set as “1”.

Absorption, Distribution and Excretion
Peak blood levels are obtained within 2 to 4 hours of oral administration. The rate and extent of absorption are formulation dependent.
Most of the drug is excreted in the unconverted form in the urine.
75% OF DOSE /ADMIN TO DOGS/ ...EXCRETED IN URINE & 25% IN BILE. 60%...AS HYDROXYLATED DERIV...& FURTHER PRODUCTS OF OXIDN...& UNIDENTIFIED POLAR METABOLITES ARE ALSO PRESENT IN URINE.
RENAL CLEARANCES OF.../THIAZIDE DIURETICS/ ARE...HIGH. MOST COMPOUNDS ARE RAPIDLY EXCRETED WITHIN 3-6 HR. BENDROFLUMETHIAZIDE & POLYTHIAZIDE HAVE LONGER DURATION OF ACTION THAT IS CORRELATED WITH SLOWER EXCRETION... SAME IS TRUE OF...METOLAZONE.

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Metabolism / Metabolites
Not substantially metabolized. 70-95% is excreted unchanged in urine via glomerular filtration and active tubular secretion. Undergoes enterohepatic recycling.
SEVENTY-FIVE PER CENT OF DOSE /ADMIN TO DOGS/ IS EXCRETED IN URINE & 25% IN BILE. 60%...AS HYDROXYLATED DERIV...& FURTHER PRODUCTS OF OXIDN...& UNIDENTIFIED POLAR METABOLITES ARE ALSO PRESENT IN URINE.

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Biological Half-Life
Approximately 14 hours.
LONG T/2 VALUE...IN DOGS IS RELATED TO TRANSPORT MECHANISM IN BLOOD & PARTITION BETWEEN FORMED ELEMENTS & SERUM PROTEINS.
...METOLAZONE IS EXCRETED BY GLOMERULAR FILTRATION & TUBULAR SECRETION IN DOGS, ALTHOUGH DRUG:CREATININE CLEARANCE RATIO IS LOWER THAN FOR MOST OTHER DIURETICS. T/2 IN DOGS OF 5-6 HR IS INFLUENCED BY EXTENSIVE BINDING TO ERYTHROCYTES & PLASMA PROTEINS, & POSSIBLY TO TISSUE PROTEINS.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the amount of metolazone in breastmilk. Intense diuresis with large doses may decrease breastmilk production. Other diuretics in low doses are preferred over metolazone.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information on metolazone was not found as of the revision date. Intense diuresis with thiazides and thiazide-like diuretics, fluid restriction and breast binding have been used to suppress postpartum lactation. The added contribution of the diuretic to these measures, which are effective in suppressing lactation, has not been studied. There are no data on the effects of diuretics on established, ongoing lactation.

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Protein Binding
50-70% bound to erythrocytes, up to 33% bound to plasma proteins, 2-5% of the drug in circulation is unbound

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Interactions
...METOLAZONE...CHEMICALLY & PHARMACOLOGICALLY RELATED TO THIAZIDE DIURETICS & ELEVATE SERUM URATE LEVELS. ...MAY...INTERACT WITH PROBENECID.
USE OF THIAZIDE DIURETIC MAY IMPAIR CONTROL OF DIABETES IN PT TREATED...WITH ORAL HYPOGLYCEMIC AGENTS OR INSULIN, BUT ONLY TO LIMITED EXTENT. /THIAZIDE DIURETICS/
METOLAZONE...STRUCTURALLY RELATED TO THIAZIDE DIURETICS, CAN CAUSE HYPOKALEMIA, & MAY BE EXPECTED TO INTERACT WITH CARDIAC GLYCOSIDES.
THIAZIDES ENHANCE ANTIHYPERTENSIVE ACTION OF GUANETHIDINE... /THIAZIDE DIURETICS/
For more Interactions (Complete) data for METOLAZONE (6 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 MICE ORAL MORE THAN 5000 MG/KG
LD50 MICE INTRAPERITONEAL MORE THAN 1500 MG/KG

[1]. Metolazone, a diuretic agent. Am Heart J. 1976 Feb;91(2):262-3.

[2]. A new antihypertensive: metolazone treatment of hypertension. J Kans Med Soc. 1977 Jul;78(7):337-9, 342.

[3]. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1. Biochem Pharmacol. 2014 Nov 15;92(2):389-402.

[4]. Beneficial effects of metolazone in a rat model of preeclampsia. J Pharmacol Exp Ther. 2006 Sep;318(3):1027-32.

Metolazone is a quinazoline that consists of 1,2,3,4-tetrahydroquinazolin-4-one bearing additional methyl, 2-tolyl, sulfamyl and chloro substituents at positions 2, 3, 6 and 7 respectively. A quinazoline diuretic, with properties similar to thiazide diuretics. It has a role as a diuretic, an antihypertensive agent and an ion transport inhibitor. It is a member of quinazolines, an organochlorine compound and a sulfonamide.
A quinazoline-sulfonamide that is considered a thiazide-like diuretic which is long-acting so useful in chronic renal failure. It also tends to lower blood pressure and increase potassium loss.
Metolazone is a Thiazide-like Diuretic. The physiologic effect of metolazone is by means of Increased Diuresis.
Metolazone is a long acting, quinazoline-based sulfonamide thiazide-like, diuretic. Similar to the thiazides, metolazone acts on the distal convoluted tubule (DCT) and inhibits the sodium-chloride symporter, thereby preventing sodium and chloride reabsorption and excretion of water.
A quinazoline-sulfonamide derived DIURETIC that functions by inhibiting SODIUM CHLORIDE SYMPORTERS.

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Drug Indication
For the treatment of hypertension, alone or in combination with other antihypertensive drugs of a different class.
FDA Label

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Mechanism of Action
The actions of metolazone result from interference with the renal tubular mechanism of electrolyte reabsorption. Metolazone acts primarily to inhibit sodium reabsorption at the cortical diluting site and to a lesser extent in the proximal convoluted tubule. Sodium and chloride ions are excreted in approximately equivalent amounts. The increased delivery of sodium to the distal tubular exchange site results in increased potassium excretion. Metolazone does not inhibit carbonic anhydrase. The antihypertensive mechanism of action of metolazone is not fully understood but is presumed to be related to its saluretic and diuretic properties.
IT ACTS PRIMARILY TO INHIBIT SODIUM REABSORPTION AT CORTICAL DILUTING SITE & IN PROXIMAL CONVOLUTED TUBULE. SODIUM & CHLORIDE IONS ARE EXCRETED IN APPROX EQUAL AMT; INCR POTASSIUM EXCRETION MAY ALSO OCCUR. DIURETIC POTENCY APPROXIMATES THAT OF THIAZIDES.

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Therapeutic Uses
Antihypertensive Agents; Diuretics, Sulfamyl
DIURETIC & ANTIHYPERTENSIVE DRUG. ... METOLAZONE IS INDICATED FOR EDEMA ACCOMPANYING CONGESTIVE HEART FAILURE, RENAL DISEASE INCL NEPHROTIC SYNDROME, & OTHER CONDITIONS OF DIMINISHED RENAL FUNCTION.
METOLAZONE MAY BE USEFUL IN PT WITH GLOMERULAR FILTRATION RATE LESS THAN 20 ML/MIN & IN UNRESPONSIVE PT, IF TREATED CONCURRENTLY WITH FUROSEMIDE.
RANGE OF OPTIMALLY EFFECTIVE ORAL DIURETIC DOSE IN MAN: 25-100 MG/DAY; RELATIVE ORAL NATRIURETIC MAXIMAL RESPONSE IN MAN: 1.8. /FROM TABLE/

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Drug Warnings
IT IS CONTRAINDICATED IN ANURIA, HEPATIC COMA, KNOWN ALLERGY OR HYPERSENSITIVITY, PREGNANCY, & NURSING MOTHERS. ... MORE EXTENSIVE CLINICAL EXPERIENCE IS REQUIRED BEFORE DEFINITIVE EVALUATION CAN BE MADE.

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Pharmacodynamics
Metolazone is a quinazoline diuretic, with properties generally similar to the thiazide diuretics. A proximal action of metolazone has been shown in humans by increased excretion of phosphate and magnesium ions and by a markedly increased fractional excretion of sodium in patients with severely compromised glomerular filtration. This action has been demonstrated in animals by micropuncture studies.

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In summary, we identified the diuretic drug MET (metolazone) that induces mRNA and protein expression of CYP3A4 and MDR1 by activating PXR and may also recruit the PXR coactivator SRC-1. We also identified different functional groups of MET that are important for PXR activation. These results will help our understanding of MET’s role in drug metabolism. The discovery of MET as an activator of hPXR suggests that it should be used cautiously in patients undergoing combination therapy. [4]

C16H16CLN3O3S

365.83

365.06

C, 52.53; H, 4.41; Cl, 9.69; N, 11.49; O, 13.12; S, 8.76

17560-51-9

Metolazone-d7;2714484-71-4; 50869-23-3 (sodium); 17560-51-9

4170

White to off-white solid powder

1.4±0.1 g/cm3

613.6±65.0 °C at 760 mmHg

252-254°C

324.9±34.3 °C

0.0±1.8 mmHg at 25°C

1.629

1.57

2

5

2

24

594

0

O=S(C1=CC2=C(NC(C)N(C3=CC=CC=C3C)C2=O)C=C1Cl)(N)=O

AQCHWTWZEMGIFD-UHFFFAOYSA-N

InChI=1S/C16H16ClN3O3S/c1-9-5-3-4-6-14(9)20-10(2)19-13-8-12(17)15(24(18,22)23)7-11(13)16(20)21/h3-8,10,19H,1-2H3,(H2,18,22,23)

7-chloro-2-methyl-3-(2-methylphenyl)-4-oxo-1,2-dihydroquinazoline-6-sulfonamide

SR 720-22; SR-720-22; 17560-51-9; Zaroxolyn; Mykrox; Diulo; Oldren; Metenix; Metalozone; trade names: Zytanix, Zaroxolyn, Mykrox, Diulo, Oldren; SR720-22;

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: ≥ 2.75 mg/mL (7.52 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 27.5 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: ≥ 2.75 mg/mL (7.52 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 27.5 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: ≥ 2.75 mg/mL (7.52 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 27.5 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.)