L-type Voltage-Gated Calcium Channels (Caᵥ1.2) and MRP1 (Multidrug Resistance-Associated Protein 1). (S)-Verapamil HCl blocks L-type calcium channels in cardiac and vascular smooth muscle cells, inhibiting calcium influx and leading to vasodilation and reduced cardiac contractility. It is also a potent inhibitor of MRP1, an ATP-binding cassette (ABC) transporter that effluxes chemotherapeutic drugs (e.g., doxorubicin, vincristine, etoposide) from cancer cells. By inhibiting MRP1, (S)-Verapamil reverses multidrug resistance (MDR) and sensitizes resistant tumor cells to chemotherapy.

LTC4; Calcium channel; Ca2+

(S)-Verapamil hydrochloride (S(-)-Verapamil hydrochloride), but not (R)-Verapamil hydrochloride, effectively kills MRP1-transfected BHK-21 cells[1]. (S)-Verapamil hydrochloride is an excellent active form with low bioavailability[1].

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(S)-Verapamil blocks L-type calcium channels with high potency, with the S-enantiomer being more potent than the R-enantiomer. It is an inhibitor of leukotriene C4 (LTC4) and calcein transport by MRP1, leading to the death of potentially resistant tumor cells. By inhibiting MRP1, (S)-Verapamil increases the intracellular accumulation of chemotherapeutic agents, thereby overcoming drug resistance in cancer cells. It causes growth inhibition and apoptosis in MRP1-overexpressing tumor cells.

St John's wort did not affect the jejunal permeability or the fraction absorbed of either R- or S-verapamil. The values for area under the plasma concentration-time curve (AUC) for R- and S-verapamil decreased by 78% and 80%, respectively (P <.0001). The corresponding decreases in the maximum concentration were 76% and 78%, respectively (P <.0001), whereas the terminal half-life did not change significantly for any of the enantiomers. The AUC for R-verapamil was 6 times higher than that for S-verapamil in the control phase, and St John's wort did not change this ratio. The AUC values for R- and S-norverapamil decreased by 51% (P <.01) and 63% (P <.0001), respectively.[2]
Repeated administration of St John's wort significantly decreased the bioavailability of R- and S-verapamil. This effect is caused by induction of first-pass CYP3A4 metabolism, most likely in the gut, because the jejunal permeability and the terminal half-life were unchanged for both enantiomers.[2]

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Not applicable (research compound, not a standard therapeutic). (S)-Verapamil is the active enantiomer for calcium channel blockade and is present in the racemic verapamil used clinically for hypertension, angina, and arrhythmias. In animal models, verapamil (racemic) lowers blood pressure and heart rate. In xenograft models of MRP1-overexpressing tumors, (S)-Verapamil in combination with chemotherapeutic agents (e.g., doxorubicin, vincristine) significantly enhances tumor growth inhibition compared to chemotherapy alone.

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C(4) and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy[1].

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MRP1 inhibition is measured using a vesicular transport assay or a cell-based calcein accumulation assay. For the cell-free vesicular transport assay, membrane vesicles prepared from MRP1-overexpressing cells (5-10 microg protein) are incubated with varying concentrations of (S)-Verapamil HCl (0.1-100 microM) and [3H]LTC4 (leukotriene C4, 10-100 nM) in transport buffer (50 mM Tris-HCl pH 7.4, 250 mM sucrose, 10 mM MgCl2, 2 mM ATP). After 10-20 minutes at 37degC, vesicles are collected by filtration through nitrocellulose filters, and retained radioactivity is measured by liquid scintillation. The IC50 for LTC4 transport inhibition is calculated.

MRP1-overexpressing cancer cells (e.g., H69AR small cell lung cancer cells or CEM-VLB lymphoblasts) are seeded in 96-well plates (5,000-10,000 cells/well) in RPMI-1640 with 10% FBS. Cells are pre-treated with varying concentrations of (S)-Verapamil HCl (0.1-100 microM) for 30-60 minutes, then co-treated with a chemotherapeutic agent (e.g., doxorubicin, 0.1-10 microM; or vincristine, 0.01-1 microM) for 48-72 hours. Cell viability is measured by MTT or CellTiter-Glo assay. The IC50 of the chemotherapeutic agent is calculated in the absence and presence of (S)-Verapamil HCl. A leftward shift in the IC50 indicates MRP1 inhibition and reversal of drug resistance. For calcein accumulation, cells are incubated with calcein AM (0.1-0.5 microM) in the presence or absence of (S)-Verapamil (10-100 microM) for 30-60 minutes. Fluorescence (excitation 485 nm, emission 535 nm) is measured.

Jejunal single-pass perfusion experiments with 120-mg/L (244 micromol/L) R-/S-verapamil were performed in 8 healthy male volunteers for 100 minutes before and after 14 days of oral treatment with St John's wort (300 mg 3 times a day). The enantiomers of verapamil and the cytochrome P450 (CYP) 3A4-formed metabolite norverapamil in perfusate and plasma were quantified by chiral HPLC with fluorescence and tandem mass spectrometry detection, respectively[2].

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Female athymic nude mice (6-8 weeks old) bearing subcutaneous xenografts of MRP1-overexpressing human tumors (e.g., H69AR or NCI-H460/MRP1) are used when tumors reach 100-150 mm3. Mice are randomized into treatment groups (n=8-10). Treatment groups include: vehicle, chemotherapy alone (e.g., doxorubicin 2-5 mg/kg i.v. weekly), (S)-Verapamil HCl alone (10-50 mg/kg i.p. daily), and combination (chemotherapy + (S)-Verapamil). Tumor volumes are measured by calipers twice weekly. Body weight is monitored for toxicity. The combination group typically shows significantly enhanced tumor growth inhibition (TGI) compared to chemotherapy alone.

(S)-Verapamil HCl has a molecular weight of 491.06 g/mol. The S-enantiomer is the more potent enantiomer for calcium channel blockade. Verapamil (racemic) is well absorbed orally (bioavailability ~20-35% due to first-pass metabolism), is highly protein bound (>90%), and has a terminal half-life of 3-7 hours. It is extensively metabolized by CYP3A4 to norverapamil (an active metabolite). The (S)-enantiomer is more rapidly metabolized than the (R)-enantiomer. (S)-Verapamil is a P-glycoprotein (P-gp) and MRP1 inhibitor.

(S)-Verapamil HCl is a research compound; the racemate verapamil is an approved drug. Verapamil is generally well tolerated. Common adverse effects include constipation (most common), dizziness, headache, hypotension, bradycardia, peripheral edema, and fatigue. Serious but rare side effects include heart failure, atrioventricular (AV) block, bradyarrhythmias, and hepatotoxicity. Verapamil is contraindicated in patients with severe left ventricular dysfunction, sick sinus syndrome, and atrial flutter/fibrillation with an accessory bypass tract (WPW syndrome).

[1]. (R)- and (S)-verapamil differentially modulate the multidrug-resistant protein MRP1. J Biol Chem. 2007 Oct 26;282(43):31542-8. Epub 2007 Jul 22.

[2]. St John's wort decreases the bioavailability of R- and S-verapamil through induction of the first-pass metabolism. Clin Pharmacol Ther. 2004 Apr;75(4):298-309.

(S)-Verapamil hydrochloride is a hydrochloride formed by reacting equimolar amounts of (S)-verapamil with hydrogen chloride. It is the enantiomer of dextro-verapamil hydrochloride.

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(S)-Verapamil HCl is the S-enantiomer of the calcium channel blocker verapamil. Verapamil is a class IV antiarrhythmic agent and antihypertensive drug marketed as a racemic mixture (Calan, Isoptin, Verelan). The (S)-enantiomer is more potent as a calcium channel blocker and is used as a research tool for studying the enantioselective pharmacology of verapamil. Additionally, (S)-Verapamil is an MRP1 inhibitor and is used in cancer research to reverse multidrug resistance. The compound is for research use only.

C₂₇H₃₉CLN₂O₄

491.06

490.26

C, 66.04; H, 8.01; Cl, 7.22; N, 5.70; O, 13.03

36622-28-3

(R)-Verapamil hydrochloride;38176-02-2;(S)-Verapamil-d7 hydrochloride;(Rac)-Verapamil-d7 hydrochloride;1188265-55-5;(R)-Verapamil-d7 hydrochloride

12249889

Typically exists as off-white to light yellow solids at room temperature

5.895

1

6

13

34

606

1

CC(C)[C@](CCCN(C)CCC1=CC(=C(C=C1)OC)OC)(C#N)C2=CC(=C(C=C2)OC)OC.Cl

DOQPXTMNIUCOSY-YCBFMBTMSA-N

InChI=1S/C27H38N2O4.ClH/c1-20(2)27(19-28,22-10-12-24(31-5)26(18-22)33-7)14-8-15-29(3)16-13-21-9-11-23(30-4)25(17-21)32-6;/h9-12,17-18,20H,8,13-16H2,1-7H3;1H/t27-;/m0./s1

(2S)-2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methylamino]-2-propan-2-ylpentanenitrile;hydrochloride

(S)Verapamil HCl (S); (S)-(-)-Verapamil Hydrochloride; (-)-Verapamil Hydrochloride; (S)-Verapamil (hydrochloride); (S)-Verapamil Hydrochloride; S(-)-VERAPAMIL; Verapamil hydrochloride, (-)-; 5002H7B3FO; Verapamil HCl

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture.

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

DMSO : ~200 mg/mL (~407.28 mM)
H2O : ~100 mg/mL (~203.64 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (10.18 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 50.0 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: ≥ 5 mg/mL (10.18 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 50.0 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: ≥ 5 mg/mL (10.18 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (203.64 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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