Calcium channel; Permeability-glycoprotein (P-gp); CYP3A4[1]

EverFluor FL Verapamil (EFV) inhibits TR-iBRB2 cells in a concentration-dependent manner, while Verapamil inhibits them in a concentration-inhibitory manner with an IC50 of 98.0 μM [4].

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Functional Analysis of EverFluor FL Verapamil (EFV) Uptake by TR-iBRB2 Cells [4]
The function of EFV uptake was investigated in TR-iBRB2 cells, and the linear increase in EFV uptake was observed for at least 10 min with an initial uptake rate of 65.3 ± 6.7 μL/(min·mg protein) (Fig. 4a). EFV uptake was significantly reduced by 57.8% at 4°C (Fig. 4a), and no significant change in EFV uptake was seen in the experiment with buffer in which Na+ was replaced by Li+ or K+ (Fig. 4b). The effect of the extracellular and intracellular pH on EFV uptake was investigated in TR-iBRB2 cells. The uptake of EFV at pH 6.4 and 8.4 exhibited no significant difference from that at pH 7.4 (Fig. 4c), whereas the acute treatment of TR-iBRB2 cells with NH4Cl produced a significant reduction in EFV uptake by 34% (Fig. 4d).

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Inhibition Analysis of EverFluor FL Verapamil (EFV) Uptake by TR-iBRB2 Cells [4]
The in vitro distribution analysis suggested the transport of EFV at the inner and outer BRB. In particular, the inner BRB has been known to nourish two-thirds of the retinal tissue. Therefore, the inhibitory effect of several compounds on EFV uptake by TR-iBRB2 cells was investigated (Table I), and cationic drugs, including desipramine, imipramine, propranolol and verapamil, markedly inhibited the uptake of EFV by more than 47%. Furthermore, cationic drugs, including quinidine, pyrilamine, and timolol, moderately inhibited EFV uptake by more than 25%, while no significant effect was produced by cimetidine, clonidine, amantadine, acetazolamide, choline, tetraethylammonium (TEA), 1-methyl-4-phenylpyridinium (MPP+), L-carnitine, serotonin and p-aminohippuric acid (PAH). In addition, the inhibition analysis of EFV uptake showed a concentration-dependent inhibition of EFV uptake by verapamil with an IC50 of 98.0 μM (Fig. 4e).

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Purpose: To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).
Methods: EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.
Results: The signal of EverFluor FL Verapamil (EFV) was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker® Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50 of 98.0 μM.
Conclusions: Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB [4].

In atrial fibrillation, Verapamil (facial) can be used to control the atrioventricular nodal response and avoid atrioventricular reentrant tachycardia [2]. An intravenous injection of Verapamil was given into the anterior chest region's femoral vein. Within 45 minutes following coronary artery closure, the incidence of ventricular arrhythmias, such as ventricular tachycardia (VT), ventricular fibrillation (VF), and premature ventricular contractions (PVCs), was considerably reduced by verapamil (1 mg/kg). An ischemic heart resulted in a considerable increase in the overall arrhythmia score. The administration of 1 mg/kg of verapamil effectively inhibited the rise in overall cardiovascular-induced arrhythmia scores [5].

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The antiarrhythmic effects of Verapamil were observed before it was appreciated that it was a calcium ion-antagonist. Intravenous Verapamil is highly effective in the termination of paroxysmal reciprocating atrioventricular tachycardia, whether associated with preexcitation or involving the atrioventricular node alone. It consistently slows and regularises the ventricular response in atrial fibrillation, and usually increases the degree of AV-nodal block in atrial flutter though it occasionally induces a return to sinus rhythm. Given orally it is useful for the prophylaxis of atrioventricular reentry tachycardia, and also in modulating the atrioventricular nodal response in atrial fibrillation. Favourable response in ventricular tachycardia is exceptional and then seen in specific benign varieties. Verapamil is the agent of choice for the termination of paroxysmal supraventricular tachycardia.[2]

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Intervention: The patients were treated with either metoprolol (Seloken ZOC 200 mg o.d.) or Verapamil (Isoptin Retard 240 b.i.d.). Acetylsalicylic acid, ACE inhibitors, lipid lowering drugs and long acting nitrates were allowed in the study. End points: Death, non-fatal cardiovascular events including acute myocardial infarction, incapacitating or unstable angina, cerebrovascular or peripheral vascular events. Psychological variables reflecting quality of life i.e. psychosomatic symptoms, sleep disturbances and an evaluation of overall life satisfaction. Results: Combined cardiovascular events did not differ and occurred in 30.8% and 29.3% of metoprolol and Verapamil treated patients respectively. Total mortality in metoprolol and verapamil treated patients was 5.4 and 6.2%, respectively. Cardiovascular mortality was 4.7% in both groups. Non-fatal cardiovascular events occurred in 26.1 and 24.3% of metoprolol and verapamil-treated patients, respectively. Psychosomatic symptoms and sleep disturbances were significantly improved in both treatment groups. The magnitudes of change were small and did not differ between treatments. Life satisfaction did not change on either drug. Withdrawals due to side effects occurred in 11.1 and 14.6% respectively. Conclusion: This long term study indicates that both drugs are well tolerated and that no difference was shown on the effect on mortality, cardiovascular end points and measures of quality of life.[3]

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The present study was to test the hypothesis that anti-arrhythmic properties of Verapamilmay be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of Verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by Verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein [5].

Methods: EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively. Results: The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker® Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50 of 98.0 μM[4].

The antiarrhythmic effects of Verapamil were observed before it was appreciated that it was a calcium ion-antagonist. Intravenous Verapamil is highly effective in the termination of paroxysmal reciprocating atrioventricular tachycardia, whether associated with preexcitation or involving the atrioventricular node alone. It consistently slows and regularises the ventricular response in atrial fibrillation, and usually increases the degree of AV-nodal block in atrial flutter though it occasionally induces a return to sinus rhythm. Given orally it is useful for the prophylaxis of atrioventricular reentry tachycardia, and also in modulating the atrioventricular nodal response in atrial fibrillation. Favourable response in ventricular tachycardia is exceptional and then seen in specific benign varieties. Verapamil is the agent of choice for the termination of paroxysmal supraventricular tachycardia[2].

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Confocal Microscopy of TR-iBRB2 Cells and RPE-J Cells [4]
TR-iBRB2 cells and RPE-J cells were immortalized rat retinal capillary endothelial cells and retinal pigment epithelial cells to be used as the inner and outer BRB model cell lines, and were seeded at 5 × 103 and 7 × 103 cells/well on BioCoat™ Collagen I Cellware 8-well culture slide, respectively, and cultured at 33°C under 5% CO2/air. After a 48 h cultivation, the uptake assay was initiated by adding extracellular fluid (ECF)-buffer containing EverFluor FL Verapamil (EFV) (1 μM) or LTR (300 nM for TR-iBRB2 cells, 600 nM for RPE-J cells), and these concentrations were set by referring previous reports. The assay was terminated at a designated time by washing cells with ice-cold ECF buffer three times. After fixation of the cells with 4% paraformaldehyde, the cells were subjected to confocal microscope observation with LSM700 as reported elsewhere.

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Cell Uptake Study [4]
In vitro uptake analysis using TR-iBRB2 cells was conducted by referring to previous reports, and cells were cultured on collagen-coated 24-well plates at 33°C under 5% CO2/air. Uptake assay was started by adding ECF-buffer containing EverFluor FL Verapamil (EFV) (1 μM in 200 μL) at 37°C, and was terminated by washing the wells three times with ice-cold ECF-buffer. After adding ECF-buffer (200 μL/well), cells were homogenized in an ultrasonic homogenizer, and the cell protein contents and the fluorescence intensity of EFV was measured with a multi-mode microplate reader system. EFV uptake was expressed as the cell-to-medium (C/M) ratio by means of Eq. 1. EverFluor FL Verapamil (EFV) uptake in the presence of inhibitors was expressed as the fluorescence intensity ratio (FI ratio) by means of Eq. 2. The nonlinear least-square regression analysis program, MULTI, was used for the determination of the 50% inhibitory concentration (IC50) for verapamil in EverFluor FL Verapamil (EFV) uptake, and the data were fitted to Eq. 3. In the in vitro inhibition analysis, the concentration of inhibitors was set to be 500 μM by referring our previous report on verapamil transport by TR-iBRB2 cells, P and Pmax are the FI ratios with and without inhibitors, and Pmin is the inhibitor-insensitive FI ratio with inhibitor. [I] and n are the inhibitor concentration and the Hill coefficient, respectively.

The present study was to test the hypothesis that anti-arrhythmic properties of Verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of Verapamil . Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of Verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after Verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein[5].

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\n\nCommon Carotid Artery Infusion Analysis [4]
\nIn vivo distribution analysis of EverFluor FL Verapamil (EFV) to the retina was conducted by modifying the in situ brain perfusion method reported previously. In the anesthetized Wistar rats with pentobarbital (50 mg/kg), the right external carotid artery was ligated by a silk thread. After ligating the right common carotid artery, polyethylene tube was inserted into the right common carotid artery just below the bifurcation of the external carotid artery, and fixed by silk thread. The dosage conditions for fluorescent compounds were verified by referring previous reports that examined conditions without toxicity. Ringer-HEPES solution containing EverFluor FL Verapamil (EFV) (400 μg/3.5 mL), Rho-D (4 mg/3.5 mL) or LTR (400 μg/3.5 mL) warmed at 37°C was infused into the pterygopalatine artery and the internal carotid artery at a constant flow rate (0.85 mL/min) with an infusion pump, and this flow rate was set to avoid damaging the barrier structure with consideration for the blood-flow rate of the retina (0.7 mL/(min·g retina)). At the end of the infusion, the rats were decapitated, and their right eyeballs were immediately collected to be soaked in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 3 h, followed by soaking in PBS containing 30% sucrose at 4°C. The tissues were then fixed in the optimal cutting temperature compound, and tissue slices were prepared by means of a cryostat. Tissue slices mounted on glass slides were treated with 4′,6-diamidino-2-phenylindole (DAPI) and VECTASHIELD mounting medium, to be examined with a confocal microscope as described elsewhere. The excitation wavelength of 488 nm was used for EverFluor FL Verapamil (EFV), and 543 nm was used for LTR and Rho-D.\n

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\nIn vivo Arrhythmia Study [5]
\n\nVerapamil (1 mg/kg) was injected i.v. into a femoral vein 10 min prior to ischemia. A sham group underwent the same surgical procedures, except the suture underneath the LAD was left untied.\n\nIn another series of experiment, arrhythmia was induced by Bay K8644, an L-type calcium channel agonist, at a dose of 0.1 mg/kg given i.v. into the FV. Verapamil (1 mg/kg) was administered 10 min prior to Bay K8644. All injections were performed within 30 sec.\n

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\nHeart Isolation and Perfusion [5]
\nEach heart underwent an initial 10 min of baseline normal perfusion and was subjected to perfusion at 37°C for 45 min. The hearts were then randomly divided into three groups: Control group (normal calcium perfusion) (1.5 mmol/L), high calcium group (high calcium perfusion) (3.3 mmol/L) and Verapamil group (high calcium plus verapamil perfusion) (3.3 mmol/L calcium +3 µmol/L Verapamil ). For measurement of arrhythmias, the ECG was continuously monitored during the entire perfusion period and the incidence of arrhythmias was evaluated.\n

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\nMeasurement of ECG and Determination of Arrhythmia Score [5]
\nAnti-arrhythmic properties of Verapamil were determined in an animal model of ischemia-induced arrhythmia or in the presence of Bay K8644 or heptanol or Gap 26, respectively. The occurrence of cardiac arrhythmias throughout the 45 min was compared by ECG recording. For analysis of arrhythmia in Langendorff-perfused rat heart, each rat heart was continuously monitored with a positive electrode attached to the heart and a negative electrode to the aorta. After 10 min of a baseline normal perfusion period, incidences of arrhythmias under different concentrations of Ca2+ in the initial 45 min of perfusion period were compared. To enable a good quantitative comparison, 45 min of an ischemia period were divided into 15 3-min intervals in an in vivo arrhythmia evaluation and 45 min of a perfusion period was divided into 15 3-min intervals in an ex vivo arrhythmia investigation. Arrhythmia scores were evaluated as described previously. PVC ≤10/3-min period was recorded as 0; 10–50 PVC/3-min period was recorded as 1; ≥50 PVC/3-min period was recorded as 2; 1 episode of VF/3-min period was recorded as 3; 2–5 episodes of VF/3-min period was recorded as 4; and ≥5 episodes of VF/3-min period was recorded as 5.\n

Absorption, Distribution and Excretion
Oral verapamil has an absorption rate exceeding 90%, but its bioavailability is only 20% to 30% due to rapid biotransformation after first-pass metabolism in the portal circulation. Absorption kinetic parameters depend primarily on the specific formulation of verapamil. Immediate-release verapamil reaches peak plasma concentration (Tmax) within 1–2 hours after administration, while the Tmax of sustained-release formulations is typically between 6 and 11 hours. AUC and Cmax values also depend on the formulation. Immediate-release verapamil administered every 6 hours results in plasma concentrations between 125 and 400 ng/mL. For the sustained-release formulation, the steady-state AUC_0-24h and Cmax values for the R-isomer were 1037 ng∙h/ml and 77.8 ng/mL, respectively, while those for the S-isomer were 195 ng∙h/ml and 16.8 ng/mL, respectively. Notably, verapamil exhibits highly stereoselective absorption kinetics—after a single oral dose of immediate-release verapamil every 8 hours, the relative systemic bioavailability of the S-enantiomer compared to the R-enantiomer was 13% at steady state and 18% at steady state. Approximately 70% of the administered dose is excreted in the urine as metabolites within 5 days, and ≥16% is excreted in the feces. Approximately 3%–4% of the drug is excreted unchanged in the urine. The steady-state volume of distribution for the R-enantiomer of verapamil is approximately 300 L, and for the S-enantiomer, approximately 500 L. After 3 weeks of continuous treatment, the systemic clearance of R-verapamil was approximately 340 mL/min, and that of S-verapamil was approximately 664 mL/min. Notably, there appears to be a significant difference in apparent oral clearance between single-dose and multiple-dose administration. After a single dose of verapamil, the apparent oral clearance of R-verapamil was approximately 1007 mL/min, and that of S-verapamil was approximately 5481 mL/min; after 3 weeks of continuous treatment, the apparent oral clearance of R-verapamil was approximately 651 mL/min, and that of S-verapamil was approximately 2855 mL/min.
/Breast Milk/ Verapamil may be present in breast milk.
/Breast Milk/ Verapamil is secreted into breast milk. After a daily dose of 240 mg, the concentration of verapamil in breast milk was approximately 23% of the maternal serum concentration. The infant's serum verapamil concentration was 2.1 ng/mL, but became undetectable (<1 ng/mL) 38 hours after discontinuation. …In another case, a mother was taking 80 mg three times daily for hypertension for four weeks prior to the determination of verapamil concentrations in her serum and breast milk. The steady-state concentrations of verapamil and its metabolite norvertapamil in breast milk were 25.8 ng/mL and 8.8 ng/mL, respectively. These values represent 60% and 16% of the plasma concentrations, respectively. Researchers estimated that breastfed infants ingested less than 0.01% of the mother's dose. Verapamil and its metabolites were not detected in the infant's plasma. A study investigated the pharmacokinetics and hemodynamics of verapamil combined with triamcinolone acetonide in 20 hypertensive patients (aged 29–71 years), 10 of whom also had fatty liver disease. These patients received once-daily oral administration of extended-release capsules containing 180 mg verapamil and 1 mg triamcinolone for 7 days. For verapamil, there were no statistically significant differences in peak plasma concentration (Cmax) (110.5 vs 76.5 μg/L), area under the plasma curve (AUC) at 0-24 hours (1260.6 vs 941.2 μg/L·h), and elimination half-life (9.8 vs 9.2 h) between patients with and without fatty liver. An open-label, randomized, single-dose study aimed to investigate the effect of food on the bioavailability of extended-release (SR) verapamil hydrochloride (Isoptin). The study included 12 healthy volunteers (aged 19–65 years) who received a 240 mg extended-release formulation either on an empty stomach or after a meal, or a regular formulation on an empty stomach. The results showed that although the elimination half-life of extended-release verapamil remained unchanged, the time to peak concentration was prolonged, and the area under the concentration-time curve (AUC) was 80% of that of the conventional formulation. Co-administration with food extended the time to peak concentration from 7.3 ± 3.4 hours to 11.7 ± 6.3 hours, but had little effect on the peak concentration, half-life, or AUC of extended-release verapamil. For more complete data on absorption, distribution, and excretion of verapamil (21 items in total), please visit the HSDB record page. Metabolites/Metabolites Verapamil is primarily metabolized in the liver, with up to 80% of the administered dose cleared via first-pass metabolism—interestingly, this first-pass metabolism appears to clear the S-enantiomer of verapamil more quickly than the R-enantiomer. The remaining parent drug undergoes O-demethylation, N-dealkylation, and N-demethylation via the cytochrome P450 enzyme system, generating various metabolites. Norvelapamil is one of the major circulating metabolites of verapamil. It is a product of N-demethylation metabolism of verapamil via CYP2C8, CYP3A4, and CYP3A5, and its cardiovascular activity is approximately 20% of that of verapamil. Another major metabolic pathway of verapamil is N-dealkylation metabolism via CYP2C8, CYP3A4, and CYP1A2, generating the metabolite D-617. Both norvelapamil and D-617 can be further metabolized into various secondary metabolites by other CYP isoenzymes. CYP2D6 and CYP2E1 also participate in the metabolic pathway of verapamil, but their effects are minor. Secondary metabolic pathways of verapamil include O-demethylation via CYP2C8, CYP2C9, and CYP2C18 to generate D-703, and O-demethylation via CYP2C9 and CYP2C18 to generate D-702. Several steps in the verapamil metabolic pathway exhibit stereoselectivity for the S-enantiomers of specific substrates, including the formation of metabolite D-620 by CYP3A4/5 and metabolite D-617 by CYP2C8. Metabolites: The major metabolite is norvertapamil, with an elimination half-life very similar to the parent compound, ranging from 4 to 8 hours. Verapamil is primarily metabolized by the liver. Due to significant first-pass effect in the liver, bioavailability in normal subjects does not exceed 20% to 35%. Twelve metabolites have been reported. The major metabolite is norvertapamil; other metabolites are various N- and O-dealkylated metabolites. Elimination via exposure pathways: Kidney: Approximately 70% of the administered dose is excreted in the urine as metabolites within 5 days, of which 3-4% is excreted unchanged. Feces: Approximately 16% of the ingested dose is excreted in the feces as metabolites within 5 days. Breast milk: Verapamil may be present in breast milk.
Verapamil produces the following metabolites in dogs: 5-(3,4-dimethoxyphenylethylamino)-2-(3,4-dimethoxyphenyl)-2-isopropylpentanonitrile; 2-(3,4-dimethoxyphenyl)-5-(n-(4-hydroxy-3-methoxyphenylethyl)methylamino)-2-isopropylpentanonitrile and 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-methylaminopentanonitrile. The latter was also found in rats. /Excerpt from table/ /Unspecified salts/
Verapamil and its major metabolite, norvertapamil, have been shown to be both mechanistic inhibitors and substrates of CYP3A and have been reported to exhibit nonlinear pharmacokinetic characteristics in clinical settings. The metabolic clearance of verapamil and norvertapamil and their effects on CYP3A activity were first determined in mixed human liver microsomes. The results showed that the S-isomers of verapamil and norvertapamil were more readily metabolized than their R-isomers, and their inhibition of CYP3A activity was stereoselective, with the S-isomer exhibiting stronger inhibitory activity than the R-isomer. This study established a semi-physiological pharmacokinetic model (semi-PBPK) capable of characterizing mechanism-based autoinhibition and predicting the stereoselective pharmacokinetic characteristics of verapamil and norvertapamil after single or multiple oral administrations. The simulation results were satisfactory, indicating that the established semi-PBPK model can simultaneously predict the pharmacokinetic characteristics of S-verapamil, R-verapamil, S-norvertapamil, and R-norvertapamil. Furthermore, this study also investigated the effect of autoinhibition on the accumulation of verapamil and norvertapamil after 38 oral administrations of verapamil extended-release tablets (240 mg, once daily). The predicted cumulative fold was approximately 1.3–1.5 times, close to the observed 1.4–2.1 times. Finally, the established semi-PBPK model was further applied to predict drug interactions (DDIs) between verapamil and three other CYP3A substrates (midazolam, simvastatin, and cyclosporine A). The predictions were also successful, demonstrating the significant advantage of the established semi-PBPK model, which incorporates self-inhibition, in predicting DDIs with CYP3A substrates. The biotransformation pathway of the widely used calcium channel blocker verapamil was investigated using electrochemistry (EC) coupled with liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Oxidation phase I metabolism was simulated in a simple amperometric thin-layer cell equipped with a boron-doped diamond (BDD) working electrode. Based on accurate mass data and additional MS/MS experiments, the structure of the electrochemically generated metabolites was elucidated. We demonstrate that the most important metabolites of all calcium antagonists, including norvertapamil (formed by N-demethylation), can be easily simulated using this purely instrumental technique. Furthermore, some newly reported metabolic reaction products, such as carbene alcoholamine or imine methylates, also become accessible. We compared the results of the electrochemical method (EC) with conventional in vitro studies, which were performed using incubation experiments on rat and human liver microsomes (RLMs, HLMs). Both methods showed good agreement with the EC/LC/MS data. Therefore, it can be seen that the electrochemical method is well-suited for simulating the oxidative metabolism of verapamil. In conclusion, this study confirms that EC/LC/MS can be a powerful tool for drug discovery and development when used in conjunction with existing in vitro or in vivo methods.
The mechanistic inactivation (MBI) of verapamil on cytochrome P450 (CYP) 3A and the resulting drug interactions have been studied in vitro, but the clinically known inhibitory effect of verapamil on its own metabolic clearance—i.e., the self-inhibition of verapamil metabolism—has not yet been reproduced in vitro. This study aimed to evaluate the efficacy of gel-embedded rat hepatocytes in reflecting this metabolic autoinhibition, with hepatocyte monolayers serving as a control. Despite similar concentration- and time-dependent curves, significant differences were observed in the autoinhibition of verapamil metabolism between the two culture models. First, gel-embedded hepatocytes were more sensitive to this inhibition, likely due to their higher CYP3A activity, detectable by the production rates of 6β-hydroxytestosterone and 1'-hydroxymidazolam. Furthermore, the inhibitory effects of ketoconazole and verapamil on CYP3A activity, as well as the reduction in verapamil's intrinsic clearance rate (CL(int)) by ketoconazole, were only observed in gel-embedded hepatocytes. Thus, the autoinhibitory effect of CYP3A on verapamil metabolism was confirmed only in gel-embedded hepatocytes, and not observed in monolayer hepatocytes. All these results suggest that gel-embedded hepatocytes better reflect the metabolic autoinhibition of verapamil compared to hepatocyte monolayers, and therefore serve as a suitable system for studying drug metabolism.
The known metabolites of verapamil include 2-(3,4-dimethoxyphenyl)acetaldehyde, norvertapamil, D-702, M9 (D-703), and D-617.
Elimination pathway: Approximately 70% of the administered dose is excreted as metabolites in the urine within 5 days, and 16% or more is excreted in the feces. Approximately 3% to 4% of the drug is excreted unchanged in the urine.
Half-life: 2.8–7.4 hours.
Single-dose studies of immediate-release verapamil have shown an elimination half-life of 2.8 to 7.4 hours, which can be prolonged to 4.5 to 12.0 hours with repeated dosing. The elimination half-life is also prolonged in patients with hepatic impairment (14 to 16 hours) and elderly patients (approximately 20 hours). After intravenous administration of verapamil, the distribution phase half-life is approximately 4 minutes, followed by the terminal elimination phase with a half-life of 2 to 5 hours.
This study investigated the pharmacokinetics of verapamil and its metabolite norvertapamil in 10 patients with portal hypertension (aged 19-69 years) and 6 healthy subjects (aged 21-69 years). All subjects were given 80 mg of verapamil hydrochloride (Isoptin) orally. The terminal elimination half-life of verapamil in the control group was 210 hours, while that in the patient group was 1384 hours.
Toxicokinetic studies in the two patients showed that the plasma half-lives were 7.9 hours and 13.2 hours, respectively, and the systemic clearance rates were 425 mL/min and 298 mL/min, respectively. ...

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In vivo distribution analysis of verapamil (EFV) in the retina [4]
The distribution of EFV in the retina was studied by carotid artery perfusion method. Under confocal microscopy, EFV (green) signals were uniformly detected from the internal limiting membrane (ILM) to the outer plexiform layer (OPL) (Fig. 1a), while the fluorescence signal of Rho-D (a cell-impermeable substrate) was weak (data not shown). In addition, strong EFV fluorescence signals were detected in the photoreceptor outer segment (POS) (Fig. 1a), and punctate signals of EFV were partially detected in the retinal pigment epithelium (RPE) (Fig. 1b, arrows). In vitro distribution analysis of EverFluor FL verapamil (EFV) [4]
The subcellular localization of EFV in internal and external blood-retinal barrier (BRB) model cell lines was studied using confocal microscopy. In TR-iBRB2 cells (an in vitro model cell line of internal BRB), the fluorescence signal of EFV was distributed throughout the cell, while the fluorescence signal of LTR was punctate, indicating that the subcellular distribution pattern of EFV was different from that of LTR (Fig. 2a). Under acute treatment with NH4Cl, the subcellular localization of EFV did not change significantly, while the punctate distribution pattern of LTR decreased (Fig. 2b). In addition, in the presence of bafloxacin A1, an inhibitor of vacuolar H+-ATPase (V-ATPase), the uptake of EFV by TR-iBRB2 cells did not change significantly (Fig. 2c). [4] In RPE-J cells, the LTR was observed to have a punctate distribution pattern, which decreased after acute treatment with NH4Cl (Fig. 3). EFV signal was distributed throughout the cell in a partially punctate distribution pattern, which merged with the punctate distribution pattern of LTR (Fig. 3a, arrow), and this partially punctate distribution pattern decreased after acute treatment with NH4Cl (Fig. 3).

Toxicity Summary
Identification and Uses: Verapamil is the first-line drug for the prevention and treatment of paroxysmal supraventricular tachycardia. Verapamil has been shown to be effective in treating angina. Verapamil can be used as an alternative treatment for mild to moderate hypertension. Human Studies: Verapamil has a vasodilatory effect on the vascular system. Toxicity usually occurs 1 to 5 hours after oral administration. After intravenous injection, symptoms appear within minutes. Major cardiovascular symptoms include: bradycardia and atrioventricular block (82% of cases), hypotension and cardiogenic shock (78% of cases), and cardiac arrest (18% of cases). Pulmonary edema may occur. Altered consciousness and seizures may occur, which are associated with low cardiac output. Nausea and vomiting may occur. Shock and hyperglycemia may lead to metabolic acidosis. Verapamil is a calcium channel blocker that inhibits the entry of calcium ions into cardiovascular cells through calcium channels. Verapamil reduces the intensity of calcium currents and slows the recovery rate of channels. Verapamil reduces peripheral vascular and coronary artery resistance, but its vasodilatory effect is weaker than nifedipine. Conversely, its cardiac effects are more pronounced than nifedipine. At the dose required to produce arterial vasodilation, verapamil's negative chronotropic, negative transductive, and negative inotropic effects are far stronger than nifedipine. At toxic doses, verapamil's inhibition of calcium channels leads to three main effects: hypotension due to arterial vasodilation, cardiogenic shock due to negative inotropic effects, bradycardia, and atrioventricular block. Verapamil's therapeutic effects on hypertension and angina pectoris stem from its vasodilatory effect on systemic and coronary arteries. Verapamil's antiarrhythmic effect arises from its direct action, delaying impulse conduction in the atrioventricular node. Toxic reactions can occur with an intake of 1 gram of verapamil. In vitro experiments used the micronucleus assay (MN assay) to test verapamil in human peripheral blood lymphocytes. The results showed that the micronucleus frequency increased after all treatments. Fluorescence in situ hybridization (FISH) analysis showed that verapamil, used alone or in combination with ritodrine, had a greater aneuploidy-inducing effect than chromosome breakage-inducing effect. Animal experiments: Verapamil induced atrial fibrillation in normal dogs. In a pig model, verapamil toxicity was observed after an average infusion of 0.6 ± 0.12 mg/kg verapamil, manifested as a 45% decrease in mean arterial pressure from baseline. The mean plasma verapamil concentration produced at this dose was 728.1 ± 155.4 μg/L. In the pig model, hypertonic sodium bicarbonate reversed the hypotension and decreased cardiac output caused by severe verapamil toxicity. Ecotoxicity studies: This study investigated the effects of long-term exposure to verapamil at concentrations of 0.29, 0.58, and 1.15 mg/L on mutagenicity, hematologic parameters, and oxidase activity in the liver of Nile tilapia (Oreochromis niloticus) for 60 days. The results showed that exposure led to a significant increase in peripheral blood cell micronuclei. Levels of oxidative stress biomarkers (lipid peroxidation and carbonyl proteins) were elevated. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were also increased. In other experiments, exposure of fish to sublethal concentrations of verapamil (0.14, 0.29, and 0.57 mg/L) for 15, 30, 45, and 60 days resulted in inhibition of acetylcholinesterase activity in the brain and muscle of fish. At the end of the study, the transcriptional levels of catalase (CAT), superoxide dismutase (SOD), and heat shock protein 70 (hsp70) were upregulated in both tissues. Behavioral changes such as dyspnea and loss of balance were observed in goldfish (Carassius auratus), confirming the cardiovascular toxicity induced by high doses of verapamil. In addition to its effects on the cardiovascular system, verapamil also affects the nervous system by altering paraalbumin expression. Acute exposure to verapamil significantly reduced heart rate in carp (Cyprinus carpio) embryos and juveniles. In chronic toxicity studies in giant fleas (D. magna), exposure to 4.2 mg/L verapamil adversely affected multiple parameters, including survival rate, body length, time to first reproduction, and litter size per female. During a short-term 24-hour exposure, verapamil downregulated the expression of CYP4 and CYP314 genes. During a long-term 21-day exposure, verapamil significantly reduced the expression level of the Vtg gene, a biomarker of reproductive capacity in oviparous animals. Verapamil inhibits voltage-dependent calcium channels. Specifically, its effect on cardiac L-type calcium channels leads to decreased myocardial contractility and heart rate, thereby reducing heart rate and blood pressure. The mechanism of action of verapamil in treating cluster headaches is believed to be related to its calcium channel blocking effect, but the specific channel subtypes involved are currently unclear.

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Toxicity Data
LD50: 8 mg/kg (intravenous injection, mice) (A308)Drug Interactions
Drug Interactions: Protein-bound drugs
Drug Interactions: Beta-adrenergic blockers
Drug Interactions: Digoxin
Drug Interactions: Antihypertensive drugs
For more (complete) interaction data (42 in total) for verapamil, please visit the HSDB record page.

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Non-human toxicity values
Mouse intraperitoneal LD50: 68 mg/kg /Verapamil hydrochloride/
Rat intraperitoneal LD50: 67 mg/kg /Verapamil hydrochloride/
Rat oral LD50: 114 mg/kg /Verapamil hydrochloride/
Mouse intravenous LD50: 7.6 mg/kg /Verapamil hydrochloride/
For more complete non-human toxicity data for verapamil (out of 14), please visit the HSDB records page.

[1]. Verapamil as a culprit of palbociclib toxicity. J Oncol Pharm Pract. 2019 Apr;25(3):743-746.

[2]. Krikler DM. Verapamil in arrhythmia. Br J Clin Pharmacol. 1986;21 Suppl 2:183S-189S.

[3]. Effects of metoprolol vs verapamil in patients with stable angina pectoris. The Angina Prognosis Study in Stockholm (APSIS). Eur Heart J. 1996 Jan;17(1):76-81.

[4]. Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier. Pharm Res. 2018 Mar 12;35(5):93.

[5]. Anti-arrhythmic effect of Verapamil is accompanied by preservation of cx43 protein in rat heart. PLoS One. 2013 Aug 12;8(8):e71567.

Therapeutic Uses
Antiarrhythmic drugs; calcium channel blockers; vasodilators. ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure being studied); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Verapamil hydrochloride is included in the database. Oral calcium channel blockers are considered the first-line treatment for Prinzmetal angina. For patients with unstable angina who do not respond well to, cannot tolerate, or have contraindications to beta-blockers and nitrates, and who do not have severe left ventricular dysfunction, pulmonary edema, or other contraindications, non-dihydropyridine calcium channel blockers (e.g., diltiazem, verapamil) are also recommended if persistent or persistent myocardial ischemia is present. In treating unstable or chronic stable angina, verapamil appears to be as effective as beta-adrenergic blockers (e.g., propranolol) and/or oral nitrates. For unstable or chronic stable angina, verapamil can reduce the frequency of attacks, decrease the dosage of sublingual nitroglycerin, and improve exercise tolerance. /US Product Label Includes/
Verapamil is used for the rapid conversion of paroxysmal supraventricular tachycardia (PSVT) to sinus rhythm, including tachycardias associated with Wolff-Parkinson-White syndrome or Lown-Ganong-Levine syndrome; it is also used to control rapid ventricular rates in unpre-excited atrial flutter or atrial fibrillation. The American College of Cardiology/American Heart Association/Heart Rhythm Society (ACC/AHA/HRS) guidelines for the management of adult patients with supraventricular tachycardia recommend verapamil for the treatment of various supraventricular tachycardias (e.g., atrial flutter, junctional tachycardia, focal atrial tachycardia, atrioventricular nodal reentrant tachycardia (AVNRT)). Typically, intravenous verapamil is used for acute treatment, while oral verapamil is used for sustained treatment of these arrhythmias. /US Product Label Includes/
For more complete data on the therapeutic uses of verapamil (14 types), please visit the HSDB record page.

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Drug Warnings
...Concomitant use of verapamil and beta-blockers may be dangerous in patients with impaired left ventricular function if...myocardial function decreases by 10-15%. /Unspecified Salt/
...Absolute contraindications to verapamil (acute myocardial infarction, complete atrioventricular block, cardiogenic shock, manifest heart failure)...It should not be administered concurrently with beta-adrenergic blockers, or used within 3 times the half-life of a beta-adrenergic blocker. /Unspecified Salt/
The basic physiological action of verapamil may lead to serious adverse reactions. /Unspecified Salt/
Maternal use generally compatible with breastfeeding: Verapamil: Signs or symptoms reported by the infant or effects on lactation: None. /From Table 6/ /Unspecified Salt/
For more complete data on drug warnings for verapamil (of 23), please visit the HSDB records page.

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Pharmacodynamics
Verapamil is an L-type calcium channel blocker with antiarrhythmic, antianginal, and antihypertensive effects. Immediate-release verapamil has a relatively short duration of action, requiring 3 to 4 doses daily, but sustained-release formulations are also available for once-daily administration. Because verapamil is a negative inotropic agent (i.e., it reduces myocardial contractility), it should not be used in patients with severe left ventricular dysfunction or hypertrophic cardiomyopathy, as the decrease in contractility caused by verapamil may increase the risk of exacerbating these pre-existing conditions.

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2-(3,4-Dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propyl-2-yl)valerate is a tertiary amine compound, 3,4-dimethoxyphenylethylamine, in which the hydrogen atom bonded to the nitrogen atom is replaced by a methyl group and a 4-cyano-4-(3,4-dimethoxyphenyl)-5-methylhexyl group. It is a tertiary amine compound, aromatic ether, polyether, and nitrile. Verapamil is a benzylalkylamine calcium channel blocker used to treat hypertension, arrhythmias, and angina. It was the first calcium channel antagonist to be used clinically in the early 1960s. It belongs to the non-dihydropyridine class of calcium channel blockers, which includes drugs such as diltiazem and flunarizine, but its chemical structure is unrelated to other cardioactive drugs. Verapamil is administered as a racemic mixture containing equal amounts of its S- and R-enantiomers, which have different pharmacological effects—the S-enantiomer is approximately 20 times more potent than the R-enantiomer, but is also metabolized more quickly. Verapamil is a calcium channel blocker. Its mechanism of action is as a calcium channel antagonist, cytochrome P450 3A4 inhibitor, cytochrome P450 3A inhibitor, and P-glycoprotein inhibitor. Verapamil is a first-generation calcium channel blocker used to treat hypertension, angina, and supraventricular arrhythmias. Verapamil is associated with a low incidence of elevated serum enzymes during treatment and rare cases of clinically significant acute liver injury. Verapamil has been reported in Teichospora striata and Schisandra chinensis, with relevant data available. According to the LOTUS Natural Products Database, verapamil is a phenylalkylamine calcium channel blocker. Verapamil inhibits the transmembrane influx of extracellular calcium ions into myocardial and vascular smooth muscle cells, leading to dilation of major coronary and systemic arteries and reducing myocardial contractility. This drug also inhibits the drug efflux pump P-glycoprotein, which is overexpressed in some multidrug-resistant tumors and may therefore enhance the efficacy of certain antitumor drugs. Verapamil is a small molecule drug, with clinical trials up to Phase IV (covering all indications). It was first approved in 1981 and currently has 4 approved indications and 16 investigational indications. Verapamil has only been detected in individuals who have taken the drug. Verapamil is a calcium channel blocker, belonging to class IV antiarrhythmic drugs. [PubChem] Verapamil inhibits voltage-dependent calcium channels. Specifically, verapamil's action on L-type calcium channels in the heart leads to decreased myocardial contractility and heart rate, thereby reducing heart rate and blood pressure. The mechanism of action of verapamil in treating cluster headaches is thought to be related to its calcium channel blocking effect, but it is currently unclear which specific channel subtypes are involved. [PubChem] Calcium channel antagonists are highly toxic. Early identification is crucial in the management of poisoning. Calcium channel antagonists are commonly prescribed medications, and overdose can lead to serious complications and death. Any patient with an overdose presenting with unexplained hypotension and conduction abnormalities should be suspected of having ingested such drugs. Patients with underlying hepatic or renal dysfunction who are receiving therapeutic doses should be monitored for toxicity risks. (A7844)
A class IV antiarrhythmic drug, belonging to the calcium channel blocker class.

C27H38N2O4

454.61

454.283

C, 71.34; H, 8.43; N, 6.16; O, 14.08

52-53-9

Verapamil hydrochloride;152-11-4; 38321-02-7 (dexverapamil)

2520

Viscous, pale yellow oil

1.1±0.1 g/cm3

586.2±50.0 °C at 760 mmHg

25°C

308.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.526

3.9

0

6

13

33

606

0

CC(C)C(CCCN(C)CCC1=CC(=C(C=C1)OC)OC)(C#N)C2=CC(=C(C=C2)OC)OC

SGTNSNPWRIOYBX-UHFFFAOYSA-N

InChI=1S/C27H38N2O4/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

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

NSC-135784; NSC 135784; VERAPAMIL; 52-53-9; Iproveratril; Dilacoran; Vasolan; Isoptimo; Isoptin; Verapamilo; Verapamil; Verapamilum; D-365;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~219.97 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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 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 (5.50 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 25.0 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: ≥ 2.5 mg/mL (5.50 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.)