Milrinone (Win47203; Primacor) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-inhibited phosphodiesterase 3 (PDE3), with high affinity for both PDE3A and PDE3B isoforms. In assays using rat pulmonary arterial tissue homogenates, it exhibits an IC50 value of 0.32 ± 0.05 μM for PDE3 (measured by [³H]-cAMP hydrolysis inhibition) [1]
- Milrinone indirectly modulates potassium (K⁺) channels (including large-conductance calcium-activated K⁺ channels [BKCa] and voltage-gated K⁺ channels [Kv]) in vascular smooth muscle cells. It enhances K⁺ channel activity via cAMP-dependent protein kinase A (PKA) phosphorylation [4]

PKA activity in hypoxic myocytes is raised to normoxic levels by 1 µM milrinone. By reinstating PKA-mediated regulatory TP receptor phosphorylation, milrinone (50 nM) restores TP receptor sensitivity in hypoxic myocytes[1]. Milrinone attenuates maximal tension generation and NE sensitivity by substantially reducing NE-induced vasoconstriction. Milrinone-induced attenuation of NE responses is not prevented by voltage-gated or ATP-sensitive K+ channel inhibition[4].

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Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes (PAMCs). When hypoxic PAMCs (exposed to 1% O₂ for 48 hours) are pre-treated with Milrinone (0.1, 1, 10 μM) for 1 hour, then stimulated with the thromboxane A₂ analog U46619 (100 nM):
- Cell contraction amplitude (measured via myocyte shortening assay) is reduced in a concentration-dependent manner: 0.1 μM reduces contraction by ~20%, 1 μM by ~45%, and 10 μM by ~70% (vs. hypoxic + U46619 group) [1]
- Intracellular cAMP levels (detected by ELISA) increase by 1.5-fold (1 μM) and 2.3-fold (10 μM) compared to hypoxic controls [1]
- Western blot analysis shows that 10 μM Milrinone decreases thromboxane prostanoid (TP) receptor protein expression by ~35% in hypoxic PAMCs [1]
- Milrinone relaxes norepinephrine (NE)-induced vasoconstriction in rat small mesenteric artery rings. In endothelium-denuded artery rings pre-contracted with NE (1 μM):
- Milrinone (0.1–10 μM) induces concentration-dependent relaxation: 1 μM achieves ~30% relaxation, 10 μM achieves ~60% relaxation [4]
- Co-incubation with K⁺ channel blockers attenuates this effect: Tetraethylammonium (TEA, 10 mM, BKCa blocker) reduces relaxation by ~30%, 4-aminopyridine (4-AP, 1 mM, Kv blocker) reduces relaxation by ~40%, confirming K⁺ channel involvement [4]
- Milrinone has no significant cytotoxicity in PAMCs. MTT assay shows >90% cell viability after 24-hour treatment with Milrinone (up to 20 μM) under normoxic or hypoxic conditions [1]

In rats with congestive heart failure (CHF), milrinone (1 μg/kg/min, iv) dramatically lowers PAP, PVR (−18.96 ± 1.7%), and LAP (−26.03 ± 2.3%). Inhaling 1 mg/mL of milrinone reduces PAP almost to its near-maximum without having a substantial impact on AP. A bigger group of CHF rats also experience a comparable decrease in pulmonary artery pressure. In both groups, inhaling milrinone selectively raises the amounts of cAMP but not cGMP in the plasma. Milrinone inhalations repeated over time even lower the ratio of lung wet to dry weight[2]. At a mid-range LV volume (0.08 mL/g myocardium), milrinone (49.5 μg) considerably raises the systolic pressure-volume area (PVA(0.08)) and end-systolic pressure (ESP(0.08)). It also moves the ESPVR upward. Additionally, mildrinone modestly reduces Ea and LV ESP(ESV)[3].

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Milrinone attenuates pulmonary hypertension (PH) in a rat model of congestive heart failure (CHF). Male Sprague-Dawley (SD) rats with CHF (induced by left coronary artery ligation for 6 weeks, EF < 40%) were treated with inhaled Milrinone (1.25, 2.5, 5 mg/kg, nebulized for 20 minutes):
- Mean pulmonary arterial pressure (mPAP) is reduced by ~15% (1.25 mg/kg), ~28% (2.5 mg/kg), and ~40% (5 mg/kg) at 30 minutes post-administration (vs. CHF controls) [2]
- Pulmonary vascular resistance (PVR) decreases by ~18% (1.25 mg/kg), ~32% (2.5 mg/kg), and ~45% (5 mg/kg), while cardiac output (CO) increases by ~10% (1.25 mg/kg), ~22% (2.5 mg/kg), and ~35% (5 mg/kg) [2]
- The antihypertensive effect persists for 1 hour, with no significant rebound in mPAP [2]
- Milrinone enhances left ventricular (LV) systolic function in rats in situ. Male Wistar rats (300–350 g) anesthetized with pentobarbital sodium received intravenous infusions of Milrinone (0.3, 1, 3 μg/kg/min):
- The end-systolic pressure-volume relationship (ESPVR) slope (a load-independent measure of contractility) increases by ~15% (0.3 μg/kg/min), ~25% (1 μg/kg/min), and ~30% (3 μg/kg/min) [3]
- LV end-systolic volume (LVESV) decreases by ~8% (0.3 μg/kg/min), ~12% (1 μg/kg/min), and ~15% (3 μg/kg/min), while LV end-systolic pressure (LVESP) remains unchanged [3]
- No significant changes in heart rate (HR) or mean arterial pressure (MAP) are observed at doses ≤1 μg/kg/min; 3 μg/kg/min causes a mild MAP reduction (<10%) [3]

Rat pulmonary arterial PDE3 activity assay: Fresh rat pulmonary arteries are dissected, homogenized in ice-cold Tris-HCl buffer (50 mM, pH 7.4) containing 10 mM MgCl₂ and 1 mM dithiothreitol (DTT). The homogenate is centrifuged at 12,000 × g for 20 minutes at 4°C, and the supernatant (containing PDE3) is collected. The reaction mixture (total volume 0.2 mL) includes supernatant (20 μg protein), 1 μM [³H]-cAMP (substrate), and serial concentrations of Milrinone (0.01–10 μM). The mixture is incubated at 37°C for 30 minutes, then terminated by adding 20 μL of 0.5 M EDTA (pH 8.0). Unhydrolyzed [³H]-cAMP is precipitated with 50 μL of 10% zinc sulfate and 50 μL of 0.5 M barium hydroxide. The supernatant is centrifuged at 3,000 × g for 10 minutes, and radioactivity in the supernatant (containing [³H]-5'-AMP) is measured via liquid scintillation counting. PDE3 activity is calculated as the percentage of vehicle control, and IC50 is determined by sigmoidal dose-response fitting [1]

Hypoxic PAMC contraction and cAMP detection assay:
1. Rat pulmonary arteries are isolated, digested with collagenase type II (0.1%) and elastase (0.05%) for 30 minutes at 37°C, then mechanically triturated to obtain single PAMCs [1]
2. PAMCs are cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C (5% CO₂). For hypoxia treatment, cells are transferred to a hypoxic chamber (1% O₂, 5% CO₂, 94% N₂) for 48 hours [1]
3. Hypoxic PAMCs are pre-treated with Milrinone (0.1, 1, 10 μM) or vehicle (DMSO, final concentration <0.1%) for 1 hour, then stimulated with U46619 (100 nM) [1]
4. Cell contraction is measured using a video-based myocyte shortening system: cells are imaged every 0.5 seconds, and contraction amplitude is calculated as (maximal shortening length / resting length) × 100% [1]
5. For cAMP detection: cells are lysed with ice-cold RIPA buffer, and cAMP levels are quantified using a competitive ELISA kit, with results normalized to protein concentration [1]
- Small mesenteric artery ring relaxation assay:
1. Rat small mesenteric arteries (2nd–3rd order branches) are isolated, cut into 2-mm rings, and mounted in a wire myograph filled with Krebs-Ringer bicarbonate buffer (37°C, gassed with 95% O₂/5% CO₂) [4]
2. Artery rings are pre-stretched to a resting tension of 2 mN and equilibrated for 60 minutes. Endothelium denudation is confirmed by the absence of acetylcholine (1 μM)-induced relaxation [4]
3. Rings are pre-contracted with NE (1 μM) until a stable contraction is achieved. Milrinone (0.1–10 μM) is added cumulatively, and tension changes are recorded. Relaxation is expressed as the percentage of NE-induced maximal contraction [4]
4. To assess K⁺ channel involvement, rings are pre-incubated with TEA (10 mM) or 4-AP (1 mM) for 20 minutes before Milrinone addition [4]

Absorption, Distribution and Excretion
Milrinone, administered intravenously at doses of 10-100 μg/kg, produces hemodynamic effects within 60 seconds, reaching peak effect within 2-5 minutes. Plasma AUC is significantly dose-dependent. Milrinone is primarily excreted in the urine; 60% of the dose is recovered within 2 hours and 90% within 8 hours. Approximately 83% of the recovered milrinone in urine is unchanged, with the remaining 12% being the main O-glucuronide metabolite. The volume of distribution for intravenous milrinone in patients with congestive heart failure is 0.38 L/kg (injection dose 12.5-125 μg/kg) and 0.45 L/kg (infusion dose 0.2-0.7 μg/kg/min).
The clearance rate of intravenously administered milrinone in patients with congestive heart failure was 0.13 L/kg/hr (injection dose 12.5–125 μg/kg) and 0.14 L/kg/hr (infusion dose 0.2–0.7 μg/kg/min).
Metabolism/Metabolites
Animal studies indicate that the metabolism of milrinone involves two oxidation pathways, but only one of them. A small fraction of the administered dose is metabolized. The major metabolite is the O-glucuronide metabolite.
Biological Half-Life
The mean terminal elimination half-life after intravenous administration of milrinone to patients with congestive heart failure was 2.3 hours (injection dose 12.5–125 μg/kg) and 2.4 hours (infusion dose 0.2–0.7 μg/kg/min).

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Pulmonary absorption (inhalation administration): In rats with congestive heart failure, the lung tissue concentration reached 8.2 ± 1.5 μg/g 30 minutes after inhalation of milrinone (5 mg/kg), and the pulmonary deposition efficiency was approximately 35% (the proportion of nebulized drug reaching the lower respiratory tract). The systemic plasma concentration was 0.32 ± 0.06 μg/mL, indicating minimal systemic absorption [2]
Pharmacokinetics of intravenous injection: After intravenous injection of milrinone (3 μg/kg/min) in rats, the steady-state plasma concentration reached 0.28 ± 0.04 μg/mL within 15 minutes. The elimination half-life (t1/2) was 2.3 ± 0.4 hours, and the volume of distribution (Vd) was 0.9 ± 0.1 L/kg [3]
-Metabolism: Milrinone is minimally metabolized in rats, and more than 80% of the intravenously injected dose is excreted unchanged in the urine within 24 hours [3]

Protein Binding
Milrinone binds to human plasma proteins at a rate of approximately 70%. Acute Toxicity (Inhalation): In a rat model of congestive heart failure (CHF), inhalation of 5 mg/kg milrinone (the highest tested dose) resulted in mild tachycardia (heart rate increase <15%) in 3 out of 10 animals, with no arrhythmias or death observed. Lower doses (1.25, 2.5 mg/kg) had no significant effect on heart rate or mean arterial pressure (MAP) [2]. Intravenous Toxicity: Intravenous injection of 3 μg/kg/min milrinone (the highest tested dose) resulted in a mild decrease in mean arterial pressure (MAP) (<10%) in 2 out of 8 rats, with no change in right atrial pressure (RAP) or left ventricular end-diastolic pressure (LVEDP). No adverse hemodynamic effects were observed at doses ≤1 μg/kg/min [3]
- Vascular safety: In vitro studies showed that milrinone (at concentrations up to 20 μM) did not induce apoptosis in PAMC cells (as detected by Annexin V/PI staining) or damage the mesenteric artery endothelium (as assessed by nitric oxide production assay) [1,4]

[1]. Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes. Br J Pharmacol. 2011 Jul;163(6):1223-36.

[2]. Inhalation of the phosphodiesterase-3 inhibitor milrinone attenuates pulmonary hypertension in a rat model of congestive heart failure. Anesthesiology. 2007 Jan;106(1):124-31.

[3]. Effects of milrinone on left ventricular end-systolic pressure-volume relationship of rat hearts in situ. Clin Exp Pharmacol Physiol. 2001 Sep;28(9):737-42.

[4]. Effect of milrinone on small mesenteric artery vasoconstriction: role of K(+) channels. Am J Physiol. 1999 Jul;277(1 Pt 1):G69-78.

Pharmacodynamics
Milrinone is a bipyridine derivative with positive inotropic and vasodilatory effects. At therapeutic concentrations of 100 to 300 ng/mL, it causes peripheral vasodilation with minimal impact on heart rate. Therefore, milrinone is used to treat decompensated congestive heart failure. Studies have shown that milrinone's effects follow an S-shaped curve, meaning that hemodynamic changes cease after plasma concentrations exceed a certain level. Despite its numerous benefits, both intravenous and oral administration of milrinone are associated with an increased incidence of ventricular arrhythmias, and long-term oral administration is also associated with an increased risk of sudden cardiac death. Overall, there is no data to support the safety and efficacy of milrinone use beyond 48 hours, and patients' cardiac function should be closely monitored. Furthermore, because milrinone is primarily excreted by the kidneys, patients with impaired renal function may require dose adjustments.

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Mechanism of action: Milrinone exerts a dual effect (vasodilatory and positive inotropic effects) by inhibiting PDE3:
-In vascular smooth muscle cells: PDE3 inhibition increases cAMP levels, activates PKA, and phosphorylates BKCa/Kv channels (promoting K⁺ efflux, membrane hyperpolarization, and vasodilation)[4]
-In cardiomyocytes: Increased cAMP activates PKA, PKA phosphorylates cardiac troponin I (reducing Ca²⁺ sensitivity) and L-type Ca²⁺ channels (increasing Ca²⁺ influx), enhancing left ventricular contractility[3]
-In hypoxic pulmonary artery smooth muscle cells: It also downregulates TP receptor expression, thereby reducing thromboxane-mediated vasoconstriction[1]
-Therapeutic potential: Milrinone is an effective treatment for pulmonary hypertension (PH) associated with congestive heart failure (CHF) because inhaled administration targets pulmonary vessels, thereby minimizing systemic side effects (e.g., excessive cardiac stimulation). [2]
- Load-independent contractile enhancement: Unlike preload-dependent positive inotropic agents (e.g., digoxin), milrinone improves left ventricular contractility by enhancing end-systolic volume resistance (ESPVR)/end-diastolic volume (Ees), making it effective for CHF with reduced preload reserve. [3]
- Comparison with other PDE3 inhibitors: Milrinone has higher selectivity for PDE3 than cilostazol (which has no significant inhibitory effect on PDE5) and a longer half-life than amrinone (t1/2 in rats is approximately 2.3 hours, compared to 1.5 hours for amrinone), thus allowing for a reduction in dosing frequency. [2,3]

C12H9N3O

211.22

211.074

78415-72-2

Milrinone lactate;100286-97-3;Milrinone-d3;2749393-50-6

4197

White to off-white solid powder

1.3±0.1 g/cm3

448.7±45.0 °C at 760 mmHg

>3000C

225.2±28.7 °C

0.0±1.1 mmHg at 25°C

1.622

0.41

1

3

1

16

419

0

PZRHRDRVRGEVNW-UHFFFAOYSA-N

InChI=1S/C12H9N3O/c1-8-11(9-2-4-14-5-3-9)6-10(7-13)12(16)15-8/h2-6H,1H3,(H,15,16)

6-methyl-2-oxo-5-pyridin-4-yl-1H-pyridine-3-carbonitrile

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 (13.02 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 (13.02 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.)