Sustained release of milrinone delivered via microparticles in a rodent model of myocardial infarction.

OBJECTIVE: The aim of the present study was to construct a new drug delivery system for milrinone using microparticles. This novel technology enhances drug bioavailability and decreases toxicity, with future implications for the treatment of end-stage heart failure. METHODS: Polylactic-co-glycolic acid microparticles (PLGA-MPs) loaded with milrinone were prepared using a double emulsion-solvent evaporation technique. In vitro release kinetics was evaluated at physiologic conditions. A total of 24 female Lewis rats underwent left coronary artery ligation. One week after ligation, all rats were randomized to 1 of 3 groups (n=8 per group). Group I received an intravenous injection of PLGA-MPs alone; group II, a bolus intravenous injection of milrinone; and group III an intravenous injection of milrinone-PLGA-MPs. All injections were administrated slowly by way of the tail vein over 10 minutes. Transthoracic echocardiography, noninvasive heart rate monitoring, and blood pressure measurements were performed at different predetermined intervals before and for 24 hours after the injection. All rats survived for 24 hours and were then killed by euthanasia. Serum plasma was taken for cytokine assays and determination of milrinone levels using high-performance liquid chromatography. RESULTS: Group III had a significantly greater left ventricular ejection fraction at 90 minutes and 3, 6, and 12 hours after treatment compared with the other groups. The milrinone plasma level was significantly greater in group III than in the other groups (group I, 0 ng/mL; group II, 1.7±2.4 ng/mL; group III, 9.1±2.2 ng/mL; P<.05). The intercellular adhesion molecule and cytokine-induced neutrophil chemoattractant-1 levels were significantly lower in group III than in the other 2 groups (P<.05). CONCLUSIONS: Drug encapsulation using microparticles can prolong the effects of milrinone. We propose a new strategy for future drug delivery in patients with end-stage heart failure.

Persistent cAMP signaling by TSH receptors revealed by phosphodiesterase inhibition.

BACKGROUND: It is controversial whether persistent signaling by the thyrotropin (TSH) receptor (TSHR) is cell-type specific. We reported persistent TSHR signaling in human embryonic kidney 293 (HEK293) cells expressing human TSHRs (HEK-TSHRs), whereas another group reported persistent signaling in mouse thyroid follicles but not in HEK293 cells. Herein, we test this hypothesis directly. METHODS: We used two methods to measure persistent signaling in HEK-TSHRs and confirm our previous observations. In Method 1, we used a chemiluminescent immunoassay to measure intracellular cAMP accumulation over 30-60 min by adding a phosphodiesterase inhibitor to the incubation medium. In Method 2, we used an intracellular biosensor to record cAMP levels continuously. RESULTS: Using Method 1, we show that TSHR signals persistently in human thyrocytes and human osteosarcoma U2OS-TSHR cells. Using Method 1 in HEK-TSHRs, we show that after 5 min, the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) increases cAMP to 2.5 pmol/well, TSH increases cAMP to 1.6 pmol/well, but IBMX added 30 min after TSH withdrawal increases cAMP to 105 pmol/well. Using Method 2 in HEK-TSHRs, we confirm that without IBMX, TSH causes a transient increase in cAMP and 30 min after TSH withdrawal, IBMX increases cAMP in cells pretreated with TSH more rapidly and to a higher level than IBMX added to cells not pre-exposed to TSH. Lastly, using Method 2, we show that in HEK-TSHRs phosphodiesterases types 3 and 4 are involved in degrading cAMP as the specific inhibitors Rolipram and Milrinone expose persistent TSHR signaling. CONCLUSIONS: We conclude that persistent TSHR activation occurs in human thyrocytes, U2OS-TSHR cells and HEK-TSHRs; it is not cell-type specific but is revealed by inhibiting phosphodiesterases.

Neurite outgrowth mediated by translation elongation factor eEF1A1: a target for antiplatelet agent cilostazol.

Cilostazol, a type-3 phosphodiesterase (PDE3) inhibitor, has become widely used as an antiplatelet drug worldwide. A recent second Cilostazol Stroke Prevention Study demonstrated that cilostazol is superior to aspirin for prevention of stroke after an ischemic stroke. However, its precise mechanisms of action remain to be determined. Here, we report that cilostazol, but not the PDE3 inhibitors cilostamide and milrinone, significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Furthermore, specific inhibitors for the endoplasmic reticulum protein inositol 1,4,5-triphosphate (IP(3)) receptors and several common signaling pathways (PLC-γ, PI3K, Akt, p38 MAPK, and c-Jun N-terminal kinase (JNK), and the Ras/Raf/ERK/MAPK) significantly blocked the potentiation of NGF-induced neurite outgrowth by cilostazol. Using a proteomics analysis, we identified that levels of eukaryotic translation elongation factor eEF1A1 protein were significantly increased by treatment with cilostazol, but not cilostamide, in PC12 cells. Moreover, the potentiating effects of cilostazol on NGF-induced neurite outgrowth were significantly antagonized by treatment with eEF1A1 RNAi, but not the negative control of eEF1A1. These findings suggest that eEF1A1 and several common cellular signaling pathways might play a role in the mechanism of cilostazol-induced neurite outgrowth. Therefore, agents that can increase the eEF1A1 protein may have therapeutic relevance in diverse conditions with altered neurite outgrowth.