Zinc(II)-bis(dipicolylamine) (Zn-BDPA) coordination complexes selectively target the surfaces of dead and dying mammalian cells, and they have promise as molecular probes for imaging cell death. A necessary step toward eventual clinical imaging applications is the development of next-generation Zn-BDPA complexes with enhanced affinity for the cell death membrane biomarker, phosphatidylserine (PS). This study employed an iterative cycle of library synthesis and screening, using a novel rapid equilibrium dialysis assay, to discover a modified Zn-BDPA structure with high and selective affinity for vesicles containing PS. The lead structure was converted into a deep-red fluorescent probe and its targeting and imaging performance was compared with an unmodified control Zn-BDPA probe. The evaluation process included a series of FRET-based vesicle titration studies, cell microscopy experiments, and rat tumor biodistribution measurements. In all cases, the modified probe exhibited comparatively higher affinity and selectivity for the target membranes of dead and dying cells. The results show that this next-generation deep-red fluorescent Zn-BDPA probe is well suited for preclinical molecular imaging of cell death in cell cultures and animal models. Furthermore, it should be possible to substitute the deep-red fluorophore with alternative reporter groups that enable clinically useful, deep-tissue imaging modalities, such as MRI and nuclear imaging.
Treprostinil (TRE), a prostanoid analogue approved in the USA for the treatment of pulmonary arterial hypertension, requires continuous infusion or multiple dosing sessions per day for inhaled and oral routes of administration due to its short half-life. The inhaled drug is known to induce adverse systemic and local effects including headache, nausea, cough, and throat irritation which may be due at least in part to transiently high drug concentrations in the lungs and plasma immediately following administration [1]. To ameliorate these side effects and reduce dosing frequency we designed an inhaled slow-release TRE formulation. TRE was chemically modified to be an alkyl prodrug (TPD) which was then packaged into a lipid nanoparticle (LNP) carrier. Preclinical screening in a rat model of hypoxia-induced pulmonary vasoconstriction led to selection of a 16-carbon alkyl ester derivative of TRE. The TPD-LNP demonstrated approximately 10-fold lower TRE plasma C compared to inhaled TRE solution while maintaining an extended vasodilatory effect. The favorable PK profile is attributed to gradual dissociation of TPD from the LNP and subsequent conversion to TRE. Together, this sustained presentation of TRE to the lungs and plasma is consistent with a once- or twice-daily dosing schedule in the absence of high C-associated adverse events which could provide patients with an improved treprostinil therapy.
There is a clinical need for imaging technologies that can accurately detect cell death in a multitude of pathological conditions. Zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to associate with the anionic phosphatidylserine that is exposed on the surface of dead and dying cells, and fluorescent monovalent Zn2BDPA probes are successful cell death imaging agents. This present study compared the membrane targeting ability of two structurally related deep-red fluorescent probes, Bis-Zn2BDPA-SR and Tetra-Zn2BDPA-SR, with two and four appended Zn2BDPA units, respectively. Vesicle and cell microscopy studies indicated that a higher number of Zn2BDPA targeting units improved probe selectivity for phosphatidylserine-rich vesicles, and increased probe localization at the plasma membrane of dead and dying cells. The fluorescent probes were also tested in three separate animal models, 1) necrotic prostate tumor rat model, 2) thymus atrophy mouse model, and 3) traumatic brain injury mouse model. In each case there was more Tetra-Zn2BDPA-SR accumulation at the site of cell death than Bis-Zn2BDPA-SR. The results indicate that multivalent Zn2BDPA probes are promising molecules for effective imaging of cell death processes in cell culture and in living subjects.
This article describes the preclinical pharmacology and pharmacokinetics (PK) of hexadecyl-treprostinil (C16TR), a prodrug of treprostinil (TRE), formulated in a lipid nanoparticle (LNP) for inhalation as a pulmonary vasodilator. C16TR showed no activity (>10 M) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E receptor 2, prostaglandin D receptor 1, prostaglandin I receptor, and prostaglandin E receptor 4; TRE potently bound to each of these prostanoid receptors. C16TR had no effect (up to 200 nM) on platelet aggregation induced by ADP in rat blood. In hypoxia-challenged rats, inhaled C16TR-LNP produced dose-dependent (0.06-6 g/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6g/kg) was active at earlier times but lost its effect by 3 hours. Single- and multiple-dose PK studies of inhaled C16TR-LNP in rats showed proportionate dose-dependent increases in TRE and area under the curve (AUC) for both plasma and lung; similar results were observed for dog plasma levels in single-dose PK studies. In both species, inhaled C16TR-LNP yielded prolonged plasma TRE levels and a lower plasma TRE compared with inhaled TRE. Inhaled C16TR-LNP was well tolerated in rats and dogs; TRE-related side effects included cough, respiratory tract irritation, and emesis and were seen only after high inhaled doses of C16TR-LNP in dogs. In guinea pigs, inhaled TRE (30 g/ml) consistently produced cough, but C16TR-LNP (30g/ml) elicited no effect. These results demonstrate that C16TR-LNP provides long-acting pulmonary vasodilation, is well tolerated in animal studies, and may necessitate less frequent dosing than inhaled TRE with possibly fewer side effects.
INS1009 is a long acting pulmonary vasodilator prodrug of treprostinil (TRE) that is formulated in a lipid nanoparticle for inhaled delivery by nebulization. This study examined the ability of INS1009 to inhibit vasoconstriction in the pulmonary vasculature of rats and dogs and the extent to which local activity within the lung contributes to its activity. Rats received a single dose of INS1009 by nose-only inhalation or were given a continuous intravenous (i.v.) infusion of TRE, followed by an i.v. challenge of the thromboxane mimetic pulmonary vasoconstrictor U46619 and the increase in pulmonary arterial pressure (PAP) was measured. In beagle dogs, INS1009 was given by inhalation via face mask and TRE was given by continuous i.v. infusion; vasoconstriction was then induced by inhaled hypoxia with reduction of FIO to 0.10. Changes in the dog's right ventricular pulse pressure (RVPP) were measured using implanted telemetry probes. Blood samples were collected in rats and dogs immediately after the challenge to measure the plasma TRE concentration. Exposure of rats to inhaled INS1009 (0.5, 3.0 and 20.9 μg/kg) inhibited the U46619-induced increase in PAP at all doses up to 6 h with statistically significant inhibition up to 24 h with the pooled dose-response data. The concentration of TRE in the plasma at which PAP was reduced by 50% was approximately 60-fold lower for INS1009 (EC = 0.08 ng/mL) as compared to i.v. TRE (EC = 4.9 ng/mL). In dogs, INS1009 (2.7-80.9 μg/kg) inhibited the hypoxia-induced increase in RVPP at all doses up to 6 h with activity once again observed with the pooled dose-response of 10 μg/kg and higher at 24 h. The concentration of TRE in the plasma at which RVPP was reduced by 50% was approximately 550-fold lower for INS1009 (EC = 0.0075 ng/mL) as compared to i.v. TRE (EC = 4.1 ng/mL). These studies, in two species and by two different pulmonary vasoconstrictor challenges, demonstrate that inhaled INS1009 not only has long-acting vasodilatory effects but also that the local activity within the lung contributes to this response. Therefore, INS1009 may offer the opportunity to effect pulmonary vasodilation for long periods but with substantially lower systemic exposure than infused TRE.
An admixture of zinc(II)-bis(dipicolylamine) receptor with covalently attached paramagnetic relaxation agent and fluorine-labeled phosphate indicator enables 19F NMR detection of phosphorylated analytes with amplified switched-on signal intensity.
5-Aminolevulinic acid (5-ALA), a prodrug of Protoporphyrin IX (PpIX), is used for photodynamic therapy of several medical conditions, and as an adjunct for fluorescence guided surgery. The clinical problem of patient photosensitivity after systemic administration could likely be ameliorated if the 5-ALA was delivered more selectivity to the treatment site. Liposomal formulations are inherently attractive as targeted delivery vehicles but it is hard to regulate the spatiotemporal release of aqueous contents from a liposome. Here, we demonstrate chemically triggered leakage of 5-ALA from stealth liposomes in the presence of cell culture. The chemical trigger is a zinc(II)-dipicolylamine (ZnBDPA) coordination complex that selectively targets liposome membranes containing a small amount of anionic phosphatidylserine. Systematic screening of several ZnBDPA complexes uncovered a compound with excellent performance in biological media. Cell culture studies showed triggered release of 5-ALA from stealth liposomes followed by uptake into neighboring mammalian cells and intracellular biosynthesis to form fluorescent PpIX.
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