Preclinical Investigation of a Lipoglycopeptide Dry Powder Inhalation Therapy for the Treatment of Pulmonary MRSA Infection.

The increased prevalence of pulmonary methicillin-resistant Staphylococcus aureus (MRSA) infection in patients living with cystic fibrosis (CF) is concerning due to a correlation with reduced life expectancy and lack of available treatment options. RV94 is a next generation lipoglycopeptide designed for pulmonary delivery that preclinically demonstrated high potency against MRSA in planktonic and protected colonies and improved pulmonary clearance relative to same class molecules. Here, RV94 was formulated into a dry powder for inhalation (DPI) to investigate the localized treatment of pulmonary MRSA presented in a potentially more convenient dosage form. RV94 DPI was generated using a spray-drying process with 12.5 wt% trileucine and demonstrated aerosol characteristics (2.0 µm MMAD and 73% FPF) predictive of efficient pulmonary deposition. In vivo PK from a single dose of RV94 DPI delivered by inhalation to rats yielded lung levels (127 µg/g) much greater than the MRSA minimum inhibitory concentration (0.063 µg/mL), low systemic levels (0.1 µg/mL), and a lung t1/2 equal to 3.5 days. In a rat acute pulmonary MRSA model, a single dose of RV94 DPI delivered by inhalation either up to seven days prior to or 24 h after infection resulted in a statistically significant reduction in lung MRSA titer.

Assessment of Inhaled Treprostinil Palmitil, Inhaled and Intravenous Treprostinil, and Oral Selexipag in a Sugen/Hypoxia Rat Model of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP), a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE), has beneficial effects in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension (PAH) that compare favorably to the oral phosphodiesterase 5 inhibitor (PDE5) sildenafil. In this study in male Sprague-Dawley rats, a dry powder formulation of TP (TPIP) was compared with inhaled and intravenous TRE and oral selexipag to evaluate inhibition of hemodynamic and pathologic changes in the lungs and heart induced by Su/Hx challenge. Su (20 mg/kg) was injected subcutaneously followed by 3 weeks of Hx (10% O2/balance N2) and then initiation of test article administration over 5 weeks with room air breathing. Hemodynamics and histopathology were measured at the end of the study. Su/Hx challenge approximately doubled the mean pulmonary arterial blood pressure (mPAP) and the Fulton index, decreased cardiac output (CO), doubled the wall thickness and muscularization of the small (10-50 µm) and medium (51-100 µm) sized pulmonary arteries, and increased the percentage of obliterated pulmonary blood vessels. Even though inhaled TRE (65 µg/kg, 4× daily), intravenous TRE (810 ng/kg/min), and oral selexipag (30 mg/kg, twice daily) provided some beneficial effects against the Su/Hx challenge, the overall benefit was generally greater with TPIP at high dose (117 µg/kg, once daily). These results demonstrate that TPIP compares favorably to inhaled and intravenous TRE and oral selexipag with respect to inhibition of the pathophysiological changes induced by Su/Hx challenge in rats. SIGNIFICANCE STATEMENT: Treprostinil palmitil (TP) is a long-acting pulmonary vasodilator prodrug of treprostinil (TRE) formulated for inhaled administration by dry powder [treprostinil palmitil inhalation powder (TPIP)]. Comparison of the activity of TPIP, inhaled and intravenous TRE, and oral selexipag in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension demonstrated that each of these drugs exert protection against the hemodynamic and histopathological changes induced by the Su/Hx challenge, with the greatest effect on these changes produced by TPIP.

Strategies to Overcome Biological Barriers Associated with Pulmonary Drug Delivery.

While the inhalation route has been used for millennia for pharmacologic effect, the biological barriers to treating lung disease created real challenges for the pharmaceutical industry until sophisticated device and formulation technologies emerged over the past fifty years. There are now several inhaled device technologies that enable delivery of therapeutics at high efficiency to the lung and avoid excessive deposition in the oropharyngeal region. Chemistry and formulation technologies have also emerged to prolong retention of drug at the active site by overcoming degradation and clearance mechanisms, or by reducing the rate of systemic absorption. These technologies have also been utilized to improve tolerability or to facilitate uptake within cells when there are intracellular targets. This paper describes the biological barriers and provides recent examples utilizing formulation technologies or drug chemistry modifications to overcome those barriers.

Treprostinil palmitil inhibits the hemodynamic and histopathological changes in the pulmonary vasculature and heart in an animal model of pulmonary arterial hypertension.

Treprostinil palmitil (TP) is a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE). In this study, TP was delivered by inhalation (treprostinil palmitil inhalation suspension, TPIS) in a rat Sugen 5416 (Su)/hypoxia (Hx) model of pulmonary arterial hypertension (PAH) to evaluate its effects on hemodynamics, pulmonary vascular remodeling, and cardiac performance and histopathology. Male Sprague-Dawley rats received Su (20 mg/kg, s.c), three weeks of Hx (10% O2) and 5 or 10 weeks of normoxia (Nx). TPIS was given during the 5-10 week Nx period after the Su/Hx challenge. Su/Hx increased the mean pulmonary arterial blood pressure (mPAP) and right heart size (Fulton index), reduced cardiac output (CO), stroke volume (SV) and heart rate (HR), and increased the thickness and muscularization of the pulmonary arteries along with obliteration of small pulmonary vessels. In both the 8- and 13-week experiments, TPIS at inhaled doses ranging from 39.6 to 134.1 µg/kg, QD, dose-dependently improved pulmonary vascular hemodynamics, reduced the increase in right heart size, enhanced cardiac performance, and attenuated most of the histological changes induced by the Su/Hx challenge. The PDE5 inhibitor sildenafil, administered at an oral dose of 50 mg/kg, BID for 10 weeks, was not as effective as TPIS. These results in Su/Hx challenged rats demonstrate that inhaled TPIS may have superior effects to oral sildenafil. We speculate that the improvement of the pathobiology in this PAH model induced by TPIS involves effects on pulmonary vascular remodeling due to the local effects of TRE in the lungs.

Recent advances in prodrug-based nanoparticle therapeutics.

Extensive research into prodrug modification of active pharmaceutical ingredients and nanoparticle drug delivery systems has led to unprecedented levels of control over the pharmacological properties of drugs and resulted in the approval of many prodrug or nanoparticle-based therapies. In recent years, the combination of these two strategies into prodrug-based nanoparticle drug delivery systems (PNDDS) has been explored as a way to further advance nanomedicine and identify novel therapies for difficult-to-treat indications. Many of the PNDDS currently in the clinical development pipeline are expected to enter the market in the coming years, making the rapidly evolving field of PNDDS highly relevant to pharmaceutical scientists. This review paper is intended to introduce PNDDS to the novice reader while also updating those working in the field with a comprehensive summary of recent efforts. To that end, first, an overview of FDA-approved prodrugs is provided to familiarize the reader with their advantages over traditional small molecule drugs and to describe the chemistries that can be used to create them. Because this article is part of a themed issue on nanoparticles, only a brief introduction to nanoparticle-based drug delivery systems is provided summarizing their successful application and unfulfilled opportunities. Finally, the review's centerpiece is a detailed discussion of rationally designed PNDDS formulations in development that successfully leverage the strengths of prodrug and nanoparticle approaches to yield highly effective therapeutic options for the treatment of many diseases.

Development and Preclinical Evaluation of New Inhaled Lipoglycopeptides for the Treatment of Persistent Pulmonary Methicillin-Resistant Staphylococcus aureus Infections.

Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.

Characterisation of cough evoked by inhaled treprostinil and treprostinil palmitil.

Cough is induced by inhaled prostacyclin analogues including treprostinil (TRE), and, at higher doses, treprostinil palmitil (TP), a prodrug of TRE. In this report, we have investigated mechanisms involved in TRE- and TP-induced cough, using a dry powder formulation of TP (TPIP) to supplement previous data obtained with an aqueous suspension formulation of TP (TPIS). Experiments in guinea pigs and rats investigated the prostanoid receptor subtype producing cough and whether it involved activation of sensory nerves in the airways and vasculature. Experiments involved treatment with prostanoid, tachykinin and bradykinin receptor antagonists, a cyclooxygenase inhibitor and TRE administration to the isolated larynx or intravenously. In guinea pigs, cough with inhaled TRE (1.23 µg·kg-1) was not observed with an equivalent dose of TPIP and required higher inhaled doses (12.8 and 35.8 µg·kg-1) to induce cough. TRE cough was blocked with IP and tachykinin NK1 receptor antagonists but not with EP1, EP2, EP3, DP1 or bradykinin B2 antagonists or a cyclooxygenase inhibitor. TRE administered to the isolated larynx or intravenously in rats produced no apnoea or swallowing, whereas citric acid, capsaicin and hypertonic saline had significant effects. The mechanisms inducing cough with inhaled TRE likely involves the activation of prostanoid IP receptors on jugular C-fibres in the tracheobronchial airways. Cough induced by inhaled dry powder and nebulised formulations of TP occurs at higher inhaled doses than TRE, presumably due to the slow, sustained release of TRE from the prodrug resulting in lower concentrations of TRE at the airway sensory nerves.

Development and Characterization of Treprostinil Palmitil Inhalation Aerosol for the Investigational Treatment of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP) is a prodrug of treprostinil (TRE), a pulmonary vasodilator that has been previously formulated for inhaled administration via a nebulizer. TP demonstrates a sustained presence in the lungs with reduced systemic exposure and prolonged inhibition of hypoxia-induced pulmonary vasoconstriction in vivo. Here, we report on re-formulation efforts to develop a more convenient solution-based metered-dose inhaler (MDI) formulation of TP, a treprostinil palmitil inhalation aerosol (TPIA) that matches the pharmacokinetic (PK) and efficacy profile of a nebulized TP formulation, treprostinil palmitil inhalation suspension (TPIS). MDI canisters were manufactured using a two-stage filling method. Aerosol performance, formulation solubility, and chemical stability assays were utilized for in vitro evaluation. For in vivo studies, TPIA formulations were delivered to rodents using an inhalation tower modified for MDI delivery. Using an iterative process involving evaluation of formulation performance in vitro (TP and excipient solubility, chemical stability, physical stability, and aerosol properties) and confirmatory testing in vivo (rat PK and efficacy, guinea pig cough), a promising formulation was identified. The optimized formulation, TPIA-W, demonstrates uniform in vitro drug delivery, a PK profile suitable for a once-daily administration, efficacy lasting at least 12 h in a hypoxic challenge model, and a significantly higher cough threshold than the parent drug treprostinil.

Treprostinil palmitil, an inhaled long-acting pulmonary vasodilator, does not show tachyphylaxis with daily dosing in rats.

BACKGROUND: Treprostinil palmitil (TP) is an inhaled long-acting pulmonary vasodilator prodrug of treprostinil (TRE) that has been formulated for delivery as a suspension (treprostinil palmitil inhalation suspension; TPIS) and as a dry powder (treprostinil palmitil inhalation powder; TPIP). In humans, tachyphylaxis is frequently observed with continuous intravenous (IV) or subcutaneous (SC) infusion of TRE and requires dosage escalation to maintain activity. The aim of the present study was to determine whether tachyphylaxis occurs with repeat daily administration of inhaled TPIS. METHODS: Experiments were performed in male Sprague-Dawley rats prepared with a telemetry probe implanted into the right ventricle to measure the change in right ventricular pulse pressure (ΔRVPP) induced by exposure to a 10% oxygen gas mixture. TPIS (6 mL) at concentrations of 0.25, 0.5, and 1 mM was given by nose-only inhalation using an Aeroneb Pro nebulizer, either as a single administration or daily for 16 or 32 consecutive days. In studies involving consecutive daily administrations of TPIS, the delivered TP dosage was 140.3 µg/kg at 1 mM and ranged from 40.2 to 72.2 µg/kg at 0.5 mM. A separate cohort of telemetered rats received continuous IV infusion of TRE via an Alzet mini-pump at a dosage rate of 250 ng/kg/min for 16 days. Blood and lung tissue samples were obtained, and the concentration of TRE in the plasma and TRE and TP in the lungs were measured approximately 1 h after TPIS administration. RESULTS: Dose-response studies with TPIS administered as a single administration inhibited the hypoxia-induced increase in RVPP in both a concentration-dependent (0.25, 0.5, and 1 mM) and time-dependent (1-24 h) manner. TPIS, given QD or BID at inhaled doses ranging from 40.2 to 140.3 µg/kg for 16 or 32 consecutive days, produced statistically significant (P < .05) inhibition of the increase of RVPP due to hypoxia over the full duration of the dosing periods. By contrast, the inhibition of the hypoxia-induced increase in RVPP observed with IV TRE infusion (250 ng/kg/min) disappeared after 16 days of infusion. The plasma concentrations of TRE were significantly higher after IV TRE (range, 2.85-13.35 ng/mL) compared to inhaled TPIS (range, 0.22-0.73 ng/mL) CONCLUSIONS: There was no evidence of tachyphylaxis with repeat daily dosing of TPIS for a period of up to 32 days. The absence of tachyphylaxis with TPIS is likely related to its local vasodilatory effects within the lungs, combined with an absence of sustained high plasma concentrations of TRE.

An overview of the biology of a long-acting inhaled treprostinil prodrug.

Treprostinil (TRE) is a prostanoid analog pulmonary vasodilator drug marketed with subcutaneous, intravenous (i.v.), oral, and inhaled routes of administration for the treatment of pulmonary arterial hypertension (PAH). Due to its short half-life, TRE requires either continuous infusion or multiple dosing, which exacerbates its side effects. Therefore, a long-acting prostanoid analog that maintains the positive attributes of TRE but has fewer TRE-related side effects could be of clinical benefit. In this report, we describe the discovery, preclinical development, and biology of the TRE ester prodrug, treprostinil palmitil (TP), which is formulated in a lipid nanoparticle (LNP) for administration as a nebulized inhaled suspension (TPIS). In screening assays focused on the conversion of prodrug to TRE, TP (16 carbon alkyl chain) had the slowest rate of conversion compared with short-alkyl chain TRE prodrugs (i.e., 2-8 carbon alkyl chain). Furthermore, TP is a pure prodrug and possesses no inherent binding to G-protein coupled receptors including prostanoid receptors. Pharmacokinetic studies in rats and dogs demonstrated that TPIS maintained relatively high concentrations of TP in the lungs yet had a low maximum plasma concentrations (Cmax) of both TP and, more importantly, the active product, TRE. Efficacy studies in rats and dogs demonstrated inhibition of pulmonary vasoconstriction induced by exposure to hypoxic air or i.v.-infused U46619 (thromboxane mimetic) over 24 h with TPIS. Cough was not observed with TPIS at an equivalent dose at which TRE caused cough in guinea pigs and dogs, and there was no evidence of desensitization to the inhibition of pulmonary vasoconstriction in rats with repeat inhaled dosing. TPIS was also more efficacious than i.v.-infused TRE in a sugen/hypoxia rat model of PAH to inhibit pulmonary vascular remodeling, an effect likely driven by local activities of TRE within the lungs. TPIS also demonstrated antifibrotic and anti-inflammatory activity in the lungs in rodent models of pulmonary fibrosis and asthma. In a phase 1 study in healthy human participants, TPIS (referred to as INS1009) had a lower plasma TRE Cmax and fewer respiratory-related side effects at equimolar doses compared with inhaled TRE. We have now formulated TP as an aerosol powder for delivery by a dry powder inhaler (referred to as treprostinil palmitil inhalation powder-TPIP), and as an aerosol solution in a fluorohydrocarbon solvent for delivery by a metered dose inhaler. These options may reduce drug administration time and involve less device maintenance compared with delivery by nebulization.

Inhaled Treprostinil-Prodrug Lipid Nanoparticle Formulations Provide Long-Acting Pulmonary Vasodilation.

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 Cmax 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 Cmax-associated adverse events which could provide patients with an improved treprostinil therapy.

Inhaled hexadecyl-treprostinil provides pulmonary vasodilator activity at significantly lower plasma concentrations than infused treprostinil.

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 FIO2 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 (EC50 = 0.08 ng/mL) as compared to i.v. TRE (EC50 = 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 (EC50 = 0.0075 ng/mL) as compared to i.v. TRE (EC50 = 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.

Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug.

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 µM) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E2 receptor 2, prostaglandin D2 receptor 1, prostaglandin I2 receptor, and prostaglandin E2 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 µg/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6 µg/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 Cmax 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 Cmax 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 µg/ml) consistently produced cough, but C16TR-LNP (30 µg/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.

Evaluation of [¹¹¹In]-labeled zinc-dipicolylamine tracers for SPECT imaging of bacterial infection.

PURPOSE: This study prepared three structurally related zinc-dipicolylamine (ZnDPA) tracers with [(111)In] labels and conducted biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging studies of a mouse leg infection model. PROCEDURES: Two monovalent tracers, ZnDPA-[(111)In]DTPA and ZnDPA-[(111)In]DOTA, each with a single zinc-dipicolylamine targeting unit, and a divalent tracer, Bis(ZnDPA)-[(111)In]DTPA, with two zinc-dipicolylamine units were prepared. Organ biodistribution and SPECT and CT imaging studies were performed on living mice with a leg infection created by injection of clinically relevant Gram positive Streptococcus pyogenes. Fluorescent and luminescent Eu(3+)-labeled versions of these tracers were also prepared and used to measure relative affinity for the exterior membrane surface of bacterial cells and mimics of healthy mammalian cells. RESULTS: All three (111)In-labeled radiotracers were prepared with a radiopurity of >90 %. The biodistribution studies showed that the two monovalent tracers were cleared from the body through the liver and kidney, with retained percentage injected dose for all organs of <8 % at 20 h and infected leg target to non-target ratio (T/NT) ratio of ≤3.0. Clearance of the divalent tracer from the bloodstream was slower and primarily through the liver, with a retained percentage injected dose for all organs <37 % at 20 h and T/NT ratio rising to 6.2 after 20 h. The SPECT/CT imaging indicated the same large difference in tracer pharmacokinetics and higher accumulation of the divalent tracer at the site of infection. CONCLUSIONS: All three [(111)In]-ZnDPA tracers selectively targeted the site of a clinically relevant mouse infection model that could not be discerned by visual external inspection of the living animal. The highest target selectivity, observed with a divalent tracer equipped with two zinc-dipicolylamine targeting units, compares quite favorably with the imaging selectivities previously reported for other nuclear tracers that target bacterial cell surfaces. The tracer pharmacokinetics depended heavily on tracer molecular structure suggesting that it may be possible to rapidly fine tune the structural properties for optimized in vivo imaging performance and clinical translation.

Chemically triggered release of 5-aminolevulinic acid from liposomes.

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.

¹9F NMR indicator displacement assay using a synthetic receptor with appended paramagnetic relaxation agent.

An admixture of zinc(II)-bis(dipicolylamine) receptor with covalently attached paramagnetic relaxation agent and fluorine-labeled phosphate indicator enables (19)F NMR detection of phosphorylated analytes with amplified switched-on signal intensity.

Library synthesis, screening, and discovery of modified Zinc(II)-Bis(dipicolylamine) probe for enhanced molecular imaging of cell death.

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.

Enhanced cell death imaging using multivalent zinc(II)-bis(dipicolylamine) fluorescent probes.

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.

Selective non-covalent triggered release from liposomes.

A zinc(II)-dipicolylamine coordination complex selectively associates with anionic liposomes, including sterically protected PEGylated liposomes, and causes rapid leakage of encapsulated contents.