Biodistribution of Native and Nanoformulated Innate Defense Regulator Peptide 1002.

Innate defense regulator-1002 (IDR-1002) is a synthetic peptide with promising immunomodulatory and antibiofilm properties. An appreciable body of work exists around its mechanism of action at the cellular and molecular level, along with its efficacy across several infection and inflammation models. However, little is known about its absorption, distribution, and excretion in live organisms. Here, we performed a comprehensive biodistribution assessment with a gallium-67 radiolabeled derivative of IDR-1002 using nuclear tracing techniques. Various dose levels of the radiotracer (2-40 mg/kg) were administered into the blood, peritoneal cavity, and subcutaneous tissue, or instilled into the lungs. The peptide was well tolerated at all subcutaneous and intraperitoneal doses, although higher levels were associated with delayed absorption kinetics and precipitation of the peptide within the tissues. Low intratracheal doses were rapidly absorbed systemically, and small increases in the dose level were lethal. Intravenous doses were rapidly cleared from the blood at lower levels, and upon escalation, were toxic with a high proportion of the dose accumulating within the lung tissue. To improve biocompatibility and prolong its circulation within the blood, IDR-1002 was further formulated onto high molecular weight hyperbranched polyglycerol (HPG) polymers. Constructs prepared at 5:1 and 10:1 peptide-to-polymer ratios were colloidally stable, maintained the biological profile of the peptide payload and helped reduce red blood cell lysis. The 5:1 construct circulated well in the blood, but higher peptide loading was associated with rapid clearance by the reticuloendothelial system. Many peptides face pharmacokinetic and biocompatibility challenges, but formulations such as those with HPG have the potential to overcome these limitations.

Biodistribution of the cationic host defense peptide LL-37 using SPECT/CT.

Human cathelicidin LL-37, a cationic host defense peptide (CHDP), has several important physiological roles, including antimicrobial activity, immune modulation, and wound healing, and is a being investigated as a therapeutic candidate for several indications. While the effects of endogenously produced LL-37 are well studied, the biodistribution of exogenously administered LL-37 are less known. Here we assess the biodistribution of a gallium-67 labeled variant of LL-37 using nuclear imaging techniques over a 48 h period in healthy mice. When administered as an intravenous bolus just over 20 µg, the LL-37-based radiotracer was rapidly cleared from the blood, largely by the liver, while an appreciable fraction of the dose temporarily distributed to the lungs. When administered subcutaneously at the same dose level, the radiotracer was absorbed systemically following a two-phase kinetic model and was predominately cleared renally. Uptake into sites rich in immune cells, such as the lymph nodes and the spleen, was observed for both routes of administration. Scans of free gallium-67 were also performed as controls. Important preclinical insights into the biodistribution of exogenously administered LL-37 were gained from this study, which can aid in the understanding of this and related cationic host-defense peptides.

Biodistribution and toxicity of innate defense regulator 1018 (IDR-1018).

Innate defense regulators (IDRs) are synthetic host-defense peptides (HDPs) with broad-spectrum anti-infective properties, including immunomodulatory, anti-biofilm and direct antimicrobial activities. A lack of pharmacokinetic data about these peptides hinders their development and makes it challenging to fully understand how they work in vivo since their mechanism of action is dependent on tissue concentrations of the peptide. Here, we set out to define in detail the pharmacokinetics of a well-characterized IDR molecule, IDR-1018. To make the peptide traceable, it was radiolabeled with the long-lived gamma-emitting isotope gallium-67. After a series of bench-top characterizations, the radiotracer was administered to healthy mice intravenously (IV) or subcutaneously (SQ) at various dose levels (2.5-13 mg/kg). Nuclear imaging and ex-vivo biodistributions were used to quantify organ and tissue uptake of the radiotracer over time. When administered as an IV bolus, the distribution profile of the radiotracer changed as the dose was escalated. At 2.5 mg/kg, the peptide was well-tolerated, poorly circulated in the blood and was cleared predominantly by the reticuloendothelial system. Higher doses (7 and 13 mg/kg) as an IV bolus were almost immediately lethal due to respiratory arrest; significant lung uptake of the radiotracer was observed from nuclear scans of these animals, and histological examination found extensive damage to the pulmonary vasculature and alveoli. When administered SQ at a dose of 3 mg/kg, radiolabeled IDR-1018 was rapidly absorbed from the site of injection and predominately cleared renally. Apart from the SQ injection site, no other tissue had a concentration above the minimum inhibitory concentration that would enable this peptide to exert direct antimicrobial effects against most pathogenic bacteria. Tissue concentrations were sufficient, however, to disrupt microbial biofilms and alter the host immune response. Overall, this study demonstrated that the administration of synthetic IDR peptide in vivo is best suited to local administration which avoids some of the issues associated with peptide toxicity that are observed when administered systemically by IV injection, an issue that will have to be addressed through formulation.

Preparation of Heat-Denatured Macroaggregated Albumin for Biomedical Applications Using a Microfluidics Platform.

Albumin is widely used in pharmaceutical applications to alter the pharmacokinetic profile, improve efficacy, or decrease the toxicity of active compounds. Various drug delivery systems using albumin have been reported, including microparticles. Macroaggregated albumin (MAA) is one of the more common forms of albumin microparticles, which is predominately used for lung perfusion imaging when labeled with radionuclide technetium-99m (99mTc). These microparticles are formed by heat-denaturing albumin in a bulk solution, making it very challenging to control the size and dispersity of the preparations (coefficient of variation, CV, ∼50%). In this work, we developed an integrated microfluidics platform to create more tunable and precise MAA particles, the so-called microfluidic-MAA (M2A2). The microfluidic chips, prepared using off-stoichiometry thiol-ene chemistry, consist of a flow-focusing region followed by an extended and water-heated curing channel (85 °C). M2A2 particles with diameters between 70 and 300 µm with CVs between 10 and 20% were reliably prepared by adjusting the flow rates of the dispersed and continuous phases. To demonstrate the pharmaceutical utility of M2A2, particles were labeled with indium-111 (111In) and their distribution was assessed in healthy mice using nuclear imaging. 111In-M2A2 behaved similarly to 99mTc-MAA, with lung uptake predominately observed early on followed by clearance over time by the reticuloendothelial and renal systems. Our microfluidic chip represents an elegant and controllable method to prepare albumin microparticles for biomedical applications.

Hybrid Metal-Phenol Nanoparticles with Polydopamine-like Coating for PET/SPECT/CT Imaging.

The validation of metal-phenolic nanoparticles (MPNs) in preclinical imaging studies represents a growing field of interest due to their versatility in forming predesigned structures with unique properties. Before MPNs can be used in medicine, their pharmacokinetics must be optimized so that accumulation in nontargeted organs is prevented and toxicity is minimized. Here, we report the fabrication of MPNs made of a coordination polymer core that combines In(III), Cu(II), and a mixture of the imidazole 1,4-bis(imidazole-1-ylmethyl)-benzene and the catechol 3,4-dihydroxycinnamic acid ligands. Furthermore, a phenolic-based coating was used as an anchoring platform to attach poly(ethylene glycol) (PEG). The resulting MPNs, with effective hydrodynamic diameters of around 120 nm, could be further derivatized with surface-embedded molecules, such as folic acid, to facilitate in vivo targeting and multifunctionality. The prepared MPNs were evaluated for in vitro plasma stability, cytotoxicity, and cell internalization and found to be biocompatible under physiological conditions. First, biomedical evaluations were then performed by intrinsically incorporating trace amounts of the radioactive metals 111In or 64Cu during the MPN synthesis directly into their polymeric matrix. The resulting particles, which had identical physicochemical properties to their nonradioactive counterparts, were used to perform in vivo single-photon emission computed tomography (SPECT) and positron emission tomography (PET) in tumor-bearing mice. The ability to incorporate multiple metals and radiometals into MPNs illustrates the diverse range of functional nanoparticles that can be prepared with this approach and broadens the scope of these nanoconstructs as multimodal preclinical imaging agents.

Monosized Polymeric Microspheres Designed for Passive Lung Targeting: Biodistribution and Pharmacokinetics after Intravenous Administration.

Local as well as systemic therapy is often used to treat bacterial lung infections. Delivery of antibiotics to the vascular side of infected lung tissue using lung-targeting microspheres (MS) is a good alternative to conventional administration routes, allowing for localized high levels of antibiotics. This delivery route can also complement inhaled antibiotic therapy, especially in the case of compromised lung function. We prepared and characterized monodisperse poly(lactic-co-glycolic acid) (PLGA) MS loaded with levofloxacin using a flow-focusing glass microfluidic chip. In vitro characterization showed that the encapsulated LVX displayed a biphasic controlled release during 5 days and preserved its antibacterial activity. The MS degradation was investigated in vitro by cross-sectioning the MS using a focused ion beam scanning electron microscope and in vivo by histological examination of lung tissue from mice intravenously administered with the MS. The MS showed changes in the surface morphology and internal matrix, whereas the degradation in vivo was 3 times faster than that in vitro. No effect on the viability of endothelial and lung epithelial cells or hemolytic activity was observed. To evaluate the pharmacokinetics and biodistribution of the MS, complete quantitative imaging of the 111indium-labeled PLGA MS was performed in vivo with single-photon emission computed tomography imaging over 10 days. The PLGA MS distributed homogeneously in the lung capillaries. Overall, intravenous administration of 12 µm PLGA MS is suitable for passive lung targeting and pulmonary therapy.

Dual SPECT imaging of 111In and 67Ga to simultaneously determine in vivo the pharmacokinetics of different radiopharmaceuticals: a quantitative tool in pre-clinical research.

Dual-isotope (DI) studies offer a number of advantages in pre-clinical imaging. These include: reducing study times when compared with sequential scans, reducing the number of animals required for any given study, and most importantly, producing images perfectly registered in space and time that provide simultaneous information about two distinct body functions. The ability of single photon emission computed tomography (SPECT) to measure and differentiate energies of the emitted photons makes it well suited for DI imaging. However, since scattered photons originating from one radioisotope may be detected in the energy window of the other and thus degrade image quality and quantitative accuracy, scatter and crosstalk corrections must be applied. The decay characteristics of 111In and 67Ga, which are suitable for quantitative DI imaging for up to 2 weeks post-injection, led us to investigate the performance of simultaneous 111In/67Ga SPECT imaging using a small-animal pre-clinical scanner. A series of phantom experiments were performed to investigate image quality and accuracy of activity quantification in 111In/67Ga images acquired with three different collimators and reconstructed from different photopeak combinations. The triple energy window (TEW) method was used to correct for scatter and crosstalk. Based on these phantom studies, the optimal selection of collimator and energy window settings was determined. When using these optimal settings, submillimeter-size structures were distinguishable in the reconstructed images and quantification errors below 20% were achieved for both isotopes. The optimal parameters were subsequently applied to an in vivo animal study. The determination of the distinct pharmacokinetic profiles of two polymer radiopharmaceuticals injected simultaneously, but by different administration routes (intravenously and intraperitoneally) into a single animal demonstrated the feasibility of simultaneous 111In/67Ga SPECT.