Person:

Caravan, Peter

Background & Aims: Liver biopsy, the current clinical gold standard for fibrosis assessment, is invasive and has sampling errors, and is not optimal for screening, monitoring, or clinical decision-making. Fibrosis is characterized by excessive accumulation of extracellular matrix proteins including type I collagen. We hypothesize that molecular magnetic resonance imaging (MRI) with a probe targeted to type I collagen could provide a direct and non-invasive method of fibrosis assessment. Methods: Liver fibrosis was induced in rats with diethylnitrosamine and in mice with carbon tetrachloride. Animals were imaged prior to and immediately following i.v. administration of either collagen-targeted probe EP-3533 or non-targeted control Gd-DTPA. Magnetic resonance (MR) signal washout characteristics were evaluated from T1 maps and T1-weighted images. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for gadolinium and hydroxyproline. Results: EP-3533-enhanced MR showed greater signal intensity on delayed imaging (normalized signal enhancement mice: control = 0.39 ± 0.04, fibrotic = 0.55 ± 0.03, p <0.01) and slower signal washout in the fibrotic liver compared to controls (liver t1/2 = 51.3 ± 3.6 vs. 42.0 ± 2.5 min, p <0.05 and 54.5 ± 1.9 vs. 44.1 ± 2.9 min, p <0.01 for fibrotic vs. controls in rat and mouse models, respectively). Gd-DTPA-enhanced MR could not distinguish fibrotic from control animals. EP-3533 gadolinium concentration in the liver showed strong positive correlations with hydroxyproline levels (r = 0.74 (rats), r = 0.77 (mice)) and with Ishak scoring (r = 0.84 (rats), r = 0.79 (mice)). Conclusions: Molecular MRI of liver fibrosis with a collagen-specific probe identifies fibrotic tissue in two rodent models of disease.

Background & Aims The gold standard in assessing liver fibrosis is biopsy despite limitations like invasiveness and sampling error and complications including morbidity and mortality. Therefore, there is a major unmet medical need to quantify fibrosis non-invasively to facilitate early diagnosis of chronic liver disease and provide a means to monitor disease progression. The goal of this study was to evaluate the ability of several magnetic resonance imaging (MRI) techniques to stage liver fibrosis.

Methods A gadolinium (Gd)-based MRI probe targeted to type I collagen (termed EP-3533) was utilized to non-invasively stage liver fibrosis in a carbon tetrachloride (CCl4) mouse model and the results were compared to other MRI techniques including relaxation times, diffusion, and magnetization transfer measurements.

Results The most sensitive MR biomarker was the change in liver:muscle contrast to noise ratio (ΔCNR) after EP-3533 injection. We observed a strong positive linear correlation between ΔCNR and liver hydroxyproline (i.e. collagen) levels (r = 0.89) as well as ΔCNR and conventional Ishak fibrosis scoring. In addition, the area under the receiver operating curve (AUR0C) for distinguishing early (Ishak ⩽3) from late (Ishak ⩾4) fibrosis was 0.942 ± 0.052 (p <0.001). By comparison, other MRI techniques were not as sensitive to changes in fibrosis in this model.

Conclusions We have developed an MRI technique using a collagen-specific probe for diagnosing and staging liver fibrosis, and validated it in the CCl4 mouse model. This approach should provide a better means to monitor disease progression in patients.

In aqueous solution, Gd3+ and thiacalix[4]arene-p-tetrasulfonate (TCAS) form the complex [Gd3TCAS2]7–, in which a trinuclear Gd3+ core is sandwiched by two TCAS ligands. Acid-catalyzed dissociation reactions, as well as transmetalation and ligand exchange with physiological concentrations of Zn2+ and phosphate, showed [Gd3TCAS2]7– to be extremely inert compared to other Gd complexes. Luminescence lifetime measurements of the Tb analogue Tb3TCAS2 allowed estimation of the mean hydration number q to be 2.4 per Tb ion. The longitudinal relaxivity of [Gd3TCAS2]7– (per Gd3+) was r1 = 5.83 mM–1 s–1 at 20 Hz (37 °C, pH 7.4); however, this relaxivity was limited by an extremely slow water exchange rate that was 5 orders of magnitude slower than the Gd3+ aqua ion. Binding to serum albumin resulted in no relaxivity increase owing to the extremely slow water exchange kinetics. The slow dissociation and water exchange kinetics of [Gd3TCAS2]7– can be attributed to the very rigid coordination geometry.

Here, we describe the synthesis of the single amino acid chelator DOTAlaP and four of its derivatives. The corresponding gadolinium(III) complexes were investigated for their kinetic inertness, relaxometric properties at a range of fields and temperatures, water exchange rate, and interaction with human serum albumin (HSA). Derivatives with one inner-sphere water (q = 1) were determined to have a mean water residency time between 8 and 6 ns in phoshate-buffered saline at 37 °C. The corresponding europium complexes were also formed and used to obtain information on the hydration number of the corresponding coordination complexes. Two complexes capable of binding HSA were also synthesized, of which one, Gd(5b), contains no inner-sphere water, while the other derivative, Gd(4b), is a mixture of ca. 15% q =1 and 85% q = 0. In the presence of HSA, the latter displayed a very short mean water residency time (τM310 = 2.4 ns) and enhanced relaxivity at intermediate and high fields. The kinetic inertness of Gd(4b) with respect to complex dissociation was decreased compared to its DOTAla analogue but still 100-fold more inert than [Gd(BOPTA)(H2O)]2–. Magnetic resonance imaging in mice showed that Gd(4b) was able to provide 38% better vessel to muscle contrast compared to the clinically used HSA binding agent MS-325.

A library of 10 Mn-containing complexes capable of switching reversibly between the Mn(II) and Mn(III) oxidation states was prepared and evaluated for potential usage as MRI reporters of tissue redox activity. We synthesized N-(2-hydroxybenzyl)-N,N′,N′-ethylenediaminetriacetic acid (HBET) and N-(2-hydroxybenzyl-N,N′,N′-trans-1,2-cyclohexylenediaminetriacetic acid (CyHBET) ligands functionalized (−H, −OMe, −NO2) at the 5-position of the aromatic ring. The Mn(II) complexes of all ligands and the Mn(III) complexes of the 5-H and 5-NO2 functionalized ligands were synthesized and isolated, but the Mn(III) complexes with the 5-OMe functionalized ligands were unstable. 1H relaxivity of the 10 isolable complexes was measured at pH 7.4 and 37 °C, 1.4 T. Thermodynamic stability, pH-dependent complex speciation, hydration state, water exchange kinetics of the Mn(II) complexes, and pseudo-first order reduction kinetics of the Mn(III) complexes were studied using a combination of pH-potentiometry, UV–vis spectroscopy, and 1H and 17O NMR measurements. The effects of ligand structural and electronic modifications on the Mn(II/III) redox couple were studied by cyclic voltammetry. The Mn(II) complexes are potent relaxation agents as compared to the corresponding Mn(III) species with [MnII(CyHBET)(H2O)]2– exhibiting a 7.5-fold higher relaxivity (3.3 mM–1 s–1) than the oxidized form (0.4 mM–1 s–1). At pH 7.4, Mn(II) exists as a mixture of fully deprotonated (ML) and monoprotonated (HML) complexes and Mn(II) complex stability decreases as the ligands become more electron-releasing (pMn for 10 μM [MnII(CyHBET–R′)(H2O)]2– decreases from 7.6 to 6.2 as R′ goes from −NO2 to −OMe, respectively). HML speciation increases as the electron-releasing nature of the phenolato-O donor increases. The presence of a water coligand is maintained upon conversion from HML to ML, but the water exchange rate of ML is faster by up to 2 orders of magnitude (kex310 for H[MnII(CyHBET)(H2O)]− and [MnII(CyHBET)(H2O)]2– are 1.2 × 108 and 1.0 × 1010 s–1, respectively). The Mn(II/III) redox potential can be tuned over a range of 0.30 V (E1/2 = 0.27–0.57 V) through electronic modifications to the 5-substituent of the aromatic ligand component. However, care must be taken in tuning the ligand electronics to avoid Mn(III)–ligand autoredox. Taken together, these results serve to establish criteria for optimizing Mn(III) versus Mn(II) relaxivity differentials, complex stability, and Mn(II/III) redox potential.

Background: Despite recent advances in cancer therapy, the treatment of bone tumors remains a major challenge. A possible underlying hypothesis, limitation, and unmet need may be the inability of therapeutics to penetrate into dense bone mineral, which can lead to poor efficacy and high toxicity, due to drug uptake in healthy organs. The development of nanostructured formulations with high affinity for bone could be an interesting approach to overcome these challenges. Purpose To develop a liposomal formulation with high affinity for hydroxyapatite and the ability to release doxorubicin (DOX) in an acidic environment for future application as a tool for treatment of bone metastases. Materials and methods Liposomes were prepared by thin-film lipid hydration, followed by extrusion and the sulfate gradient-encapsulation method. Liposomes were characterized by average diameter, ζ-potential, encapsulation percentage, X-ray diffraction, and differential scanning calorimetry. Release studies in buffer (pH 7.4 or 5), plasma, and serum, as well as hydroxyapatite-affinity in vitro analysis were performed. Cytotoxicity was evaluated by MTT assay against the MDA-MB-231 cell line, and biodistribution was assessed in bone metastasis-bearing animals. Results: Liposomes presented suitable diameter (~170 nm), DOX encapsulation (~2 mg/mL), controlled release, and good plasma and serum stability. The existence of interactions between DOX and the lipid bilayer was proved through differential scanning calorimetry and small-angle X-ray scattering. DOX release was faster when the pH was in the range of a tumor than at physiological pH. The bone-targeted formulation showed a strong affinity for hydroxyapatite. The encapsulation of DOX did not interfere in its intrinsic cytotoxicity against the MDA-MB-231 cell line. Biodistribution studies demonstrated high affinity of this formulation for tumors and reduction of uptake in the heart. Conclusion: These results suggest that bone-targeted pH-sensitive liposomes containing DOX can be an interesting strategy for selectively delivering this drug into bone-tumor sites, increasing its activity, and reducing DOX-related toxicity.

Fibrosis with excessive amounts of type I collagen is a hallmark of many solid tumours, and fibrosis is a promising target in cancer therapy, but tools for its non-invasive quantification are missing. Here we used magnetic resonance imaging with a gadolinium-based probe targeted to type I collagen (EP-3533) to image and quantify fibrosis in pancreatic ductal adenocarcinoma. An orthotopic syngeneic mouse model resulted in tumours with 2.3-fold higher collagen level compared to healthy pancreas. Animals were scanned at 4.7 T before, during and up to 60 min after i.v. injection of EP-3533, or of its non-binding isomer EP-3612. Ex-vivo quantification of gadolinium showed significantly higher uptake of EP-3533 compared to EP-3612 in tumours, but not in surrounding tissue (blood, muscle). Uptake of EP-3533 visualized in T1-weighted MRI correlated well with spatial distribution of collagen determined by second harmonic generation imaging. Differences in the tumour pharmacokinetic profiles of EP-3533 and EP-3612 were utilized to distinguish specific binding to tumour collagen from non-specific uptake. A model-free pharmacokinetic measurement based on area under the curve was identified as a robust imaging biomarker of fibrosis. Collagen-targeted molecular MRI with EP-3533 represents a new tool for non-invasive visualization and quantification of fibrosis in tumour tissue.

Despite established clinical utilisation, there is an increasing need for safer, more inert gadolinium-based contrast agents, and for chelators that react rapidly with radiometals. Here we report the syntheses of a series of chiral DOTA chelators and their corresponding metal complexes and reveal properties that transcend the parent DOTA compound. We incorporated symmetrical chiral substituents around the tetraaza ring, imparting enhanced rigidity to the DOTA cavity, enabling control over the range of stereoisomers of the lanthanide complexes. The Gd chiral DOTA complexes are shown to be orders of magnitude more inert to Gd release than [GdDOTA]−. These compounds also exhibit very-fast water exchange rates in an optimal range for high field imaging. Radiolabeling studies with (Cu-64/Lu-177) also demonstrate faster labelling properties. These chiral DOTA chelators are alternative general platforms for the development of stable, high relaxivity contrast agents, and for radiometal complexes used for imaging and/or therapy.

Chemotherapy-induced pain is a dose-limiting condition that affects 30% of patients undergoing chemotherapy. We found that the gut microbiota promotes the development of chemotherapy-induced mechanical hyperalgesia. Oxaliplatin-induced mechnical hyperalgesia was reduced in germ-free mice and in those mice pretreated with antibiotics. Restoration of the microbiota of germ-free mice abrogated this protection. These effects appear to be mediated, in part, by TLR4 expressed on hematopoietic cells, including macrophages.