Development and validation of LC-MS/MS method for determination of DNDI-VL-2098 in mouse, rat, dog and hamster blood.

Aim: To develop a bioanalytical method to support pharmacokinetic evaluation of DNDI-VL-2098 in mouse, rat, dog and hamster following oral administration. Results & methodology: A robust LC-MS/MS bioanalytical method was developed to quantify DNDI-VL-2098. DNDI-VL-2098 showed time-dependent recovery loss in acetonitrile precipitated plasma in all species. Acid-lysed whole blood was identified as a matrix in which recovery was stable over time. A two-step extraction procedure was used, with protein precipitation followed by liquid-liquid extraction with methyl tert-butyl ether. The assay was validated in the dynamic range of 5-5000 ng/ml for mouse, rat and dog blood, and a fit-for-purpose method was developed for hamster. Conclusion: A specific LC-MS/MS assay for DNDI-VL-2098 was developed and validated in hemolyzed blood.

Validation of a sensitive simultaneous LC-MS/MS method for the quantification of novel anti-cancer thiazolidinedione and quinazolin-4-one derivatives in rat plasma and its application in a rat pharmacokinetic study.

Thiazolidinediones and quinazolin-4-ones compounds, previously known for their activity against Type 2 diabetes and antifungal activity respectively, are currently being investigated for their anti-cancer activity. The determination of pharmacokinetic parameters for these two classes of compounds using a simultaneous chromatographic method with a low detection limit is a challenge. In this study, a highly sensitive and simultaneous LC-MS/MS-based bioanalytical method was developed and validated in rat plasma for the estimation of four novel anti-cancer compounds, BIT-15-67 and BNT-11, belonging to the Thiazolidinedione class, and BNUA-108 and BNUA-48, from the quinazolin-4-one class. The analytes were extracted from plasma samples by protein precipitation and separated on a short reverse phase Hypersil Phenyl BDS, 50 × 4.6 mm, 2.4 µm column at a column oven temperature of 40 °C. An isocratic mobile phase, a 20:80 (v/v) mixture of 5 mM ammonium acetate solution and acetonitrile containing 0.1% formic acid, was used for the elution at a flow rate of 0.4 mL/min. The analytes and internal standard, sulfaphenazole, were quantified in the multiple reaction monitoring mode using positive electrospray ionization with specific pair of mass by charge ratio. All standard validation parameters were assessed as per current bioanalytical method validation guidelines in rat plasma. The area response for the four analytes was found to be linear over the concentration range of 1.00 to 1000 ng/mL in rat plasma. The signal to noise at LLOQ of 1 ng/mL was adequate for application to different pre-clinical studies. The intra- and inter-day precision were <11% and accuracy deviated -1.8 to 9.60% from the nominal. The mean recovery was high (about 90%) and consistent for all the analytes over the linear dynamic range of the method. This simple, robust and validated method can be employed to determine the rat plasma concentrations of the four selected anticancer compounds in preclinical studies such as the pharmacodynamic and the pharmacokinetic studies including tissue distribution and excretion, and the toxicokinetic studies. In this study, pharmacokinetic parameters were determined using this method for all the four compounds individually following intravenous administration in rats.

T2 relaxation time is related to liver fibrosis severity.

BACKGROUND: The grading of liver fibrosis relies on liver biopsy. Imaging techniques, including elastography and relaxometric, techniques have had varying success in diagnosing moderate fibrosis. The goal of this study was to determine if there is a relationship between the T2-relaxation time of hepatic parenchyma and the histologic grade of liver fibrosis in patients with hepatitis C undergoing both routine, liver MRI and liver biopsy, and to validate our methodology with phantoms and in a rat model of liver fibrosis. METHODS: This study is composed of three parts: (I) 123 patients who underwent both routine, clinical liver MRI and biopsy within a 6-month period, between July 1999 and January 2010 were enrolled in a retrospective study. MR imaging was performed at 1.5 T using dual-echo turbo-spin echo equivalent pulse sequence. T2 relaxation time of liver parenchyma in patients was calculated by mono-exponential fit of a region of interest (ROI) within the right lobe correlating to histopathologic grading (Ishak 0-6) and routine serum liver inflammation [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)]. Statistical comparison was performed using ordinary logistic and ordinal logistic regression and ANOVA comparing T2 to Ishak fibrosis without and using AST and ALT as covariates; (II) a phantom was prepared using serial dilutions of dextran coated magnetic iron oxide nanoparticles. T2 weighed imaging was performed by comparing a dual echo fast spin echo sequence to a Carr-Purcell-Meigboom-Gill (CPMG) multi-echo sequence at 1.5 T. Statistical comparison was performed using a paired t-test; (III) male Wistar rats receiving weekly intraperitoneal injections of phosphate buffer solution (PBS) control (n=4 rats); diethylnitrosamine (DEN) for either 5 (n=5 rats) or 8 weeks (n=4 rats) were MR imaged on a Bruker Pharmascan 4.7 T magnet with a home-built bird-cage coil. T2 was quantified by using a mono-exponential fitting algorithm on multi-slice multi echo T2 weighted data. Statistical comparison was performed using ANOVA. RESULTS: (I) Histopathologic evaluation of both rat and human livers demonstrated no evidence of steatosis or hemochromatosis There was a monotonic increase in mean T2 value with increasing degree of fibrosis (control 65.4±2.9 ms, n=6 patients); mild (Ishak 1-2) 66.7±1.9 ms (n=30); moderate (Ishak 3-4) 71.6±1.7 ms (n=26); severe (Ishak 5-6) 72.4±1.4 ms (n=61); with relatively low standard error (~2.9 ms). There was a statistically significant difference between degrees of mild (Ishak <4) vs. moderate to severe fibrosis (Ishak >4) (P=0.03) based on logistic regression of T2 and Ishak, which became insignificant (P=0.07) when using inflammatory markers as covariates. Expanding on this model using ordinal logistic regression, there was significance amongst all 4 groups comparing T2 to Ishak (P=0.01), with significance using inflammation as a covariate (P=0.03) and approaching statistical significance amongst all groups by ANOVA (P=0.07); (II) there was a monotonic increase in T2 and statistical significance (ANOVA P<0.0001) between each rat subgroup [phosphate buffer solution (PBS) 25.2±0.8, DEN 5-week (31.1±1.5), and DEN 9-week (49.4±0.4) ms]; (III) the phantoms that had T2 values within the relevant range for the human liver (e.g., 20-100 ms), demonstrated no statistical difference between two point fits on turbo spin echo (TSE) data and multi-echo CPMG data (P=0.9). CONCLUSIONS: The finding of increased T2 with liver fibrosis may relate to inflammation that may be an alternative or adjunct to other noninvasive MR imaging based approaches for assessing liver fibrosis.

Molecular imaging of fibrin in a breast cancer xenograft mouse model.

RATIONALE AND OBJECTIVES: Fibrin deposition has been indicated within the stroma of a majority of solid tumors. Here we assess the feasibility of using the established fibrin-specific probe EP-2104R for noninvasive imaging of fibrin in the context of breast cancer. METHODS: EP-2104R, untargeted gadopentetate dimeglumine (Gd-DTPA), and a newly synthesized nonfibrin binding control linear peptide (CLP) were compared using steady-state and dynamic contrast-enhanced magnetic resonance imaging in a breast cancer xenograft mouse model at 9.4 T. RESULTS: EP-2104R transiently enhanced both tumor core and tumor periphery, but only the enhancement in the tumor periphery persisted even 90 minutes after EP-2104R administration. However, untargeted Gd-DTPA and CLP are not retained in the tumor periphery. The half-life of EP-2104R in the tumor periphery (103 ± 18 minutes) is significantly longer (P < 0.05) than that of either Gd-DTPA (29.6 ± 2.4 minutes) or CLP (42.4 ± 1.5 minutes), but the rate of clearance is similar for all the 3 probes from the tumor core. The presence of high concentrations of fibrin in the tumor periphery was corroborated using immunohistochemistry with a fibrin-specific antibody. CONCLUSIONS: The persistent enhancement observed in the tumor periphery with EP-2104R is likely a result of its fibrin-specific binding rather than its size and demonstrates the feasibility of EP-2104R for molecular imaging of fibrin in tumor stroma.

Discrete bimodal probes for thrombus imaging.

Here we report a generalizable solid/solution-phase strategy for the synthesis of discrete bimodal fibrin-targeted imaging probes. A fibrin-specific peptide was conjugated with two distinct imaging reporters at the C- and N-termini. In vitro studies demonstrated retention of fibrin affinity and specificity. Imaging studies showed that these probes could detect fibrin over a wide range of probe concentrations by optical, magnetic resonance, and positron emission tomography imaging.

Molecular MR imaging of liver fibrosis: a feasibility study using rat and mouse models.

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 t(1/2)=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.

Noninvasive detection of neural progenitor cells in living brains by MRI.

The presence of pericytes in brain regions undergoing repair is evident of the recruitment of bone marrow-derived multipotent regenerative cells to the neurovascular unit during angiogenesis. At present, post mortem sampling is the only way to identify them. Therefore, such cell typing is inadequate for preserving neural progenitor cells for any meaningful stem cell therapy. We aimed to target cerebral pericytes in vivo using dual gene transcript-targeted MRI (GT-tMRI) in male C57black6 mice after a 60-min bilateral carotid artery occlusion (BCAO). We attached superparamagnetic iron oxide nanoparticles (SPIONs) to phosphorothioate-modified micro-DNA that targets actin or nestin mRNA. Because BCAO compromises the blood-brain barrier (BBB) and induces expression of α-smooth muscle (αSM)-actin and nestin antigens by pericytes in new vessels, we delivered pericyte-specific magnetic resonance contrast agents (SPION-actin or SPION-nestin at 4 mg Fe/kg) by i.p. injection to C57black6 mice that had experienced BCAO. We demonstrated that the surge in cerebral iron content by inductively coupled plasma-mass spectrometry matched the increase in the frequency of relaxivity. We also found that SPION-nestin was colocalized in αSM- actin- and nestin-expressing pericytes in BCAO-treated C57black6 or transgenic mice [B6.Cg-Tg(CAG-mRFP1) 1F1Hadj/J, expressing red fluorescent protein by actin promoter]. We identified pericytes in the repair patch in living brains after BCAO with a voxel size of 0.03 mm(3). The presence of electron-dense nanoparticles in vascular pericytes in the region of BBB injury led us to draw the conclusion that GT-tMRI can noninvasively reveal neural progenitor cells during vascularization.

Bimodal thrombus imaging: simultaneous PET/MR imaging with a fibrin-targeted dual PET/MR probe--feasibility study in rat model.

PURPOSE: To image thrombus by using magnetic resonance (MR) imaging and positron emission tomography (PET) simultaneously in a rat arterial thrombus model with a dual PET/MR probe. MATERIALS AND METHODS: Animal studies were approved by the institutional animal use committee. A dual PET/MR probe was synthesized by means of partial exchange of gadolinium for copper 64 ((64)Cu) in the fibrin-targeted MR probe EP-2104R. A preformed 25-mm thrombus was injected into the right internal carotid artery of a rat. Imaging was performed with a clinical 3.0-T MR imager with an MR-compatible human PET imager. Rats (n = 5) were imaged prior to and after systemic administration of the dual probe by using simultaneous PET/MR. The organ distribution of (64)Cu and gadolinium was determined ex vivo (n = 8), 2 hours after injection by using well counting and inductively coupled plasma mass spectrometry, respectively. Signal intensity ratios (SIRs) between the thrombus-containing and contralateral vessel were computed from PET images and MR data before and after probe administration. RESULTS: The dual probe was synthesized with greater than 98% radiochemical purity. Thrombus enhancement was observed in all five animals at both MR (SIR([postprobe])/SIR([preprobe]) = 1.71 ± 0.35, P = .0053) and PET (SIR = 1.85 ± 0.48, P = .0087) after injection of the dual PET/MR probe. Ex vivo analysis at 2 hours after injection showed the highest (64)Cu and gadolinium concentrations, after the excretory organs (kidney and liver), to be in the thrombus. CONCLUSION: A fibrin-targeted dual PET/MR probe enables simultaneous, direct MR and PET imaging of thrombus.

Targeted probes for cardiovascular MRI.

Molecular MRI plays an important role in studying molecular and cellular processes associated with heart disease. Targeted probes that recognize important biomarkers of atherosclerosis, apoptosis, necrosis, angiogenesis, thrombosis and inflammation have been developed. This review discusses the properties of chemically different contrast agents including iron oxide nanoparticles, gadolinium-based nanoparticles or micelles, discrete peptide conjugates and activatable probes. Numerous examples of contrast agents based on these approaches have been used in preclinical MRI of cardiovascular diseases. Clinical applications are still under investigation for some selected agents with highly promising initial results. Molecular MRI shows great potential for the detection and characterization of a wide range of cardiovascular diseases, as well as for monitoring response to therapy.

Molecular MRI of intracranial thrombus in a rat ischemic stroke model.

BACKGROUND AND PURPOSE: Intracranial thrombus is a principal feature in most ischemic stroke, and thrombus location and size may correlate with outcome and response to thrombolytic therapy. EP-2104R is a fibrin-specific molecular MR agent that has previously been shown to enhance extracranial and venous sinus thrombi in animal models and, recently, in clinical trials. In this study, we examined whether this fibrin-specific MR probe could noninvasively characterize intracranial arterial thrombi. METHODS: Embolic stroke was induced in adult rats by occlusion of the right internal carotid artery with an aged thrombus. We used diffusion-weighted imaging, time of flight angiography, and high-resolution 3-dimensional T1-weighted MRI at 4.7 T before and after use of contrast agents EP-2104R (n=6) and gadopentetate dimeglumine (n=5). RESULTS: In all animals, MR angiography revealed a flow deficit and diffusion-weighted imaging showed hyperintensity consistent with ischemia. Using EP-2104R-enhanced MRI, we saw occlusive thrombi and vessel wall enhancement in all 6 animals with high contrast to noise relative to blood, whereas gadopentetate dimeglumine-injected animals showed no occlusive thrombus or vessel wall enhancement. The concentration of gadolinium in the thrombus after EP-2104R was 18 times that in the blood pool. CONCLUSIONS: EP-2104R-enhanced MRI successfully identifies intracranial thrombus in a rat embolic stroke model.

Titanium(IV) complexes with N,N'-dialkyl-2,3-dihydroxyterephthalamides and 1-hydroxy-2(1H)-pyridinone as siderophore and tunichrome analogues.

The aqueous chemistry of Ti(IV) with biological ligands siderophores and tunichromes is modeled by using N,N'-dialkyl-2,3-dihydroxyterephthalamides (alTAMs), analogues of catecholamide-containing biomolecules, and 1-hydroxy-2(1H)-pyridinone (1,2-HOPO), an analogue of hydroxamate-containing biomolecules. Both types of ligands stabilize Ti(IV) with respect to hydrolytic precipitation, and afford tractable complexes. Complexes with the methyl derivative of alTAM, meTAM, are characterized by using mass spectrometry and UV/vis spectroscopy. Complexes with etTAM are characterized by the same techniques as well as X-ray crystallography, cyclic voltammetry, and spectropotentiomeric titration. The ESI mass spectra of these complexes in water show both 1:2 and 1:3 metal/ligand species. The X-ray crystal structure of a 1:2 complex, K(2)[Ti(etTAM)(2)(OCH(3))(2)].2CH(3)OH (1), is reported. The midpoint potential for reduction of 1 dissolved in solution is -0.98 V. A structure for a 1:3 Ti/etTAM species, Na(2)[Ti(etTAM)(3)] demonstrates the coordination and connectivity in that complex. Spectropotentiometric titrations in water reveal three metal-containing species in solution between pH 3 and 10. 1,2-HOPO supports Ti(IV) complexes that are stable and soluble in aqueous solution. The bis-HOPO complex [Ti(1,2-HOPO)(2)(OCH(3))(2)] (5) was characterized by X-ray crystallography and by mass spectrometry in solution, and the tris-HOPO dimer [(1,2-HOPO)(3)TiOTi(1,2-HOPO)(3)] (6) was characterized by X-ray crystallography. Taken together, these experiments explore the characteristics of complexes that may form between siderophores and tunichromes with Ti(IV) in biology and in the environment, and guide efforts toward new, well characterized aqueous Ti(IV) complexes. By revealing the identities and some characteristics of complexes that form under a variety of conditions, these studies further our understanding of the complicated nature of aqueous titanium coordination chemistry.

Influence of molecular parameters and increasing magnetic field strength on relaxivity of gadolinium- and manganese-based T1 contrast agents.

Simulations were performed to understand the relative contributions of molecular parameters to longitudinal (r(1)) and transverse (r(2)) relaxivity as a function of applied field, and to obtain theoretical relaxivity maxima over a range of fields to appreciate what relaxivities can be achieved experimentally. The field-dependent relaxivities of a panel of gadolinium and manganese complexes with different molecular parameters, water exchange rates, rotational correlation times, hydration state, etc. were measured to confirm that measured relaxivities were consistent with theory. The design tenets previously stressed for optimizing r(1) at low fields (very slow rotational motion; chelate immobilized by protein binding; optimized water exchange rate) do not apply at higher fields. At 1.5 T and higher fields, an intermediate rotational correlation time is desired (0.5-4 ns), while water exchange rate is not as critical to achieving a high r(1). For targeted applications it is recommended to tether a multimer of metal chelates to a protein-targeting group via a long flexible linker to decouple the slow motion of the protein from the water(s) bound to the metal ions. Per ion relaxivities of 80, 45, and 18 mM(-1) s(-1) at 1.5, 3 and 9.4 T, respectively, are feasible for Gd(3+) and Mn(2+) complexes.

Isolation and characterization of the iron-binding properties of a primitive monolobal transferrin from Ciona intestinalis.

Transferrins are bilobal glycoproteins responsible for iron binding, transport, and delivery in many higher organisms. The two homologous lobes of transferrins are thought to have evolved by gene duplication of an ancestral monolobal form. In the present study, a 37.7-kDa primitive monolobal transferrin (nicatransferrin, or nicaTf) from the serum of the model ascidian species Ciona intestinalis was isolated by using an immobilized iron-affinity column and characterized by using mass spectrometry and N-terminal sequencing. The protein binds one equivalent of iron(III) and exhibits an electron paramagnetic resonance spectrum that is anion-dependent. The UV/vis spectrum of nicaTf has a shoulder at 330 nm in both the iron-depleted and the iron-replete forms, but does not display the approximately 460 nm tyrosine-to-iron charge transfer band common to vertebrate serum transferrins under the conditions investigated. This result suggests that iron may adopt a different binding mode in nicaTf compared with the more highly evolved transferrin proteins. This difference in binding mode could have implications for the physiological role of the protein in the ascidian. The genome of C. intestinalis has genes for both a monolobal and a bilobal transferrin, and the sequences of both proteins are discussed in light of the known features of vertebrate serum transferrins as well as other transferrin homologs.

Aqueous spectroscopy and redox properties of carboxylate-bound titanium.

The aqueous chemistry of Ti(III) and Ti(IV) in two different chemical environments is investigated given its relevance to environmental, materials, and biological chemistry. Complexes of titanium with the carboxylate ligands citrate and oxalate, found ubiquitously in Nature, were synthesized. The redox properties were studied by using cyclic voltammetry. All the titanium citrate redox couples are quasi-reversible. Electrospray mass spectrometry of the Ti(III) citrate solution shows the presence of a 1:2 Ti/cit complex in solution, in contrast to the predominant 1:3 Ti/cit complex with Ti(IV). The change in the coordination of the ligand to the metal on reduction may explain the quasi-reversible behavior of the electrochemistry. The redox potentials for Ti(IV) citrate in water vary with pH. At pH 7, the approximate E(1/2) is less than -800 mV. This stated change in redox properties is considered in light of the previously reported Ti(IV) citrate solution speciation. Analogous speciation behavior is suggested from the EPR spectroscopy of Ti(III) citrate aqueous solutions. The g tensors are deduced for several pH-dependent species from the simulated data. The X-ray crystal structure of a Ti(III)(2) oxalate dimer Ti(2)(mu-C(2)O(4))(C(2)O(4))(2)(H(2)O)(6).2H(2)O (3), which crystallizes from water below pH 2, is reported. Complex 3 crystallizes in a monoclinic P2(1)/c space group with a = 9.5088(19) Angstroms, b = 6.2382(12) Angstroms, c = 13.494(3) Angstroms, V = 797.8(3) Angstroms(3), and Z = 2. The infrared spectroscopy, EPR spectroscopy, and cyclic voltammetry on complex 3 are reported. The cyclic voltammetry shows an irreversible redox couple approximately -196 mV which likely corresponds to the Ti(IV)(2)/Ti(III)Ti(IV) couple. The EPR spectroscopy on solid complex 3 shows a typical S = 1 triplet-state spectrum. The solid follows non-Curie behavior, and the antiferromagnetic coupling between the two metal centers is determined to be -37.2 cm(-1). However, in solution the complex follows Curie behavior and supports a Ti(III)Ti(IV) oxidation state for the dimer.

Titanium(IV) citrate speciation and structure under environmentally and biologically relevant conditions.

The water-soluble complexes of Ti(IV) with citrate are of interest in environmental, biological, and materials chemistry. The aqueous solution speciation is revealed by spectropotentiometric titration. From pH 3-8, given at least three equivalents of ligand, 3:1 citrate/titanium complexes predominate in solution with successive deprotonation of dangling carboxylates as the pH increases. In this range and under these conditions, hydroxo- or oxo-metal species are not supported by the data. At ligand/metal ratios between 1:1 and 3:1, the data are difficult to fit, and are consistent with the formation of such hydroxo- or oxo- species. Stability constants for observed species are tabulated, featuring log beta-values of 9.18 for the 1:1 complex [Ti(Hcit)](+), and 16.99, 20.41, 16.11, and 4.07 for the 3:1 complexes [Ti(H(2)cit)(3)](2-), [Ti(H(2)cit)(Hcit)(2)](4-), [Ti(Hcit)(2)(cit)](6-), and [Ti(cit)(3)](8-), respectively (citric acid = H(4)cit). Optical spectra for the species are reported. The complexes exhibit similar yet distinct spectra, featuring putative citrate-to-Ti(IV) charge-transfer absorptions (lambda(max) approximately 250-310 nm with epsilon approximately 5000-7000 M(-)(1) cm(-1)). The prevailing 3:1 citrate/titanium ratio in solution is supported by electrospray mass spectrometry data. The X-ray crystal structure of a fully deprotonated tris-citrate complex Na(8)[Ti(C(6)H(4)O(7))(3)].17H(2)O (1) (or Na(8)[Ti(cit)(3)].17H(2)O) that crystallizes from aqueous solution at pH 7-8 is reported. Compound 1 crystallizes in the triclinic space group P, with a = 11.634(2) Angstroms, b = 13.223(3) Angstroms, c = 13.291(3) Angstroms, V = 1982.9(7) Angstroms(3), and Z = 2.