Ashfaq Mahmood

In vivo near-infrared fluorescence imaging of osteoblastic activity

In vertebrates, the development and integrity of the skeleton requires hydroxyapatite (HA) deposition by osteoblasts. HA deposition is also a marker of, or a participant in, processes as diverse as cancer and atherosclerosis. At present, sites of osteoblastic activity can only be imaged in vivo using γ-emitting radioisotopes. The scan times required are long, and the resultant radioscintigraphic images suffer from relatively low resolution. We have synthesized a near-infrared (NIR) fluorescent bisphosphonate derivative that exhibits rapid and specific binding to HA in vitro and in vivo. We demonstrate NIR light–based detection of osteoblastic activity in the living animal, and discuss how this technology can be used to study skeletal development, osteoblastic metastasis, coronary atherosclerosis, and other human diseases.

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1(-/-) mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

Histone deacetylase 6–mediated selective autophagy regulates COPD-associated cilia dysfunction

Chronic obstructive pulmonary disease (COPD) involves aberrant airway inflammatory responses to cigarette smoke (CS) that are associated with epithelial cell dysfunction, cilia shortening, and mucociliary clearance disruption. Exposure to CS reduced cilia length and induced autophagy in vivo and in differentiated mouse tracheal epithelial cells (MTECs). Autophagy-impaired (Becn1+/- or Map1lc3B-/-) mice and MTECs resisted CS-induced cilia shortening. Furthermore, CS increased the autophagic turnover of ciliary proteins, indicating that autophagy may regulate cilia homeostasis. We identified cytosolic deacetylase HDAC6 as a critical regulator of autophagy-mediated cilia shortening during CS exposure. Mice bearing an X chromosome deletion of Hdac6 (Hdac6-/Y) and MTECs from these mice had reduced autophagy and were protected from CS-induced cilia shortening. Autophagy-impaired Becn1-/-, Map1lc3B-/-, and Hdac6-/Y mice or mice injected with an HDAC6 inhibitor were protected from CS-induced mucociliary clearance (MCC) disruption. MCC was preserved in mice given the chemical chaperone 4-phenylbutyric acid, but was disrupted in mice lacking the transcription factor NRF2, suggesting that oxidative stress and altered proteostasis contribute to the disruption of MCC. Analysis of human COPD specimens revealed epigenetic deregulation of HDAC6 by hypomethylation and increased protein expression in the airways. We conclude that an autophagy-dependent pathway regulates cilia length during CS exposure and has potential as a therapeutic target for COPD.

Technepine: A high-affinity 99mtechnetium probe to label the dopamine transporter in brain by SPECT imaging

Increasing evidence suggests that the dopamine transporter, localized on dopamine neurons, is a marker for a number of physiological and pathological states (KaufmanandMadras, 1991,1993; Madras et al., 1990a, Schoemaker et al., 1985; Singer et al., 1991). With the development of sensitive probes, brainimaging and mea- surement of the transporter have become feasible in re- cent years (Brownell et al., in press; Innis et al., 1991; Frost et al., 1993; Madras et al., 1991; Morns et al., sub- mitted; Seibyl et al., 1995; van Dyke et a1.,1995; Wonget al., 1993,1995). Drugs of many chemical classes, includ- ing cocaine, bind to the dopamine transporter (Seeman, 1993). Nevertheless, effective imaging agents have been developed almost exclusively from the phenyltropane analogue of cocaine WIN 35,428 or CFT, a potent dopa- mine transport inhibitor (Clarke et al., 1973; Heikkila et al., 1979). The impetus for developing [llC]WIN 35,428 as a PET ligand (Hantraye et al., 1992; Madras, 1994; Madras et al., 1991, 1994; Wong et al., 1993; Meltzer et al., 1993) and y-emitting analogues for SPECT imaging (e.g., RTI-55, the 4-iodophenyl analogue of WIN 35,428, Canfield et al., 1990; Boja et al., 1991; Innis et al., 1991) arose directly from our observations of the binding of WIN 35,428 to the dopamine transporter. Unlike previ- ous dopamine transport inhibitors (noncocaine conge- ners) proposed for brain imaging(Kuhar et a1.,1990), the radiolabeled form of WIN 35,428 binds to the dopamine transporter in brain striatum with very low levels of non- specific binding (Madras et al., 1989a,b) and distributes principally to dopamine-rich regions of brain, as we re- ported in 1989 (Canfield et al., 1989) and subsequently (Canfield et al., 1990; Kaufman et al., 1991; Kaufman and Madras, 1992). SPECT imaging techniques are more practical than PET for routine clinical studies because of the lesser

Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element–binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Melanin-Targeted Preclinical PET Imaging of Melanoma Metastasis

Dialkylamino-alkyl-benzamides possess an affinity for melanin, suggesting that labeling of such benzamides with 18F could potentially produce melanin-targeted PET probes able to identify melanotic melanoma metastases in vivo with high sensitivity and specificity. Methods: In this study, N-[2-(diethylamino)ethyl]-4-18F-fluorobenzamide (18F-FBZA) was synthesized via a 1-step conjugation reaction. The σ-receptor binding affinity of 19F-FBZA was determined along with the in vitro cellular uptake of radiofluorinated 18F-FBZA in B16F10 cells. In vivo distribution and small-animal PET studies were conducted on mice bearing B16F10 melanoma, A375M amelanotic melanoma, and U87MG tumors, and comparative studies were performed with 18F-FDG PET in the melanoma models. Results: In vitro, uptake of 18F-FBZA was significantly higher in B16F10 cells treated with l-tyrosine (P < 0.001). In vivo, 18F-FBZA displayed significant tumor uptake; at 2 h, 5.94 ± 1.83 percentage injected dose (%ID) per gram was observed in B16F10 tumors and only 0.75 ± 0.09 %ID/g and 0.56 ± 0.13 %ID/g was observed in amelanotic A375M and U87MG tumors, respectively. Lung uptake was significantly higher in murine lungs bearing melanotic B16F10 pulmonary metastases than in normal murine lungs (P < 0.01). Small-animal PET clearly identified melanotic lesions in both primary and pulmonary metastasis B16F10 tumor models. Coregistered micro-CT with small-animal PET along with biopsies further confirmed the presence of tumor lesions in the mouse lungs. Conclusion: 18F-FBZA specifically targets primary and metastatic melanotic melanoma lesions with high tumor uptake and may have translational potential.

[99mTcOAADT]-(CH2)2-NEt2: A Potential Small-Molecule Single-Photon Emission Computed Tomography Probe for Imaging Metastatic Melanoma

Evaluation of [99mTc]oxotechnetium(V) complexes of the amine-amide-dithiol (AADT) chelates containing tertiary amine substituents as small-molecule probes for the diagnostic imaging of metastatic melanoma has shown that technetium-99m-labeled AADT-(CH2)2-NEt2 (99mTc-1) has the highest tumor uptake and other favorable biological properties. We have, therefore, assessed this agent in a more realistic metastatic melanoma model in which, after i.v. tail injection, a highly invasive melanoma cell line, B16F10, forms pulmonary tumor nodules in normal C57BL6 mice. Small melanotic lesions develop in the lungs and, on histologic examination, appear as small black melanoma colonies, increasing in size and number with time after tumor cell injection. Groups of mice received tumor cell inocula of 2 x 10(5), 4 x 10(5), or 8 x 10(5) B16F10 cells; 14 days later, 2 hours after 99mTc-1 administration, lung uptake of 2.83 +/- 0.21%, 3.63 +/- 1.07%, and 4.92 +/- 1.61% injected dose per gram of tissue (% ID/g), respectively, was observed, compared with normal lung uptake of 2.13 +/- 0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1 accumulation was seen 17 days after tumor cell inoculation as the lung lesions grew. These in vivo studies coupled with additional in vitro and ex vivo assessment show that 99mTc-1 has high and specific uptake in melanoma metastases in lungs and can potentially follow the temporal growth of these tumors.

Adsorption of metallic radionuclides on plastic phantom walls

Metal radionuclide solutions at neutral pH adhere to plastic containers. Adsorption of radionuclides on the walls of phantoms leads to a nonuniform activity distribution, which could adversely affect imaging studies, as well as phantom-based validations of absorbed dose calculations used in radioimmunotherapy, requiring accurate knowledge of the underlying activity distribution. In the work reported here, the authors determined the degree of metal chelation required to minimize metallic radionuclide oxide formation and adsorption on phantom walls in order to yield more reliable experimental data for validating image-based dosimetry. Using hollow spherical plastic phantoms, the authors evaluated three different radionuclides, I-131, In-111, and Y-90, in solutions containing three different concentrations of the chelator, ethylenediaminetetraacetate (EDTA). Adsorption to plastic walls was determined using microSPECT imaging and/or by counting aliquots of solutions. Reconstructed images and measurements of I-131 activity showed that it was uniformly distributed within all spheres; however, images of In-111 in 0.25-microM EDTA indicated that the activity concentration near the wall was much higher than that in the middle of the sphere. The decrease in activity concentration near the center of the spheres was approximately 47%. Y-90 in 0.25-microM EDTA behaved similarly; the activity concentration of Y-90 decreased by 46%. For an In-111 or Y-90 radioactivity concentration of 0.74 MBq/mL, a 2.5-microM EDTA solution was required to achieve a uniform distribution, suggesting that, under our experimental conditions, approximately 700 EDTA molecules were required for each radiometal atom to prevent precipitation and adsorption on poly(methylmethacrylate). For certain radiometals, e.g., In-111 or Y-90, adequate chelation is essential to achieve uniform activity concentration values and homogeneous distribution within the phantom compartments.

The synthesis and characterization of [M(C8H5N2N=NH)3](BPh4), where M = Tc or Re. Tris-diazene chelate complexes of technetium(I) and rhenium(I)

Abstract The reaction of ammonium pertechnetate or tetrabutylammonium perrhenate with the chelating organohydrazine hydralazine hydrochloride gives the Tc( I ) (or Re( I )) cationic tris-chelate complex [M(C 8 H 5 N 2 N=NH) 3 ] + , which is isolated as the tetraphenylborate salt. These dark green complexes are freely soluble in ethanol but decompose in solution in the absence of excess ligand. The infrared spectra of these complexes show no peaks associated with ν(M=O), nor associated with ν(C=N) or ν(N=N), indicative of exclusively chelated organodiazene ligation. A weak absorption near 3250 cm −1 is associated with ν(N-H) from the organodiazene ligands. The FAB(+) mass spectra of the complexes show the free parent ions for both metals. For the technetium complex M + = 573 m/z and for the rhenium complex M + =661 m/z . The technetium complexs mass spectrum also shows a peak of 417 m/z associated with a fragment from the loss of one of the three chelated diazene ligands. The 99 Tc NMR spectroscopy shows the technetium complexes resonance occurring in the established Tc(V) region, at ∼1620 ppm. This downfield shift is attributed to a paramagnetic shielding effect. These complexes are electrochemically active, with each complex exhibiting a pair of reversible oxidations. One of the two observed processes is likely ligand based, since it occurs at very similar E 1/2 values for both the rhenium and technetium complexes.

Performance of a triple-detector, multiple-pinhole SPECT system with iodine and indium isotopes

SPECT imaging with isotopes other than /sup 99m/Tc is useful for developing new molecular probes for in-vivo imaging. We have recently modified a standard 3-head gamma camera system for /spl mu/SPECT with 6 pinhole tungsten collimators using a 3D-OSEM reconstruction algorithm. We have previously reported on the /sup 99m/Tc performance of this /spl mu/SPECT imager. We have recently measured the performance of the /spl mu/SPECT system for imaging with /sup 125/I, /sup 123/I, and /sup 111/In. The smallest of the interchangeable apertures (0.8-mm) was used for all tests. Point sources consisted of droplets or ion exchange beads with /sup 123/I and /sup 111/In. The imaging of /sup 125/I was simulated using the 30-keV X-rays from /sup 123/I. The volume source was a 30 cc syringe containing 25 ml. Point and volume projection data were acquired separately. The projection data sets of point and volumes were scaled and added before reconstruction. The pinhole-collimated /sup 123/I energy spectrum demonstrated 18% contribution from high-energy photons detected in the 159-keV, 20% wide window, whereas a low-energy parallel-hole collimator allowed 60% contamination. The point-source sensitivity at 159 keV was comparable to that measured previously for /sup 99m/Tc (18 cpm/kBq), and was 3.6 times higher than that measured in the X-ray region (30-keV, 50% window). Point sensitivity for /sup 111/In was 39 cpm/kBq. Volume sensitivities were 1.4 cpm/kBq at 30 keV, 4.3 cpm/kBq at 159 keV and 14 cpm/kBq for /sup 111/In. With 1 iteration, the average FWHM resolution at 30 keV was 1.05 mm, at a background noise level of 40%. Average resolution for /sup 123/I and /sup 111/In were both measured to be 1.2 mm at a 10% noise level with 1 iteration and decreased to 0.9 mm at 25% noise level and 3 iterations. The /spl mu/SPECT system performs comparably for /sup 99m/Tc, /sup 123/I and /sup 111/In and acceptably for /sup 125/I. New iodinated probes and mice containing /sup 111/In are now being imaged with the system.