Radiosynthesis and Early Evaluation of a Positron Emission Tomography Imaging Probe [18F]AGAL Targeting Alpha-Galactosidase A Enzyme for Fabry Disease.

Success of gene therapy relies on the durable expression and activity of transgene in target tissues. In vivo molecular imaging approaches using positron emission tomography (PET) can non-invasively measure magnitude, location, and durability of transgene expression via direct transgene or indirect reporter gene imaging in target tissues, providing the most proximal PK/PD biomarker for gene therapy trials. Herein, we report the radiosynthesis of a novel PET tracer [18F]AGAL, targeting alpha galactosidase A (α-GAL), a lysosomal enzyme deficient in Fabry disease, and evaluation of its selectivity, specificity, and pharmacokinetic properties in vitro. [18F]AGAL was synthesized via a Cu-catalyzed click reaction between fluorinated pentyne and an aziridine-based galactopyranose precursor with a high yield of 110 mCi, high radiochemical purity of >97% and molar activity of 6 Ci/µmol. The fluorinated AGAL probe showed high α-GAL affinity with IC50 of 30 nM, high pharmacological selectivity (≥50% inhibition on >160 proteins), and suitable pharmacokinetic properties (moderate to low clearance and stability in plasma across species). In vivo [18F]AGAL PET imaging in mice showed high uptake in peripheral organs with rapid renal clearance. These promising results encourage further development of this PET tracer for in vivo imaging of α-GAL expression in target tissues affected by Fabry disease.

Multiparametric magnetic resonance imaging/magnetic resonance elastography assesses progression and regression of steatosis, inflammation, and fibrosis in alcohol-associated liver disease.

BACKGROUND: Magnetic resonance imaging (MRI) and MRI-based elastography (MRE) are the most promising noninvasive techniques in assessing liver diseases. The purpose of this study was to evaluate an advanced multiparametric imaging method for staging disease and assessing treatment response in realistic preclinical alcohol-associated liver disease (ALD). METHODS: We utilized four different preclinical mouse models in our study: Model 1-mice were fed a fast-food diet and fructose water for 48 weeks to induce nonalcoholic fatty liver disease; Model 2-mice were fed chronic-binge ethanol (EtOH) for 10 days or 8 weeks to induce liver steatosis/inflammation. Two groups of mice were treated with interleukin-22 at different time points to induce disease regression; Model 3-mice were administered CCl4 for 2 to 4 weeks to establish liver fibrosis followed by 2 or 4 weeks of recovery; and Model 4-mice were administered EtOH plus CCl4 for 12 weeks. Mouse liver imaging biomarkers including proton density fat fraction (PDFF), liver stiffness (LS), loss modulus (LM), and damping ratio (DR) were assessed. Liver and serum samples were obtained for histologic and biochemical analyses. Ordinal logistic regression and generalized linear regression analyses were used to model the severity of steatosis, inflammation, and fibrosis, and to assess the regression of these conditions. RESULTS: Multiparametric models with combinations of biomarkers (LS, LM, DR, and PDFF) used noninvasively to predict the histologic severity and regression of steatosis, inflammation, and fibrosis were highly accurate (area under the curve > 0.84 for all). A three-parameter model that incorporates LS, DR, and ALT predicted histologic fibrosis progression (r = 0.84, p < 0.0001) and regression (r = 0.79, p < 0.0001) as measured by collagen content in livers. CONCLUSION: This preclinical study provides evidence that multiparametric MRI/MRE can be used noninvasively to assess disease severity and monitor treatment response in ALD.

fMRI study of the role of glutamate NMDA receptor in the olfactory adaptation in rats: Insights into cellular and molecular mechanisms of olfactory adaptation.

Olfactory adaptation, characterized by attenuation of response to repeated odor stimulations or continuous odor exposure, is an intrinsic feature of olfactory processing. Adaptation can be induced by either "synaptic depression" due to depletion of neurotransmitters, or "enhanced inhibition" onto principle neurons by local inhibitory interneurons in olfactory structures. It is not clear which mechanism plays a major role in olfactory adaptation. More importantly, molecular sources of enhanced inhibition have not been identified. In this study, olfactory responses to either repeated 40-s stimulations with interstimulus intervals (ISI) of 140-s or 30-min, or a single prolonged 200-s stimulus were measured by fMRI in different naïve rats. Olfactory adaptations in the olfactory bulb (OB), anterior olfactory nucleus (AON), and piriform cortex (PC) were observed only with repeated 40-s odor stimulations, and no olfactory adaptations were detected during the prolonged 200-s stimulation. Interestingly, in responses to repeated 40-s odor stimulations in the PC, the first odor stimulation induced positive activations, and odor stimulations under adapted condition induced negative activations. The negative activations suggest that "sparse coding" and "global inhibition" are the characteristics of olfactory processing in PC, and the global inhibition manifests only under an adapted condition, not a naïve condition. Further, we found that these adaptations were NMDA receptor dependent; an NMDA receptor antagonist (MK801) blocked the adaptations. Based on the mechanism that glutamate NMDA receptor plays a role in the inhibition onto principle neurons by interneurons, our data suggest that the olfactory adaptations are caused by enhanced inhibition from interneurons. Combined with the necessity of the interruption of odor stimulation to observe the adaptations, the molecular source for the enhanced inhibition is most likely an increased glutamate release from presynaptic terminals due to glutamate over-replenishment during the interruption of odor stimulation. Furthermore, with blockage of the adaptations, the data reveal that orbital, medial & prefrontal, and cingulate cortices (OmPFC) are involved in the olfactory processing.

fMRI study of olfaction in the olfactory bulb and high olfactory structures of rats: Insight into their roles in habituation.

Cerebral blood volume (CBV) fMRI with ultrasmall superparamagnetic iron oxide particles (USPIO) as a contrast agent was used to investigate olfactory processing in rats. fMRI data were acquired in sixteen 0.75-mm coronal slices covering the olfactory bulb (OB) and higher olfactory regions (HOR), including the anterior olfactory nucleus and piriform cortex. For each animal, multiple consecutive fMRI measurements were made during a 3-h experiment session, with each measurement consisting of a baseline period, an odorant stimulation period, and a recovery period. Two different stimulation paradigms with a stimulation period of 40s or 80s, respectively, were used to study olfactory processing. Odorant-induced CBV increases were robustly observed in the OB and HOR of each individual animal. Olfactory adaptation, which is characterized by an attenuation of responses to continuous exposure or repeated stimulations, has different characteristics in the OB and HOR. For adaptation to repeated stimuli, while it was observed in both the OB and HOR, CBV responses in the HOR were attenuated more significantly than responses in the OB. In contrast, within each continuous 40-s or 80-s odor exposure, CBV responses in the OB were stable and did not show adaptation, but the CBV responses in the HOR were state dependent, with no adaptation during initial exposures, but significant adaptation during following exposures. These results support previous reports that HOR plays a more significant role than OB in olfactory habituation. The technical approach presented in this study should enable more extensive fMRI studies of olfactory processing in rats.

Micro-CT imaging: Developing criteria for examining fetal skeletons in regulatory developmental toxicology studies - A workshop report.

During the past two decades the use and refinements of imaging modalities have markedly increased making it possible to image embryos and fetuses used in pivotal nonclinical studies submitted to regulatory agencies. Implementing these technologies into the Good Laboratory Practice environment requires rigorous testing, validation, and documentation to ensure the reproducibility of data. A workshop on current practices and regulatory requirements was held with the goal of defining minimal criteria for the proper implementation of these technologies and subsequent submission to regulatory agencies. Micro-computed tomography (micro-CT) is especially well suited for high-throughput evaluations, and is gaining popularity to evaluate fetal skeletons to assess the potential developmental toxicity of test agents. This workshop was convened to help scientists in the developmental toxicology field understand and apply micro-CT technology to nonclinical toxicology studies and facilitate the regulatory acceptance of imaging data. Presentations and workshop discussions covered: (1) principles of micro-CT fetal imaging; (2) concordance of findings with conventional skeletal evaluations; and (3) regulatory requirements for validating the system. Establishing these requirements for micro-CT examination can provide a path forward for laboratories considering implementing this technology and provide regulatory agencies with a basis to consider the acceptability of data generated via this technology.

Functional imaging of olfaction by CBV fMRI in monkeys: insight into the role of olfactory bulb in habituation.

Cerebral blood volume (CBV) fMRI with superparamagnetic iron oxide nanoparticles (USPIO) as contrast agent was used to investigate the odorant-induced olfaction in anesthetized rhesus monkeys. fMRI data were acquired in 24 axial slices covering the entire brain, with isoamyl-acetate as the odor stimulant. For each experiment, multiple fMRI measurements were made during a 1- or 2-h period, with each measurement consisting of a baseline period, a stimulation period, and a recovery period. Three different stimulation paradigms with a stimulation period of 1 min, 2 min, or 8 min, respectively, were used to study the olfactory responses in the olfactory bulb (OB). Odorant-induced CBV increases were observed in the OB of each individual monkey. The spatial and temporal activation patterns were reproducible within and between animals. The sensitivity of CBV fMRI in OB was comparable with the sensitivities reported in previous animal fMRI studies. The CBV responses during the 1-min, 2-min, or 8-min odor stimulation period were relatively stable, and did not show attenuation. The amplitudes of CBV response to the repeated stimuli during the 1- or 2-h period were also stable. The stable CBV response in the OB to both continuous and repeated odor stimuli suggests that the OB may not play a major role in olfactory habituation. The technical approach described in this report can enable more extensive fMRI studies of olfactory processing in OB of both humans and non-human primates.

Micro-computed tomography imaging and analysis in developmental biology and toxicology.

Micro-computed tomography (micro-CT) is a high resolution imaging technique that has expanded and strengthened in use since it was last reviewed in this journal in 2004. The technology has expanded to include more detailed analysis of bone, as well as soft tissues, by use of various contrast agents. It is increasingly applied to questions in developmental biology and developmental toxicology. Relatively high-throughput protocols now provide a powerful and efficient means to evaluate embryos and fetuses subjected to genetic manipulations or chemical exposures. This review provides an overview of the technology, including scanning, reconstruction, visualization, segmentation, and analysis of micro-CT generated images. This is followed by a review of more recent applications of the technology in some common laboratory species that highlight the diverse issues that can be addressed.

Gene therapy for cross-correction of somatic organs and the CNS in mucopolysaccharidosis II in rodents and non-human primates.

Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.

Continuing education course #1: non-invasive imaging as a problem-solving tool and translational biomarker strategy in toxicologic pathology.

The continuing education course "Non-Invasive Imaging as a Problem-Solving Tool and Translational Biomarker Strategy in Toxicologic Pathology" provided a thorough overview of commonly used imaging modalities and the logistics required for integration of small animal imaging into toxicologic pathology. Non-invasive imaging (NIN) is gaining acceptance as an important modality in toxicologic pathology. This technology allows nonterminal, time-course evaluation of functional and morphologic endpoints and can be used to translate biomarkers between preclinical animal models and human patients. NIN can support drug development as well as basic research in academic or industrial environments. An initial overview of theoretical principles was followed by focused presentations on magnetic resonance imaging (MRI)/magnetic resonance microscopy (MRM), positron emission tomography (PET)/single proton emission computed tomography (SPECT), ultrasonography (US, primarily focused on echocardiography), optical (bioluminescent) imaging, and computed tomography (CT). The choice of imaging modality will depend on the research question and the needed resolution.

Treatment of mammary carcinomas in HER-2 transgenic mice through combination of genetic vaccine and an agonist of Toll-like receptor 9.

PURPOSE: Oligodeoxynucleotides containing unmethylated CpG dinucleotides induce innate and adaptive immunity through Toll-like receptor 9 (TLR9). In the present study, we have examined the ability of a novel agonist of TLR9, called immunomodulatory oligonucleotide (IMO), to enhance effects of a HER-2/neu plasmid DNA electroporation/adenovirus (DNA-EP/Ad) vaccine. EXPERIMENTAL DESIGN: BALB/NeuT mice were treated with DNA-EP vaccine alone, IMO alone, or the combination of two agents starting at week 13, when all mice showed mammary neoplasia. Tumor growth and survival were documented. Antibody and CD8+ T-cell responses were determined. Peptide microarray analysis of sera was carried out to identify immunoreactive epitopes. Additionally, microCT and microPET imaging was carried out in an advanced-stage tumor model starting treatment at week 17 in BALB/NeuT mice. RESULTS: The combination of DNA-EP and IMO resulted in significant tumor regression or delay to tumor progression. 2-Deoxy-2-[18F]fluoro-D-glucose microPET and microCT imaging of mice showed reduced tumor size in the DNA-EP/IMO combination treatment group. Mice treated with the combination produced greater antibody titers with IgG2a isotype switch and antibody-dependent cellular cytotoxicity activity than did mice treated with DNA-EP vaccine. An immunogenic B-cell linear epitope, r70, within the HER-2 dimerization domain was identified through microarray analysis. Heterologous DNA-EP/Ad vaccination combined with IMO increased mice survival. CONCLUSION: The combination of HER-2/neu genetic vaccine and novel agonist of TLR9 had potent antitumor activity associated with antibody isotype switch and antibody-dependent cellular cytotoxicity activities. These results support possible clinical trials of the combination of DNA-EP/Ad-based cancer vaccines and IMO.

Micro-computed tomographic evaluation of fetal skeletal changes induced by all-trans-retinoic acid in rats and rabbits.

BACKGROUND: Our laboratory has been conducting positive control studies to evaluate the utility of micro-computed tomography (micro-CT) for qualitative evaluation of fetal skeletal morphology. All-trans-retinoic acid (atRA) was used to produce a different spectrum of defects compared to our previous studies with boric acid and hydroxyurea. METHODS: Groups of five mated Crl:CD(SD) female rats each were administered vehicle or atRA (2.5-50 mg/kg) on GD 10, and groups of four mated Dutch Belted rabbits each were dosed with vehicle or atRA (6.25-25 mg/kg) on GD 9. Cesarean sections were performed on GD 21 and 28, respectively. Following external examination the viscera were removed and fetuses scanned in a micro-CT imaging system. Fetuses were subsequently stained with alizarin red. Skeletal morphology was evaluated by each method without the knowledge of treatment group. Total bone mineral content (BMC) of each fetus was quantitated using the micro-CT images. RESULTS: In rats there were dose-related increases in the incidence of extra lumbar vertebra and non-dose-related increases in supernumerary ribs at all dose levels. There were decreases in mean number of ossified sacrocaudal vertebra at ≥ 5 mg/kg, and increases in skull bone malformations at ≥ 10 mg/kg. Rabbits were less sensitive on a mg/kg basis since skeletal malformations and a decrease in mean number of ossified sacrocaudal vertebra were observed only in the 25-mg/kg group. Micro-CT evaluation detected essentially the same incidence of skeletal abnormalities as seen in alizarin red-stained rat and rabbit fetuses. BMC analysis showed a trend toward slight decreases in atRA-treated rats, but no notable changes in rabbits. CONCLUSIONS: These results add support to our previous work that demonstrates that micro-CT imaging can effectively assess rat and rabbit fetal skeletal morphology.

Micro-computed tomography and alizarin red evaluations of boric acid-induced fetal skeletal changes in Sprague-Dawley rats.

BACKGROUND: Assessment of developmental toxicity has historically included assessment of fetal skeletal morphology after alizarin red staining. X-ray micro-computed tomography (micro-CT) produces high-resolution images of skeletal structures and was investigated as an alternative method. METHODS: Groups of 5 mated Crl:CD (SD) female rats each were administered vehicle or boric acid (40 to 500 mg/kg/day) from GD 6 through 11. On GD 21, all live fetuses were weighed, euthanized, and viscera removed. Each litter was placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 sec ( approximately 20 litters per hour) and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 min later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. RESULTS: Micro-CT evaluation of fetal rat skeletons detected essentially the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. The specific skeletal abnormalities that did not match exactly involved the smallest skeletal elements with minimal degrees of ossification (i.e., cervical ribs, hypoplastic 13(th) ribs, supernumerary ribs, the 5(th) sternebra, and numbers of caudal vertebrae), but the differences did not impact the overall conclusions. Additional measures such as femur length were easily measured by micro-CT. CONCLUSIONS: These results indicate that micro-CT imaging can effectively assess rat fetal skeletal structures, and for those laboratories with this resource, it may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.

Evaluation of hydroxyurea-induced fetal skeletal changes in Dutch belted rabbits by micro-computed tomography and alizarin red staining.

BACKGROUND: This laboratory has been investigating the utility of X-ray micro-computed tomography (micro-CT) to produce high-resolution, 3D images of skeletal structures in common laboratory species. The present investigation uses micro-CT evaluation of skeletons from rabbit fetuses exposed to the known teratogen, hydroxyurea. METHODS: Groups of 4-6 mated Dutch Belted female rabbits each were administered vehicle or hydroxyurea (62.5 to 500 mg/kg) once on GD 12. On GD 28, all live fetuses were weighed, euthanized, and viscera removed. Up to 7 fetuses per litter were placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 seconds, and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 minutes later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. RESULTS: Except for a few isolated cases, micro-CT evaluation detected the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. Skeletal elements that are very small (e.g., caudal-most vertebrae, metacarpal no. 1) or those with a minimal degree of ossification were occasionally not observed with micro-CT. However, this difference did not impact the overall study conclusions. Femur length was easily measured by micro-CT. CONCLUSIONS: These results indicate that micro-CT imaging can effectively assess rabbit fetal skeletal structures, and for those laboratories with this resource, may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.

Role of genetic background in determining phenotypic severity throughout postnatal development and at peak bone mass in Col1a2 deficient mice (oim).

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disease characterized by extreme bone fragility. Although fracture numbers tend to decrease post-puberty, OI patients can exhibit significant variation in clinical outcome, even among related individuals harboring the same mutation. OI most frequently results from mutations in type I collagen genes, yet how genetic background impacts phenotypic outcome remains unclear. Therefore, we analyzed the phenotypic severity of a known proalpha2(I) collagen gene defect (oim) on two genetic backgrounds (congenic C57BL/6J and outbred B6C3Fe) throughout postnatal development to discern the phenotypic contributions of the Col1a2 locus relative to the contribution of the genetic background. To this end, femora and tibiae were isolated from wildtype (Wt) and homozygous (oim/oim) mice of each strain at 1, 2 and 4 months of age. Femoral geometry was determined via muCT prior to torsional loading to failure to assess bone structural and material biomechanical properties. Changes in mineral composition, collagen content and bone turnover were determined using neutron activation analyses, hydroxyproline content and serum pyridinoline crosslinks. muCT analysis demonstrated genotype-, strain- and age-associated changes in femoral geometry as well as a marked decrease in the amount of bone in oim/oim mice of both strains. Oim/oim mice of both strains, as well as C57BL/6J (B6) mice of all genotypes, had reduced femoral biomechanical strength properties compared to Wt at all ages, although they improved with age. Mineral levels of fluoride, magnesium and sodium were associated with biomechanical strength properties in both strains and all genotypes at all ages. Oim/oim animals also had reduced collagen content as compared to Wt at all ages. Serum pyridinoline crosslinks were highest at two months of age, regardless of strain or genotype. Strain differences in bone parameters exist throughout development, implicating a role for genetic background in determining biomechanical strength. Age-associated improvements indicate that oim/oim animals partially compensate for their weaker bone material, but never attain Wt levels. These studies indicate the importance of genetic background in determining phenotypic severity, but the presence of the proalpha2(I) collagen gene defect and age of the animal are the primary determinants of phenotypic severity.

TLD assessment of mouse dosimetry during microCT imaging.

Advances in laboratory animal imaging have provided new resources for noninvasive biomedical research. Among these technologies is microcomputed tomography (microCT) which is widely used to obtain high resolution anatomic images of small animals. Because microCT utilizes ionizing radiation for image formation, radiation exposure during imaging is a concern. The objective of this study was to quantify the radiation dose delivered during a standard microCT scan. Radiation dose was measured using thermoluminescent dosimeters (TLDs), which were irradiated employing an 80 kVp x-ray source, with 0.5 mm A1 filtration and a total of 54 mA s for a full 360 deg rotation of the unit. The TLD data were validated using a 3.2 cm3 CT ion chamber probe. TLD results showed a single microCT scan air kerma of 78.0 +/- 5.0 mGy when using a poly(methylmethacrylate) (PMMA) anesthesia support module and an air kerma of 92.0 +/- 6.0 mGy without the use of the anesthesia module. The validation CT ion chamber study provided a measured radiation air kerma of 81.0 +/- 4.0 mGy and 97.0 +/- 5.0 mGy with and without the PMMA anesthesia module, respectively. Internal TLD analysis demonstrated an average mouse organ radiation absorbed dose of 76.0 +/- 5.0 mGy. The author's results have defined x-ray exposure for a routine microCT study which must be taken into consideration when performing serial molecular imaging studies involving the microCT imaging modality.

fMRI study of the role of glutamate NMDA receptor in the olfactory processing in monkeys.

Studies in rodents show that olfactory processing in the principal neurons of olfactory bulb (OB) and piriform cortex (PC) is controlled by local inhibitory interneurons, and glutamate NMDA receptor plays a role in this inhibitory control. It is not clear if findings from studies in rodents translate to olfactory processing in nonhuman primates (NHPs). In this study, the effect of the glutamate NMDA receptor antagonist MK801 on odorant-induced olfactory responses in the OB and PC of anesthetized NHPs (rhesus monkeys) was investigated by cerebral blood volume (CBV) fMRI. Isoamyl-acetate was used as the odor stimulant. For each NHP, sixty fMRI measurements were made during a 4-h period, with each 4-min measurement consisting of a 1-min baseline period, a 1-min odor stimulation period, and a 2-min recovery period. MK801 (0.3 mg/kg) was intravenously delivered 1 hour after starting fMRI. Before MK801 injection, olfactory fMRI activations were observed only in the OB, not in the PC. After MK801 injection, olfactory fMRI activations in the OB increased, and robust olfactory fMRI activations were observed in the PC. The data indicate that MK801 enhances the olfactory responses in both the OB and PC. The enhancement effects of MK801 are most likely from its blockage of NMDA receptors on local inhibitory interneurons and the attenuation of the inhibition onto principal neurons. This study suggests that the mechanism of local inhibitory control of principal neurons in the OB and PC derived from studies in rodents translates to NHPs.

Assessment of near-infrared fluorophores to study the biodistribution and tumor targeting of an IL13 receptor α2 antibody by fluorescence molecular tomography.

Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17-34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

Melanoma therapy via peptide-targeted {alpha}-radiation.

PURPOSE: The therapeutic efficacy of a unique melanoma-targeting peptide conjugated with an in vivo generated alpha-particle-emitting radionuclide was evaluated in the B16/F1 mouse melanoma animal model. alpha-Radiation is densely ionizing, resulting in high concentrations of destructive radicals and irreparable DNA double-strand breaks. This high linear energy transfer overcomes radiation-resistant tumor cells and oxygen effects resulting in potentially high therapeutic indices in tumors such as melanoma. EXPERIMENTAL DESIGN: The melanoma targeting peptide, 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA)-Re(Arg(11))CCMSH, was radiolabeled with (212)Pb, the parent of (212)Bi, which decays via alpha and beta decay. Biodistribution and therapy studies were done in the B16/F1 melanoma-bearing C57 mouse flank tumor model. RESULTS: (212)Pb[DOTA]-Re(Arg(11))CCMSH exhibited rapid tumor uptake and extended retention coupled with rapid whole body disappearance. Radiation dose delivered to the tumor was estimated to be 61 cGy/muCi (212)Pb administered. Treatment of melanoma-bearing mice with 50, 100, and 200 muCi of (212)Pb[DOTA]-Re(Arg(11))CCMSH extended their mean survival to 22, 28, and 49.8 days, respectively, compared with the 14.6-day mean survival of the placebo control group. Forty-five percent of the mice receiving 200 muCi doses survived the study disease-free. CONCLUSIONS: Treatment of B16/F1 murine melanoma-bearing mice with (212)Pb[DOTA]-Re(Arg(11))CCMSH significantly decreased tumor growth rates resulting in extended mean survival times, and in many cases, complete remission of disease. (212)Pb-DOTA-Re(Arg(11))CCMSH seems to be a very promising radiopharmaceutical for targeted radionuclide therapy of melanoma.

Odanacatib, effects of 16-month treatment and discontinuation of therapy on bone mass, turnover and strength in the ovariectomized rabbit model of osteopenia.

Odanacatib (ODN) a selective and reversible cathepsin K inhibitor, inhibits bone resorption, increases bone mass and reduces fracture risk in women with osteoporosis. A 16-month (~7-remodeling cycles) study was carried out in treatment mode to assess the effects of ODN versus ALN on bone mass, remodeling status and biomechanical properties of lumbar vertebrae (LV) and femur in ovariectomized (OVX) rabbits. This study also evaluated the impact of discontinuing ODN on these parameters. Rabbits at 7.5months post-OVX were dosed for 16-months with ODN (7.5µM·h0-24, in food) or ALN (0.2mg/kg/wk, s.c.) and compared to vehicle-treated OVX- (OVX+Veh) or Sham-operated animals. After 8months, treatment was discontinued in half of the ODN group. ODN treatment increased in vivo LV aBMD and trabecular (Tb) vBMD until reaching plateau at month 12 by 16% and 23% vs. baseline, respectively, comparable levels to that in Sham and significantly above OVX+Veh. LV BMD was also higher in ALN that plateaued around month 8 to levels below that in ODN or Sham. ODN treatment resulted in higher BMD, structure and improved biomechanical strength of LV and central femur (CF) to levels similar to Sham. ALN generally showed less robust efficacy compared to ODN. Neither ODN nor ALN influenced material properties at these bone sites following ODN or ALN treatment for 7 remodeling cycles in rabbits. ODN and ALN persistently reduced the bone resorption marker urinary helical peptide over study duration. While ALN reduced the bone formation marker BSAP, ODN treatment did not affect this marker. ODN also preserved histomorphometry-based bone formation indices in LV trabecular, CF endocortical and intracortical surfaces, at the levels of OVX+Veh. Discontinuation of ODN returned bone mass, structure and strength parameters to the comparable respective levels in OVX+Veh. Together, these data demonstrate efficacy and bone safety profile of ODN and suggests the potential long-term benefits of this agent over ALN with respect to accrued bone mass without long-term effects on bone formation.

Considerations for conducting imaging studies in support of developmental toxicology studies for regulatory submission.

Preclinical imaging technologies are increasingly being applied to developmental toxicology studies in drug development to determine potential compound toxicity. Although most of these studies are conducted in a non-regulatory setting, there is interest in performing these imaging studies under applicable regulations, for example Good Laboratory Practices (GLP), to support regulatory decisions concerning drug safety. This manuscript will describe regulations and processes to consider when bringing an imaging technology into GLP compliance.