Molecular Probes in Disease and a Potential Role in Biodosimetry During Nuclear Threat and Disaster.

A Short Talk by Dr. Rao V. L. Papineni Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525, USA.
rao.papineni1@carestreamhealth.com

Abstract
Small molecules like bisphophonates bind to bones with high affinity and regulate homeostasis in bone remodeling orchestrated by osteoclasts. Bones responsible for multiple physiological functions also provide a pillar of support to several maladies. Breast, lung, and prostate cancer cells metastasize frequently at bone resulting in debilitating pain, which is also one of the major complications in a majority of patients. Bisphosphonates apart from being a potent inhibitor of bone resorption, also provides pain relief and improves bone health. However these medications result in complications such as bisphosphonate-related osteonecrosis of the jaw (BRONJ). Our continued efforts in investigating site-selective skeletal targeting of bisphosphonates are primarily to establish modes to overcome such drug-related side-effects. Small molecular probes are used here to demonstrate both the targeting, and for non-invasive imaging of bone related physiological activities. In vivo non-invasive imaging of mice was performed using Optical, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Xray Computed Tomography (CT). Using the approaches described above we show how bisphosphanate analogs can be utilized for both therapeutic and diagnostic purposes.
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Molecular Imaging of Radiation Related Normal Tissue Toxicity

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N H 640 LSS Xsight dye

640 LSS Xsight dye

Bisphophonate analogs the new class of theragnostic genre of drugs?

NIR Dye Conjugated Bisphosphonate- A Bone Binding Agent. Mouse pup was administered (gavage) with Kodak Large stokes shift dye conjugated- Alendronate (0.25 nmol) . Post 24 hrs, NIRF imaging was performed - EX: 655 EM: 750; no binning; 300 sec exp; 80mm FOV

Irradiated mice Control Mice (0 Gy)

Unshielded hind-limb Three sets of athymic nude mice (n=3) were subjected to 0, 5 Gy, and 10 Gy X-ray irradiation focally over the entire right hind limb. NIR dye conjugated bisphosphonate (Osteosense) was injected intravenously to assess the changes in the bone binding ability of the molecular probe. The mice were imaged from different angles by automatic rotation using a commercially available in vivo multispectral-FX imaging system attached with multimodal animal rotation system (MARS). Non-invasive near-infrared fluorescence (NIRF) imaging indicated a significant increase in NIR signals at the growing regions of the long bones of irradiated mice likely due to osteoradionecrosis related changes, and/or radiation induced bone morphological changes.
TORSIONAL SUPPORT APPARATUS AND METHOD FOR CRANIOCAUDAL ROTATION OF ANIMALS Patent app number: 20100022866 Gilbert Feke Rao Papineni et al

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Theragnostics: Therapeutics with Diagnostics Seek, See, Subdue and Salve

Can localized vibration loading enhance bisphosphonate binding?

Vibration loading, was performed on multiple sets of mice subjecting them to vibration frequency of 70 Hz for five min (5 times/week) duration at the right thigh region. After the 21-day exercise regime, Osteosense was injected intravenously prior to the day of near-IR fluorescence imaging. NMRI-Foxn1nu adult mice was used for the studies. Near-IR Fluorescence (NIRF) and X-ray Imaging. The animals were imaged with a commercially available imaging system - bench-top multimodal (X-ray and optical) imaging apparatusCarestream in-Vivo Multispectral FX Pro.

Small Molecules in SPECT/Optical/CT/Planar X-ray Imaging of Bone

Optical/X-ray
Localized Drug Targeting at Lumbar Vertebrae by Vibration Loading

Targeting-Bioligand

Optical X-ray PET MRI/MRS Ultrasound

Drugs Peptides siRNA DNA Cells radiation

Drugs Cells Biomolecules Nutriceuticals

Vibration Protocol:

Multiple (A)

1 time (B)

Placebo (C)

Courtesy Dr. Michael Bolognese

A B C A B C A B C

Bisphoshonates
pamidronate Mevalonate
Farnesyl diphosphate synthase
Lumbar Knee NT-Thigh

Net Intensity- Ratio

Multiple Vibration 2.56 1.06 1.22 Single Vibration 1.75 0.68 1.1 Control

1 1 1

Farnesyl diphosphate

A B C Lumbar spine Targeting (fold increase) : 3.34 +/- 0.96 (n=3; Std Dev.)

www.ncbi.nlm.nih.gov

Bisphosphonates by preventing protein prenylation inhibit osteoclast function. Thus form a potent drugs for bone loss.

Near-infrared (NIR) fluorescence imaging of LNCaP cells grown orthotopically in nude mice.
On going work in support of the proposed concepts. 1 mCi of Tc99m-MDP (SPECT), 0.2mCi of Na18F (PET), or 4 nmoles of Osteosense 750 (optical Imaging) injected in mice and imaged on the Albira tri-modal PET/SPECT/CT imaging system. The optical whole body imaging of the mouse was carried out on a Carestream In-Vivo MS FX PRO imaging system equipped with a Multimodal Animal Rotation System (MARS) ( Sean Orton et al.) Near IR-Dye Conjugated Bisphosphonates Injected intra-tumoral (orthotopic prostrate) and excised after18 hrs.

Geranyl geranyl diphosphate

NIR Dye

NIR-Alendronate

NIR Dye

NIR-Alendronate

emedicine.medscape.com

Persistent Drug Binding at the site.

X-ray Image Ex Vivo Tumor Imaging. The excised tumors of control and NIRalendronate injected mice were imaged (NIRF:exp: 60 sec; 80 FOV). The X-ray (upper left) and NIRF-X-ray overlay (upper right) are shown. The fluorescence data is plotted to the right.

Apoptosis

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is one of the side-effects- a condition found in patients who have received intravenous and oral forms of alendronate. Other effects include peculiar bone fractures ensues with prolonged bisphosphonate therapy.

4 nmol Bisphosphonate-NIR

NIR Dye NIR-Alendronate

Near-IR Fluorescence

Carestream is a trademark of Carestream Health, Inc. Carestream Molecular Imaging is a division of Carestream Health, Inc. Although the Carestream In-Vivo Multispectral Imaging System FX can be used for in vivo and in vitro molecular imaging of materials, researchers should be aware that the methods of preparing and viewing the materials for molecular imaging may be subject to various patent rights.

PET/CT

SPECT/CT

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Carestream Health, Inc.
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Summary
The induction of localized bisphosphonate targeting by induced vibration provides the opportunity to use such methods as bisphoshonate therapeutic aid and overcome side-effects resulting from prolonged bisphosphonate use. NIR - tricarbocyanine, cyclic enamine dye conjugated alendronate is a potential tumor theragnostic agent - bisphosphonates has been shown to prevent cancer cell proliferation and prevent metastasis. Screening of high affinity analogs of bisphosphonates for tumor binding activity is now feasible with the noninvasive approach demonstrated here. Bisphosphonate molecular probes is capable of determining radiation induced sensitive changes in bone structure. The property can be utilized in biodosimetry for radiation disasters or in radiation threat. These theragnostic agents can also be used for non invasive monitoring of the progression of therapy.

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"Molecular Imaging - Wisdom To See For Maladies To Flee" Dr. Rao V. L. Papineni