23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity.

Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit.

Comparison of cobinamide to hydroxocobalamin in reversing cyanide physiologic effects in rabbits using diffuse optical spectroscopy monitoring.

Our purpose is to compare cobinamide to hydroxocobalamin in reversing cyanide (CN)-induced physiologic effects in an animal model using diffuse optical spectroscopy (DOS). Cyanide poisoning is a major threat worldwide. Cobinamide is a novel molecule that can bind two molecules of cyanide, has a much higher binding affinity than hydroxocobalamin, and is more water soluble. We investigated the ability of equimolar doses of cobinamide and hydroxocobalamin to reverse the effects of cyanide exposure in an animal model monitored continuously by DOS. Cyanide toxicity was induced in 16 New Zealand white rabbits by intravenous infusion. Animals were divided into three groups: controls (n=5) received saline following cyanide, hydroxocobalamin (N=6) following cyanide, and cobinamide (N=5) following cyanide. Cobinamide caused significantly faster and more complete recovery of oxy- and deoxyhemoglobin concentrations in cyanide-exposed animals than hydroxocobalamin- or saline-treated animals, with a recovery time constant of 13.8+/-7.1 min compared to 75.4+/-25.1 and 76.4+/-42.7 min, for hydroxocobalamin- and saline-treated animals, respectively (p<0.0001). This study indicates that cobinamide more rapidly and completely reverses the physiologic effects of cyanide than equimolar doses of cobalamin at the dose used in this study, and CN effects and response can be followed noninvasively using DOS.

Intramuscular cobinamide sulfite in a rabbit model of sublethal cyanide toxicity.

STUDY OBJECTIVE: Exposure to cyanide in fires and industrial exposures and intentional cyanide poisoning by terrorists leading to mass casualties is an ongoing threat. Current treatments for cyanide poisoning must be administered intravenously, and no rapid treatment methods are available for mass casualty cyanide exposures. Cobinamide is a cobalamin (vitamin B(12)) analog with an extraordinarily high affinity for cyanide that is more water-soluble than cobalamin. We investigate the use of intramuscular cobinamide sulfite to reverse cyanide toxicity-induced physiologic changes in a sublethal cyanide exposure animal model and determine the ability of an intramuscular cobinamide sulfite injection to rapidly reverse the physiologic effects of cyanide toxicity. METHODS: New Zealand white rabbits were given 10 mg sodium cyanide intravenously over 60 minutes. Quantitative diffuse optical spectroscopy and continuous-wave near-infrared spectroscopy monitoring of tissue oxyhemoglobin and deoxyhemoglobin concentrations were performed concurrently with blood cyanide level measurements and cobinamide levels. Immediately after completion of the cyanide infusion, the rabbits were injected intramuscularly with cobinamide sulfite (n=6) or inactive vehicle (controls, n=5). RESULTS: Intramuscular administration led to rapid mobilization of cobinamide and was extremely effective at reversing the physiologic effects of cyanide on oxyhemoglobin and within deoxyhemoglobin extraction. Recovery time to 63% of their baseline values in the central nervous system occurred within a mean of 1,032 minutes in the control group and 9 minutes in the cobinamide group, with a difference of 1,023 minutes (95% confidence interval 116 to 1,874 minutes). In muscle tissue, recovery times were 76 and 24 minutes, with a difference of 52 minutes (95% confidence interval 7 to 98 minutes). RBC cyanide levels returned toward normal significantly faster in cobinamide sulfite-treated animals than in control animals. CONCLUSION: Intramuscular cobinamide sulfite rapidly and effectively reverses the physiologic effects of cyanide poisoning, suggesting that a compact cyanide antidote kit can be developed for mass casualty cyanide exposures.

Broadband diffuse optical spectroscopy assessment of hemorrhage- and hemoglobin-based blood substitute resuscitation.

Hemoglobin-based oxygen carriers (HBOCs) are solutions of cell-free hemoglobin (Hb) that have been developed for replacement or augmentation of blood transfusion. It is important to monitor in vivo tissue hemoglobin content, total tissue hemoglobin [THb], oxy- and deoxy-hemoglobin concentrations ([OHb], [RHb]), and tissue oxygen saturation (S(t)O(2)=[OHb][THb]x100%) to evaluate effectiveness of HBOC transfusion. We designed and constructed a broadband diffuse optical spectroscopy (DOS) prototype system to measure bulk tissue absorption and scattering spectra between 650 and 1000 nm capable of accurately determining these tissue hemoglobin component concentrations in vivo. Our purpose was to assess the feasibility of using DOS to optically monitor tissue [OHb], [RHb], S(t)O(2), and total tissue hemoglobin concentration ([THb]=[OHb]+[RHb]) during HBOC infusion using a rabbit hypovolemic shock model. The DOS prototype probe was placed on the shaved inner thigh muscle of the hind leg to assess concentrations of [OHb], [RHb], [THb], as well as S(t)O(2). Hemorrhagic shock was induced in intubated New Zealand white rabbits (N=6) by withdrawing blood via a femoral arterial line to 20% blood loss (10-15 cckg). Hemoglobin glutamer-200 (Hb-200) 1:1 volume resuscitation was administered following the hemorrhage. These values were compared against traditional invasive measurements, serum hemoglobin concentration (sHGB), systemic blood pressure, heart rate, and blood gases. DOS revealed increases of [THb], [OHb], and tissue hemoglobin oxygen saturation after Hb-200 infusion, while blood total hemoglobin values continued did not increase; we speculate, due to hyperosmolality induced hemodilution. DOS enables noninvasive in vivo monitoring of tissue hemoglobin and oxygenation parameters during shock and volume expansion with HBOC and potentially enables the assessment of efficacy of resuscitation efforts using artificial blood substitutes.

Detection and monitoring of early airway injury effects of half-mustard (2-chloroethylethylsulfide) exposure using high-resolution optical coherence tomography.

Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.

In vivo three-dimensional imaging of normal tissue and tumors in the rabbit pleural cavity using endoscopic swept source optical coherence tomography with thoracoscopic guidance.

The purpose of this study was to develop a dynamic tunable focal distance graded-refractive-index lens rod-based high-speed 3-D swept-source (SS) optical coherence tomography (OCT) endoscopic system and demonstrate real-time in vivo, high-resolution (10-microm) imaging of pleural-based malignancies in an animal model. The GRIN lens-based 3-D SS OCT system, which images at 39 fps with 512 A-lines per frame, was able to capture images of and detect abnormalities during thoracoscopy in the thoracic cavity, including the pleura, chest wall, pericardium, and the lungs. The abnormalities were confirmed by histological evaluation and compared to OCT findings. The dynamic tunable focal distance range and rapid speed of the probe and SS prototype OCT system enabled this first-reported application of in vivo 3-D thoracoscopic imaging of pleural-based malignancies. The imaging probe of the system was found to be easily adaptable to various sites within the thoracic cavity and can be readily adapted to other sites, including rigid airway endoscopic examinations.

In vivo optical coherence tomography detection of differences in regional large airway smoke inhalation induced injury in a rabbit model.

Smoke inhalation injury causes acute airway injury that may result in airway compromise with significant morbidity and mortality. We investigate the ability of high resolution endobronchial optical coherence tomography (OCT) to obtain real-time images for quantitatively assessing regional differences between upper tracheal versus lower tracheal and bronchial airway injury responses to smoke inhalation in vivo using a prototype spectral domain (SLD)-OCT system we constructed, and flexible fiber optic probes. 33 New Zealand White rabbits are intubated and mechanically ventilated. The treatment groups are exposed to inhaled smoke. The OCT probe is introduced through the endotracheal tube and maintained in place for 5 to 6 h. Images of airway mucosa and submucosa are obtained at baseline and at specified intervals postexposure. Starting within less than 15 min after smoke inhalation, there is significant airway thickening in the smoke-exposed animals. This is maintained over 5 h of imaging studies. The lower tracheal airway changes, correlating closely with carboxyhemoglobin levels, are much greater than upper tracheal changes. Significant differences are seen in lower trachea and bronchi after acute smoke inhalation compared to upper trachea as measured in vivo by minimally invasive OCT. OCT is capable of quantitatively detecting regional changes in airway swelling following inhalation injury.

Development of a rabbit pleural cancer model by using VX2 tumors.

Primary and secondary pleural cancer remains an important clinical problem, with research progress limited by the lack of a suitable moderate- to large-sized (3 to 4 kg) animal model of pleural cancer. Many potential pleura-based imaging and treatment modalities cannot be investigated sufficiently by using currently available small murine animal models because their pleural space is not comparable to that of humans and therefore does not allow for the use of standard thoracoscopic techniques. Here we describe the development of a reproducible model of pleural malignancy in moderate-sized immunocompetent rabbits. Under thoracoscopic guidance, 9-15 x 10(6) VX2 carcinoma cells were inoculated into the plural space of 3 to 4 kg New Zealand white rabbits that had undergone gentle pleural abrasion. Malignant tumor involvement developed on the visceral and parietal pleural surfaces in an average of 2 to 4 wk. This novel pleural tumor model induction method likely will facilitate a broad range of investigations of pleural cancer diagnostics and therapeutics.

Detection of acute smoke-induced airway injury in a New Zealand white rabbit model using optical coherence tomography.

Optical coherence tomography (OCT) is a micron scale high-resolution optical technology that can provide real-time in vivo images noninvasively. The ability to detect airway mucosal and submucosal injury rapidly will be valuable for a range of pulmonary applications including assessment of acute inhalation smoke and burn injury. OCT has the potential ability to monitor the progression of airway injury changes including edema, hyperemia, and swelling, which are critical clinical components of smoke-inhalation injury. New Zealand white male rabbits exposed to cold smoke from standardized unbleached burned cotton administered during ventilation were monitored for 6 h using a 1.8-mm diameter flexible fiberoptic longitudinal probe that was inserted through the endotracheal tube. The thickness of the epithelial, mucosal, and submucosal layers of the rabbit trachea to the tracheal cartilage was measured using a prototype superluminescent diode OCT system we constructed. OCT was able to detect significant smoke-injury-induced increases in the thickness of the tracheal walls of the rabbit beginning very shortly after smoke administration. Airway wall thickness increased to an average of 120% (+/-33%) of baseline values by 5 h following exposure. OCT is capable of providing real-time, noninvasive images of airway injury changes following smoke exposure. These studies suggest that OCT may have the ability to provide information on potential early indicators of impending smoke-inhalation-induced airway compromise.

Non-invasive in vivo diffuse optical spectroscopy monitoring of cyanide poisoning in a rabbit model.

The objective of this study is to establish a cyanide toxicity animal model and to investigate the ability of broadband diffuse optical spectroscopy (DOS) to non-invasively monitor physiological changes that occur during the development of cyanide toxicity in a rabbit model. Broadband DOS combines multi-frequency frequency-domain photon migration (FDPM) with time-independent near-infrared spectroscopy (NIRS) to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1000 nm. Serum cyanide concentration and arterial and venous blood gas analysis at pre- and post-cyanide infusion were presented. To investigate the ability of DOS to non-invasively monitor physiologic changes occurring during development of CN toxicity, tissue concentrations of deoxyhemoglobin [Hb-R], oxyhemoglobin [Hb-O2], cytochrome c oxidase oxidized state [CcO_Ox] and reduced state [CcO_Re] were determined from absorption spectra acquired in 'real time' during cyanide infusions (NaCN 6 mg/60 ml normal saline) in six pathogen-free New Zealand white rabbits. During cyanide infusion, in vivo tissue oxygen saturation increased ( approximately 10%). In addition, broadband DOS was able to detect a concurrent increase in [CcO_Re] and decrease in [CcO_Ox]. Changes in tissue scattering properties in all six animals were detected during these events, confirming the need for DOS-based methods over traditional NIR spectroscopy to obtain accurate results.

Potential interference by hydroxocobalamin on cooximetry hemoglobin measurements during cyanide and smoke inhalation treatments.

STUDY OBJECTIVE: Concentrated aqueous solutions of hydroxocobalamin (OHCob) are administered intravenously for cyanide poisoning victims, many of whom also have concurrent smoke inhalation. Because of its intense light absorbance in visible wavelengths (absorption peak at 532 nm), we investigate potential interference effects of OHCob on total hemoglobin concentration (tHb), carboxyhemoglobin (COHb), methemoglobin (MetHb), and oxyhemoglobin (Hb-O2) cooximetry measurement values in blood. METHODS: In vivo cooximetry measurements were conducted with 3 specific pathogen-free white New Zealand rabbits (3.80+/-0.21 kg) during the intravenous infusion of OHCob (625 mg during a 100-minute period). Resultant changes in tHb, Hb-O2, COHb, and MetHb values were measured and correlated with respect to estimated in vivo OHCob concentrations. In vitro measurements were conducted with rabbit blood to confirm in vivo measurements. RESULTS: The introduction of OHCob clearly interfered with the cooximetry measurements of each of the hemoglobin component fractions in whole blood and resulted in altered measurement values from the baseline values. The presence of OHCob in blood interferes with cooximetry measurements of COHb, MetHb, and Hb-O2. The increase in measured COHb fraction with increasing concentrations of OHCob was most notable. CONCLUSION: The presence of OHCob in blood interferes with cooximetry measurements of COHb, MetHb, and Hb-O2. These effects need to be considered during OHCob treatment of cyanide poisoning, particularly in smoke inhalation victims with potential for concurrent carbon monoxide exposure, because it may lead to potentially erroneous reported COHb levels.

Feasibility of three-dimensional optical coherence tomography and optical Doppler tomography of malignancy in hamster cheek pouches.

OBJECTIVE: Hamster cheek pouches (HCP) with various degrees of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced dysplasia and malignancies were imaged with OCT/ODT in vivo and in vitro to assess the potential for three-dimensional high-resolution optical localization of airway malignancy. BACKGROUND DATA: Optical coherence tomography (OCT)/optical doppler tomography (ODT) provide potential capability for real-time in vivo high-resolution (2-20 microm) cross-sectional imaging of tissues and spatially resolved blood flow in microvasculature for pathology diagnostics. METHODS: DMBA was applied to the right side of the cheek pouch (HCP), and mineral oil (control) to the left side three times weekly for 10-18 weeks in Syrian Golden Hamsters using a standard protocol for malignancy induction. HCP were imaged in vivo with OCT/ODT as well as in vitro post-excision, using a prototype 1310-nm broadband superluminescent diode-based OCT/ODT device constructed in our laboratory. Three-dimensional images were constructed, and compared to standard and three-dimensional histology hematoxylin and eosin staining. RESULTS AND CONCLUSION: OCT imaging offered exceptional resolution of the HCP to depths of 1-2 mm and confirmed ability to detect dysplasia and malignancy. Three-dimensional OCT images were readily constructed, allowing visualization of extent and localization of tumor margins. ODT demonstrated increased vascularity in the area of neoplasia. OCT/ODT is a promising new technology for oral airway diagnostics.