Safety Evaluation of an Alpha-Emitter Bismuth-213 Labeled Antibody to (1→3)-ß-Glucan in Healthy Dogs as a Prelude for a Trial in Companion Dogs with Invasive Fungal Infections.

Background: With the limited options available for therapy to treat invasive fungal infections (IFI), radioimmunotherapy (RIT) can potentially offer an effective alternative treatment. Microorganism-specific monoclonal antibodies have shown promising results in the experimental treatment of fungal, bacterial, and viral infections, including our recent and encouraging results from treating mice infected with Blastomyces dermatitidis with 213Bi-labeled antibody 400-2 to (1→3)-ß-glucan. In this work, we performed a safety study of 213Bi-400-2 antibody in healthy dogs as a prelude for a clinical trial in companion dogs with acquired invasive fungal infections and later on in human patients with IFI. Methods: Three female beagle dogs (≈6.1 kg body weight) were treated intravenously with 155.3, 142.5, or 133.2 MBq of 213Bi-400-2 given as three subfractions over an 8 h period. RBC, WBC, platelet, and blood serum biochemistry parameters were measured periodically for 6 months post injection. Results: No significant acute or long-term side effects were observed after RIT injections; only a few parameters were mildly and transiently outside reference change value limits, and a transient atypical morphology was observed in the circulating lymphocyte population of two dogs. Conclusions: These results demonstrate the safety of systemic 213Bi-400-2 administration in dogs and provide encouragement to pursue evaluation of RIT of IFI in companion dogs.

Mitochondrial dysfunction contributes to the cytotoxicity of some 3,5-bis(benzylidene)-4-piperidone derivatives in colon HCT-116 cells.

The objectives of this study are to investigate the possible ways by which the curcumin analogs 2a and 2b exert their antiproliferative properties. The analogs 2a and 2b have submicromolar IC(50) values towards human HCT-116 colon cancer cells but are far less toxic to human non-malignant CRL-1790 colon cells. Both compounds affected a number of mitochondrial functions in HCT-116 cells namely increasing the intracellular concentrations of reactive oxygen species, inhibiting oxygen consumption and decreasing the mitochondrial membrane potential. These molecules also produced swelling of isolated rat liver mitochondria, supporting a mitochondrial mechanism of cytotoxicity. Both compounds reacted with glutathione in the presence of glutathione S-transferase π and hence they may be classified as thiol alkylators.

Doxorubicin Cytotoxicity in Differentiated H9c2 Cardiomyocytes: Evidence for Acute Mitochondrial Superoxide Generation.

Although a mitochondrial redox-cycling superoxide-generating mechanism for the cardiotoxicity of doxorubicin was suggested from experiments with isolated mitochondria, its occurrence and contribution to cytotoxicity in intact cardiomyocytes is not fully established. Therefore, we determined the immediate and delayed effects of doxorubicin on the generation of reactive oxygen species (ROS) and cytotoxicity in differentiated H9c2 cardiomyocytes. Although relatively short incubations (3 or 6 h) with 1 or 5 µM doxorubicin did not acutely decrease cell survival, exposure to 5 µM doxorubicin for 3 h was sufficient to cause a significant delayed decrease in cell survival after an additional 24 h without doxorubicin. Mitochondrial superoxide generation was observed to increase within 30 min of incubation with 5 µM doxorubicin. Increased intracellular ROS generation, decreased mitochondrial metabolic activity, and decreased mitochondrial membrane potential (MMP) were observed after more extended periods (6-12 h). Overall, these observations support that the toxicity of doxorubicin to differentiated cardiomyocytes involves acute mitochondrial superoxide generation with subsequent intracellular ROS generation, mitochondrial dysfunction, and cell death.

Radioimmunotherapy of Blastomycosis in a Mouse Model With a (1→3)-ß-Glucans Targeting Antibody.

Invasive fungal infections (IFI) cause devastating morbidity and mortality, with the number of IFIs more than tripling since 1979. Our laboratories were the first to demonstrate that radiolabeled microorganism-specific monoclonal antibodies are highly effective for treatment of experimental fungal, bacterial and viral infections. Later we proposed to utilize surface expressed pan-antigens shared by major IFI-causing pathogens such as beta-glucans as RIT targets. Here we evaluated in vivo RIT targeting beta-glucan in Blastomyces dermatitidis which causes serious infections in immunocompromised and immunocompetent individuals and in companion dogs. B. dermatitidis cells were treated with the 400-2 antibody to (1→3)-ß-glucans radiolabeled with the beta-emitter 177Lutetium (177Lu) and alpha-emitter 213Bismuth (213Bi) and the efficacy of cell kill was determined by colony forming units (CFUs). To determine the antigen-specific localization of the 400-2 antibody in vivo, C57BL6 mice were infected intratracheally with 2 × 105 B. dermatitidis cells and given 111In-400-2 antibody 24 h later. To evaluate the killing of B. dermatitidis cells with RIT, intratracheally infected mice were treated with 150 µCi 213Bi-400-2 and their lungs analyzed for CFUs 96 h post-infection. 213Bi-400-2 proved to be more effective in killing B. dermatitidis cells in vitro than 177Lu-400-2. Three times more 111In-400-2 accumulated in the lungs of infected mice, than in the non-infected ones. 213Bi-400-2 lowered the fungal burden in the lungs of infected mice more than 2 logs in comparison with non-treated infected controls. In conclusion, our results demonstrate the ability of an anti-(1-3)-beta-D-glucan antibody armed with an alpha-emitter 213Bi to selectively kill B. dermatitidis cells in vitro and in vivo. These first in vivo results of the effectiveness of RIT targeting pan-antigens on fungal pathogens warrant further investigation.

Photocatalytic degradation of phenazopyridine contaminant in soil with direct solar light.

Photocatalytic degradation of waste pharmaceutics, with solar radiation, is described here as a feasible method to purify pre-contaminated soils. Phenazopyridine has been used as a model soil contaminant. Two different nano-size powders have been first examined as catalysts, namely commercial TiO2 (anatase) and commercial ZnO. As the ZnO showed higher catalytic efficiency, the study was then focused on it. The commercial ZnO powder was then compared with lab-prepared ZnO powder, and the latter shows relatively higher efficiency. The ZnO was used in two different ways. In one way, dry ZnO catalyst powder was spread onto the soil, while in the other way the ZnO was sprayed onto the soil surface by a wet spray method. The spray technique shows slightly higher efficiency, in addition to being easier to apply at future large scale. Depending on conditions and type of photocatalyst used, up to 90% contaminant removal can be achieved in 30 min. In case of photocatalysis experiments, the reacted contaminant molecules undergo complete degradation with no detectable side reaction organic products. Possible evaporation or escape of organic contaminant, or other possibly resulting organics, is ruled out by a series of control experiments. Photodegradation process takes place only at the catalytic sites on the soil surface, where contaminant molecules that diffuse from the soil bulk are completely degraded. Other useful organisms inside the soil are not affected as they are kept away from catalyst sites. A plausible mechanism is proposed for the degradation process.

Evaluation of novel highly specific antibodies to cancer testis antigen Centrin-1 for radioimmunoimaging and radioimmunotherapy of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of pancreatic malignancies, and has median survival of <6 months. There is an urgent need for diagnostic and therapeutic options for PDAC. Centrin1 (CETN1) is a novel member of Cancer/Testis Antigens, with a 25-fold increase of CETN1 gene expression in PDX from PDAC patients. The absence of selective anti-CETN1 antibodies is hampering CETN1 use for diagnosis and therapy. Here we report the generation of highly specific for CETN1 antibodies and their evaluation for radioimmunoimaging and radioimmunotherapy (RIT) of experimental PDAC. METHODS: The antibodies to CETN1 were generated via mice immunization with immunogenic peptide distinguishing CETN1 from CETN2. Patient tumor microarrays were used to evaluate the binding of the immune serum to PDAC versus normal pancreas. The antibodies were tested for their preferential binding to CETN1 over CETN2 by ELISA. Mice bearing PDAC MiaPaCa2 xenografts were imaged with microSPECT/CT and treated with 213 Bi- and 177 Lu-labeled antibodies to CETN1. RESULTS: Immune serum bind to 50% PDAC cases on patient tumor microarrays with no specific binding to normal pancreas. Antibodies demonstrated preferential binding to CETN1 versus CETN2. Antibody 69-11 localized to PDAC xenografts in mice in vivo and ex vivo. RIT of PDAC xenografts with 213 Bi-labeled antibodies was effective, safe, and CETN1-specific. CONCLUSIONS: The results demonstrate the ability of these novel antibodies to detect CETN1 both in vitro and in vivo; as well, the RIT treatment of experimental PDAC when radiolabeled with 213 Bi is highly efficient and safe. Further evaluation of these novel reagents for diagnosis and treatment of PDAC is warranted.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors.

Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors.

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Radioimmunotherapy as a Novel Approach in HIV, Bacterial, and Fungal Infectious Diseases.

In the past several decades, many antimicrobial agents have been used in treating different fungal, bacterial, and viral infections. However, these agents have faced challenges such as pronounced side-effect profiles and pathogen resistance. In addition, a cure for many chronic infections such as human immunodeficiency virus (HIV) has not been achieved, and the incidence of opportunistic infections in immunocompromised patients has increased significantly in the past decades. Therefore, an alternative strategy for combating these infections is needed. Radioimmunotherapy (RIT) has been proposed to be a valuable tool in the management of such infections. The side-effects associated with RIT are minimal as the targeted antigens are only expressed on microbial or infected cells. RIT demonstrated impressive potency in eradicating pathogens in animal models and patient samples. Cryptococcus neoformans, HIV, and Bacillus anthracis are few examples of infections for which RIT has been an effective treatment using radionuclides such as bismuth-213 (213Bi) or rhenium-188 (188Re).

Optimizing photo-mineralization of aqueous methyl orange by nano-ZnO catalyst under simulated natural conditions.

BACKGROUND: Photo-degradation of organic contaminants into non-hazardous mineral compounds is emerging as a strategy to purify water and environment. Tremendous research is being done using direct solar light for these purposes. In this paper we report on optimum conditions for complete mineralization of aqueous methyl orange using lab-prepared ZnO nanopowder catalyst under simulated solar light. RESULTS: Nano-scale ZnO powder was prepared in the lab by standard methods, and then characterized using electronic absorption spectra, photolumenscence emission (PL) spectra, XRD, and SEM. The powder involved a wurtzite structure with ~19 nm particles living in agglomerates. Photo-degradation progressed faster under neutral or slightly acidic conditions which resemble natural waters. Increasing catalyst concentration increased photodegradation rate to a certain limit. Values of catalyst turn over number and degradation percentage increased under higher light intensity, whereas the quantum yield values decreased. The photocatalytic efficiency of nano-ZnO powders in methyl orange photodegradation in water with solar light has been affected by changing the working conditions. More importantly, the process may be used under natural water conditions with pH normally less than 7, with no need to use high concentrations of catalyst or contaminant. The results also highlight the negative impact of possible high concentrations of CO2 on water purification processes. Effects of other added gaseous flows to the reaction mixture are also discussed. CONCLUSION: ZnO nano-particles are useful catalyst for complete mineralization of organic contaminants in water. Photo-degradation of organic contaminants with ZnO nano-particles, methyl orange being an example, should be considered for future large scale water purification processes under natural conditions.

Diaminobenzene schiff base, a novel class of DNA minor groove binder.

Molecules that target the deoxyribonucleic acid (DNA) minor groove are relatively sequence specific and they can be excellent carrier structures for cytotoxic chemotherapeutic compounds which can help to minimize side effects. Two novel isomeric derivatives of diaminobenzene Schiff base [N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene (2MJ) and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene (2MH)] were analyzed for their DNA minor groove binding (MGB) ability using viscometry, UV and fluorescence spectroscopy, computational modeling and clonogenic assay. The result shows that 2MJ and 2MH are strong DNA MGBs with the latter being more potent. 2MH can form interstrand hydrogen bond linkages at its oxygens with N3 of adenines. Changing the 2-hydroxy-3-methoxybenzylidene binding position to the 1,3 location on the diaminobenzene structure (2MJ) completely removed any viable hydrogen bond formation with the DNA and caused significant decrease in binding strength and minor groove binding potency. Neither compound showed any significant cytotoxicity towards human breast, colon or liver cancer cell lines.