A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog.

Nitrosylcobalamin (NO-Cbl), a novel vitamin B12 analog and anti-tumor agent, functions as a biologic 'Trojan horse', utilizing the vitamin B12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis® RP-amide (150 mm × 4.6 mm, 5 µm) column at 35 °C with a mobile phase (1.0 mL min-1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.

Antimicrobial properties of diethylamine NONOate, a nitric oxide donor, against Escherichia coli: a pilot study.

The emergence of SARS-CoV-2, the causative agent of COVID-19, highlights the increasing need for new and effective antiviral and antimicrobial agents. The FDA has recently banned several active ingredients used in hand sanitizers, including triclosan and benzethonium chloride. Nitric oxide (NO) is involved in the innate immune response and is a major component of macrophage-mediated attack on foreign viruses and bacteria. The specific aim of this study was to assess the antibacterial effects of 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NONOate) against Escherichia coli (E. coli). A bacterial growth assay was compared to an adenosine triphosphate (ATP) activity assay at various time points to assess effects of DEA-NONOate on E. coli growth. A UV/Vis spectrophotometer was used to determine concentration of E. coli by measuring optical density (OD) at 630 nm. A luminescent assay was used to measure ATP activity correlating to viable cells. DEA-NONOate at a concentration of 65 mM was able to inhibit the growth of E. coli with the same efficacy as 1 µg ml-1 concentration of ciprofloxacin. Both the OD and ATP assays demonstrated a 99.9% reduction in E. coli. Both a 1 µg ml-1 concentration of ciprofloxacin and a 65 mM concentration of DEA-NONOate achieved 99.9% inhibition of E. coli, verified using both optical density measurement of bacterial cultures in 96 well plates and a luminescent ATP activity assay. The bactericidal effects of DEA-NONOate against E. coli is proof-of-concept to pursue evaluation of nitric oxide-based formulations as antimicrobial and antiviral agents as hand sanitizers.

Immunohistochemical quantification of the cobalamin transport protein, cell surface receptor and Ki-67 in naturally occurring canine and feline malignant tumors and in adjacent normal tissues.

Cancer cells have an obligate need for cobalamin (vitamin B12) to enable DNA synthesis necessary for cellular replication. This study quantified the immunohistochemical expression of the cobalamin transport protein (transcobalamin II; TCII), cell surface receptor (transcobalamin II-R; TCII-R) and proliferation protein (Ki-67) in naturally occurring canine and feline malignant tumors, and compared these results to expression in corresponding adjacent normal tissues. All malignant tumor tissues stained positively for TCII, TCII-R and Ki-67 proteins; expression varied both within and between tumor types. Expression of TCII, TCII-R and Ki-67 was significantly higher in malignant tumor tissues than in corresponding adjacent normal tissues in both species. There was a strong correlation between TCII and TCII-R expression, and a modest correlation between TCII-R and Ki-67 expression in both species; a modest association between TCII and Ki-67 expression was present in canine tissues only. These results demonstrate a quantifiable, synchronous up-regulation of TCII and TCII-R expression by proliferating canine and feline malignant tumors. The potential to utilize these proteins as biomarkers to identify neoplastic tissues, streamline therapeutic options, evaluate response to anti-tumor therapy and monitor for recurrent disease has important implications in the advancement of cancer management for both human and companion animal patients.

Immunohistochemical quantification of the vitamin B12 transport protein (TCII), cell surface receptor (TCII-R) and Ki-67 in human tumor xenografts.

BACKGROUND/AIM: Cancer cells have an essential demand for vitamin B12 (cobalamin) to enable cellular replication. The present pilot study quantified the immunohistochemical expression of vitamin B12 transport protein (Transcobalamin II; TCII), cell surface receptor (Transcobalamin II-R; TCII-R) and proliferation protein (Ki-67) in human tumor xenografts. MATERIALS AND METHODS: Tissue microarray slides containing 34 xenograft tumor tissues were immunohistochemically stained using TCN2 (anti-TCII), CD320 (anti-TCII-R) and MIB-1 (anti-Ki-67) antibodies. Representatively stained areas of all slides were digitally imaged and protein expression was quantified using ImageJ software plugins. RESULTS: All xenograft tumor tissues stained positively for TCII, TCII-R and Ki-67 proteins; expression varied both within and between tumor types. Correlation between TCII/TCII-R and Ki-67 expression was not significant in xenograft tissues. CONCLUSION: Proliferating cancer cells express measurable levels of TCII and TCII-R. Immunohistochemical quantification of these markers may be useful as a tool for detection of tumors, tailored selection of anti-tumor therapies and surveillance for evidence of recurrent disease.

Pharmacokinetics of intravenous nitrosylcobalamin, an antitumor agent, in healthy Beagle dogs: a pilot study.

BACKGROUND/AIM: Nitrosylcobalamin (NO-Cbl) is a cobalamin-based anti-tumor agent. This study evaluated the pharmacokinetic parameters of NO-Cbl following intravenous administration in dogs. MATERIALS AND METHODS: Four dogs received 10 mg/kg, 20 mg/kg and 40 mg/kg intravenous bolus doses of NO-Cbl, with a 14-day washout period between doses. Blood samples were collected at baseline and post-dosing, and noncompartmental pharmacokinetic parameters were determined. RESULTS: Average peak serum concentrations of 2265, 5523 and 13,866 pg/mL were achieved following single-dose bolus intravenous administration of 10 mg/kg, 20 mg/kg and 40 mg/kg of NO-Cbl respectively. The average area under the curve was 12,697 h × pg/mL, 24,497 h × pg/mL and 44,976 h × pg/mL respectively, with an average elimination half-life of 16.2 h, 13.5 h and 13.1 h respectively. CONCLUSION: These results can be used to determine the dose and dosing intervals for clinical trials evaluating NO-Cbl in humans and companion animals.

Serum folate: a pharmacodynamic biomarker of intracellular nitrosylcobalamin activity following intravenous administration in dogs.

BACKGROUND/AIM: Cobalamin and folate are interdependent co-factors of the methionine synthase pathway. This study evaluated the effect of intravenously-administered nitrosylcobalamin (NO-Cbl), a vitamin B12 analog, on serum folate concentrations in healthy dogs. MATERIALS AND METHODS: Four dogs received a 10-mg/kg, 20-mg/kg and 40-mg/kg intravenous bolus dose of NO-Cbl, with a 14-day washout period between doses. Blood samples were collected at baseline and post-dosing, and serum cobalamin and folate concentrations were measured. RESULTS: For each dose, serum cobalamin concentrations were inversely correlated with serum folate concentrations. Spearman rank correlation co-efficient values were -0.976 (10 mg/kg, p<0.0096), and -1.0 (20 mg/kg, p<0.008; 40 mg/kg, p<0.0046). CONCLUSION: Cellular uptake of NO-Cbl, following intravenous administration exerted a biological effect on folate, similar to that previously described for other vitamin B12 analogs. Serum folate concentration may serve as a pharmacodynamic biomarker of intracellular nitrosylcobalamin activity following intravenous administration.