Nitrosyl-Cobinamide, a New and Direct Nitric Oxide–Releasing Drug Effective In Vivo

Abstract

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi-stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice

“…In a separate experiment, 12 week-old intact female mice (six mice per group) were randomized to receive either vehicle or NO-Cbi on the same dose and schedule for five weeks. This NO-Cbi dose did not significantly reduce systolic blood pressure (< 10 mm Hg), consistent with our previous report, 33 and the NO-Cbi-treated mice showed no signs of toxicity and had similar weight gain during the experiment as the corresponding vehicle-treated groups. Double calcein labeling was performed by intraperitoneal injection of calcein (25 mg/kg) at seven and four days before euthanasia.…”

“…10, 30, 32 Here, we examined the skeletal effects of a novel NO donor, nitrosyl-cobinamide (NO-Cbi), derived from the vitamin B 12 precursor cobinamide; it releases NO directly, without biotransformation or generation of reactive oxygen species. 33–36 We found that NO-Cbi enhanced trabecular bone mass in intact and OVX mice by enhancing osteoblast activity and inhibiting osteoclast differentiation.…”

Cobinamides Are Novel Coactivators of Nitric Oxide Receptor That Target Soluble Guanylyl Cyclase Catalytic Domain

“…It is unlikely that physiological concentrations of circulating cobinamides (Ͻ30 pM) or B 12 vitamins (ϳ300 pM) (Hardlei and Nexo, 2009) have any effects on sGC func- Because of high affinity to cyano groups, cobinamides are used for cyanide detoxification (Broderick et al, 2006) and doses of 800 mg/kg are deemed safe for laboratory animals (Brenner et al, 2010). Previous studies reported that nitrosyl-cobinamide functions as an NO donor (Broderick et al, 2007), increasing vasodilation and vasodilator-stimulated phosphoproteinphosphorylation, presumably through NO-dependent activation of sGC. Data presented in the current report suggest that some of these effects may be, in fact, due to the direct NO-independent activation of sGC by the cobinamide macrocycle itself.…”

Nitrosyl-cobinamide (NO-Cbi), a new nitric oxide donor, improves wound healing through cGMP/cGMP-dependent protein kinase

“…In PtK2 cells, a single application of NO-Cbi was sufficient to significantly improve wound healing, while in the other three cell types several low dose drug applications were necessary. Multiple drug administrations were needed, likely because NO-Cbi has a half-life of about 90 min in tissue culture medium [25]. Studies are in progress on different formulations to increase the half-life of NO-Cbi, however, application of NO-Cbi to a wound three or four times a day would not obviate its potential medical use.…”

“…A cobinamide solution was thoroughly deoxygenated using argon, the cobalt was reduced from +3 to +2 valence state using a two-molar excess of ascorbic acid, and NO gas (99.99% pure, Matheson Gas Co.) was bubbled through the reduced cobinamide solution [25]. Concentrated stock solutions of NO-Cbi were diluted in deoxygenated sterile water, and the diluted NO-Cbi was added to cells using a Hamilton syringe (Hamilton, Reno, NV).…”