Oral Nitrite in Adults With Metabolic Syndrome and Hypertension

Cardiovascular disease remains the leading cause of death in the United States and worldwide. Several studies have demonstrated that fruit and vegetable rich diets significantly reduced blood pressure and reduced the risk of ischemic stroke and cardiovascular disease in general, the exact mechanisms remain poorly understood. Preclinical and clinical research over the last decade has revealed the important vasoprotective effects of nitrates and nitrites with regards to reduction in blood pressure, vascular inflammation and endothelial dysfunction. More recent findings suggest that inorganic nitrate and nitrite therapy may be involved in the regulation of glucose-insulin homeostasis.

For this reason, development of an oral formulation of nitrite salt represents a rational avenue of exploration for the treatment of cardiovascular diseases, whereby nitrite would ensure rapid acting effects upon absorption which can be further oxidized to nitrate via the enterosalivary circulation pathway. In this pathway, about 25% of circulating nitrate is concentrated in the saliva and reduced to nitrite by commensal mouth bacteria with nitrate reductase enzymes. The proposal is the first human study to investigate the inorganic nitrite effects (in any form) on insulin sensitivity in a patient population. This is the second human trial using orally delivered nitrite (previously as aqueous solution).

In the initial phase of the study, step up dosing and frequency of oral sodium nitrite to 40 mg three times daily occurred with no serious adverse events. After three subjects completed the study intervention on sodium nitrite 20 mg three times daily for 2 weeks followed by 40 mg three times daily for the remaining 10 weeks with no serious adverse events, all subjects in this current phase of the trial (n=20) began the 12-week study intervention with 40 mg three times daily. At the same time, in person monitoring visits (which included brief physical exams, directly observed nitrite dosing, secondary outcome measure assessment of methemoglobin level and blood pressure, interval histories, medication compliance review, symptom review and dispensing of study drug) were spaced from weekly intervals to subjects alternating weekly in person visits with phone visits.

• 14Nitrogen Sodium Nitrite (Drug), also known as sodium nitrite: oral formulation of sodium nitrite 40 mg three times a day for 12 weeks

Primary outcomes

• Change in Insulin Stimulated Glucose Disposal Over 12 Week Study Period [measured at 0 (baseline) and at 12 weeks]: Change in insulin stimulated glucose disposal was assessed during the 4-hour hyperinsulinemic euglycemic clamp at end of the 12 weeks and was compared to baseline (pre-nitrite). Insulin stimulated glucose disposal (mg/min) was calculated in the final 20 minutes of the 4-hour clamp at steady state and expressed as mg per kg lean body mass (as measured by DEXA) per minute. HumuLIN R regular insulin was infused at a rate of 40 microUnits/m2/min. A non-radioactive glucose isotope dilution (Isotec, Inc) was delivered as a primed, then constant infusion of the 98+% enriched stable isotope of D-glucose \[6,6-D2\] (0.22 umol x kg-1, 17.6 umol x kg-1 prime). Plasma glucose was clamped between 85-95 mg/dL with a variable infusion of 20% dextrose in water. The rate of dextrose infusion was adjusted based on arterialized plasma glucose measurements obtained every 5 minutes.

Secondary outcomes

• Peak Change in Systolic Blood Pressure Over 12 Week Study Period [measured at 0 (baseline) and bi-weekly over 12 weeks]: Peak change in systolic blood pressure (SBP) on sodium nitrite compared to baseline. Subjects performed bi-weekly blinded automated blood pressures with a Dinamap DPC200X (GE Medical Systems Information Technology) in triplicate at study visits beginning 30 minutes after directly observed nitrite dosing. Five minute intervals were maintained between measurements while in the supine position with legs uncrossed and sitting upright in a hospital bed in a quiet room. The final 2 of 3 SBP measurements were averaged.
• Peak Change in Diastolic Blood Pressure Over 12 Week Study Period [measured at 0 (baseline) and bi-weekly over 12 weeks]: Peak change in diastolic blood pressure (DBP) on sodium nitrite compared to baseline. Subjects performed bi-weekly blinded automated blood pressures with a Dinamap DPC200X (GE Medical Systems Information Technology) in triplicate at study visits beginning 30 minutes after directly observed nitrite dosing. Five minute intervals were maintained between measurements while in the supine position with legs uncrossed and sitting upright in a hospital bed in a quiet room. The final 2 of 3 DBP measurements were averaged.
• Peak Change in Mean Arterial Pressure Over 12 Week Study Period [measured at 0 (baseline) and bi-weekly over 12 weeks]: Peak change in mean arterial pressure (MAP) on sodium nitrite compared to baseline. Subjects performed bi-weekly blinded automated blood pressures with a Dinamap DPC200X (GE Medical Systems Information Technology) in triplicate at study visits beginning 30 minutes after directly observed nitrite dosing. Five minute intervals were maintained between measurements while in the supine position with legs uncrossed and sitting upright in a hospital bed in a quiet room. The final 2 of 3 MAP measurements were averaged.
• Peak Change in Methemoglobin Over 12 Week Study Period [measured at 0 (baseline) and bi-weekly over 12 weeks]: Peak change in methemoglobin on sodium nitrite compared to baseline. Methemoglobin was assessed bi-weekly at study visits 30 minutes after directly observed nitrite dosing using noninvasive co-oximetry (Masimo Corp, Irvine, CA).

Sex: All, ages: 18 Years to 60 Years

Inclusion Criteria:

* Age 18-60 years
* Body mass index (BMI) greater than or equal to 30 kg/m\^2
* Hypertension: defined as systolic blood pressure (SBP) greater than or equal to 130 and/or diastolic blood pressure (DBP) greater than or equal to 85 mm Hg
* Waist circumference: greater than 102 cm in men, greater than 88 cm in women

Exclusion Criteria:

* Positive urine pregnancy test or breastfeeding
* Concurrent use of medications affecting glucose metabolism (oral hypoglycemics, insulin, atypical antipsychotics)
* Recent addition or change in dosing of hormonal contraceptive medications \[oral contraceptive pill (OCP), intrauterine device (IUD), DepoProvera\]
* Current use of greater than or equal to 3 anti-hypertensive agents regardless of blood pressure control or normotensive on a single or double agent
* Current use of phosphodiesterase-5 inhibitors or organic nitrates
* Not stable on treatments for the prior three months or not planning to remain on current dose of medications for blood pressure, contraception, etc.
* Known chronic psychiatric or medical conditions including diabetes, liver or kidney disease or obesity syndromes
* Thyroid-stimulating hormone (TSH) greater than 8 milli-International unit/mL
* Smoker
* Anemia (central lab hemoglobin less than 11g/dL)

Participant flow

Outcome results

• Change in Insulin Stimulated Glucose Disposal Over 12 Week Study Period (Primary), Mean, mg/kg lean body mass/minute: 14Nitrogen Sodium Nitrite 0.76 +/- 0.58 (p 0.2068)
• Peak Change in Systolic Blood Pressure Over 12 Week Study Period (Secondary), Mean, mmHg: 14Nitrogen Sodium Nitrite -10.45 +/- 3.01 (p 0.0074)
• Peak Change in Diastolic Blood Pressure Over 12 Week Study Period (Secondary), Mean, mmHg: 14Nitrogen Sodium Nitrite -13.05 +/- 2.2 (p <0.0001)
• Peak Change in Mean Arterial Pressure Over 12 Week Study Period (Secondary), Mean, mmHg: 14Nitrogen Sodium Nitrite -12.00 +/- 2.15 (p <0.0001)
• Peak Change in Methemoglobin Over 12 Week Study Period (Secondary), Mean, percentage of total hemoglobin: 14Nitrogen Sodium Nitrite 0.42 +/- 0.11 (p 0.0127)

Adverse events

Time frame: Adverse event (AE) collection occurred throughout 12 weeks of study drug treatment until 1 month after completing study drug.

Other adverse events by term

Limitations and caveats: The pre-drug clamp isotope data for 1 subject (used to determine insulin stimulated glucose disposal) was uninterpretable, despite repeating the mass spectrometry assay. Subject's isotope data was removed from analysis (n=19 analysis sample size).

• Results publication: Hughan KS, Levine A, Helbling N, Anthony S, DeLany JP, Stefanovic-Racic M, Goodpaster BH, Gladwin MT. Effects of Oral Sodium Nitrite on Blood Pressure, Insulin Sensitivity, and Intima-Media Arterial Thickening in Adults With Hypertension and Metabolic Syndrome. Hypertension. 2020 Sep;76(3):866-874. doi: 10.1161/HYPERTENSIONAHA.120.14930. Epub 2020 Aug 3. PMID 32755471

• Study Protocol and Statistical Analysis Plan (PDF, 2017-05-25)
• Informed Consent Form (PDF, 2018-06-05)

• Kara S Hughan, MD, Principal investigator, University of Pittsburgh
• Mark Gladwin, MD, Study director, University of Pittsburgh