In Vitro Analysis of N-Nitrosodimethylamine (NDMA) Formation From Ranitidine Under Simulated Gastrointestinal Conditions

Key Points Question Does ranitidine convert to N-nitrosodimethylamine (NDMA), a probable human carcinogen, in simulated gastric fluid at physiologic pH levels and nitrite concentrations? Findings In this in vitro study of 150-mg ranitidine tablets added to simulated gastric fluid, NDMA did not form when gastric nitrite concentrations were at the upper range of physiologic or at nitrite concentrations as much as 50-fold greater than the upper range. Meaning In this study, 150-mg ranitidine tablets did not convert to NDMA in simulated gastric fluid with physiologic nitrite concentrations.


Introduction
Ranitidine is a histamine 2 (H2) receptor antagonist that inhibits gastric parietal cell acid secretion for the treatment of gastroesophageal reflux and peptic ulcer disease. 1 The ranitidine drug product, sold under the brand name Zantac, was approved for human use in 1983 and became a widely used stomach acid inhibitor with more than 18 million prescriptions written in 2018. 2 However, many countries withdrew ranitidine drug products from their markets after N-nitrosodimethylamine (NDMA), a probable human carcinogen, was detected and observed to increase over time under normal storage conditions to amounts greater than the acceptable daily intake. [3][4][5][6] In addition, ranitidine was proposed to convert to NDMA in vivo, as described in an article by Braunstein et al titled "Analysis of Ranitidine-Associated N-Nitrosodimethylamine Production Under Simulated Physiologic Conditions." 7 However, that article did not provide any justification for the physiologic relevance of the nitrite (NO 2 ) concentrations studied (1000 to 50 000 μmol/L). The proposed mechanism for ranitidine 8 to form NDMA involves 2 separate nitrite-dependent steps, and the reaction rate for the second step is proportional to the concentration of the NDMA precursor dimethylamine and the square of the concentration of protonated nitrite. 9 Thus, pH level, ranitidine concentration, and nitrite concentration are key reaction conditions that will affect whether NDMA will be formed.
Review of the literature revealed that 25 μmol/L of nitrite (ie, 40-fold lower than the lowest concentration studied by Braunstein et al 7 ) had been considered the upper range of normal in the acidic fasting stomach based on published clinical studies. 10 However, that assessment was performed in 1985, and an updated evaluation of physiologic nitrite concentrations was warranted, with additional consideration of the potential effect of food; of medications, such as H2 blockers and proton pump inhibitors; and among specific patient groups. The current in vitro study characterized the potential formation of NDMA following the addition of ranitidine to simulated gastric fluid using different combinations of fluid volume, pH, and nitrite concentration, which included physiologic levels.

Methods
Physiological boundary conditions for in vitro studies on ranitidine were based on previous clinical studies. In vitro experiments were performed by adding 150-mg ranitidine tablets to simulated gastric or intestinal fluid with varying pH, fluid volume, and nitrite concentration. This study did not involve patients; thus, it was not under the purview of an institutional review board. The eMethods in the Supplement describe materials used.

Selecting Boundary Conditions for Physiologic Gastric Nitrite Concentrations
eFigure 1 and eTable 1 in the Supplement summarize information from 26 clinical studies reporting simultaneous measurements of gastric nitrite concentration and pH from a variety of patient populations, receiving different medications, and tested in fasted and fed states. Most studies were published in the 1970s or 1980s, when the nitrite content in many foods was significantly higher than today and in an era before proton pump inhibitors and treatment for Helicobacter pylori. The more limited medical treatment options led to a much higher rate of surgical treatment for ulcers, which altered gastric anatomy and physiology, resulting in higher gastric nitrite concentrations. In addition, most studies measured nitrite concentration with the Griess reaction, which was described in the nineteenth century and does not match the sensitivity or accuracy of newer technologies. With this context, certain studies are of higher quality than others, and analytical measurement error or imprecision may contribute to individual patient outliers. Studies using improved analytical methods and nitrite data by 1-unit pH increases are highlighted.  Figure 1A shows the reported minimum, maximum, and mean nitrite concentrations as well as the calculated 95th percentile of the population for each 1-unit increment of pH. 11 Nitrite concentration increased with pH. The 95th percentile nitrite concentration was less than 1 μmol/L at pH levels less than 4, less than 10 μmol/L at pH levels 4 to 5, less than 100 μmol/L at pH levels greater than 5 to 6.99, and less than 200 μmol/L at pH levels of 7 or greater. As the reaction to form NDMA depends on the nitrite being protonated, Figure 1B shows the protonated nitrite concentration based on nitrite's pK a , which is the negative log of the acid dissociation constant (K a ) value. The lower the pK a , the stronger the acid. For nitrite, the pK a value is 3.3, which means that at pH 3.3, 50% of nitrite will be protonated and 50% will be deprotonated. In Figure 1, with pH levels of less than 5, increases in total nitrite concentration are offset by the decreased amount of nitrite that is protonated, such that protonated nitrite remains approximately constant. With pH levels greater than 5, protonated nitrite concentration decreases rapidly as pH increases. eTable 1 and eFigure 1 in the Supplement summarize all studies with paired fasting nitrite and pH data by patient or study group.

Association of Meals With Gastric Nitrite and pH
eTable 2 in the Supplement summarizes clinical studies that contain data on nonfasting gastric nitrite concentration and pH. Two studies 12,13 reported the association of concentrated potassium nitrate solution boluses administered via nasogastric tube on gastric nitrite concentration. After nitrate is absorbed from the intestines, secretion into saliva occurs in a concentrated form that can then be converted to nitrite by nitrate-reducing bacteria on the back of the tongue. However, a bolus of nitrate solution is not reflective of physiology that occurs after consuming nitrate in food, as the meal will increase pH as well as the volume of food and liquids in the stomach, diluting the concentration of salivary nitrite that enters the stomach.
Regarding the effect of actual meals, 1 study 14,15 from 45 years ago reported the effect of an extremely high-nitrite meal including canned luncheon meat from a time prior to modern regulations and technology that reduced nitrite in cured meats. 16 Four other studies [17][18][19][20] assessed gastric pH and nitrite every 30 or 60 minutes for 24 hours, thus including data from the fasting and fed state.  Milton-Thompson et al 19 reported median and upper range data from healthy volunteers before, during, and after cimetidine administration combined and binned by 1-unit pH increases (eFigure 2A in the Supplement). 19 The upper range of nitrite concentration was approximately 1 μmol/L at pH levels less than 2, less than 20 μmol/L at pH levels less than 5, and less than 50 μmol/L at pH levels less than 7. Hall et al 17 also reported data by 1-unit pH in patients with a history of Pólya partial gastrectomy or pernicious anemia as well as control participants (eFigure 2B in the Supplement). 17 The 95th percentile nitrite concentration was less than 10 μmol/L at pH levels of less than 2, less than 100 μmol/L until pH levels of 5, and approximately 100 μmol/L at pH levels of 5 to 7. Note that Pólya partial gastrectomy resulted in significantly altered anatomy and physiology, predisposing patients to higher nitrite concentrations, similar to Billroth II partial gastrectomy. 21 With both studies, 17,19 the protonated nitrite concentration decreased substantially at pH levels greater than 4 (eFigure 2 in the Supplement). Overall, these studies provide a conservative upper bound for gastric fluid nitrite amounts at a pH level of less than 6 of approximately 100 μmol/L. liquids empties throughout the digestive period. 24 After the incubation, 1-mL sample aliquots were taken, and 0.1 mL of 1 mol/L NaOH was added to prevent further potential NDMA formation (eMethods in the Supplement). Sample aliquots were kept at 4°C before analysis for NDMA using the liquid chromatography with high-resolution mass spectrometer (LC-HRMS) method.

LC-HRMS Method
An LC-HRMS analytical procedure was developed and validated for quantifying NDMA per regulatory guidelines. This is described in the eMethods in the Supplement.

Statistical Analysis
NDMA data are presented as mean and SD of 3 independent samples run in duplicate. Data were collected and analyzed in Excel 2002 (Microsoft Corp).

JAMA Network Open | Pharmacy and Clinical Pharmacology
Because the nitrite concentration was matched between the prior 50 mL simulated gastric fluid experiments and these experiments with 250 mL, the total amount of nitrite in simulated gastric fluid was 5 times greater in the 250 mL experiments. As 1 ranitidine tablet was added to both the 50 mL and 250 mL experiments, the ratio of nitrite to ranitidine molecules was 5 times greater in the 250 mL experiments compared with the 50 mL experiments.

Discussion
The results of this in vitro study suggest that ranitidine does not convert to NDMA in the human stomach or small intestine under physiological conditions. At a pH of 1.2 with 50 mL simulated gastric fluid representative of the fasted stomach, ranitidine conversion to NDMA was not detected until the nitrite concentration was 10 000 μmol/L, which is approximately 18 000-fold greater than the clinically observed 95th percentile at pH levels less than 2 ( Figure 1). 11 When increasing the simulated gastric fluid volume to 250 mL, as may occur when consuming 200 mL of water, conversion of ranitidine to NDMA was detected when gastric nitrite was 5000 μmol/L instead of 10 000 μmol/L.
However, this scenario is unlikely to be encountered in patients, as consuming water will increase pH and decrease the gastric nitrite concentration by dilution. Furthermore, 5000 μmol/L is approximately 600-fold greater than the 95th percentile of gastric nitrite at pH values of less than 2 from patients with 24-hour (combined fed and fasted) gastric fluid measurements (Figure 4 and eFigure 2 in the Supplement). 17 As shown in Figure 4 and consistent with the results of this study, Zeng and Mitch 8 also reported NDMA formation from ranitidine only at 5000 μmol/L nitrite and greater. In contrast, Braunstein Increased gastric nitrite concentrations have been proposed to be because of increased nitrateto-nitrite reducing bacteria at higher pHs in the stomach. However, nitrite is known to be more stable at higher pH, 31 and if more nitrite is present in gastric juice at a higher pH, then as pH decreases, nitrite will also decrease. Interestingly, when study participants in Mowat et al, 12 [35][36][37] In this study, as well as in the study by Gillatt et al, 10 conditions. When that was done with ranitidine in this study, in vitro formation of NDMA did not occur.

Limitations
The in vitro model used in this study has inherent limitations and does not reflect all aspects of human physiology, but it does provide a method for assessing the potential for physiologic nitrite reactions with drugs in the gastric fluid to lead to NDMA formation. The in vitro studies performed here did not include gastric conditions in the presence of a meal aside from experiments at higher pH levels that occur following a meal, which led to decreased formation of NDMA. In addition, the lack of conversion of ranitidine to NDMA at simulated physiological conditions observed here was further supported by the accompanying randomized, placebo-controlled clinical trial, 38 which demonstrated that ranitidine did not increase urinary excretion of NDMA across 2 different diets with, 1 containing large quantities of cured meats.

Conclusions
In this in vitro study of ranitidine tablets added to simulated gastric fluid with different nitrite concentrations, ranitidine conversion to NDMA was not detected until nitrite was 5000 μmol/L, which is 50-fold greater than the upper range of physiologic gastric nitrite concentrations at acidic pH. These findings suggest that ranitidine is not converted to NDMA in gastric fluid at physiologic conditions and are further supported by the results of a recent clinical trial. 38