Bumetanide as a Model NDSRI Substrate: N-nitrosobumetanide Impurity Formation and its Inhibition in Bumetanide Tablets.

Nitrosamine compounds are classified as potential human carcinogens, the origin of these impurities can be broadly classified in two categories, nitrosamine impurity found in drug products that are not associated with the Active Pharmaceutical Ingredient (API), such as N-nitrosodimethylamine (NDMA) or nitrosamine impurities associated with the API, such as nitrosamine drug substance-related impurities (NDSRIs). The mechanistic pathway for the formation of these two classes of impurities can be different and the approach to mitigate the risk should be tailored to address the specific concern. In the last couple of years number of NDSRIs have been reported for different drug products. Though, not the only contributing factor for the formation of NDSIRs, it is widely accepted that the presence of residual a nitrites/nitrates in the components used in the manufacturing of the drug products can be the primary contributor to the formation of NDSRIs. Approaches to mitigate the formation of NDSRIs in drug products include the use of antioxidants or pH modifiers in the formulation. The primary objective of this work was to evaluate the role of different inhibitors (antioxidants) and pH modifiers in tablet formulations prepared in-house using bumetanide (BMT) as a model drug to mitigate the formation of N-nitrosobumetanide (NBMT). A multi-factor study design was created, and several bumetanide formulations were prepared by wet granulation with and without sodium nitrite spike (100 ppm) and different antioxidants (ascorbic acid, ferulic acid or caffeic acid) at three concentrations (0.1%, 0.5% or 1% of the total tablet weight). Formulations with acidic and basic pH were also prepared using 0.1 N hydrochloric acid and 0.1 N sodium bicarbonate, respectively. The formulations were subjected to different storage (temperature and humidity) conditions over 6 months and stability data was collected. The rank order of N-nitrosobumetanide inhibition was highest with alkaline pH formulations, followed by formulations with ascorbic acid, caffeic acid or ferulic acid present. In summary, we hypothesize that maintaining a basic pH or the addition of an antioxidant in the drug product can mitigate the conversion of nitrite to nitrosating agent and thus reduce the formation of bumetanide nitrosamines.

Rapid Quantitation of Four Nitrosamine Impurities in Angiotensin Receptor Blocker Drug Substances.

Starting in July 2018, the FDA alerted patients and health care professionals to the recall of ARBs such as valsartan by several pharmaceutical companies because of their potential contamination with carcinogenic nitrosamine impurities, including: (1) N-nitrosodimethylamine (NDMA), (2) N-nitrosodiethylamine (NDEA), (3) N-nitrosoethylisopropylamine (NEIPA), (4) N-nitrosodiisopropylamine (NDIPA), (5) N-nitrosodibutylamine (NDBA) and (6) N-nitroso-N-methyl-4-aminobutyric acid (NMBA). The FDA initiated a laboratory investigation to develop analytical procedures to test multiple lots of marketed ARB drugs to determine the possible presence of carcinogenic impurities and, if present, quantitate the levels of these impurities. Here the FDA laboratory developed and validated an automated micro-solid phase extraction MS/MS method, where all the analytes are not separated prior to elution to the MS, to simultaneously quantify NEIPA, NDIPA, NDBA and NMBA in ARB drug substances with an instrument sample analysis time of 12 seconds. The method was validated according to the ICH Q2(R1) guideline, and was determined to be specific, accurate, precise and linear over the corresponding nitrosamine analytical ranges. The method has been successfully implemented to quantitate the four nitrosamine impurities in 129 generic losartan, valsartan, olmesartan, irbesartan and telmisartan drug substance samples from 32 lots; and 32 losartan and valsartan drug product samples from 6 lots.

A headspace-gas chromatography method for isopropanol determination in warfarin sodium products as a measure of drug crystallinity.

Coumadin® a nd s everal generic products of warfarin s odium (WS) contain the crystalline form (clathrate) in which WS and isopropanol (IPA) are associated in a 2:1 molar ratio. IPA is critical in maintaining the WS crystalline structure. Physicochemical properties of the drug and drug product may change when the crystalline drug transforms to amorphous form. A headspace-gas chromatography (HS-GC) method was developed and validated for IPA determination in the WS drug product. n-propanol (NPA) was used as internal standard and the method was validated for specificity, system suitability, linearity, accuracy, precision, range, limits of detection and quantification, and robustness. The method was specific, with good resolution between IPA and NPA peaks. Chromatographic parameters (retention time, IPA/NPA area ratio, tailing factor, theoretical plates, USP symmetry, capacity factor, selectivity and resolution) were consistent over three days of validation. The analytical method was linear from 2-200 µg mL-1 (0.1- 10 % IPA present in the drug product). LOD and LOQ were 0.1 and 2 µg mL-1, respectively. Accuracy at low (2 µg mL-1) and high (200 µg mL-1) IPA concentrations of the calibration curve was 103.3-113.3 and 98.9-102.2 % of the nominal value, resp. The validated method was precise, as indicated by the RSD value of less than 2 % at three concentration levels of the calibration curve. The method reported here was utilized to determine accurately and precisely the IPA content in in-house formulations and commercial products. In summary, IPA determination by HS-GC provides an indirect measure of WS crystallinity in the drug product. Nevertheless, it should be confirmed by another analytical method since IPA from the drug substance is not distinguishable from IPA that may be present outside the drug crystals in a dosage form when prepared by wet granulation with IPA.

Pharmaceutical characterization and thermodynamic stability assessment of a colloidal iron drug product: iron sucrose.

The study objective was to evaluate the thermodynamic stability of iron sucrose complexes as determined by molecular weight (m.w.) changes. The first part of the study focused on the effect of thermal stress, pH, electrolyte or excipient dilution on the stability of a colloidal iron drug product. Part two focused on the physical and chemical evaluation of the colloidal nature of iron sucrose using a series of characterization experiments: ultracentrifugation, dialysis, particle size, zeta potential, and osmotic pressure analysis. A validated Taguchi-optimized high performance gel permeation chromatography method was used for m.w. determinations. Results indicate m.w. of the iron sucrose complex remained unchanged after excipient dilution, ultracentrifugation, dialysis, and electrolyte dilution. Electrolyte dilution studies indicated the lyophilic nature of the iron sucrose colloid with a particle size of 10nm and zeta potential of 0 mV. The complex deformed at low pH and reformed back at the formulation pH. The complex is stable under mild-to-moderate temperature <50°C but aggregates following prolonged exposure to high temperatures >70°C. In conclusion, the resistance of the complex to breakdown by electrolytic conditions, excipient dilution, ultracentrifugation and the reversible complexation after alteration of formulation pH suggest iron sucrose is a lyophilic colloid in nature and lyophilic colloidals are thermodynamically stable.

Thermodynamic stability assessment of a colloidal iron drug product: sodium ferric gluconate.

A high performance gel permeation chromatography (HP-GPC) method was developed, validated and used to determine the molecular weight (MW) of sodium ferric gluconate following various stress conditions. The intra-day accuracy (90-103%), intra-day precision (1.5-2.7%), inter-day accuracy (91-105%), inter-day precision (1.3-3.2%) were within acceptable range stated in FDA guidance. The MW of sodium ferric gluconate remained unchanged after: (1) autoclaving (121 degrees C), (2) moderate thermal stress (30 days at 50 degrees C or 7 days at 70 and 90 degrees C), (3) excipient dilution, (4) basic buffer dilution (pH of 8 and 9), (5) ultracentrifugation, (6) dialysis, and (7) electrolyte dilution. However sodium ferric gluconate showed signs of instability at higher temperatures (>90 degrees C) after 30 days and at pH of 10-11. Sodium ferric gluconate was found to be a lypophilic colloidal solution with an average particle size of 10 nm and a zeta potential of -13 mV. The colloid osmotic pressure was 3.5 mmHg and remained unchanged after moderate thermal stress. Additionally, in-house drug products with similar MW to sodium ferric gluconate were produced by three different synthetic procedures, suggesting that this colloidal iron drug product might be thermodynamically stable.

Effects of amitriptyline and fluoxetine upon the in vitro proliferation of tumor cell lines.

Previous publications have suggested that commonly prescribed antidepressants have the potential to stimulate the proliferation of extant tumors in human and rodent in vivo and in vitro models. The direct effects of amitriptyline and fluoxetine were evaluated in assays that detect different aspects of proliferative responses at pharmacologically relevant drug concentrations. Three in vitro assays of cellular proliferation and clonal growth were used with human (MCF7, PA-1 and LS174T) and murine (B16.f10, C-3 and B16.f1) tumor cell lines. The cells were exposed to amitriptyline or fluoxetine (0.001-100 microM) for different time periods (1-7 days) and at varying serum concentrations (0.1-15%). Amitriptyline and fluoxetine failed to significantly stimulate tumor cell proliferation, DNA synthesis, or colony formation. Both drugs inhibited B16.f10 colony growth at concentrations above 5 microM along with significant suppression of DNA synthesis in B16.f10 and C-3 cells at 30 microM. Although there were generally no effects on cell proliferation by the drugs in the microtiter tetrazolium assay, several rare instances of stimulation were noted. Amitriptyline and fluoxetine were consistent in their lack of effect or inhibition with the human or murine tumor cell lines in conventional in vitro assays of cell proliferation and clonogenicity in optimal or suboptimal culture conditions.