Endothelial dysfunction and myocardial injury after major emergency abdominal surgery: a prospective cohort study.

BACKGROUND: Preoperative endothelial dysfunction is a predictor of myocardial injury and major adverse cardiac events. Non-cardiac surgery is known to induce acute endothelial changes. The aim of this explorative cohort study was to assess the extent of systemic endothelial dysfunction after major emergency abdominal surgery and the potential association with postoperative myocardial injury. METHODS: Patients undergoing major emergency abdominal surgery were included in this prospective cohort study. The primary outcome was the change in endothelial function expressed as the reactive hyperemia index from 4-24 h after surgery until postoperative day 3-5. The reactive hyperemia index was assessed by non-invasive digital pulse tonometry. Secondary outcomes included changes in biomarkers of nitric oxide metabolism and bioavailability. All assessments were performed at the two separate time points in the postoperative period. Clinical outcomes included myocardial injury within the third postoperative day and major adverse cardiovascular events within 30 days of surgery. RESULTS: Between October 2016 and June 2017, 83 patients were included. The first assessment of the endothelial function, 4-24 h, was performed 15.8 (SD 6.9) hours after surgery and the second assessment, postoperative day 3-5, was performed 83.7 (SD 19.8) hours after surgery. The reactive hyperemia index was suppressed early after surgery and did not increase significantly; 1.64 (95% CI 1.52-177) at 4-24 h after surgery vs. 1.75 (95% CI 1.63-1.89) at postoperative day 3-5, p = 0.34. The L-arginine/ADMA ratio, expressing the nitric oxide production, was reduced in the perioperative period and correlated significantly with the reactive hyperemia index. A total of 16 patients (19.3%) had a major adverse cardiovascular event, of which 11 patients (13.3%) had myocardial injury. The L-arginine/ADMA ratio was significantly decreased at 4-24 h after surgery in patients suffering myocardial injury. CONCLUSION: This explorative pathophysiological study showed that acute systemic endothelial dysfunction was present early after major emergency abdominal surgery and remained unchanged until day 3-5 after the procedure. Early postoperative disturbances in the nitric oxide bioavailability might add to the pathogenesis of myocardial injury. This pathophysiological link should be confirmed in larger studies. TRIAL REGISTRATION: clinicaltrials.gov no. NCT03010969.

The effect of melatonin on endothelial dysfunction in patients after acute coronary syndrome: The MEFACS randomized clinical trial.

Endothelial dysfunction (ED) precedes acute coronary syndrome. Oxidative stress results in ED but is reversible. Melatonin is aside from being a circadian hormone, also an antioxidant. The aim of this study was to investigate whether 25 mg melatonin administered for twelve weeks following acute coronary syndrome (ACS) could improve ED. In this placebo-controlled randomized trial, ED was measured as reactive hyperemia index (RHI) at baseline, day 14, and day 84. The effect was assessed using a generalized estimating equation adjusted for the baseline RHI. As secondary outcome, the concentrations of three biomarkers were measured: l-arginine, asymmetric dimethylarginine, and uric acid. Thirty-one patients were included in the study. The intention-to-treat analysis of the primary outcome had 26 patients due to missing data. The estimated marginal mean difference in RHI at day 14 and day 84 between the groups was 0.15 (95% CI: 0.29-0.01, P = .039) in favor of the placebo group. No significant differences in the biomarker concentrations were found. Melatonin treatment after ACS did not improve but may have aggravated ED. The significant difference between groups was in favor of placebo, but this might be due to the effect of missing data or uneven distribution of comorbidities.

Peri-operative endothelial dysfunction in patients undergoing minor abdominal surgery: An observational study.

BACKGROUND: Myocardial injury after noncardiac surgery is common and associated with major adverse cardiac events. Surgery induces acute endothelial dysfunction, which might be central in the pathophysiology of myocardial injury; however, the relationship between surgical stress and endothelial function remains incompletely understood. OBJECTIVES: This study aimed to assess the acute peri-operative changes in endothelial function after minor elective abdominal surgery. DESIGN: A prospective, observational, single-centre study. SETTING: A university hospital from February 2016 to January 2017. PATIENTS: Sixty patients undergoing elective minor abdominal surgery. MAIN OUTCOME MEASURES: The change in endothelial function, expressed as the reactive hyperaemia index (RHI), was assessed by non-invasive digital pulse tonometry. RHI, biomarkers of nitric oxide bioavailability and oxidative stress were assessed prior to and 4 h after surgery. RESULTS: RHI decreased significantly from 1.93 [95% confidence interval (95% CI 1.78 to 2.09)] before surgery to 1.76 (95% CI 1.64 to 1.90), P = 0.03, after surgery. The nitric oxide production, L-arginine/asymmetric dimethylarginine, decreased significantly from a ratio of 213.39 (95% CI 188.76 to 241.2) to a ratio of 193.3 (95% CI 171.82 to 217.54), P = 0.03. Plasma biopterins increased significantly after surgery, while the ratio between tetrahydrobiopterin and dihydrobiopterin was unchanged. Total ascorbic acid decreased significantly after surgery (P < 0.001), while its oxidation ratio was unchanged. CONCLUSION: Elective minor abdominal surgery impaired systemic endothelial function early after surgery. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02690233.

Effect of High Inspiratory Oxygen Fraction on Endothelial Function in Healthy Volunteers: A Randomized Controlled Crossover Pilot Study.

It has been suggested that high inspiratory oxygen concentrations during anesthesia may be associated with higher postoperative mortality due to endothelial dysfunction. A randomized controlled crossover study was conducted with 25 healthy male volunteers. They inhaled an oxygen concentration of 30% and 80%. The endothelial function was assessed using noninvasive digital pulse amplitude tonometry (EndoPAT) supported by endothelial biomarkers. The difference in endothelial function between the 2 treatments was 0.05 (95% confidence interval, -0.36 to 0.27; P = .77). Endothelial biomarkers were unaffected. Inhalation of a high oxygen fraction in healthy volunteers did not result in a significant reduction of endothelial function.

A Long-Term Energy-Rich Diet Increases Prefrontal BDNF in Sprague-Dawley Rats.

Findings of the effect of high-fat feeding including "Cafeteria Diets" (CAF) on brain-derived neurotrophic factor (BDNF) in the hippocampus (HIP) and prefrontal cortex (PFC) in rodents are conflicting. CAF is a non-standardized, highly palatable energy-rich diet composed by everyday food items for human consumption and is known to induce metabolic syndrome and obesity in rats. However, the highly palatable nature of CAF may counteract a negative effect of chronic stress on anticipatory behavior and synaptic plasticity in the hippocampus, hence represent a confounding factor (e.g., when evaluating functional effects on the brain). This study investigated the effects of a chronic, restricted access to CAF on BDNF, monoamine neurotransmitters, and redox imbalance in HIP and PFC in male rats. Our results show that CAF induced BDNF and its receptor TrkB in PFC compared to the controls (p < 0.0005). No differences in monoamine neurotransmitters were detected in either PFC or HIP. CAF increased dehydroascorbic acid and decreased malondialdehyde in PFC (p < 0.05), suggesting an early redox imbalance insufficient to induce lipid peroxidation. This study supports that a chronic CAF on a restricted schedule increases BDNF levels in the PFC of rats, highlighting that this may be a suboptimal feeding regime when investigating the effects of diet-induced obesity in the brain and emphasizing this as a point of attention when comparing the findings.

Depleted Myocardial Coenzyme Q10 in Cavalier King Charles Spaniels with Congestive Heart Failure Due to Myxomatous Mitral Valve Disease.

Congestive heart failure (CHF) has been associated with depleted myocardial coenzyme Q10 (Q10) concentrations in human patients. The aim of this study was to investigate associations between myocardial Q10 concentrations and myxomatous mitral valve disease (MMVD) severity in dogs. Furthermore, citrate synthase (CS) activity was analysed to determine if a reduction in myocardial Q10 was associated with mitochondrial depletion in the myocardium. Thirty Cavalier King Charles spaniels (CKCS) in MMVD stages B1 (n = 11), B2 (n = 5) and C (n = 14) according to the American College of Veterinary Internal Medicine (ACVIM) guidelines and 10 control (CON) dogs of other breeds were included. Myocardial Q10 concentration was analysed in left ventricular tissue samples using HPLC-ECD. CKCS with congestive heart failure (CHF; group C) had significantly reduced Q10 concentrations (median, 1.54 µg/mg; IQR, 1.36-1.94), compared to B1 (2.76 µg/mg; 2.10-4.81, p < 0.0018), B2 (3.85 µg/mg; 3.13-4.46, p < 0.0054) and CON dogs (2.8 µg/mg; 1.64-4.88, p < 0.0089). CS activity was comparable between disease groups. In conclusion, dogs with CHF due to MMVD had reduced myocardial Q10 concentrations. Studies evaluating antioxidant defense mechanisms as a therapeutic target for treatment of CHF in dogs are warranted.

The counterregulatory response to hypoglycaemia in the pig.

The domestic pig is commonly used as animal model in the pharmaceutical development of new therapeutics for treatment of diabetes. Since a formal definition of hypoglycaemia only exists in humans, the purpose of this study was to assess the counterregulatory response in the domestic pig at glucose levels known to induce symptoms of hypoglycaemia in humans. Six pigs were included in hyperinsulinaemic glucose clamps with plasma glucose targets of 2, 3 and 5 mmol/L in a cross-over design, and the associated glucose counterregulatory response was assessed by measuring glucose kinetics and levels of glucagon, c-peptide, catecholamines, cortisol and growth hormone. Results showed that the 2 and 3 vs 5 mmol/L clamps significantly decreased and increased the secretion of c-peptide and glucagon, respectively (P < .05). This finding was associated with increased rate of glucose appearance (Ra ) and decreased rate of glucose disappearance (Rd ) (P < .001). No marked differences in the catecholamine, growth hormone or cortisol response were observed. Consequently, like humans, pigs respond to hypoglycaemia by decreasing the pancreatic output of insulin while increasing that of glucagon, with increased glucose mobilization and decreased glucose disposal as a result. The hypoglycaemic clamps did not result in a marked secretion of the other counterregulatory hormones.

Pharmacokinetics of Repeated Oral Dosing with Coenzyme Q10 in Cavalier King Charles Spaniels with Myxomatous Mitral Valve Disease.

Coenzyme Q10 (Q10) is a mitochondrial cofactor and an antioxidant with the potential to combat oxidative stress in heart failure. This study aims to determine the pharmacokinetics of repeated oral dosing of Q10 in Cavalier King Charles Spaniels (CKCS) with spontaneous myxomatous mitral valve disease (MMVD) and to evaluate echocardiographic parameters, circulating cardiac biomarkers, and quality of life (QoL) after treatment. The study is a randomized, placebo-controlled, single-blinded crossover study. Nineteen CKCS with MMVD were randomized to receive 100 mg Q10 (ubiquinone) bi-daily for three weeks, then placebo (or in reverse order). Clinical examination, blood sampling, echocardiography, and QoL assessment were performed before and after each treatment phase. Q10 plasma concentrations were determined in plasma using a validated high-performance liquid chromatography method using electrochemical detection (HPLC-ECD). Eighteen CKCS were included in the analyses. Total plasma concentration of Q10 increased significantly (p < 0.0001) from baseline (median, 0.92 µg/mL; interquartile range (IQR), 0.70-1.26) to after treatment (median, 3.51 µg/mL; IQR, 2.30-6.88). Thirteen dogs reached the threshold of a total plasma Q10 concentration of ≥2.0 µg/mL. The average half-life (T1/2) of Q10 was 2.95 days (IQR, 1.75-4.02). No significant differences were observed in clinical MMVD severity, and the owner perceived QoL between Q10 and placebo treatment. The solubilized Q10 formulation was well-tolerated in the dogs. Individual variation in plasma concentrations was observed following oral treatment. A long-term placebo-controlled trial is warranted in dogs with MMVD to determine long-term efficacy on the clinical severity of MMVD.

Determination of Reduced and Oxidized Coenzyme Q10 in Canine Plasma and Heart Tissue by HPLC-ECD: Comparison with LC-MS/MS Quantification.

Coenzyme Q10 (Q10) plays an important role in mammals for energy production in the mitochondria, and as a potent antioxidant. Oxidation ratio (% oxidized in relation to total Q10) has been proposed as an important biomarker. A sensitive and reproducible HPLC-ECD method was developed for determination of reduced and oxidized Q10 in canine plasma and heart tissue. Chromatographic separation was achieved in 10 min using a Waters Nova-pak C18 column and a mobile phase with lithium perchlorate in ethanol/methanol/2-propanol. The validation showed satisfying results. Excellent linear correlation was found (r2 > 0.9997), intra- and inter-day precisions were below 6.5% (n = 5) and recoveries were between 89 and 109% (n = 5). Sensitivity stated as Lower Limit of Quantification (LLOQ) was 10 nM. Acceptable stability of both extracted and un-extracted samples was observed. The plasma concentration range of total Q10 was found to be between 0.64 and 1.24 µg/mL. Comparison with a developed LC-MS/MS method showed a correlation of r = 0.85 for reduced Q10 and r = 0.60 for oxidized Q10 (N = 17). However, average results were around 30% lower for ubiquinol using the LC-MS/MS method as compared with the HPLC-ECD analysis. The two methods are therefore not considered to be interchangeable.

Atorvastatin and Vitamin E Accelerates NASH Resolution by Dietary Intervention in a Preclinical Guinea Pig Model.

Despite affecting millions of patients worldwide, no pharmacological treatment has yet proved effective against non-alcoholic steatohepatitis (NASH) induced liver fibrosis. Current guidelines recommend lifestyle modifications including reductions in dietary energy intake. Recently, therapy with atorvastatin and vitamin E (vitE) has been recommended, although clinical studies on the resolution of hepatic fibrosis are inconclusive. Targeting NASH-induced hepatic end-points, this study evaluated the effects of atorvastatin and vitE alone or in combination with a dietary intervention in the guinea pig NASH model. Guinea pigs (n = 72) received 20 weeks of high fat feeding before allocating to four groups: continued HF feeding (HF), HF diet with atorvastatin and vitE (HF+), low-fat diet (LF) and low-fat with atorvastatin and vitE (LF+), for four or eight weeks of intervention. Both LF and LF+ decreased liver weight, cholesterol and plasma dyslipidemia. LF+ further improved hepatic histopathological hallmarks (p < 0.05), liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (p < 0.05) and reduced the expression of target genes of hepatic inflammation and fibrosis (p < 0.05), underlining an increased effect on NASH resolution in this group. Collectively, the data support an overall beneficial effect of diet change, and indicate that atorvastatin and vitE therapy combined with a diet change act synergistically in improving NASH-induced endpoints.

Comparison of Three Sample Preparation Procedures for the Quantification of L-Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma Using HPLC-FLD.

Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were compared regarding the quantification of L-arginine and ADMA in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). The samples were analysed by using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The analytical performance of (A) was comparable with that of (C), demonstrating recoveries of >90%, coefficient of variations (CVs, %) of <8, and a resolution (Rs ) between ADMA and symmetric dimethylarginine (SDMA) of 1.2. (B) was disregarded due to recoveries below 75%. (A) was validated with good results regarding linearity (>0.994), precision (<5%), and sensitivity (lower limit of quantification (LLOQ)) of 0.14 µM and 12 nM for L-arginine and ADMA, respectively. Due to the simplicity and speed of procedure (A), this approach may serve as preferred sample preparation of human plasma samples before HPLC-FLD in providing important information regarding elevated ADMA concentrations.

Spatial Memory Dysfunction Induced by Vitamin C Deficiency Is Associated with Changes in Monoaminergic Neurotransmitters and Aberrant Synapse Formation.

Vitamin C (vitC) is important in the developing brain, acting both as an essential antioxidant and as co-factor in the synthesis and metabolism of monoaminergic neurotransmitters. In guinea pigs, vitC deficiency results in increased oxidative stress, reduced hippocampal volume and neuronal numbers, and deficits in spatial memory. This study investigated the effects of 8 weeks of either sufficient (923 mg vitC/kg feed) or deficient (100 mg vitC/kg feed) levels of dietary vitC on hippocampal monoaminergic neurotransmitters and markers of synapse formation in young guinea pigs with spatial memory deficits. Western blotting and high performance liquid chromatography (HPLC) were used to quantify the selected markers. VitC deficiency resulted in significantly reduced protein levels of synaptophysin (p = 0.016) and a decrease in 5-hydroxyindoleacetic acid/5-hydroxytryptamine ratio (p = 0.0093). Protein expression of the N-methyl-d-aspartate receptor subunit 1 and monoamine oxidase A were reduced, albeit not reaching statistical significance (p = 0.0898 and p = 0.067, respectively). Our findings suggest that vitC deficiency induced spatial memory deficits might be mediated by impairments in neurotransmission and synaptic development.

Long term Westernized diet leads to region-specific changes in brain signaling mechanisms.

Western diets, high in fat and energy, are associated with cognitive deficits in humans and animal models, but the underlying mechanisms are not fully elucidated. This includes whether diet-induced dyslipidemia per se negatively impacts brain signaling. Here we investigate the effects of dyslipidemia induced by two high fat diets with or without high sucrose on hippocampal and frontal cortical oxidative stress, brain-derived neurotrophic factor (BDNF) and down-stream markers of synaptic plasticity, as well as alterations in monoaminergic neurotransmitter levels. A high fat diet was associated with decreased antioxidant status (vitamin C), increased serotonin in the frontal cortex, and increased ratio of phosphorylated Ca2+/calmodulin-dependent protein kinase II in the hippocampus, while a high fat and sucrose diet decreased levels of vitamin C in the frontal cortex and BDNF in the hippocampus. Markers of dyslipidemia correlated significantly with cerebral vitamin C levels, monoaminergic neurotransmitters and metabolites in the frontal cortex, but not in the hippocampus. Thus, a high fat diet caused regional alterations in antioxidant levels, neurochemistry and molecular markers in the non-obese dyslipidemic guinea pig.

Liraglutide Decreases Hepatic Inflammation and Injury in Advanced Lean Non-Alcoholic Steatohepatitis.

Although commonly associated with obesity, non-alcoholic fatty liver disease (NAFLD) is also present in the lean population representing a unique disease phenotype. Affecting 25% of the world's population, NAFLD is associated with increased mortality especially when progressed to non-alcoholic steatohepatitis (NASH). However, no approved pharmacological treatments exist. Current research focuses mainly on NASH associated with obesity, leaving the effectiveness of promising treatments in lean NASH virtually unknown. This study therefore aimed to evaluate the effect of liraglutide (glucagon-like peptide 1 analogue) and dietary intervention, alone and in combination, in guinea pigs with non-obese NASH. After 20 weeks of high-fat feeding (20% fat, 15% sucrose, 0.35% cholesterol), 40 female guinea pigs were block-randomized based on weight into four groups receiving one of four treatments for 4 weeks: continued high-fat diet (HF, control), high-fat diet and liraglutide treatment (HFL), chow diet (4% fat, 0% sucrose, 0% cholesterol; HFC) or chow diet and liraglutide treatment (HFCL). High-fat feeding induced NASH with severe fibrosis. Liraglutide decreased inflammation (p < 0.05) and hepatocyte ballooning (p < 0.05), while increasing hepatic α-tocopherol (p = 0.0154). Dietary intervention did not improve liver histopathology significantly, but decreased liver weight (p = 0.004), plasma total cholesterol (p = 0.0175), LDL-cholesterol (p = 0.0063), VLDL-cholesterol (p = 0.0034), hepatic cholesterol (p < 0.0001) and increased hepatic vitamin C (p = 0.0099). Combined liraglutide and dietary intervention induced a rapid weight loss, necessitating periodical liraglutide dose adjustment/discontinuation, limiting the strength of the findings from this group. Collectively, this pre-clinical study supports the beneficial effect of liraglutide on NASH and extends this notion to lean NASH.

Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain.

In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical detection provided limits of quantifications (LOQs) between 3.6 and 12nM. Within the linear range, obtained recoveries were from 90.9±9.9 to 120±14% and intra-day and inter-day precisions found to be less than 5.5% and 12%, respectively. The analytical method was applicable for quantification of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7pmol per 2 million cells intracellularly, but only the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid in frontal cortex, as compared to cerebellum. The chemical turnover in frontal cortex tissue of guinea pig was for serotonin successfully predicted from the turnover observed in the frontal cortex cell culture. In conclusion, the present analytical method shows high precision, accuracy and sensitivity and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry.

Structure elucidation and quantification of impurities formed between 6-aminocaproic acid and the excipients citric acid and sorbitol in an oral solution using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

Concentrated solutions containing 6-aminocaproic acid and the excipients citric acid and sorbitol have been studied at temperatures of 50°C, 60°C, 70°C and 80°C as well as at 20°C. It has previously been reported that the commonly employed citric acid is a reactive excipient, and it is therefore important to thoroughly investigate a possible reaction between 6-aminocaproic acid and citric acid. The current study revealed the formation of 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid between 6-aminocaproic acid and citric acid by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). Less than 0.03% of 6-aminocaproic acid was converted to 3-hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid after 30 days of storage at 80°C. Degradation products of 6-aminocaproic acid were also observed after storage at the applied temperatures, e.g., dimer, trimer and cyclized 6-aminocaproic acid, i.e., caprolactam. No reaction products between D-sorbitol and 6-aminocaproic acid could be observed. 3-Hydroxy-3,4-dicarboxy-butanamide-N-hexanoic acid, dimer and caprolactam were also observed after storage at 20°C for 3 months. The findings imply that an oral solution of 6-aminocaproic acid is relatively stable at 20°C at the pH values 4.00 and 5.00 as suggested in the USP for oral formulations. Compliance with the ICH guideline Q3B is expected.

Evaluation of microwave oven heating for prediction of drug-excipient compatibilities and accelerated stability studies.

Microwave ovens have been used extensively in organic synthesis in order to accelerate reaction rates. Here, a set up comprising a microwave oven combined with silicon carbide (SiC) plates for the controlled microwave heating of model formulations has been applied in order to investigate, if a microwave oven is applicable for accelerated drug stability testing. Chemical interactions were investigated in three selected model formulations of drug and excipients regarding the formation of ester and amide reaction products. In the accelerated stability studies, a design of experiments (DoE) approach was applied in order to be able to rank excipients regarding reactivity: Study A: cetirizine with PEG 400, sorbitol, glycerol and propylene glycol. Study B: 6-aminocaproic acid with citrate, acetate, tartrate and gluconate. Study C: atenolol with citric, tartaric, malic, glutaric, and sorbic acid. The model formulations were representative for oral solutions (co-solvents), parenteral solutions (buffer species) and solid dosage forms (organic acids applicable for solubility enhancement). The DoE studies showed overall that the same impurities were generated by microwave oven heating leading to temperatures between 150°C and 180°C as compared to accelerated stability studies performed at 40°C and 80°C using a conventional oven. Ranking of the reactivity of the excipients could be made in the DoE studies performed at 150-180°C, which was representative for the ranking obtained after storage at 40°C and 80°C. It was possible to reduce the time needed for drug-excipient compatibility testing of the three model formulations from weeks to less than an hour in the three case studies. The microwave oven is therefore considered to be an interesting alternative to conventional thermal techniques for the investigation of drug-excipient interactions during preformulation.

Kinetics of the esterification of active pharmaceutical ingredients containing carboxylic acid functionality in polyethylene glycol: formulation implications.

Polyethylene glycols (PEGs) are attractive as excipients in the manufacture of drug products because they are water soluble and poorly immunogenic. They are used in various pharmaceutical preparations. However, because of their terminal hydroxyl groups, PEGs can participate in esterification reactions. In this study, kinetics of two active pharmaceutical ingredients, cetirizine and indomethacin possessing carboxylic acid functionality, has been studied in PEG 400 and PEG 1000 at 50 °C, 60 °C, 70 °C, and 80 °C. HPLC-UV was applied for the determination of concentrations in the kinetic studies, whereas HPLC-MS was used to identify reaction products. The esterification reactions were observed to be reversible. A second-order reversible kinetic model was applied and rate constants were determined. The rate constants demonstrated that cetirizine was esterified about 240 times faster than indomethacin at 80 °C. The shelf-life for cetirizine in a PEG 400 formulation at 25 °C expressed as t(95%) was predicted to be only 30 h. Further, rate constants for esterification of cetirizine in PEG 1000 in relation to PEG 400 decreased by a factor of 10, probably related to increased viscosity. However, it is important to be aware of this drug-excipient interaction, as it can reduce the shelf-life of a low-average molecular weight PEG formulation considerably.

Evaluation of supercritical fluid chromatography for testing of PEG adducts in pharmaceuticals.

Drug formulations containing polyethylene glycol may give rise to formation of reaction products between the aforementioned and the active pharmaceutical ingredient. Supercritical fluid chromatography has recently achieved new interest and improved instrumentation is now available. Here, supercritical fluid chromatography has been evaluated for its possible use for determination of reactions products formed between polyethylene glycol and active pharmaceutical ingredients. A mixture of polyethylene glycols with average molecular weights of 400-6000Da was separated with supercritical fluid chromatography using silica columns and carbon dioxide modified with methanol as mobile phase. Satisfactory resolution (Rs=1.2) of the individual oligomers up to a molecular weight of 1000Da was obtained using evaporative light scattering as detection technique. The active pharmaceutical ingredients, cetirizine or indomethacin were investigated in a reaction mixture containing polyethylene glycol 400 after incubation at 80°C for 120h. Polyethylene glycol esters formed upon reaction with both active pharmaceutical ingredients were observed as polymeric patterns with ultraviolet detection and identified with mass spectrometry. Cetirizine was observed to be more reactive than indomethacin. The observed difference in reactivity is due to differences in polar and steric effects between cetirizine and indomethacin. Evaporative light scattering, ultraviolet absorbance and mass spectrometric detection were investigated and each detection technique has its own advantages and disadvantages, but in order to be able to detect selected impurities in the complex mixture of impurities formed, mass spectrometry is superior.