Risk assessment report of potential impurities in cetirizine dihydrochloride.

Recently, the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) had reported on "unexpected" impurities in a couple of sartans, ranitidine, and metformin. These events led to a lot of discussion with regard to the risk assessment for the production process itself. Most of these discussions covered the field of nitrosamine impurities only, but that would be too short-sighted. One should expand that scope. It is impossible to synthesize a 100 % pure compound which holds true for all active pharmaceutical ingredients (APIs). Different synthetic routes result in different impurity profiles. Therefore, pharmacopoeias try to consider all possible impurities that can arise from different drug synthesis routes in one a single monograph for impurity assessment. However, API manufacturers cannot simply rely on the impurity profile reported in pharmacopoeias for the production of a high-quality product. They have to implement a whole risk assessment to rate the presence of impurities in the API. Here, a strategy to evaluate and minimize the load of potential risks of impurities during the manufacturing process of the drug substance cetirizine dihydrochloride within the frame of a detailed risk assessment report is demonstrated.

Investigation of tryptophan-related yellowing in parenteral amino acid solution: Development of a stability-indicating method and assessment of degradation products in pharmaceutical formulations.

Parenteral amino acid solutions containing tryptophan tend to develop a yellow colouration upon storage. Hence, the aim of the present study was to find out whether tryptophan degradation products are the reason for the yellowing. The degree of discolouration and tryptophan degradation was examined by visual examination and UV/Vis measurements with respect to oxygen presence, pH value, and duration of steam sterilization. LC-UV analyses of autoclaved tryptophan solutions indicated eight degradation products, namely R,R/R,S 2-amino-3-(oxoindolin-3-yl)propanoic acid, R,R/R,S 2-amino-3-hydroxy-2-oxoindolin-3-yl)propanoic acids, cis/trans 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, N´-formylkynurenine, and kynurenine. The proposed degradation products were confirmed by spiking of synthesized degradation products and LC-UV/MS analyses. The LC-UV analysis method was optimized and validated according to the ICH guideline Q2 (R1). Tryptophan stability in commercially available parenteral amino acid formulations was evaluated over a storing period of 12 months in two common types of primary packaging after autoclave procedure.

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 µM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

A long-time stability study of 50 drug substances representing common drug classes of pharmaceutical use.

For assurance of the quality of active pharmaceutical ingredients (APIs) used for manufacturing medicines, the analytical requirements of the European Pharmacopoeia serve as a guideline and have a binding character. Within a particular timeframe, an API is considered to comply with predefined specifications. If applicable, it can be used for the manufacturing of a finished pharmaceutical product. The objective of the study presented here was to assess the long-term stability of 50 drug substances with an age of 20-30 years or even older in some cases. The substances are part of a collection of old pharmaceuticals at the Institute for Pharmacy in Würzburg, Germany, and represent commonly used drug classes containing ß-blockers, ß-sympathomimetic drugs, anticholinergics, anti-infectives, non-steroidal anti-inflammatory drugs, antipsychotics, antihistaminic drugs, and one antiarrhythmic drug. The content and the degradation profile of the items were determined by means of potentiometric titration and liquid chromatography techniques based on pharmacopoeial approaches for impurity profiling covering all process and degradation related substances. The results of the study show that 44 out of 49 tested substances still complied with specifications of the current pharmacopoeias. For metipranolol, which is not monographed in any pharmacopoeia, a small degradation by hydrolysis was observed. In one lot of ampicillin sodium, atenolol, atropine, penbutolol, and salbutamol, at least one impurity did not meet the acceptance criteria, respectively. Some impurities were not related to degradation. However, a surprisingly high chemical stability of the old drug substances was revealed after more than two decades of storage.

Quantification of Hydrochlorothiazide and Ramipril/Ramiprilate in Blood Serum and Cerebrospinal Fluid: A Pharmacokinetic Assessment of Central Nervous System Adverse Effects.

BACKGROUND: A drug must reach the central nervous system (CNS) in order to directly cause CNS adverse effects (AEs). Our current study addressed the pharmacokinetic (PK) background of the assumption that CNS concentrations of hydrochlorothiazide (HCT) and ramiprilate may directly cause CNS AEs such as headache and drowsiness. METHODS: In neurological patients, paired serum and cerebrospinal fluid (CSF) samples were withdrawn simultaneously. Some of them were treated with HCT (n = 15, daily chronic doses 7.5-25 mg) or ramipril (n = 9, 2.5-10 mg). Total concentrations of HCT and ramiprilate were quantified in these samples. To this end, sensitive liquid chromatography/tandem mass spectrometry methods were developed. RESULTS: CSF reached 4.1% (interquartile ranges 2.5-5%) of total serum concentrations for HCT and 2.3% (1.7-5.7%) for ramiprilate, corresponding to about 11.3% and 5.5% of respective unbound serum concentrations. CONCLUSION: The PK/Pharmacodynamic characteristics of HCT and ramiprilate in the CNS are unknown. However, since the CSF levels of these agents, both free and bound, were much lower than the corresponding concentrations in serum, it is unlikely that the observed CNS AEs are mediated primarily via direct effects in the brain.

Stability and assessment of amino acids in parenteral nutrition solutions.

Sterile amino acid solutions are applied in medical care as part of Total Parenteral Nutrition systems. Typical formulations consist of variable admixtures of essential and non-essential amino acids together with carbohydrates, electrolytes, vitamins, trace element solutions and lipid emulsions. The complexity of these formulations gives rise to stability and compatibility reflections. This review focuses on amino acid stability in pure amino acid solution and name methods of assessment. Incompatibilities of amino acids with the other ingredients are matter of concern in clinical practice and evaluated for relevance.

Population pharmacokinetics of piperacillin at two dose levels: influence of nonlinear pharmacokinetics on the pharmacodynamic profile.

Piperacillin in combination with tazobactam is one of the most commonly used intravenous antibiotics. There is evidence for a possible saturable elimination of piperacillin. Therefore, the saturable elimination and its impact on the choice of optimal dosage regimens were quantified. In a randomized crossover study, 10 healthy volunteers received 1,500 mg and 3,000 mg of piperacillin as 5-min intravenous infusion. Population pharmacokinetics based on plasma and urine data were determined utilizing NONMEM and S-ADAPT. Probabilities of target attainment (PTAs) were compared for different models and dosage regimens, based on the target time of the non-protein-bound concentration above the MIC of at least 50% of the dosing interval. Total clearance of piperacillin was 18% (geometric mean ratio, 90% confidence interval, 11 to 24%) lower (P < 0.01), and renal clearance was 24% (9 to 37%) lower (P = 0.02) at the high compared to the low dose. The final model included first-order nonrenal elimination and parallel first-order and mixed-order renal elimination. Nonrenal clearance was 5.44 liter/h (coefficient of variation, 18%), first-order renal clearance was 4.42 liter/h (47%), and the maximum elimination rate of mixed-order renal elimination was 219 mg/h (84%), with a Michaelis-Menten constant of 36.1 mg/liter (112%). Compared to models with saturable elimination, a linear model predicted up to 10% lower population PTAs for high-dose short-term infusions (6 g every 8 h) and up to 4% higher population PTAs for low-dose continuous infusions (6 g/day). While renal elimination of piperacillin was saturable at therapeutic concentrations, the extent of saturation of nonrenal clearance was small. The influence of saturable elimination on PTAs for clinically relevant dosage regimens was relatively small.

Charged aerosol detection in pharmaceutical analysis.

Due to its wide field of application, sensitivity, wide range of linearity and the applicability to gradient elution, the most common detection technique for HPLC nowadays is UV/vis spectrophotometry. However, UV/vis detection comes to its limits when the analytes are lacking suitable chromophors or exhibit very different UV responses. In the past years, different types of evaporation/aerosol based HPLC detectors have been developed to fill this gap in HPLC detection. Amongst those, the corona-charged aerosol detector (CAD) is one of the most powerful and versatile representatives. In the recent past a variety of papers have been published, demonstrating the potential of the CAD in different fields of analytical chemistry. This paper is intended to provide an overview of the key performance characteristics and manifold applications for HPLC-CAD in the field of pharmaceutical analysis.

Nonlinear pharmacokinetics of piperacillin in healthy volunteers--implications for optimal dosage regimens.

AIMS: (i) To describe the first-order and mixed-order elimination pathways of piperacillin, (ii) to determine the between occasion variability (BOV) of pharmacokinetic parameters and (iii) to propose optimized dosage regimens. METHODS: We performed a five-period replicate dose study in four healthy volunteers. Each subject received 4g piperacillin as a single 5min intravenous infusion in each study period. Drug analysis was performed by HPLC. We used NONMEM and S-ADAPT for population pharmacokinetic analysis and Monte Carlo simulation to predict the probability of target attainment (PTA) with a target time of non-protein bound concentration above MIC >50% of the dosing interval. RESULTS: A model with first-order nonrenal elimination and parallel first-order and mixed-order renal elimination had the best predictive performance. For a 70kg subject we estimated 4.40lh(-1) for nonrenal clearance, 5.70lh(-1) for first-order renal clearance, 170mgh(-1) for V(max) , and 49.7mgl(-1) for K(m) for the mixed-order renal elimination. The BOV was 39% for V(max) , 117% for K(m) , and 8.5% for total clearance. A 30min infusion of 4g every 6h achieved robust (≥90%) PTAs for MICs ≤12mgl(-1) . As an alternative mode of administration, a 5h infusion of 6g every 8h achieved robust PTAs for MICs ≤48mgl(-1) . CONCLUSIONS: Part of the renal elimination of piperacillin is saturable at clinically used doses. The BOV of total clearance and volume of distribution were low. Prolonged infusions achieved better PTAs compared with shorter infusions at similar daily doses. This benefit was most pronounced for MICs between 12 and 48mgl(-1) .

Comparison of the pharmacokinetics and pharmacodynamic profile of carumonam in cystic fibrosis patients and healthy volunteers.

Our objectives were to compare the pharmacokinetics (PK) of carumonam, a monobactam, between cystic fibrosis (CF) patients and healthy volunteers and assess its pharmacodynamic profile. We studied 10 adult CF patients and 18 healthy volunteers of similar body size (dose: 2.166 g of carumonam as 15-min intravenous infusion). High performance liquid chromatography with ultraviolet detection (HPLC-UV) was used for drug analysis and NONMEM (ICON, Ellicot City, MD) for population PK and Monte Carlo simulation with targets between > or =20% and 100% free time above MIC (fT > MIC). Unscaled renal clearance was 24% higher in CF patients. Lean body mass and creatinine clearance explained the difference in average clearance and volume of distribution between both subject groups. For a daily dose of 6 g per 70 kg of total body weight, 15-min infusions q8h achieved robust (>90%) probabilities of target attainment (PTAs) (target, 60% fT > MIC) for MICs < or =3 mg/L in CF patients and < or =6 mg/L in healthy volunteers. At the same dose, 4-h infusions q8h achieved robust PTAs up to markedly higher MICs < or =8 to 12 mg/L in CF patients and < or =16 mg/L in healthy volunteers.

Bone penetration of amoxicillin and clavulanic acid evaluated by population pharmacokinetics and Monte Carlo simulation.

Amoxicillin (amoxicilline)-clavulanic acid has promising activity against pathogens that cause bone infections. We present the first evaluation of the bone penetration of a beta-lactam by population pharmacokinetics and pharmacodynamic profiling via Monte Carlo simulations. Twenty uninfected patients undergoing total hip replacement received a single intravenous infusion of 2,000 mg/200 mg amoxicillin-clavulanic acid before surgery. Blood and bone specimens were collected. Bone samples were pulverized under liquid nitrogen with a cryogenic mill, including an internal standard. The drug concentrations in serum and total bone were analyzed by liquid chromatography-tandem mass spectrometry. We used NONMEM and S-ADAPT for population pharmacokinetic analysis and a target time of the non-protein-bound drug concentration above the MIC for > or = 50% of the dosing interval for near-maximal bactericidal activity in serum. The median of the ratio of the area under the curve (AUC) for bone/AUC for serum was 20% (10th to 90th percentile for between-subject variability [variability], 16 to 25%) in cortical bone and 18% (variability, 11 to 29%) in cancellous bone for amoxicillin and 15% (variability, 11 to 21%) in cortical bone and 10% (variability, 5.1 to 21%) in cancellous bone for clavulanic acid. Analysis in S-ADAPT yielded similar results. The equilibration half-lives between serum and bone were 12 min for amoxicillin and 14 min for clavulanic acid. For a 30-min infusion of 2,000 mg/200 mg amoxicillin-clavulanic acid every 4 h, amoxicillin achieved robust (> or = 90%) probabilities of target attainment (PTAs) for MICs of < or = 12 mg/liter in serum and 2 to 3 mg/liter in bone and population PTAs above 95% against methicillin-susceptible Staphylococcus aureus in bone and serum. The AUC of amoxicillin-clavulanic acid was 5 to 10 times lower in bone than in serum, and amoxicillin-clavulanic acid achieved a rapid equilibrium and favorable population PTAs against pathogens commonly encountered in bone infections.

Penetration of moxifloxacin into bone evaluated by Monte Carlo simulation.

Moxifloxacin is a fluoroquinolone with a broad spectrum of activity and good penetration into many tissues, including bone. Penetration of moxifloxacin into bone has not yet been studied using compartmental modeling techniques. Therefore, we determined the rate and extent of bone penetration by moxifloxacin and evaluated its pharmacodynamic profile in bone via Monte Carlo simulation. Twenty-four patients (10 males, 14 females) undergoing total hip replacement received 400 mg moxifloxacin orally 2 to 7 h prior to surgery. Blood and bone specimens were collected. Bone samples were pulverized under liquid nitrogen by a cryogenic mill, including an internal standard. Drug concentrations were analyzed by high-performance liquid chromatography. We used ADAPT II (results reported), NONMEM, and WinBUGS for pharmacokinetic analysis. Monte Carlo simulation was performed to reverse engineer the necessary area under the free concentration-time curve fAUC(SERUM)/MIC in serum and total AUC(BONE)/MIC in bone for a successful clinical or microbiological outcome. The median (10% to 90% percentile for between-subject variability) of the AUC in bone divided by the AUC in serum (AUC(BONE)/AUC(SERUM)) was 80% (51 to 126%) for cortical bone and 78% (42 to 144%) for cancellous bone. Equilibration between serum and bone was rapid. Moxifloxacin achieved robust (> or = 90%) probabilities of target attainment (PTAs) in serum, cortical bone, and cancellous bone up to MICs of < or = 0.375 mg/liter based on the targets fAUC(SERUM)/MIC > or = 40 and AUC(BONE)/MIC > or = 33. Moxifloxacin showed high bone concentrations and a rapid equilibrium between bone and serum. The favorable PTAs compared to the 90%-inhibitory MIC of Staphylococcus aureus warrant future clinical trials on the effectiveness of moxifloxacin in the treatment of bone infections.

Population pharmacokinetics at two dose levels and pharmacodynamic profiling of flucloxacillin.

Flucloxacillin is often used for the treatment of serious infections due to sensitive staphylococci. The pharmacokinetic (PK)-pharmacodynamic (PD) breakpoint of flucloxacillin has not been determined by the use of population PK. Targets based on the duration of non-protein-bound concentrations above the MIC (fT(>MIC)) best correlate with clinical cure rates for beta-lactams. We compared the breakpoints for flucloxacillin between several dosage regimens. In a randomized, two-way crossover study, 10 healthy volunteers received 500 mg and 1,000 mg flucloxacillin as 5-min intravenous infusions. Drug concentrations were determined by high-pressure liquid chromatography. We used the programs WinNonlin for noncompartmental analysis and statistics and NONMEM for population PK and Monte Carlo simulation. We compared the probability of target attainment (PTA) for intermittent- and continuous-dosage regimens based on the targets of fT(>MIC)s of > or =50% and > or =30% of the dosing interval. The clearance and the volume of distribution were very similar after the administration of 500 mg and 1,000 mg flucloxacillin. We estimated renal and nonrenal clearances of 5.37 liters/h (coefficient of variation, 19%) and 2.73 liters/h (33%). For near maximal killing (target, fT(>MIC) of > or =50%) flucloxacillin showed a robust (> or =90%) PTA up to MICs of 0.75 to 1 mg/liter (PTA of 86% at 1 mg/liter) for a continuous or a prolonged infusion of 6 g/day. Short-term infusions of 6 g/day had a lower breakpoint of 0.25 to 0.375 mg/liter. The flucloxacillin PK was linear for doses of 500 mg and 1,000 mg. Prolonged and continuous infusion at a 66% lower daily dose achieved the same PK-PD breakpoints as short-term infusions. Prolonged infusion and continuous infusion are appealing options for the treatment of serious infections caused by sensitive staphylococci.

Assessment of inhibitory potency of antibiotics by MRI: apparent T2 as a marker of cell growth.

A new method to assess the antibiotic potency by MRI has been developed. Correlating 1H NMR spectra of bacterial cultures with the extracellular parameters T2, OD600, and pH, a relationship between cell growth and T2 variations was established. T2 is influenced by chemical exchange that depends on pH, composition, and concentration of the medium. Changes in the medium from bacterial metabolism are reflected in alternating T2 values. At 17.6 T, growth curves based on T2 values were measured simultaneously of several cultures of Streptococcus vestibularis. From T2 growth curves in the presence of varying concentrations of vancomycin, the minimum inhibitory concentration of the antibiotic could be determined to be 0.33+/-0.08 microM. This value was in good agreement with the result obtained by the conventional broth microdilution. In principle, T2 growth curves can be determined on a large number of cultures simultaneously and may potentially be used as a novel tool in high through-put screening of novel anti-infective substances.

Interlaboratory study of a NACE method for the determination of R-timolol content in S-timolol maleate: assessment of uncertainty.

Analyses of statistical variance were applied to evaluate the precision and practicality of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and S-timolol. Data were collected in an interlaboratory study by 11 participating laboratories located in Europe and North America. General qualitative method performance was examined using suitability descriptors (i.e. resolution, selectivity, migration times and S/N), while precision was determined by quantification of variances in the determination of R-timolol at four different impurity levels in S-timolol maleate samples. The interlaboratory trials were designed in accordance with the ISO guideline 5725-2. This allowed estimating for each sample, the different variances, i.e. between-laboratory (s2(Laboratories)), between-day (s2(Days)) and between-replicate (s2(Replicates)). The variances of repeatability (s2r) and reproducibility (s2R) were then calculated. The estimated uncertainty, derived from the precision estimates, seems to be concentration-dependent above a given threshold. This example of R-timolol illustrates how a laboratory can evaluate uncertainty in general.

Evaluation by monte carlo simulation of the pharmacokinetics of two doses of meropenem administered intermittently or as a continuous infusion in healthy volunteers.

Meropenem is a broad-spectrum carbapenem antibacterial agent. In order to optimize levels in plasma relative to the MICs, the ideal dose level and dosage regimen need to be determined. The pharmacokinetics of meropenem were studied in two groups, each comprising eight healthy volunteers who received the following doses: 500 mg as an intravenous infusion over 30 min three times a day (t.i.d.) versus a 250-mg loading dose followed by a 1,500 mg continuous infusion over 24 h for group A and 1,000 mg as an intravenous infusion over 30 min t.i.d. versus a 500-mg loading dose followed by a 3,000-mg continuous infusion over 24 h for group B. Meropenem concentrations in plasma and urine were determined by liquid chromatography-mass spectrometry/mass spectrometry and high-performance liquid chromatography with UV detection, respectively. Pharmacokinetic calculations were done by use of a two-compartment open model, and the data were extrapolated by Monte Carlo simulations for 10,000 simulated subjects for pharmacodynamic evaluation. There were no significant differences in total clearance and renal clearance between group A and group B or between the intermittent treatment and the continuous infusion. The analyses of the probability of target attainment by MIC for the high- and low-dose continuous infusions were robust up to MICs of 4 mg/liter and 2 mg/liter, respectively. The corresponding values for intermittent infusions were only 0.5 mg/liter and 0.25 mg/liter. When these observations were correlated with MICs obtained from the MYSTIC database, intermittent infusion results in adequate activity against two of the most common nosocomially acquired pathogens, Klebsiella pneumoniae and Enterobacter cloacae. However, against Pseudomonas aeruginosa, the evaluation shows a clear advantage of high-dose therapy administered as a continuous infusion. We believe that in the empirical therapy situation, the continuous-infusion mode of administration is most worth the extra efforts. We conclude that clinical trials for evaluation of the continuous infusions of meropenem in critically ill patients are warranted.

Quantitative liquid chromatography-mass spectrometry determination of isatin in urine using automated on-line extraction.

Here we describe a simple, fast and sensitive liquid chromatography/mass spectrometry method with automated on-line extraction to quantify isatin, an endogenous monoamine oxidase, and atrial natriuretic peptide inhibitor, in urine. After derivatisation of isatin to isatinoxime with hydroxylamine hydrochloride and zinc sulfate precipitation, samples were loaded on the extraction column, washed and, after activation of the column-switching valve, backflushed onto the analytical column. Using electrospray ionisation, [M+H]+ ions could be detected in the selected ion monitoring mode. The assay was linear from 5 to 5000 ng/ml (r2>0.99) and analytical recovery was >80%. Inter-assay precision for the quality control samples was less than 3% and inter-assay accuracy was within +/- 5%.