Nitrosated Active Pharmaceutical Ingredients - Lessons Learned?

The occurrence of N-nitrosodialkylamines in active pharmaceutical ingredients (APIs) and drug products in the last years was a kind of eye opener with regard to quality of drugs. We became aware of the fact that quality control tests described in the international pharmacopoeias might not be sufficient. The N-nitrosodialkylamines found were neither so-called (structurally) related substances, nor residual solvents or heavy metals; hence they were not limited by a compendial test, but by the ICH guideline M7 of mutagenic impurities. Additionally, nitrosamine drug-substance-related impurities (NDSRIs) were detected, mostly within the process of risk assessment required by regulatory authorities. Here, the APIs containing a vulnerable amino moiety had reacted with nitrites being a contaminant of an excipient. This review deals with the formation, toxicity, and mitigation of NDSRISs.

Hydrogen-deuterium (H/D) exchange reaction of acebutolol hydrochloride in D2O and CD3OD solution.

H/D exchange reactions can be observed by NMR spectroscopy of acebutolol (ACE). The results obtained showed deuterium incorporation at α-posi t ion of the carbonyl group of acebutolol, when using deuterium oxide or deuterated methanol as deuterium source and solvent. The spontaneous deuteration is proceeded by the following pathway CH3→CH2D→CHD→CD3, through a keto-enol tautomerization reaction. Furthermore, LC-MS / QTOF analyses have confirmed the proposed H/D exchange. In order to reduce the time of total deuteration observed at the acetyl group alkaline catalysts were employed.

Determination of plasma protein binding for sympathomimetic drugs by means of ultrafiltration.

Ephedrine and its diastereomer pseudoephedrine have long been used in therapy and are part of over-the-counter combination drugs against colds or allergies. Nonetheless, there is scarcely any information on their plasma protein binding in literature. Plasma protein binding is an important parameter from a pharmacokinetic and pharmacodynamic point of view and can play a crucial role in therapy. The aim of this study was to determine the extent of plasma protein binding using ultrafiltration and different types of plasma proteins like human (HSA) and bovine serum albumin (BSA) and human serum. Two orthogonal methods - continuous and discontinuous ultrafiltration - were used to confirm our findings. To get some structural and stereochemical insights into binding affinity towards plasma proteins, all four stereoisomers of ephedrine and pseudoephedrine were included in this study. Since all four stereoisomers exhibit a low affinity towards albumin, other sympathomimetic drugs of structural similarity were tested to investigate the influence of the basic character of the Ephedra alkaloids in their binding to plasma proteins.

[Pharmacokinetics and pharmacodynamics of antibiotics in intensive care]. / Pharmakokinetik und Pharmakodynamik von Antibiotika in der Intensivmedizin.

Optimized dosage regimens of antibiotics have remained obscure since their introduction. During the last two decades pharmacokinetic(PK)-pharmacodynamic(PD) relationships, originally established in animal experiments, have been increasingly used in patients. The action of betalactams is believed to be governed by the time the plasma concentration is above the minimum inhibitory concentration (MIC). Aminoglycosides act as planned when the peak concentration is a multiple of the MIC and vancomycin seems to work best when the area under the plasma vs. time curve (AUC) to MIC has a certain ratio. Clinicians should be aware that these relationships can only be an indication in which direction dosing should go. Larger studies with sufficiently high numbers of patients and particularly severely sick patients are needed to prove the concepts. In times where all antibiotics can be measured with new technologies, the introduction of therapeutic drug monitoring (TDM) is suggested for ICUs (Intensive Care Unit). The idea of a central lab for TDM of antibiotics such as PEAK (Paul Ehrlich Antibiotika Konzentrationsmessung) is supported.

Validation and application of an HPLC-CAD-TOF/MS method for identification and quantification of pharmaceutical counterions.

A generic approach for the analysis of counterions of pharmaceutical reference substances, which are established by the laboratory department of the European Pharmacopoeia (Ph. Eur.), was developed. A mixed-mode chromatography method using charged aerosol detection (CAD) published by Zhang et al. separating 25 commonly used pharmaceutical counterions was selected for this purpose. The method was validated in terms of specificity, repeatability, limits of quantification (LOQs), linearity and range according to ICH guideline Q2(R1) and the Technical Guide for the Elaboration of Monographs of the Ph. Eur. Moreover, the applicability of the method for the purpose of counterion identification and quantification in drug substances as well as for the control of inorganic ions as impurities was demonstrated using selected examples of Ph. Eur. reference standards and other samples of substances for pharmaceutical use (e.g. cloxacillin sodium, somatostatin). It was shown that for identification purposes of the parent substance as well as organic ions the chromatographic system can easily be coupled to a mass selective detector without any modification.

Possibilities and limitations of capillary electropherosis in pharmaceutical analysis.

Capillary electropherosis (CE) has been proved to be an important alternative to high-performance liquid chromatography (HPLC) in pharmaceutical analysis. However, when it comes to the analysis of compounds, e.g. impurities or metabolites, of very different polarity and water solubility CE and the related techniques come to its limits. This is demonstrated for the antipsychotic drug quetiapine and its impurities. A nonaqueous capillary electrophoresis (NACE) method was developed using a background electrolyte (BGE) composed of ammonium acetate dissolved in a mixture of acetonitrile and methanol including acetic acid to protonate the substances. The NACE method gave an excellent separation of all components. Since the conductivity of the BGE used in the NACE method is quite low and problems with current occurred, an additional aqueous capillary zone electrophoresis (CZE) method was developed for quetiapine and the two water soluble derivatives, using phosphate buffer as BGE. The method was validated with regard to repeatability and limit of detection.

Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor.

BACKGROUND AND PURPOSE: Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'. EXPERIMENTAL APPROACH: Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. KEY RESULTS: In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi /Gs signalling competence. In the orthosteric loss-of-function mutant M2 -Y104(3.33) A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. CONCLUSION AND IMPLICATIONS: Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.

How to evaluate the separation efficiency of CZE methods?

In trying to estimate the separation efficiency of Capillary Electrophoresis (CE) methods, the resolution (RS), the number of theoretical plates (N) and the peak-to-valley ratio (p/v) are often used assessment criteria. This study demonstrates that these criteria are not as suitable to describe the separation efficiency in case of Capillary Zone Electrophoresis (CZE) methods as they are for Liquid Chromatography (LC) methods. The investigations were performed by means of a validated CZE method for the evaluation of tetracyclines and their related substances. Four impurities of tetracycline hydrochloride are described in the European Pharmacopoeia. Three were found in the sample used for our investigations, i.e. epi-tetracycline formed by keto-enol-tautomerism, anhydrotetracyclin and epi-anhydrotetracyline. It could be shown that higher values of these assessment criteria like RS do not necessarily represent better separation. Thus, a discussion on the usefulness of separation selectivity and efficiency as assessment criteria for capillary electrophoresis as well as on the introduction of additional parameters is needed.

Comparable population pharmacokinetics and pharmacodynamic breakpoints of cefpirome in cystic fibrosis patients and healthy volunteers.

Cystic fibrosis (CF) patients are often reported to have higher clearances and larger volumes of distribution per kilogram of total body weight (WT) for beta-lactams than healthy volunteers. As pharmacokinetic (PK) data on cefpirome from studies of CF patients are lacking, we systematically compared its population PK and pharmacodynamic breakpoints for CF patients and healthy volunteers of similar body size. Twelve adult CF patients (median lean body mass [LBM] = 45.7 kg) and 12 healthy volunteers (LBM = 50.0 kg) received a single 10-min intravenous infusion of 2 g cefpirome. Plasma and urine concentrations were determined by high-performance liquid chromatography (HPLC). Population PK and Monte Carlo simulations were performed using NONMEM and S-ADAPT and a duration of an unbound plasma concentration above the MIC ≥ 65% of the dosing interval as a pharmacodynamic target. Unscaled clearances for CF patients were similar to those seen with healthy volunteers, and the volume of distribution was 6% lower for CF patients. Linear scaling of total clearance by WT resulted in clearance that was 20% higher (P ≤ 0.001 [nonparametric bootstrap]) in CF patients. Allometric scaling by LBM explained the differences between the two subject groups with respect to average clearance and volume of distribution and reduced the unexplained between-subject variability of renal and nonrenal clearance by 10 to 14%. For the CF patients, robust (>90%) probabilities of target attainment (PTA) were achieved by the administration of a standard dose of 2 g/70 kg WT every 12 h (Q12h) given as 30-min infusions for MICs ≤ 1.5 mg/liter. As alternative dosage regimens, a 5-h infusion of 1.33 g/70 kg WT Q8h achieved robust PTAs for MICs ≤ 8 to 12 mg/liter and a continuous infusion of 4 g/day for MICs ≤ 12 mg/liter. Prolonged infusion of cefpirome is expected to be superior to short-term infusions for MICs between 2 and 12 mg/liter.

NMR techniques in biomedical and pharmaceutical analysis.

This article focuses on the description of some of the NMR techniques used in the field of biomedical and pharmaceutical research. Indeed, the NMR method has special characteristics which make it uniquely suitable for these kinds of studies. It is non-selective so that all the low molecular weight compounds in the sample investigated are detected simultaneously in a single run. NMR also provides rich structural information which is an important asset to characterize complex mixture components. NMR is quantitative, i.e. the area of a NMR signal is directly proportional to the number of corresponding nuclei and thus, at variance with other techniques, the response factor is not dependent on the molecular structure. It is also a non-invasive tool that permits in vivo studies in humans. Compared with other techniques, NMR is significantly insensitive, which represents the main drawback of the technique. The recent technological developments of the technique have nevertheless considerably improved its sensitivity. The first part of this article presents an overview of the advantages and limitations of NMR for in vitro quantitative analysis of complex matrices in liquid or semi-solid phases. The second part deals with the NMR-based metabolomics methodology. The third part describes the in vivo clinical magnetic resonance spectroscopy techniques. The fourth part reports some examples of NMR applications in the biomedical and pharmaceutical research fields.

N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity.

Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) µM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) ≥ 300 µM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 µM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 µM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells.

Population pharmacokinetic comparison and pharmacodynamic breakpoints of ceftazidime in cystic fibrosis patients and healthy volunteers.

Despite the promising activity of ceftazidime against Pseudomonas aeruginosa and Burkholderia cepacia, there has not yet been a study that directly compared the pharmacokinetics (PK) of ceftazidime in cystic fibrosis (CF) patients and healthy volunteers by population PK methodology. We assessed the population PK and PK/pharmacodynamic (PD) breakpoints of ceftazidime in CF patients and healthy volunteers. Eight CF patients (total body weight [WT] [average +/- standard deviation] = 42.9 +/- 18.4 kg) and seven healthy volunteers (WT = 66.2 +/- 4.9 kg) received 2 g ceftazidime as a 5-min intravenous infusion. High-performance liquid chromatography (HPLC) was used for drug analysis, and NONMEM (results reported), S-ADAPT, and NPAG were used for parametric and nonparametric population PK modeling. We considered linear and allometric body size models to scale clearance and volume of distribution. Monte Carlo simulations were based on a target time of non-protein-bound plasma concentration of ceftazidime above MIC of > or =65%, which represents near-maximal killing. Unscaled total clearance was 19% lower in CF patients, and volume of distribution was 36% lower. Total clearance was 7.82 liters/h for CF patients and 6.68 liters/h for healthy volunteers with 53 kg fat-free mass. Allometric scaling by fat-free mass reduced the between-subject variability by 32% for clearance and by 18 to 26% for volume of both peripheral compartments compared to linear scaling by WT. A 30-min ceftazidime infusion of 2 g/70 kg WT every 8 h (q8h) achieved robust (> or =90%) probabilities of target attainment (PTAs) for MICs of < or =1 mg/liter in CF patients and < or =3 mg/liter in healthy volunteers. Alternative modes of administration achieved robust PTAs up to markedly higher MICs of < or =8 to 12 mg/liter in CF patients for 5-h infusions of 2 g/70 kg WT q8h and < or =12 mg/liter for continuous infusion of 6 g/70 kg WT daily.

New semiphysiological absorption model to assess the pharmacodynamic profile of cefuroxime axetil using nonparametric and parametric population pharmacokinetics.

Cefuroxime axetil is widely used to treat respiratory tract infections. We are not aware of a population pharmacokinetic (PK) model for cefuroxime axetil. Our objectives were to develop a semiphysiological population PK model and evaluate the pharmacodynamic profile for cefuroxime axetil. Twenty-four healthy volunteers received 250 mg oral cefuroxime as a suspension after a standardized breakfast. Liquid chromatography-tandem mass spectrometry was used for drug analysis, NONMEM and S-ADAPT (results reported) were used for parametric population PK modeling, and NPAG was used for nonparametric population PK modeling. Monte Carlo simulations were used to predict the duration for which the non-protein-bound-plasma concentration was above the MIC (fT(>MIC)). A model with one disposition compartment, a saturable and time-dependent drug release from the stomach, and fast drug absorption from the intestine yielded precise (r > 0.992) and unbiased curve fits and an excellent predictive performance. The apparent clearance was 21.7 liters/h (19.8% coefficient of variation [CV]) and the volume of distribution 38.7 liters (18.3% CV). Robust (>or=90%) probabilities of target attainment (PTAs) were achieved by 250 mg cefuroxime given every 12 h (q12h) or q8h for MICs of MIC) of >or=40% and for MICs of MIC) of >or=65%. For the >or=40% fT(>MIC) target, the PTAs for 250 mg cefuroxime q12h were >or=97.8% for Streptococcus pyogenes and penicillin-susceptible Streptococcus pneumoniae. Cefuroxime at 250 mg q12h or q8h achieved PTAs below 73% or 92%, respectively, for Haemophilus influenzae, Moraxella catarrhalis, and penicillin-intermediate S. pneumoniae for susceptibility data from various countries. Depending on the MIC distribution, 250 mg oral cefuroxime q8h instead of q12h should be considered, especially for more-severe infections that require near-maximal killing by cefuroxime.

Prediction of the oversulphated chondroitin sulphate contamination of unfractionated heparin by ATR-IR spectrophotometry.

The detection of a contamination of heparin with oversulphated chondroitin sulphate (OSCS) was first analysed in an unfractionated heparin batch supplied to the US API-market in April 2006. OSCS is a semi-synthetic derivative of the natural occuring glycosaminoglycan chondroitin sulphate. Moreover some spectroscopic characteristics of the substance overlap with those of heparin, so that the infrared (IR) spectra are visually difficult to distinguish whereas (1)H-NMR (Nuclear Magnetic Resonance) spectroscopy or capillary electrophoresis (CE) provides identification by a simple visual inspection of either the spectrum or the electropherogram respectively. However, applying special tools of Multivariate Data Analysis (MVA) to the IR spectra an identification of the contaminated samples is possible. In detail a rapid Attenuation Total Reflectance-Infrared (ATR-IR) measurement was selected, which does not require any sample preparation. The result (contaminated or not contaminated) is predicted within a few minutes. A method transfer to mobile ATR-IR spectrometers seems to be possible. The analysis is based on the fact that the fingerprint of the OSCS IR spectrum (1st derivative) complies with a theoretically calculated principal component in the MVA.

Quality assessment of unfractionated heparin using 1H nuclear magnetic resonance spectroscopy.

Due to problems, especially anaphylactoid reactions, raised by impure unfractionated heparin the quality assessment of heparin has to be reconsidered. Neither the USP nor the European Pharmacopoeia are able to guarantee the purity of heparin, i.e., the limitation of oversulfated chondroitin sulfate (OSCS) which was found to be the reason for the allergic adverse effects. In the first run the regulatory authorities ask for 1H NMR spectroscopic and capillary electrophoretic measurements in order to characterize the impurity profile of heparin. Using an optimized 1H NMR method the limit of detection for OSCS was found to be 0.1%. In addition, it is possible to reliably quantify both OSCS and dermatan sulfate (DS), the latter being an indicator of poor purification of the unfractionated heparin. Screening of more than 100 heparin samples collected from international markets revealed a high number of samples containing substantial amounts of DS and a number of samples containing OSCS in an amount higher than 0.1%.

Evaluation of the stability of gentamicin in different antibiotic carriers using a validated MEKC method.

The quality control of gentamicin in different antibiotic carriers, using MEKC as stability-indicating method is described. Baseline separations of gentamicin C1, C1a, C2, C2a and C2b and, furthermore the impurities and degradation products garamin (GARA), 2-deoxy-streptamine (DSA) and sisomicin (SISO) were achieved with a background electrolyte containing 20mM deoxycholic acid, 15 mM beta-cyclodextrin and 100mM tetraborate (pH 10.0). After derivatization with o-phthaldialdehyde reagent (OPA), UV detection at 340 nm was possible. The method was validated with respect to selectivity, limit of detection (LOD) and quantification (LOQ) of the impurities, linearity, accuracy, precision and robustness. Evaluation of four different antibiotic carriers stored under stability conditions according to the International Conference on Harmonization (ICH) guidelines and older pharmaceutical formulations disclosed good stability.

Determination of Oversulphated Chondroitin Sulphate and Dermatan Sulphate in unfractionated heparin by (1)H-NMR - Collaborative study for quantification and analytical determination of LoD.

Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

Systematic comparison of the population pharmacokinetics and pharmacodynamics of piperacillin in cystic fibrosis patients and healthy volunteers.

Respiratory tract infections cause 90% of premature mortality in patients with cystic fibrosis (CF). Treatment of Pseudomonas aeruginosa infection is often very problematic. Piperacillin-tazobactam has good activity against P. aeruginosa, but its pharmacokinetics (PK) in CF patients has not been compared to the PK in healthy volunteers in a controlled clinical study. Therefore, we compared the population PK and pharmacodynamics (PD) of piperacillin between CF patients and healthy volunteers. We studied 8 adult (median age, 20 years) CF patients (average total body weight [WT], 43.1 +/- 7.8 kg) and 26 healthy volunteers (WT, 71.1 +/- 11.8 kg) who each received 4 g piperacillin as a 5-min intravenous infusion. We determined piperacillin levels by high-performance liquid chromatography, and we used NONMEM for population PK and Monte Carlo simulation. We used a target time of nonprotein-bound concentration above the MIC of 50%, which represents near-maximal bacterial killing. Unscaled total clearance was 25% lower, and the volume of distribution was 31% lower in CF patients. Allometric scaling by lean body mass reduced the unexplained (random) between-subject variability in clearance by 26% compared to the variability of linear scaling by WT. A standard dosage regimen of 3 g/70 kg body WT every 4 h as a 30-min infusion (daily dose, 18 g) achieved a robust (> or =90%) probability-of-target attainment (PTA) for MICs of < or =12 mg/liter in CF patients and < or =16 mg/liter in healthy volunteers. Alternative modes of administration allowed a marked dose reduction to 9 g daily. Prolonged (4-h) infusions of 3 g/70 kg WT every 8 h and continuous infusion (daily dose, 9 g), achieved a robust PTA for MICs of < or =16 mg/liter in both groups. Piperacillin achieved PTA expectation values of 64% and 89% against P. aeruginosa infection in CF patients, based on susceptibility data from two German CF clinics.

The influence of fluoroquinolone drugs on the bacterial growth of S. epidermidis utilizing the unique potential of vibrational spectroscopy.

Increasing resistance of many antibiotics has made the design of new drugs necessary. To assist a target-oriented search for new structures and for the elucidation of the mode of action of existing drugs, powerful analytical techniques are required. In this work, vibrational spectroscopy is used to shed more light on the as-yet elusive interaction of gyrase inhibitors of the fluoroquinolone type with their biological target inside the Gram-positive bacterium Staphylococcus epidermidis by investigating whole-cell changes that occur as a result of the presence of the drug moxifloxacin. IR absorption and Raman spectra with excitation off resonance (lambda exc = 532 nm) and in resonance with the biological targets DNA and the aromatic amino acids of gyrase (lambda exc = 244 nm) were recorded for unperturbed bacteria and bacteria in varying drug concentrations (0.08, 0.16, 0.27, and 0.62 microg moxifloxacin/mL bacterial culture). The spectral changes caused by the action of the drug were analyzed with the help of statistical methods, such as hierarchical cluster analysis (HCA), principal component analysis (PCA), and Fisher's linear discriminant analysis (LDA) combined with variable selection. The wavenumbers mostly affected by the action of the drug could be assigned to protein and DNA moieties, supporting the proposed mechanisms of a tertiary complex of the fluoroquinolone, the enzyme gyrase, and DNA.

Development of an enhanced separation of erythromycin and its related substances by liquid chromatography.

A new HPLC-UV method for the determination of the impurity profile of erythromycin is developed. In contrast to the liquid chromatography described in the European Pharmacopoeia the analysis could be performed at a temperature of 25 degrees C. Erythromycin samples were analysed on an endcapped RP phase with cyanopropyl groups on the surface using gradient elution with 32 mM potassium phosphate buffer pH 8.0 and acetonitrile/methanol (75:25). The aforementioned method shows clear improvements compared to the actual method of the European Pharmacopoeia, which is less selective and sensitive.