Moheb Nasr

Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System

BACKGROUND There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

Stability, Dose Uniformity, and Palatability of Three Counterterrorism Drugs—Human Subject and Electronic Tongue Studies

PurposeThese studies evaluated the ability of common household food and drink products to mask the bitter taste of three selected anti-terrorism drugs.MethodsThree anti-terrorism drugs (doxycycline, ciprofloxacin hydrochloride, and potassium iodide) were mixed with a variety of common household food and drinks, and healthy adult volunteers evaluated the resulting taste and aftertaste. In parallel, the ASTREE Electronic Tongue was used to evaluate taste combinations. Stability of the mixtures over time was monitored, as was the dosage uniformity across preparations.ResultsFoods and drinks were identified that satisfactorily masked the bitter flavor of each drug. Dose uniformity and stability were also acceptable over the range studied, although some combinations were significantly less stable than others. The electronic tongue was able to differentiate between tastes, but ranked masking agents in a different order than human volunteers.ConclusionsDoxycycline, potassium iodide, and ciprofloxacin, which are stockpiled in solid tablet form, can conveniently be prepared into more palatable formulations, using common household foods and drinks. The electronic tongue can be used to perform an initial screening for palatability.

Determination of galactosamine impurities in heparin samples by multivariate regression analysis of their 1H NMR spectra

Heparin, a widely used anticoagulant primarily extracted from animal sources, contains varying amounts of galactosamine impurities. Currently, the United States Pharmacopeia (USP) monograph for heparin purity specifies that the weight percent of galactosamine (%Gal) may not exceed 1%. In the present study, multivariate regression (MVR) analysis of 1H NMR spectral data obtained from heparin samples was employed to build quantitative models for the prediction of %Gal. MVR analysis was conducted using four separate methods: multiple linear regression, ridge regression, partial least squares regression, and support vector regression (SVR). Genetic algorithms and stepwise selection methods were applied for variable selection. In each case, two separate prediction models were constructed: a global model based on dataset A which contained the full range (0–10%) of galactosamine in the samples and a local model based on the subset dataset B for which the galactosamine level (0–2%) spanned the 1% USP limit. All four regression methods performed equally well for dataset A with low prediction errors under optimal conditions, whereas SVR was clearly superior among the four methods for dataset B. The results from this study show that 1H NMR spectroscopy, already a USP requirement for the screening of contaminants in heparin, may offer utility as a rapid method for quantitative determination of %Gal in heparin samples when used in conjunction with MVR approaches.

Class Modeling Analysis of Heparin 1H NMR Spectral Data Using the Soft Independent Modeling of Class Analogy and Unequal Class Modeling Techniques

To differentiate heparin samples with varying amounts of dermatan sulfate (DS) impurities and oversulfated chondroitin sulfate (OSCS) contaminants, proton NMR spectral data for heparin sodium active pharmaceutical ingredient samples from different manufacturers were analyzed using multivariate chemometric techniques. A total of 168 samples were divided into three groups: (a) Heparin, [DS] ≤ 1.0% and [OSCS] = 0%; (b) DS, [DS] > 1.0% and [OSCS] = 0%; (c) OSCS, [OSCS] > 0% with any content of DS. The chemometric models were constructed and validated using two well-established methods: soft independent modeling of class analogy (SIMCA) and unequal class modeling (UNEQ). While SIMCA modeling was conducted using the entire set of variables extracted from the NMR spectral data, UNEQ modeling was combined with variable reduction using stepwise linear discriminant analysis to comply with the requirement that the number of samples per class exceed the number of variables in the model by at least 3-fold. Comparison of the results from these two modeling approaches revealed that UNEQ had greater sensitivity (fewer false positives) while SIMCA had greater specificity (fewer false negatives). For Heparin, DS, and OSCS, respectively, the sensitivity was 78% (56/72), 74% (37/50), and 85% (39/46) from SIMCA modeling and 88% (63/72), 90% (45/50), and 91% (42/46) from UNEQ modeling. Importantly, the specificity of both the SIMCA and UNEQ models was 100% (46/46) for Heparin with respect to OSCS; no OSCS-containing sample was misclassified as Heparin. The specificity of the SIMCA model (45/50, or 90%) was superior to that of the UNEQ model (27/50, or 54%) for Heparin with respect to DS samples. However, the overall prediction ability of the UNEQ model (85%) was notably better than that of the SIMCA model (76%) for the Heparin vs DS vs OSCS classes. The models were challenged with blends of heparin spiked with nonsulfated, partially sulfated, or fully oversulfated chondroitin sulfate A, dermatan sulfate, or heparan sulfate at the 1.0, 5.0, and 10.0 wt % levels. The results from the present study indicate that the combination of (1)H NMR spectral data and class modeling techniques (viz., SIMCA and UNEQ) represents a promising strategy for assessing the quality of commercial heparin samples with respect to impurities and contaminants. The methodologies show utility for applications beyond heparin to other complex products.

Combining 1H NMR spectroscopy and chemometrics to identify heparin samples that may possess dermatan sulfate (DS) impurities or oversulfated chondroitin sulfate (OSCS) contaminants

Heparin is a naturally produced, heterogeneous compound consisting of variably sulfated and acetylated repeating disaccharide units. The structural complexity of heparin complicates efforts to assess the purity of the compound, especially when differentiating between similar glycosaminoglycans. Recently, heparin sodium contaminated with oversulfated chondroitin sulfate A (OSCS) has been associated with a rapid and acute onset of an anaphylactic reaction. In addition, naturally occurring dermatan sulfate (DS) was found to be present in these and other heparin samples as an impurity due to incomplete purification. The present study was undertaken to determine whether chemometric analysis of these NMR spectral data would be useful for discrimination between USP-grade samples of heparin sodium API and those deemed unacceptable based on their levels of DS, OSCS, or both. Several multivariate chemometric methods for clustering and classification were evaluated; specifically, principal components analysis (PCA), partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and the k-nearest-neighbor (kNN) method. Data dimension reduction and variable selection techniques, implemented to avoid over-fitting the training set data, markedly improved the performance of the classification models. Under optimal conditions, a perfect classification (100% success rate) was attained on external test sets for the Heparin vs OSCS model. The predictive rates for the Heparin vs DS, Heparin vs [DS+OSCS], and Heparin vs DS vs OSCS models were 89%, 93%, and 90%, respectively. In most cases, misclassifications can be ascribed to the similarity in NMR chemical shifts of heparin and DS. Among the chemometric methods evaluated in this study, we found that the LDA models were superior to the PLS-DA and kNN models for classification. Taken together, the present results demonstrate the utility of chemometric methods when applied in combination with (1)H NMR spectral analysis for evaluating the quality of heparin APIs.

High performance liquid chromatographic analysis of erythromycin

Abstract Liquid chromatographic analysis can provide a simpler and superior alternative to microbial assay of antibiotics. A number of liquid chromatographic methods for the analysis of erythromycin have been published in the past 17 years. However, many of these methods are complex and lack the selectivity needed for the assay of erythromycin in the presence of erythromycin derivatives and impurities. We examined several C18 based stationary phase columns and developed a significantly improved C18 liquid chromatographic method for the assay of erythromycin. In comparison to a recently published polymer (poly(styrene-divinylbenzene)) stationary phase LC method, our method is simpler, more rugged, faster, and more sensitive. The developed method has been successfully used for the analysis of erythromycin in commercial bulk samples. The chromatographic assay results correlate with microbiological assay.

Effect of formulation factors on the observed bounce in cascade impactors used to measure the spray particle size of metered dose inhalers

This study was conducted to investigate the extent of particle reentrainment, or bounce, in cascade impactors used to measure the size of the spray from single and multiple doses of metered dose inhalers (MDIs), and to determine the effect of some common formulation properties on the observed bounce. MDIs were formulated as suspensions or solutions, with varying levels of surfactant. These were fired into two impactors (the Andersen Sampler Mark II and the Marple-Miller model 150), the collection surfaces of which were uncoated or coated with silicone oil or glycerin. The amount of drug from a formulation collected on each stage was determined and comparison was made between results with coated and uncoated stages. Efficacy of the coating for eliminating bounce was demonstrated by comparing single shots with multiple shots. Different ways of describing bounce effects offer varying indications of its significance. The suspension formulations (containing surfactant) all showed evidence of bounce, but solution formulations showed little or none. Silicone oil and glycerin were equally effective in eliminating bounce. Results with impactors of two different designs were similar. Particle bounce was shown to occur with a variety of formulations when impactor stages are not coated.

High Performance Liquid Chromatographic Assay of Erythromycin Salts and Esters in Bulk and Pharmaceutical Dosage Forms

Recently, several liquid chromatographic methods for the assay of erythromycin base were developed and are being considered for the routine assay of erythromycin. In this study, we developed simple and rugged liquid chromatographic methods for the assay of erythromycin salts and esters, and used these methods for the assay of erythromycin estolate, erythromycin ethylsuccinate, erythromycin stearate, erythromycin gluceptate, and erythromycin lactobionate in bulk and pharmaceutical formulations. The HPLC methods proved to be simple, sensitive, versatile, and rugged. The developed methods provide an attractive alternative to current official microbiological methods. The HPLC assay results for all commercial erythromycin products tested comply with the USP Antibiotics-Microbial assay specification.

High Performance Liquid Chromatographic Analysis of Erythromycin and Related Impurities in Pharmaceutical Formulations

Abstract In the last few years, several liquid chromatographic methods for the analysis of erythromycin have been published. Until recently, most of the published methods were either complex or lacked the selectivity needed for the assay of erythromycin in the presence of erythromycin derivatives and impurities. Recently, we developed a significantly improved C18 liquid chromatographic method for the assay of erythromycin. In comparison, our method is simpler, more rugged, faster, and more sensitive and selective than other published methods. In this investigation, the developed method was successfully used for the assay of erythromycin in several pharmaceutical formulations.