Kinetics of Residual Hydrogen Peroxide in Presence of Excipients and Preservatives

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Vaccine samples were spiked with 10 ppm hydrogen peroxide and stored at 4, 25, and 37°C for approximately 90 days with and without trace amounts of thimerosal present. Thimerosal dramatically reduced hydrogen peroxide levels in samples stored at 37°C.

Quantitation of residual hydrogen peroxide (H2O2) and evaluation of the impact on product stability is necessary as unwanted H2O2 can potentially be introduced during the manufacturing of pharmaceuticals, biologics, and vaccines. A sensitive and convenient microplate-based method with fluorescence detection for H2O2 quantitation was recently reported (Towne et al., 2004, Anal Biochem 334: 290-296).

This method was found to be highly robust and reproducible, with a level of detection of 0.015 ppm and a level of quantitation of 0.025 ppm (in water). The relatively small sample requirements and amenability for automation make this assay an attractive tool for detecting residual H2O2 levels. Without additional manipulation, the assay can be conducted on heterogeneous solutions with significant degree of turbidity, such as the presence of suspensions or aluminum-containing adjuvants.

The quantitation of H2O2 and its decomposition kinetics was also studied in presence of two common vaccine preservatives (thimerosal and phenol) and eight commonly used excipients (polyols). Over time, there is a distinct, temperature dependent decrease in H2O2 recovered in thimerosal and phenol containing samples versus non-preservative containing controls. Based on the half-life of spiked H2O2, the decay rates in eight polyols tested were found to be: ribose > sucrose > (glycerol, glucose, lactose, mannitol, sorbitol, and xylose).

Towne V, Oswald CB, Mogg R, et al. Measurement and decomposition kinetics of residual hydrogen peroxide in the presence of commonly used excipients and preservatives. J Pharm Sci. 2009; 98:3987-3996. Correspondence to Victoria Towne, Department of Bioprocess and Bioanalytical Research, Merck Research Laboratories at victoria_towne@merck.com or (215) 652-5370.

Analysis of Heparins and Potential Contaminants Using 1H-NMR and PAGE

Chemical structures of major repeat units of the sodium salts of (A) heparin, (B) chondroitin sulfate (R = SONa+; R = H, CSA; R = H; R = SONa+, CSC), (C) dermatan sulfate, and (D) oversulfated chondroitin sulfate.

In 2008, heparin (active pharmaceutical ingredient, API) lots were associated with anaphylactoid-type reactions. Oversulfated chondroitin sulfate (OSCS), a semi-synthetic glycosaminoglycan (GAG), was identified as a contaminant and dermatan sulfate (DS) as an impurity.

While DS has no known toxicity, OSCS was toxic leading to patient deaths. Heparins, prepared before these adverse reactions, needed to be screened for impurities and contaminants. Heparins were analyzed using high-field 1H-NMR spectroscopy. Heparinoids were mixed with a pure heparin and analyzed by 1H-NMR to assess the utility of 1H-NMR for screening heparin adulterants.

Sensitivity of heparinoids to deaminative cleavage, a method widely used to depolymerize heparin, was evaluated with polyacrylamide gel electrophoresis to detect impurities and contaminants, giving limits of detection (LOD) ranging from 0.1% to 5%. Most pharmaceutical heparins prepared between 1941 and 2008 showed no impurities or contaminants. Some contained DS, CS, and sodium acetate impurities.

Heparin prepared in 2008 contained OSCS contaminant. Heparin adulterated with heparinoids showed additional peaks in their high-field 1H-NMR spectra, clearly supporting NMR for monitoring of heparin API with an LOD of 0.5-10%. Most of these heparinoids were stable to nitrous acid treatment suggesting its utility for evaluating impurities and contaminants in heparin API.

Zhang Z, Li B, Suwan J, et al. Analysis of pharmaceutical heparins and potential contaminants using 1H-NMR and PAGE. J Pharm Sci. 2009;98:4017-4026.Correspondence to Robert J. Linhardt, Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, at linhar@rpi.edu or (518) 276-3404.

SIMANIM Particles for Modified-Release Delivery of Antibodies

Scanning electron micrograph of the spray-dried, IgG formulation (a), and transmission electron microscopy image of poly(lactide-co-glycolide) nanoparticles produced upon incubation of the spray-dried microparticulate formulation in aqueous media (b).

Simultaneously Manufactured Nano-In-Micro (SIMANIM) particles for the pulmonary delivery of antibodies have been prepared by the spray-drying of a double-emulsion containing human IgG (as a model antibody), lactose, poly(lactide-co-glycolide) (PLGA) and dipalmitoylphosphatidylcholine (DPPC). The one-step drying process involved producing microparticles of a diameter suitable for inhalation that upon contact with aqueous media, partially dissolved to form nanoparticles, 10-fold smaller than their original diameter.

Continuous release of the model antibody was observed for 35 days in pH 2.5 release media, and released antibody was shown to be stable and active by gel electrophoresis, field-flow fractionation and enzyme linked immunosorbent assay. Adding 1% L-leucine to the emulsion formulation, and blending SIMANIM particles with 1% magnesium stearate, achieved a fine particle fraction of 60%, when aerosolised from a simple, capsule-based, dry powder inhaler device. SIMANIM particles could be beneficial for the delivery of antibodies targeted against inhaled pathogens or other extracellular antigens, as well as having potential applications in the delivery of a wide range of other biopharmaceuticals and certain small-molecule drugs.

Kaye RS, Purewal TS, Alpar HO. Simultaneously manufactured nano-in-micro (SIMANIM) particles for dry-powder modified-release delivery of antibodies. J Pharm Sci. 2009;98:4055-4068.Correspondence to H. Oya Alpar, Centre for Drug Delivery Research, The School of Pharmacy, University of London at oya.alpar@pharmacy.ac.uk or +44-20-7753-5928.

Improved Permeation Enhancers for Transdermal Drug Delivery

Permeation profiles of melatonin in the presence of chemical penetration enhancers.

One promising way to breach the skin's natural barrier to drugs is by the application of chemicals called penetration enhancers. However, identifying potential enhancers is difficult and time consuming. We have developed a virtual screening algorithm for generating potential chemical penetration enhancers (CPEs) by integrating nonlinear, theory-based quantitative structure-property relationship models, genetic algorithms, and neural networks.

Our newly developed algorithm was used to identify seven potential CPE molecular structures. These chemical enhancers were tested for their toxicity on (a) mouse embryonic fibroblasts (MEFs) with MTT assay, and (b) porcine abdominal skin by histology using H/E staining at the end of a 48-h exposure period to the chemicals. Further, melatonin permeability in the presence of the enhancers was tested using porcine skin and Franz diffusion cells. Careful toxicity tests showed that four of the seven general CPEs were nontoxic candidate enhancers (menthone, 1-(1-adamantyl)-2-pyrrolidinone, R(+)-3-amino-1-hydroxy-2-pyrrolidinone, and 1-(4-nitro-phenyl)-pyrrolidine-2,5-dione). Further testing of these four molecules as potential melatonin-specific CPEs revealed that only menthone and 1-dodecyl-2-pyrrolidinone provided sufficient enhancement of the melatonin permeation.

The results from our permeability and toxicity measurements provide validation of the efficacy and ability of our virtual screening algorithm for generating potential chemical enhancer structures by virtual screening algorithms, in addition to providing additional experimental data to the body of knowledge.

Godavarthy SS, Yerramsetty KM, Rachakonda VK, et al. Design of improved permeation enhancers for transdermal drug delivery. J Pharm Sci. 2009; 98:4085-4099. Correspondence to Khaled A.M. Gasem, School of Chemical Engineering, Oklahoma State University at gasem@okstate.edu or (405) 744-5280.

Dissolution Profiles From Enteric-Coated Dosage Forms

Dissolution profiles of theophylline, antipyrine and acetaminophen from enteric-coated granules in simulated intestinal fluid of pH 6.8 (paddle method, 900 mL, 50 rpm). The black circle represents AS-LG-coated granules; the black triangle represents AS-MG-coated granules; and the black square represents AS-HG-coated granules. Each value represents the mean ± s.d. of six experiments.

We examined the in vitro dissolution-in vivo absorption correlation (IVIVC) for enteric-coated granules containing theophylline, antipyrine or acetaminophen as model drugs with high solubility and high permeability. More than 85% of each drug was released from granules coated with hypromellose acetate succinate (HPMCAS) (AS-LG grade, which dissolves at pH above 5.5) at a mean dissolution rate of more than 5 %/min after a lag time of less than 4 min in simulated intestinal fluid of pH 6.8. The lag time and the dissolution rate were significantly extended and reduced, respectively, when AS-LG was replaced with AS-HG (a grade of HPMCAS that dissolves at pH above 6.8). Enteric-coated granules were administered intraduodenally to anesthetized rats.

Statistical significances of differences of in vitro lag time between AS-LG- and AS-HG-coated granules were consistent with those in vivo, for all drugs. Significant differences in dissolution rates between granules also corresponded to those in absorption rates calculated using a deconvolution method, and both parameters had comparable absolute values, except in the case of antipyrine-containing granules with relatively fast dissolution rates. Thus, a good IVIVC was generally obtained; however, the exception suggests the importance of developing a dissolution test that fully reflects the in vivo situation.

Sakuma S, Ogura R, Masaoka Y, et al. Correlation between in vitro dissolution profiles from enteric-coated dosage forms and in vivo absorption in rats for high-solubility and high-permeability model drugs. J Pharm Sci. 2009; 98:4141-4152. Correspondence to Shinji Sakuma, Faculty of Pharmaceutical Sciences, Setsunan University at sakuma@pharm.setsunan.ac.jp or 81-72-866-3124.

Metronidazole Loaded Pectin Microspheres for Colon Targeting

The shape and surface morphology of pectin microspheres were studied using scanning electron microscopy. The sample was prepared by lightly sprinkling the microspheres powder on a double adhesive tape, which was stuck on aluminum stub. The stubs were then coated with gold.

A multiparticulate system having pH-sensitive property and specific enzyme biodegradability for colon-targeted delivery of metronidazole was developed. Pectin microspheres were prepared using emulsion-dehydration technique.

These microspheres were coated with Eudragit S-100 using oil-in-oil solvent evaporation method. The SEM was used to characterize the surface of these microspheres and a distinct coating over microspheres could be seen. The in vitro drug release studies exhibited no drug release at gastric pH, however continuous release of drug was observed from the formulation at colonic pH. Further, the release of drug from formulation was found to be higher in the presence of rat caecal contents, indicating the effect of colonic enzymes on the pectin microspheres.

The in vivo studies were also performed by assessing the drug concentration in various parts of the GIT at different time intervals which exhibited the potentiality of formulation for colon targeting. Hence, it can be concluded that Eudragit coated pectin microspheres can be used for the colon specific delivery of drug.