Chemical stability of naloxone products beyond their labeled expiration dates.

OBJECTIVE: The purpose of this study was to evaluate the chemical stability of previously dispensed, expired naloxone products. SETTING: When properly stored, certain products maintain stable, defined as within compendia acceptability, beyond their manufacturer's expiration date. Stockpiling life-saving medications such as the opioid overdose reversing agent naloxone nasal spray (NNS) or injection (NIJ) is of utmost importance to ensure public health emergency preparedness and response. Design/interventions/methods: After each naloxone product was stored at room temperature for several months (6-19) past their labeled expiration date, the level of active therapeutic content and the presence of degradation impurity, 2,29'-bisnaloxone, were evaluated via chromatographic separation with waters higher performance liquid chromatography integrated using the Waters X-Select CSHC-18. The effluent was detected at 229 nm. MAIN OUTCOME MEASURE: Active naloxone presence and the presence of degradation impurity, 2,29'-bisnaloxone, were evaluated. RESULTS: The mean potency of naloxone in both NNS and NIJ, up to 10 and 19 months post-expiration, respectively, is within the 90-110 percent United States Pharmacopeia acceptance limit (NNS: 102.8 ± 2.6 percent and NIJ: 106.0 ± 1.3 percent). No impurity was detected in any chromatogram of the expired products. CONCLUSION: In summary, since both NNS and NIJ were found to be chemically stable beyond 10 months of the expiration date, shelf-life extension of climate controlled, commercially available naloxone products should be further investigated as a potential cost savings measure for national strategic stockpiles, emergency medical services, hospitals, and public responders.

Turkey Hemorrhagic Enteritis Virus Can Be Titrated but Not Propagated in Chicken Embryos.

This study aimed to investigate the feasibility of propagating and titrating hemorrhagic enteritis virus (HEV) in chicken embryos. A total of 308 embryonated eggs were used. At 10 days of embryonic age, eggs were inoculated via allantoic sac or chorioallantoic membrane routes with non-heat-treated (live) HEV or heat-treated (dead) HEV or served as negative controls. Allantoic fluid retrieved at 0, 1, 3, 5, and 7 days postinoculation (dpi) was tested for HEV by quantitative PCR. Inoculation with HEV did not cause visible growth impairment or lesions in the chicken embryos. Overall, there was no difference in postinoculation mortality rates among groups sham-inoculated (6/30, 20.0%) or inoculated with live (34/252, 13.4%) or dead (3/ 26, 6.9%) HEV (P = 0.58). The amount of HEV DNA detected in allantoic fluid at 7 dpi in eggs inoculated with live virus was similar to the inoculated dose, indicating that virus propagation in chicken embryos is not efficient. No HEV DNA was detected after 3 dpi in eggs inoculated with dead virus. Inoculation of chicken embryos combined with qualitative PCR can be used for titration of HEV virus stocks and presents a high correlation with in vivo titration using chickens (R2 0.98, P = 0.007). This method may be relevant in countries in which specific-pathogen-free turkeys are unavailable and in which the importation of RP19 cells, the only cell that supports effective propagation of HEV, is not permitted.

El virus de la enteritis hemorrágica de los pavos puede ser titulado pero no propagado en embriones de pollo. Este estudio tuvo como objetivo investigar la viabilidad de propagar y titular al virus de la enteritis hemorrágica de los pavos (con las siglas en inglés HEV) en embriones de pollo. Se utilizaron un total de 308 huevos embrionados. A los 10 días de edad embrionaria, los huevos se inocularon por vía saco alantoideo o por la membrana corioalantoidea con el virus de la enteritis hemorrágica sin tratamiento térmico (vivo) o con un virus de la enteritis hemorrágica con tratamiento térmico (muerto) y algunos sirvieron como controles negativos. El fluido alantoideo recuperado a los cero, uno, tres, cinco y siete días después de la inoculación se analizó para detectar el VHE mediante un método cuantitativo de PCR. La inoculación con el virus de la enteritis hemorrágica no causó daños visibles en el crecimiento ni lesiones en los embriones de pollo. En general, no hubo diferencias en las tasas de mortalidad después de la inoculación entre los grupos con inoculación simulada (6/30, 20.0%) o inoculados con el virus vivo de la enteritis hemorrágica (34/252, 13.4%) o con el virus muerto (3/26, 6.9%) (P=0.58). La cantidad de ADN del virus fue detectada en el fluido alantoideo a los siete días después de la inoculación en huevos inoculados con virus vivos fue similar a la dosis inoculada, lo que indica que la propagación del virus en embriones de pollo no fue eficiente. No se detectó ADN del virus de la enteritis hemorrágica después de tres días después de la inoculación en huevos inoculados con virus muerto. La inoculación de embriones de pollo combinada con el método cuantitativo de PCR se puede utilizar para la titulación de lotes del virus de la enteritis hemorrágica y presenta una alta correlación con la titulación in vivo utilizando pollos (R2 0.98, P = 0.007). Este método puede ser relevante en países en los que no se dispone de pavos libres de patógenos específicos y en los que no se permite la importación de células RP19, la única célula que admite la propagación efectiva del virus de la enteritis hemorrágica.

A Simplified, Specific HPLC Method of Assaying Thiamine and Riboflavin in Mushrooms.

Mushrooms have been used as part of the average diet and as a nutraceutical for thousands of years due to their immense health benefits. The purpose of this study was to develop a simple, fast, accurate, specific, reproducible, and robust chromatographic method to identify and quantify two water-soluble vitamins: thiamine (B1) and riboflavin (B2) in mushrooms. The method employed for qualitative and quantitative analysis of these vitamins was Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) equipped with Ultraviolet-Visible (UV-Vis) Detector. The extraction process involved acid hydrolysis followed by enzymatic dephosphorylation with takadiastase enzyme. Chromatographic separation was achieved with a Shimadzu prominence HPLC system using isocratic elution mode on a Waters Xterra® MS C-18 column (4.6mm × 150mm, 5 µm) integrated with a XBridge® BEH C-18 Guard column (2.1mm × 5 mm, 5 µm). The mobile phase of this study consisted of buffer and methanol in the ratio of 80:20, where the buffer contained sodium-1-hexanesulfonate, glacial acetic acid, methanol, and pH adjusted to 3.0 with diethylamine. Vitamins were detected simultaneously at their lambda max wavelengths B1: 245nm and B2: 268nm using dual-wavelength UV detection technique to get their highest response. The proposed method was found to be specific, linear R>1.0, accurate, precise (% recovery ± SD; B1:104.45±4.5 and B2: 104.88±2.04), sensitive, (limit of detection for B1 and B2 was 0.043 and 0.029 µg/mL, respectively), and robust for mushrooms analysis. No coeluting peaks were observed at the retention time of the vitamins and all the peaks were spectrally homogenous. The standard and sample solutions were found to remain stable at cold temperature for 72 hours. In summary, our data suggest that the proposed method could be used in food industries to monitor the product quality during routine quality control purposes.

Propagation of an Avirulent Turkey Hemorrhagic Enteritis Virus Isolate in Chickens.

A series of studies were undertaken to optimize the propagation of hemorrhagic enteritis virus (HEV) in specific-pathogen-free (SPF) chickens. A total of 562 SPF chickens were orally inoculated with an Australian avirulent HEV isolate of turkey origin at 9, 14, 21, or 28 days of age with 5, 6, 7, or 8 log 10 genomic copies (GC), while 102 chickens served as uninfected controls. No clinical signs were observed in infected chickens. There was an inoculum-dose-dependent increase in the relative spleen and liver weight ( P < 0.01). Relative spleen weight 7 days post-HEV inoculation was up to 85% higher in chickens that were inoculated with 6 to 7 GC compared with controls, with no further increase at higher doses. Relative liver weight increased up to 14% in chickens inoculated with 6 GC, with no further increase. Birds inoculated with a 7 GC dose had a higher frequency of HEV DNA-positive birds (77% to 86%) than birds inoculated with lower doses (33% to 59%; P < 0.01). The most efficient dose for live passage propagation was 7 GC inoculated in 9-to-14-day-old birds, yielding an infection rate of 81%. Livers and spleens from infected birds at all doses were processed to produce a putative vaccine with a final GC recovery in the vaccine material of 8.6 GC/bird. In summary, HEV of turkey origin can be readily propagated in SPF chickens, and conditions to maximize viral retrieval were established.

Disulfiram-based disulfides as narrow-spectrum antibacterial agents.

Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic parameters over disulfiram.

Discovery of Antischistosomal Drug Leads Based on Tetraazamacrocyclic Derivatives and Their Metal Complexes.

Praziquantel (PZQ) is the only drug available for the treatment of schistosomiasis, and since its large-scale use might be associated with the onset of resistance, new antischistosomal drugs should be developed. A series of 26 synthetic tetraazamacrocyclic derivatives and their metal complexes were synthesized, characterized, and screened for antischistosomal activity by application of a phased screening program. The compounds were first screened against newly transformed schistosomula (NTS) of harvested Schistosoma mansoni cercariae, then against adult worms, and finally, in vivo using the mouse model of S. mansoni infection. At a concentration of 33 µM, incubation with a total of 12 compounds resulted in the mortality of NTS at the 62% to 100% level. Five of these showing 100% inhibition of viability of NTS at 10 µM were selected for further screening for determination of the 50 inhibitory concentrations (IC50s) against both NTS and adult worms. Against NTS, all 5 compounds showed IC50s comparable to the IC50 of the standard drug, PZQ (0.87 to 9.65 µM for the 5 compounds versus 2.20 µM for PZQ). Three of these, which are the bisquinoline derivative of cyclen and its Fe(2+) and Mn(2+) complexes, showed micromolar IC50s (1.62 µM, 1.34 µM, and 4.12 µM, respectively, versus 0.10 µM for PZQ) against adult worms. In vivo, the worm burden reductions were 12.3%, 88.4%, and 74.5%, respectively, at a single oral dose of 400 mg/kg of body weight. The Fe(2+) complex exhibited activity in vivo comparable to that of PZQ, pointing to the discovery of a novel drug lead for schistosomiasis.