Leveraging Dissolution by Autoinjector Designs.

Chemical warfare or terrorism attacks with organophosphates may place intoxicated subjects under immediate life-threatening and psychologically demanding conditions. Antidotes, such as the oxime HI-6, which must be formulated as a powder for reconstitution reflecting the molecule's light sensitivity and instability in aqueous solutions, dramatically improve recovery-but only if used soon after exposure. Muscle tremors, anxiety, and loss of consciousness after exposure jeopardize proper administration, translating into demanding specifications for the dissolution of HI-6. Reflecting the patients' catastrophic situation and anticipated desire to react immediately to chemical weapon exposure, the dissolution should be completed within ten seconds. We are developing multi-dose and single-dose autoinjectors to reliably meet these dissolution requirements. The temporal and spatial course of dissolution within the various autoinjector designs was profiled colorimetrically. Based on these colorimetric insights with model dyes, we developed experimental setups integrating online conductometry to push experiments toward the relevant molecule, HI-6. The resulting blueprints for autoinjector designs integrated small-scale rotor systems, boosting dissolution across a wide range of viscosities, and meeting the required dissolution specifications driven by the use of these drug products in extreme situations.

Multi-dimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry for detection of Clostridium botulinum type C and the produced botulinum neurotoxin type C complex.

Botulinum neurotoxin types C, D and their mosaic forms C/D and D/C produced mainly by Clostridium botulinum types C and D cause botulism in animals and belong to the most toxic substances for poultry and fish. In addition to intoxications, also toxoinfections with C. botulinum types C and D play a role that should not be underestimated, especially in veterinary medicine. Contrary to other botulinum neurotoxin complexes (BT x), the biosynthesis of these types is phage-encoded. Currently, the gold standard for neurotoxin detection in cases of clinical botulism is the mouse bioassay. In the last few years, alternatives for replacing this mouse bioassay have become increasingly interesting for the detection and characterisation of botulinum neurotoxins. Therefore, immunological techniques based mainly on antibodies, PCR or mass spectral methods have been developed. In this context, the most promising development is that of different endopeptidase assays. In our study, we were able to show that the 2D-nano-LC-MS/MS method presented by Klaubert et al. 2009 especially for detecting BT x A, B, E and F in complex culture media can also be used for detecting BT x C. The focus was therefore on transferring this method to detecting BT x C and pointing out necessary modifications of this current method. For method development, we used different culture preparations and sample conditions. To find out whether BT x C is just as stable against acetic peptic pretreatment as other BT x, we used sample preparations with and without peptic pretreatment. The decisive difference to previous publications is the detection of produced BT x C directly from culture supernatant of different strains of C. botulinum type C. In addition, we present a new approach of detecting protein fragments from C3 and C2 toxin and some specific host cell proteins of the bacterium Clostridium spp. in order to specify the carrier bacterium, therefore verifying the presence of an intact neurotoxin-encoding phage also without directly detecting BT x C and thus the possibility to produce neurotoxin. Herein, we describe a new method to examine environmental samples or suspected feed samples in cases of toxoinfections as well as finding out the causes of clinical botulism. This new approach is particularly interesting for veterinary medicine, especially for diseases like chronic botulism in cows or equine grass sickness.

Photostability of antidotal oxime HI-6, impact on drug development.

HI-6 exhibits superior efficacy in the therapy of intoxication by different highly toxic organophosphorus nerve agents. Therefore HI-6 is a promising candidate for the development of new antidotes against nerve agents. For ethical and safety reasons antidotes containing HI-6 should get marketing authorization. Active pharmaceutical ingredients of medicinal products have to fulfil regulatory conditions in terms of purity and stability. Photostability is an essential parameter in this testing strategy. HI-6 was tested under conditions of ICH Q1B 'Photostability testing of new drug substances and products'. The data showed a marked degradation of HI-6 after exposure to daylight. The mechanism of degradation could be detected as photoisomerism. The light burden dependent rate of photoisomerism was followed quantitatively. Based on these quantitative results on the amount of light induced isomeric product a pharmacological qualification was made. A standardized in vitro test showed a decreased ability of light exposed HI-6 to reactivate sarin- and paraoxon-inhibited human acetylcholinesterase. These results have an impact on the further development of antidotes containing HI-6, as light protection will probably be necessary during handling, packaging, storage and application.

Purity of antidotal oxime HI-6 DMS as an active pharmaceutical ingredient for auto-injectors and infusions.

As reactivators of inhibited acetylcholinesterase, oximes are essential antidotes in poisoning by organophosphorus compounds. Due to its superior efficacy in cases of soman, cyclosarin, and sarin poisoning, the oxime HI-6 represents a promising option for an active pharmaceutical ingredient (API) in the further development of antidote therapy for nerve agent poisoning. Developmental lots of HI-6 DMS (dimethanesulfonate) provided by different manufacturers were examined with respect to their content and purity with a view to their future use as an API. There are distinct differences in the HI-6 content from three manufacturers. With respect to purity, gradual differences arise with the known synthetic by-products as well as with unknown accompanying compounds. It became apparent that in the case of a modified synthesis using protective groups, the proportion of some synthesis by-products decreases considerably. With one exception, they are thus below the reporting threshold for API in accordance with pertinent regulatory guidelines. In HI-6, an unknown impurity always occurs, whose percentage necessitates identification due to regulations. This unknown impurity, which has not been described so far, could be identified as an isomer. These findings supply data required for the description of pharmaceutical quality in accordance with module 3 of a Common Technical Document (CTD). They thus contribute to the marketing authorization of this substance as an API for auto-injectors and infusions.

Determination of botulinum toxins after peptic sample pre-treatment by multidimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry.

Botulinum neurotoxin types A to G are produced from different strains of Clostridium botulinum. The complex neurotoxins belong to the most toxic substances known and cause botulism both in humans and animals. Botulinum toxin complexes are produced with molecular weights of 300, 500 and 900 kDa. These large protein complexes contain beside the toxic zinc protease of 150 kDa, additional neurotoxin associated proteins, which are responsible for the extreme pH and protease stability. In this study we present for the first time a rugged detection method of botulinum toxins at femtomole levels in complex culture media after peptic sample pre-treatment and 2D-nano-LC-ESI-MS-MS-technique. In contrast to other studies, we used progenitor toxins directly from culture supernatant of C. botulinum strains A, B, E and F without further purification, to simulate complex, protein-containing sample conditions. We were able to demonstrate, that peptic pre-treatment is a great challenge in reducing ubiquitous proteins as well as proteins from suspicious samples. The study also found that multidimensional chromatography leads to significant better peptide differentiation and identification in protein loaded matrices than one dimensional nano-LC-ESI-MS.

Effects of processing and of storage on the stability of pantothenic acid in sea buckthorn products (Hippophaë rhamnoides L. ssp. rhamnoides) assessed by stable isotope dilution assay.

A stable isotope dilution assay for quantification of pantothenic acid in sea buckthorn berries, juice, and concentrate using a four-fold labeled isotopologue of vitamin B5 as the internal standard was adopted using reversed phase liquid chromatography-mass spectrometry with electrospray ionization. Because of a rapid sample clean up procedure without the necessity of external calibration, this methodology permits the accurate analysis of a high number of samples within a short time. Sea buckthorn juice was stored at 25 and 40 degrees C for up to 7 days to determine the effects of storage temperature on the stability of pantothenic acid. Analysis of kinetic data suggested that the degradation follows a first-order model. The results of the experiments showed that storage of sea buckthorn juice for 7 days at ambient temperature (25 degrees C) already resulted in a significant degradation of pantothenic acid of about 18%. The processing effects of juice production and subsequent concentration revealed a decrease of about 6-7% in the juice and of 23% in the juice concentrate.