- Email: [email protected]
Atropine degradation products and trace heavy metal content in AtroPen® and ComboPen® auto-injectors
Abstracts
studies gathered from open literature have suggested that in the presence of PB-pretreatment, the choice of oxime reactivators does not influence the success in resuscitation of nerve agent poisoned subjects. Moreover, carbamate-inhibited AChE is reportedly resistant to oxime reactivation. Hence, it remains unclear whether recovery of PB pre-inhibited and aged AChE-soman conjugate occurred solely through spontaneous regeneration of PB-inhibited AChE or through oxime-assisted regeneration of PB-inhibited AChE. To answer this question, we carried out a series of in vitro reactivation studies with HI-6 (1-(4-carbamoylpyridino)methoxymethyl-2-(hydroxyiminomethyl) pyridinium dichloride), Obidoxime (1,1 -[oxybis(methylene)]-bis[4-(hydroxyimino) methyl]-pyridinium dichloride monohydrate) and 2-PAM (2-pyridinealdoxime methochloride) on human erythrocyte ghost AChE that has been subjected to PB pre-inhibition with and without soman postinhibition. In the first set of experiments, the spontaneous reactivation rate of PB inhibited AChE was compared to that obtained in the presence of oxime reactivators added after the 1h inhibition process. In the second followon experiment, PB pre-inhibited (1 h inhibition) human AChE was post-inhibited with sufficient soman so that <20% free AChE activity remained. The soman inhibition process was allowed to proceed for 30 min to ensure that aging of human AChE–soman conjugate has been achieved. Hence, any post recovery of AChE activity must arise from the AChE–PB conjugate pool. Recovery of AChE activity following oximes addition was noted and compared to the spontaneous recovery rate observed from the AChE–PB pool following soman inhibition. The latter was also compared to the spontaneous reactivation rate obtained from AChE–PB conjugate that was not subjected to soman post-inhibition. Our studies demonstrated that soman-inhibition actually triggers spontaneous regeneration of AChE–PB conjugate by shifting the equilibrium between free- and PB-bound AChE. However, only HI-6 was observed to enhance the spontaneous reactivation of PB-inhibited AChE. A similar rate enhancement was observed in PB pre-inhibited and soman post-inhibited AChE. A competitive binding mechanism is postulated involving oxime blockage of the AChE’s active site such that recarbamylation of the enzyme by free PB is minimized. From in vitro data, accelerated regeneration of AChE was evidenced at in vivo therapeutic concentrations of HI-6 (40 M). Hence, HI-6 appears to be the best therapeutic oxime reactivator, compared to obidoxime and 2-PAM, to be used in conjunction with PB pre-treatment regimen, against soman poisoning. However, animal efficacy data have to be gathered to validate the superi-
229
ority of HI-6 in enhancing the beneficial effect of PB pre-treatment. doi:10.1016/j.tox.2006.04.014 Atropine degradation products and trace heavy metal content in AtroPen® and ComboPen® autoinjectors Michael Loch, Thomas Zimmermann Central Institute of the Bundeswehr Medical Service, Munich, Laboratory Department IV—Pharmacy, Ingolst¨adter Landstr. 102, 85748 Garching-Hochbr¨uck, Germany E-mail address: [email protected] (M. Loch) The emergency medicines AtroPen® (containing atropine, 1.67 mg/0.7 ml) and ComboPen® (containing atropine (1.67 mg/2 ml), obidoxime) are part of the Bundeswehr’s central stocks of medical supplies. These medicines have no use-by date, which is why their pharmaceutical quality is checked regularly, not least to draw conclusions about storage-related changes and thus the further use of the medicines. It is known, that in the atropine-containing antidotes the proportion of tropic acid as a decomposition product of atropine sulfate is subject to remarkable changes. Furthermore the metal respective glass cylinder of the pen material (AtroPen® , ComboPen® ) may deliver heavy metal-ions. In many pharmacopoeias, standard marketing authorisations and company specifications, limit values are set for the mentioned parameters. Tropic acid is of small toxicological importance (metabolite of atropine). The toxicology of heavy metals is well known. Results: Degradation products in the Atropen® and CompoPen® were measured by high-performance liquid chromatography (HPLC) and diode array detection (DAD) using an RP-18 phase. Heavy metal investigations were done by ICP/MS. With the AtroPen® , the concentration of tropic acid increased during storage (15–25 ◦ C stockpiling) from 0.1% (after 1 year) to 1.2% (with respect to the total mass of anhydrous atropine base) after 9 years (equivalent to 2.6–28.8 g/ml). The increase seems to be linear (6 data points). Tropic acid concentrations of 0.5% (after 8–9 years of storage) and 2.9% (after 16–19 years) were measured for the ComboPen® (4 and 24 g/ml with regard to the total mass of atropine sulphate). In AtroPen® 0.05% (1.2 g/ml) of the degradation product apoatropine was verified after 1 year of storage and 0.12% (2.9 g/ml) after 5 years; whereas in ComboPen® this product was
230
Abstracts
not detected (limit of detection: 0.4 g/ml). Atropic acid was not found in the investigated samples (limit of detection: 0.3 g/ml). Preliminary investigations on heavy metal ions showed the following data: 5-year-old samples of AtroPen® (metal cylinder) had a Cd(II)—concentration of 0.0016 g/ml, a Cr(III + VI)—concentration of 1.17 g/ml, a Ni(II)—concentration of 0.35 g/ml and a Pb(II)—concentration of 0.015 g/ml. ComboPen® (16–19 years of storage, glass cylinder) contained 0.006 g/ml of Cd(II), 0.21 g/ml of Cr(III + VI), 0.01 g/ml of Ni(II), and 0.11 g/ml of Pb(II). In addition colouring of the ejected solution was visible in AtroPen® after 5 years of storage. Also visible particles were found. Their origin is under investigation. Conclusions: (1) No relevant forming of the investigated degradation products was observed. (2) There is a significant difference in the formation of the tropic acid between AtroPen® and ComboPen® . (3) Colouring and particles were found after 5 years (AtroPen® ). doi:10.1016/j.tox.2006.04.015 Two aging pathways for organophosphorus-inhibited human butyrylcholinesterase resolved by MALDITOF mass spectrometry He Li 1 , Florian Nachon 2 , Lawrence M. Schopfer 1 , Patrick Masson 2 , Oksana Lockridge 1 1 Eppley Institute, University of Nebraska Medical Cen-
ter, Omaha, NE 68198, USA; 2 Centre de Recherches du Service de Sant´e des Arm´ees, La Tronche, France Organophosphorus (OP) compounds inhibit serine hydrolases such as cholinesterases by phosphylation of the active site serine, forming a stable conjugate. The inhibited enzyme can be reactivated by nucleophilic agents. However, the covalently bound OP can also undergo a time-dependent aging process leading to a non-reactivatable enzyme. This process involves hydrolysis of a phosphorus-alkyl ligand bond (P X C, where X can be oxygen, sulfur, or nitrogen). The aging mechanisms for acetylcholinesterase (AChE) inhibited by soman or tabun; and butyrylcholinesterase (BChE) inhibited by isomalathion have been previously investigated. Soman inhibited AChE ages by hydrolyzing the O C portion of the phospho-bond; tabun, the P N portion; and isomalathion, the P S portion. Our work utilized matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry to systematically study the aging mechanism of human BChE
inhibited by OP compounds. Inhibited BChE was aged in the presence of H2 O18 and subsequently trypsinized. We identified the peptide OP conjugates through peptide mass mapping. Our results show that BChE inhibited by dichlorvos and echothiophate ages exclusively through O C bond cleavage. In contrast, isomalathion aging occurs through two pathways: the main product results from a P S bond cleavage and a minor product results from O C and/or S C bond cleavage. Supported by U.S. Army Research, Development & Engineering Command grant W911SR-04-C-0019. doi:10.1016/j.tox.2006.04.016 Nanoparticulate systems for brain delivery of oximes: First loading studies J¨urgen Kufleitner 1 , J¨urgen Briesen 2 , J¨org Kreuter 1
Hermanns 2 , Hagen
von
1 Institute of Pharmaceutical Technology, Johann Wolfgang Goethe University, D-60439 Frankfurt am Main, Germany; 2 Fraunhofer-Institut f¨ur Biomedizinische Technik (IBMT), D-66386 St. Ingbert, Germany
Purpose: Intoxication with organophosphorous nerve agents requires immediate administration of acetylcholinesterase reactivating antidotes like obidoxime or HI-6. Unfortunately, these oximes are unable to rapidly penetrate the blood-brain barrier (BBB) at physiologically relevant concentrations. The objective of this research project is to load the drugs to poly(butyl cyanoacrylate) (PBCA) nanoparticles (NP) which have previously been shown to enable brain delivery of a variety of drugs that normally cannot penetrate the blood-brain barrier freely. The aim of this study is to incorporate obidoxime dichloride and HI-6 dichloride into these particles and to evaluate the influence of different types of stabilisers on drug loading, particle size and polydispersity. The second perspective is to investigate the effects of polysorbate 80 coated PBCA-NP on confluent primary porcine brain capillary endothelial cells and to introduce a new approach of measuring trans endothelial electrical resistance (TEER) in BBB-model cell lines by means of impedance spectroscopy (IS). Methods: Particle formation was performed by anionic emulsion polymerisation of 1% (w/v) n-butyl2-cyanoacrylate in 0.01N HCl (pH 2). This solution contained 1% (w/v) of either poloxamer 188 or dextran 70,000 as stabilisers. The total drug concentration was 0.1% (w/v) with a drug-to-polymer ratio of 1:10. The oxime was added either before (method A) or 20 min
studies gathered from open literature have suggested that in the presence of PB-pretreatment, the choice of oxime reactivators does not influence the success in resuscitation of nerve agent poisoned subjects. Moreover, carbamate-inhibited AChE is reportedly resistant to oxime reactivation. Hence, it remains unclear whether recovery of PB pre-inhibited and aged AChE-soman conjugate occurred solely through spontaneous regeneration of PB-inhibited AChE or through oxime-assisted regeneration of PB-inhibited AChE. To answer this question, we carried out a series of in vitro reactivation studies with HI-6 (1-(4-carbamoylpyridino)methoxymethyl-2-(hydroxyiminomethyl) pyridinium dichloride), Obidoxime (1,1 -[oxybis(methylene)]-bis[4-(hydroxyimino) methyl]-pyridinium dichloride monohydrate) and 2-PAM (2-pyridinealdoxime methochloride) on human erythrocyte ghost AChE that has been subjected to PB pre-inhibition with and without soman postinhibition. In the first set of experiments, the spontaneous reactivation rate of PB inhibited AChE was compared to that obtained in the presence of oxime reactivators added after the 1h inhibition process. In the second followon experiment, PB pre-inhibited (1 h inhibition) human AChE was post-inhibited with sufficient soman so that <20% free AChE activity remained. The soman inhibition process was allowed to proceed for 30 min to ensure that aging of human AChE–soman conjugate has been achieved. Hence, any post recovery of AChE activity must arise from the AChE–PB conjugate pool. Recovery of AChE activity following oximes addition was noted and compared to the spontaneous recovery rate observed from the AChE–PB pool following soman inhibition. The latter was also compared to the spontaneous reactivation rate obtained from AChE–PB conjugate that was not subjected to soman post-inhibition. Our studies demonstrated that soman-inhibition actually triggers spontaneous regeneration of AChE–PB conjugate by shifting the equilibrium between free- and PB-bound AChE. However, only HI-6 was observed to enhance the spontaneous reactivation of PB-inhibited AChE. A similar rate enhancement was observed in PB pre-inhibited and soman post-inhibited AChE. A competitive binding mechanism is postulated involving oxime blockage of the AChE’s active site such that recarbamylation of the enzyme by free PB is minimized. From in vitro data, accelerated regeneration of AChE was evidenced at in vivo therapeutic concentrations of HI-6 (40 M). Hence, HI-6 appears to be the best therapeutic oxime reactivator, compared to obidoxime and 2-PAM, to be used in conjunction with PB pre-treatment regimen, against soman poisoning. However, animal efficacy data have to be gathered to validate the superi-
229
ority of HI-6 in enhancing the beneficial effect of PB pre-treatment. doi:10.1016/j.tox.2006.04.014 Atropine degradation products and trace heavy metal content in AtroPen® and ComboPen® autoinjectors Michael Loch, Thomas Zimmermann Central Institute of the Bundeswehr Medical Service, Munich, Laboratory Department IV—Pharmacy, Ingolst¨adter Landstr. 102, 85748 Garching-Hochbr¨uck, Germany E-mail address: [email protected] (M. Loch) The emergency medicines AtroPen® (containing atropine, 1.67 mg/0.7 ml) and ComboPen® (containing atropine (1.67 mg/2 ml), obidoxime) are part of the Bundeswehr’s central stocks of medical supplies. These medicines have no use-by date, which is why their pharmaceutical quality is checked regularly, not least to draw conclusions about storage-related changes and thus the further use of the medicines. It is known, that in the atropine-containing antidotes the proportion of tropic acid as a decomposition product of atropine sulfate is subject to remarkable changes. Furthermore the metal respective glass cylinder of the pen material (AtroPen® , ComboPen® ) may deliver heavy metal-ions. In many pharmacopoeias, standard marketing authorisations and company specifications, limit values are set for the mentioned parameters. Tropic acid is of small toxicological importance (metabolite of atropine). The toxicology of heavy metals is well known. Results: Degradation products in the Atropen® and CompoPen® were measured by high-performance liquid chromatography (HPLC) and diode array detection (DAD) using an RP-18 phase. Heavy metal investigations were done by ICP/MS. With the AtroPen® , the concentration of tropic acid increased during storage (15–25 ◦ C stockpiling) from 0.1% (after 1 year) to 1.2% (with respect to the total mass of anhydrous atropine base) after 9 years (equivalent to 2.6–28.8 g/ml). The increase seems to be linear (6 data points). Tropic acid concentrations of 0.5% (after 8–9 years of storage) and 2.9% (after 16–19 years) were measured for the ComboPen® (4 and 24 g/ml with regard to the total mass of atropine sulphate). In AtroPen® 0.05% (1.2 g/ml) of the degradation product apoatropine was verified after 1 year of storage and 0.12% (2.9 g/ml) after 5 years; whereas in ComboPen® this product was
230
Abstracts
not detected (limit of detection: 0.4 g/ml). Atropic acid was not found in the investigated samples (limit of detection: 0.3 g/ml). Preliminary investigations on heavy metal ions showed the following data: 5-year-old samples of AtroPen® (metal cylinder) had a Cd(II)—concentration of 0.0016 g/ml, a Cr(III + VI)—concentration of 1.17 g/ml, a Ni(II)—concentration of 0.35 g/ml and a Pb(II)—concentration of 0.015 g/ml. ComboPen® (16–19 years of storage, glass cylinder) contained 0.006 g/ml of Cd(II), 0.21 g/ml of Cr(III + VI), 0.01 g/ml of Ni(II), and 0.11 g/ml of Pb(II). In addition colouring of the ejected solution was visible in AtroPen® after 5 years of storage. Also visible particles were found. Their origin is under investigation. Conclusions: (1) No relevant forming of the investigated degradation products was observed. (2) There is a significant difference in the formation of the tropic acid between AtroPen® and ComboPen® . (3) Colouring and particles were found after 5 years (AtroPen® ). doi:10.1016/j.tox.2006.04.015 Two aging pathways for organophosphorus-inhibited human butyrylcholinesterase resolved by MALDITOF mass spectrometry He Li 1 , Florian Nachon 2 , Lawrence M. Schopfer 1 , Patrick Masson 2 , Oksana Lockridge 1 1 Eppley Institute, University of Nebraska Medical Cen-
ter, Omaha, NE 68198, USA; 2 Centre de Recherches du Service de Sant´e des Arm´ees, La Tronche, France Organophosphorus (OP) compounds inhibit serine hydrolases such as cholinesterases by phosphylation of the active site serine, forming a stable conjugate. The inhibited enzyme can be reactivated by nucleophilic agents. However, the covalently bound OP can also undergo a time-dependent aging process leading to a non-reactivatable enzyme. This process involves hydrolysis of a phosphorus-alkyl ligand bond (P X C, where X can be oxygen, sulfur, or nitrogen). The aging mechanisms for acetylcholinesterase (AChE) inhibited by soman or tabun; and butyrylcholinesterase (BChE) inhibited by isomalathion have been previously investigated. Soman inhibited AChE ages by hydrolyzing the O C portion of the phospho-bond; tabun, the P N portion; and isomalathion, the P S portion. Our work utilized matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry to systematically study the aging mechanism of human BChE
inhibited by OP compounds. Inhibited BChE was aged in the presence of H2 O18 and subsequently trypsinized. We identified the peptide OP conjugates through peptide mass mapping. Our results show that BChE inhibited by dichlorvos and echothiophate ages exclusively through O C bond cleavage. In contrast, isomalathion aging occurs through two pathways: the main product results from a P S bond cleavage and a minor product results from O C and/or S C bond cleavage. Supported by U.S. Army Research, Development & Engineering Command grant W911SR-04-C-0019. doi:10.1016/j.tox.2006.04.016 Nanoparticulate systems for brain delivery of oximes: First loading studies J¨urgen Kufleitner 1 , J¨urgen Briesen 2 , J¨org Kreuter 1
Hermanns 2 , Hagen
von
1 Institute of Pharmaceutical Technology, Johann Wolfgang Goethe University, D-60439 Frankfurt am Main, Germany; 2 Fraunhofer-Institut f¨ur Biomedizinische Technik (IBMT), D-66386 St. Ingbert, Germany
Purpose: Intoxication with organophosphorous nerve agents requires immediate administration of acetylcholinesterase reactivating antidotes like obidoxime or HI-6. Unfortunately, these oximes are unable to rapidly penetrate the blood-brain barrier (BBB) at physiologically relevant concentrations. The objective of this research project is to load the drugs to poly(butyl cyanoacrylate) (PBCA) nanoparticles (NP) which have previously been shown to enable brain delivery of a variety of drugs that normally cannot penetrate the blood-brain barrier freely. The aim of this study is to incorporate obidoxime dichloride and HI-6 dichloride into these particles and to evaluate the influence of different types of stabilisers on drug loading, particle size and polydispersity. The second perspective is to investigate the effects of polysorbate 80 coated PBCA-NP on confluent primary porcine brain capillary endothelial cells and to introduce a new approach of measuring trans endothelial electrical resistance (TEER) in BBB-model cell lines by means of impedance spectroscopy (IS). Methods: Particle formation was performed by anionic emulsion polymerisation of 1% (w/v) n-butyl2-cyanoacrylate in 0.01N HCl (pH 2). This solution contained 1% (w/v) of either poloxamer 188 or dextran 70,000 as stabilisers. The total drug concentration was 0.1% (w/v) with a drug-to-polymer ratio of 1:10. The oxime was added either before (method A) or 20 min