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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/113718 Please be advised that this information was generated on 2017-12-06 and may be subject to change. BIODEGRADATION OF 1. 2. 2-TRIMETHYLPROPYI MriHYLPHOSPHONOFLUORIDATf ( SOMAN ) Studies in human and canine serum and plasma H. С J V DE BISSCHOP BIODEGRADATION OF 1, 2,2-TRIMETHYLPROPYL METHYLPHOSPHONOFLUORIDATE (SOMAN) Studies in human and canine serum and plasma BIODEGRADATION OF 1,2,2-TRIMETHYLPROPYL METHYLPHOSPHONOFLUORIDATE ( SOMAN) Studies in human and canine serum and plasma PROEFSCHRIFT ter verkrijging van de graad van doctor in de wiskunde en de natuurwetenschappen aan de Katholieke Universiteit te Nijmegen, op gezag van de Rector Magnificus Prof. Dr. J. H. G. I. Giesbers volgens besluit van het College van Decanen in het openbaar te verdedigen op woensdag 29 October 1986 des namiddags te 1. 30 uur precies door Herbert Cyr Jean Valerie DE BISSCHOP geboren te Erwetegem ( België ) GSSrt Repro 1986 Promotores : Prof. Dr. J.M. van Rossum Prof. Dr. M.G. Bogaert (Rijksuniversiteit Gent) Co-referenten : Dr. H.P. Benschop (PML-TNO) Dr. J.L. Willems (Rijksuniversiteit Gent) ISBN 90-9001415-2 The studies presented in this thesis were performed in the Department for Nuclear, Biological and Chemical Protection of the Technical Division of the Army, Vilvoorde (Belgium) and in the Heymans Institute of Pharmacology, State Uni versity of Ghent Medical School (Belgium). Figures IV-2 and V-l, previously published in "H. С De Bisschop étal., Funda». Appi. Toxicol., 5, S175-S179 (1985)", are reproduced with pernissìon of the Publisher. Copyright Acadeiic Press., Inc. ACKNOWLEDGEMENT The author is very indebted to all persons and authorities who have contributed to the realization of this dissertation. Thanks are expressed to the directors of the Technical Division of the Army and of the Heymans Institute for Pharmacology, for their permission to carry out the necessary experiments in their facilities. Particular acknowledgement is due to Prof. Dr. M.G. Bogaert, for his general interest and suggestions, to Dr. Jan Willems, for the many years of support and for his warm friendship, to Dr. H.P. Benschop and Dr. L.P.A. De Jong, for their valuable criticism and many constructive suggestions during the preparation of the manuscript, to Dr. F. Belpaire, for general advise. The following persons deserve special thanks : Dr. E. Michiels, Dr. Vet. J.G. Maiml, Lic. E.E. Van Driessche, Ing M.L.M. Alberty, Lic. P.R.J. Van Hecke and ir. W.A.P. De Meerleer, for their careful assistance in the analytical experiments, Dr. J.P. Blancquaert and Mr. L. Vermeersch, Heymans Institute of Pharmacology, for their aid in the preparation and the blood sampling of the dogs, and Mr. K. Breckpot, Queen Astrid Military Hospital, for his care in preparing human serum and plasma pools. The author is grateful to Dr. D.A. Ligtenstein and Dr. G. Cooper for critial proofreading of part of the manuscript and for many corrections to the english text. Dr. D. Lison is acknowledged for correcting the french text. Special thanks are due to Mrs. Monique Troch for the careful typesetting, to Mr. R. De Clercq, for his assistance in the preparation of the figures, and to GSSrt, for the preparation of this book. Last but not least I thank Monique, Karl and Pieter for the patience and understanding during many evenings and week-ends used for the realisation of this dissertation. CONTENTS TERMINOLOGY AND ABBREVIATIONS XI CHAPTER I INTRODUCTION 1.1. Historical note 1 1.2. Chemical properties of soman 1 1.3. Toxicity of soman 5 1.3.1. General 5 1.3.2. Biochemical bacKground of poisoning by anti cholinesterase compounds and general thera peutic principles 5 1.3.3. Specific characteristics of the intoxication by soman 7 1.4. Biodegradation of soman 10 1.4.1. Spontaneous and catalyzed hydrolysis 13 1.4.2. Enzymatic hydrolysis 13 1.4.3. Phosphylation 15 1.5. Aim of the investigations 16 1.6. Outline of the experiments 17 CHAPTER II QUANTITATIVE DETERMINATION OF SOMAN IN SERUM OR PLASMA 11.1. Introduction 19 11.2. Elements for the design of a useful method 20 11.2.1. Separation of isomer 20 11.2.2. Sample preparation 21 11.2.3. Internal standard 22 11.2.4. Sample injection 23 11.2.5. Detector 23 11.3. Estimates of detection and determination limits 24 11.4. Materials and methods 27 11.4.1. Glasswork 27 11.4.2. Reagents and solvents 27 11.4.3. Samples 27 11.4.4. Apparatus 27 VII 'I.A.b. Ex'.raction and Clean-up 28 ! 1.4.6. Qualitative analysis 31 1Γ.4.7. Quantitative analysis 32 11.1}. Results and discussion 33 CHAPTER III DEGRADATION OF SOMAN IN HUMAN SERUM. 111.1. j'ntroducnion 49 111.2. Materials and methods 50 111.2.1. Materials Ь0 :ΐί.2.2. Adjustment of p' 50 lil.2.3. De;radatJon experiments 51 Hi..2.4. Asr.ay 51 L!1.2.Ç-. f í-eincubation 53 Iíí.2.f). Calculation of rate constants 54 IJ[.3. Hesul^s 54 Hi.3.1. Leg^adaticn in human serum without preincubation 54 111.3.2. Influence of preincubation on the degradation of soman in serum 54 i [1.3.2.1. Preincubar.ion with C(-)P(i)-soinan 54 li 1.3.2.2. Preincubation with other oroanophosphorus compounds 59 IIі.З.І. Hydrolysis of soman in serum-ultra- filtrace Г0 III.3.4. Hydrolysis of soman in preincubated serum 61 LJ.I.4. Discussion 64 CHAPTER IV STEREOSELECTIVE HYDROLYSIS OF SOMAN IN HUMAN PLASMA OR SERUM IV.1. introduction 69 IV.2. Materials and methods 69 IV.2.1. Materials 69 IV.2.2. Degradation experiments and assay 70 vu: IV.2.3. Calculai ion of hydrolysis raie con stants І .З. Result's and discussion IV.3.1. Spontaneous hydrolysis IV.3.2. Enzymatic hydrolysib ]V. 3.2.1. be termination of hydro lys ι =. rate constants LV.3.2.2. Hydrolytic activity of •"-erum fractions IV.3.2.3. Hydrolysis oí soman лзоі-.егч in humcin piasma and sr-ruri ÎV.4. Summary and conelusion a CHAPTER V IRREVERSIBLE BINDING OF SOMAN TO HUMAN SERUM PROTEINS (PHOSPHONYLATION) V.l. Introduction V.2. Materials and '.etnoas V.3. hesults and discussion .З.І. Degradation of C4 ± jP(i )-soman in ,υη,.ιπ serum V.3.I.]. Influence of initiol srman con centrati on .ЗЛ.2. Eslimcition of concentration oí binding siteb in human piasra anc serum V.3.2. Stability of pnosphonylatea proteins V.3.3. Stereoselectivity of the phosrjhonyl ition of human serum proteins V.3.3.1. ririit approach : inhibition of •ютапаіе. and application of the model of competing reactions V.3.3.2. Second approach : comparison of degradation rate constants in preincubated and in not-prein- cubated human serum V.3.3.3. Stereoselectivity of phosphony- lation : conclusions V.3.4. Identification of the irreversioie oin- ding sites m human serum CHAPTER VI DEGRADATION AND STABILIZATION OF SOMAN IN DOG SERUM OR PLASMA VI. 1. Introduction 111 VI.2. Materials and methods 112 VI.2.1. У/г ІІЛО experiments 112 VI.2.1.1. Dog serum 112 VI.2.1.2. Preincubation 112 VI.2.1.3. Degradation experiments and assay 112 VI.2.2. 'Jn. VLVû experiments 112 VI.2.2.1. Reagents 112 VI.2.2.2. Determination of soman plas ma concentrations in the dog ИЗ VI.3. Results and discussion 114 VI.3.1. Jn ь-іло degradation of soman in dog serum 114 VI.3.1.1. Phosphonylation of dog serum proteins 114 VI.3.1.2. Hydrolysis of soman isomers in dog serum 117 VI.3.1.3. Stabilization of soman in dog serum 119 VI. 3.2. Ja vj-vo study of the soman plasma con centration in the dog as a function of time : preliminary observations 120 VI.4. Conclusion 122 SUMMARY 123 SAMENVATTING 127 RESUME 131 REFERENCES 135 CURRICULUM VITAE 147 X TERMINOLOGY AND ABBREVIATIONS Acetylcholinesterase EC 3.1.1.7 (true Cholinesterase) Aliesterase EC 3.1.1.1 carboxylesterase Al umi non Aurin tricarboxylic acid ammonium salt Armine Ethyl 4-nitrophenyl ethylphospho- nate NO. "^ !-0 ос2н5 \=/ ButyrylCholinesterase Cholinesterase (pseudocholinestera- se) EC 3.1.1.8 DFP Diisopropyl phosphorofluoridate 0 F - Ρ - CH(CH3)2 осн(сн3)2 EDTA Ethylene diamine tetraacetic acid HPLC High Pressure Liquid Chromatography NMWL Normal Molecular Weight Limit Paraoxon Diethyl 4-nitrophenyl phosphate H С О - Ρ - о 5 2 , ос2н5 ΧΙ Isopropyl methylphosphonofluori date О F - Ρ - PCHtCHj, снэ Ν,Ν-dimethyl ethyl phosphoroamido- cyanidate 0 HCO - Ρ - CN 5 2 , N(CH3)2 1,2-dimethylpropyl methylphosphono- fluoridate 0 F - Ρ - 0 - CH(CH )CH(CH,), ι 3 3 2 1,3,3-trimethylbutyl methylphospho nof luoridate О F - Ρ - 0 - CH(CH JCH С(СН,), I 3 2 3 3 сн, CHAPTER I. INTRODUCTION. 1.1. HISTORICAL NOTE. During his work on organophosphorus insecticides, G. Schrader synthetized tabun in 1936 and sarin in 1939. Be cause of the high mammalian toxicity of both compounds, now commonly known as nerve agents, their possible use as chemical warfare agents was studied at the Heeres Gas schütz Laboratorien at Spandau (Berlin) and some work on the pharmacology of sarin was done at Heidelberg by R. Kuhn. In the context of this pharmacological research, soman was synthesized as a sarin analogue at the end of 1944 (Robinson, 1971). Later the compound proved to be more toxic than tabun or sarin and, in addition, soman intoxication appeared to be more resistent to therapy.