PHARMACOKINETICS OF NEOSTIGMINE AND PYRIDOSTIGMINE IN MAN

Transcription

1 PHARMACKINETICS F NESTIGMINE AND PYRIDSTIGMINE IN MAN Thesis submitted in accodance with the equiements of the Council fo National Academic Awads fo the Degee of Docto of Philosophy by Akba Dehghan, D. Phaia. School of Phamacy, Livepool Polytechnic, Byom Steet, Livepool, L3 3AF. June, 1981.

2 ABSTRACT Phamacokinetics of Neostigmine and Pyidostigmine in Man, by Akba Dehghan Most methods used fo the extaction of quatenay amines in biological fluids have been based on ion-pai extaction techniques. In this study, a sensitive and selective chomatogaphic pocedue is descibed to measue the concentation of pyidostigmine, neostigmine and thei majo metabolites in the plasma and uine. The methods involve a peliminay selective ion-pai extaction of the unchanged dugs and thei metabolites into dichloomethane and dichloomethane-acetone mixtue espectively. Quantitation is possible down to 3 ng/ml fo paent dugs and 5 ng/ml fo the metabolites. The pesent wok also descibed a modified pocedue to measue neostigmine and pyidostigmine in plasma simultaneously, using a specially synthesized pyidostigmine analogue as a common intenal make. The plasma concentation of pyidostigmine was measued afte thee diffeent doses of the quatenay amine (36.2 Ng/kg, 72.4 pg/kg and )tg/kg) wee given intavenously to twenty five sugical patients duing anaesthesia. The elation between the plasma concentation of pyidostigmine and time was invaiably expessed as a bi-exponential equation, and the data was intepeted in tems of a two compatment model. The slow disposition halflife of the dug was pogessively polonged as the dose of the dug was aised fom 36.2 Ng/kg to jig/kg. A simila incease in the half-life was obseved in coss-ove studies. The cleaance of the quatenay amine was enhanced at intemediate dose (72.4 )xg/kg) but significantly educed afte high dose (144.8 pg/kg). It was suggested that these esults may patially account fo the obseved diffeences in the duation of action of neostigmine and pyidostigmine in man. The dose of pyidostigmine used to evese non-depolaizing neuomuscula block is 4-5 times geate than neostigmine. Afte intamuscula administation of neostigmine to five myasthenic patients, the plasma concentation of the dug declined monoexponentially fom 21 ±2 no/ml to 9t1 ng/ml between 3 and 12 minutes and data was intepeted in tems of a one-compatment model. Estimates of the plasma half-life vaied fom minutes. The oal administation of pyidostigmine alone and of both neostigmine and pyidostigmine in two diffeent goups of myasthenic patients, wee also studied. Thee was a diect linea elation between aea, unde the plasma concentation - time cuve and total daily dose of pyidostigmine in the fist goup of patients ( =.95), but no such obsevation was noticed in eithe all patients (two goups; =.15) o in the second goup who wee teated with both dugs ( =-.8). It was suggested that thee might be a dug-dug inteaction between pyidostigmine and neostigmine duing oal absoption. The elation between plasma levels of pyidostigmine to clinical evaluation of muscle powe was examined in nine myasthenic patients duing teatment with pyidostigmine in doses of 6 to 18 mg pe day. Five of the nine subjects demonstated a tend towads a positive coelation and in two of them was this significant at pt.5. In addition, the pesence o absence of a possible coelation between muscle powe and plasma concentation was not elated to the duation of disease, additional pednisolone theapy o thymectomy.

3 CNTENTS Page No. PREFACE 1 CHAPTER 1 Geneal intoduction 3 CHAPTER 2 CHAPTER 3 PART NE PART TW CHAPTER 4 CHAPTER 5 CHAPTER 6 PART NE PART TW Phamacokinetics A eview on analytical techniques available fo studies of Anti-ChE dugs Development of methodology fo the detemination of neostigmine, pyidostigmine and thei metabolites in human plasma and uine Dose dependent kinetics of intavenous pyidostigmine in sugical patients Kinetics of intamuscula neostigmine in myasthenia gavis Plasma levels of neostigmine and pyidostigmine in myasthenic patients afte oal administation The elationship of plasma levels of pyidostigmine to clinical effect in patients with myasthenia gavis CHAPTER 7 Final discussion 147 APPENDICES REFERENCES ADDENDA 196

4 -1- PREFACE This thesis, compising of 7 Chaptes, pesents the develop- ment of the techniques available to measue the concentation of neostigmine and pyidostigmine in human biological fluids and also a study of the phamacokinetics of the dugs in man. In addition, clinical studies wee caied out to investigate the elation of plasma levels of pyidostigmine to clinical effects in patients with myasthenia gavis. In the fist Chapte, the known phamacological popeties and clinical use of quatenay ammonium compounds ae discussed. An intoduction to the mathematical expessions of phamacokinetics and a eview on pevious methods available fo the detemination of quatenay ammonium compounds is descibed in Chapte 2. Chapte 3 includes the development of the techniques used in the pesent investigations. The studies of phamacokinetics of pyidostigmine in sugical patients ae pesented in Chapte 4. In Chapte 5 the study of phamacokinetics of neostigmine afte intamuscula administation to myasthenic patients is descibed. Chapte 6 includes the study of phamacokinetics and phamacologic effects of pyidostigmine and neostigmine afte oal administation in patients with myasthenia gavis. The main findings and conclusions of this thesis ae summaized in the final chapte. The appendices expand some expeimental details and tabulated esults not included elsewhee in the thesis. The investigation was caied out in the Phamaceutical Chemisty section of the School of Phamacy, Livepool Polytechnic. I wish to expess my appeciation to D. K. Chan fo the consistent

5 -2- advice and encouagement he has given me whilst supevising this wok. I expess my thanks also to D. T. N. Calvey fo his advice and co-opeation in poviding human blood samples to cay out the studies in Chapte 4 and 5. I am indebted to D. D. M. Feeman and D. M. Haywad fo pemitting the study of patients admitted unde thei cae and to the patients fo thei eady co-opeation. I am gateful to D. Susan C. Davison and D. N. Hyman of the Radcliffe Infimay, xfod, fo thei joint effot in the study on clinical esponse and plasma pyidostigmine (Chapte 6). Finally, I would like to thank D. P. Deavi fo his assistance in compute pogamming.

6 -3- CHAPTER 1 GENERAL INTRDUCTIN

7 -4- CHAPTE2 NE Paus No. Anticholineetease Agents; Histoical backgound 5 The mecbanfea of action of Anti-ChE agents 7 The chemisty and stuctue-activity elationship of some evesible Gab. mate inhibitos 13 Pba acological and toxic popeties of Asti-ChE agents 15 The effects of gnatemay Anti-ME agents on neuomuscula tansmission 18 The use of Anti-ChE dugs in ayaetheaia ga'vis 19

8 -5- ANTICHLINESTERASE AGENTS HISTRICAL BACKGRUND Dugs that inhibit o inactivate acetylcholinesteases (AChE) ae called anticholinestease (anti-che) agents and thus ae potentially capable of poducing effects equivalent to continuous stimulation of cholinegic fibes thoughout the cental and peipheal nevous system. In view of the widespead distibution of cholinegic neuons, it is not supising that the anti-che agents as a goup have assumed moe extensive pactical application as toxic agents, in the fom of agicultual insecticides and potential chemical waefae "neve gases" than as dugs, nevetheless, thee ae cetain epesentatives that ae clinically useful. (See Table 1-1). Pio to the Second Wold Wa, only "evesible" anti-che agents wee geneally known, of which physostigmine (eseine) is the outstanding example. Shotly befoe and duing Wold Wa II a compaatively new class of highly toxic chemicals, the oganophosphous was developed, chiefly by Schade, of I. G. Fabenindustie. These wee fist used as agicultual insecticides and late as potential chemical wafae agents (Roelle 1975). The exteme toxicity of these compounds was found to be due to thei "ievesible" inactivation of AChE, theeby exeting thei effects fo consideably longe peiods than do the classical evesible inhibitos. Since the phamacological actions of both classes of anti-che agents ae qualitatively simila, they will be discussed as a goup, and the impotant special featues of the individual classes o compounds will be pointed out.

9 -6- Physostigmine, the fist known anti-che agent, is an alkaloid obtained fom the Calaba o odeal bean of Physostigma Venonosum Balfou, which is a peennial woody climbe gowing in topical West Afica. The calaba bean, also known as Esee nut, chop nut, o bean Etu Esee, was once used by native tibes of West Afica as an odeal poison in tials fo witchcaft. It was bought to England in 184 by Daniell who was a Bitish Medical ffice stationed in Calaba. Ealy investigations of its phamacological popeties wee induced by Chistieson (1825), Fase (1863), and Agyll-Robetson (1863). The pue alkaloid of physostigmine was isolated by Jobts and Hesse in 1864, and named physostigmine and the following yea Vee and Leven (Goldman, 1975) obtained the same alkaloid which they named as eseine. The alkaloid was used fist as a theapeutic agent in 1877 by Laqueus in the teatment of glaucoma. Polonovski and Polonovski (1923) and Stedman and Baga (1925) elucidated the chemical stuctue (see Table 1.1) of physostigmine, and its synthesis was accomplished by Julian and Pikl., (1935). Inteesting accounts of the histoy of physostigmine have been pesented by Rodin (1947), Kaczma (197) and Holmstedt (1972). As a esult of the basic eseach of Stedman and associates, (1929a, 1929b) in elucidating the chemical basis of the activity of physostigmine, Aeschlimannand Reinad (1931) systematically investigated a seies of substituted phenyl estes of alkyl cabamic acids. Neostigmine (see Table 1.1) a membe of this seies, was intoduced into theapeutics in 1931 fo its stimulant action on the intestinal tact. It was epoted independently by Remen (1932), and Walke (1935) to be

10 -7- effective in the symptomatic theapy of myasthenia gavis. Additional anti-che agents that bea a geneal stuctual esembs. lance to neostigmine wee intoduced fo the teatment of myasthenia gavis. Among these ae pyidostigmine and ambenonium. Edöphonium, a dug of simple stuctue and with an extemely bief duation of actionis used in the diagnosis and evaluation of the theapy of the same disease. It is inteesting to note that the fist account of the synthesis of a highly potent compound of the oganophosptous ChE seies, tetaethylpyophosphate (TEPP), was published by _cin'ti- Clement in 1854, ten yeas pio to the " Isolation of physostigmine. Moe emakable still as Holmstedt (1963) pointed out, is the fact that the investigato suvived to epot on the compounds taste as a few dops of the pue compound placed on the tongue would be expected to pove apidly fatal. Duing the synthesis and investigation of appoximately 2, compounds, Schade (1952) defined the stuctual equiements fo insecticidal (and as leaned subsequently, fo anti-che) activity 2f compounds in this ealy seies, paathion became the most widely employed insecticide of this class. The synthesis of seveal compounds of much geate toxicity than paathion such as Sain, Soman and Tabun esulted (Schade 1952), and these wee used as neve gases in chemical wafae. THE MECHANISM F ACTIN F ANTI-ChE AGENTS The anti-che agents ae among the elatively few dugs fo which the mechanism of action can pesently be descibed in pecise molecula tems. This can be elated diectly to thei

11 -8- oveall phamacological effects and has led to the development of useful compounds capable of evesing the actions of anti- ChE agents. The inteactions between most of the anti-che agents and the enzyme AChE diffe pimaily in quantitative espects fom the eaction between AChE_and its nomal substate acetylcholine (ACh). Fom studies initiated by Wilson and Begmann (195) and extended by Wilson and othes, it has been established that the active suface of the enzyme unit consists of two sites, an anionic and an esteatic site (see Figuei. l). At the anionic site (a negative chage pobably of the fee ionised caboxyl goup of a dicaboxylic amino acid), the postively chaged quatenay N atom of ACh is attacted by electostatic foces, and bonding is enhanced by hydophobic foces exeted on the N-methyl goups. The esteatic site consists of essentially two components located 2.5 and 5A espectively fom the anionic site; a potentially acidic function (the hydoxy goup of seine) and a nucleophilic goup (the basic N of an imidazole goup of histidine). The imidazole goup by hydogen bonding (see Figue 14) enhances the nucleophilic activity of the seine hydoxyl goup, thus enabling it to inte act with the electon deficient cabonyl C atom of ACh. A covalent bond is fomed with the poduction of an acetylated enzyme intemediate and the elease of choline (Figuel"la. ). The electon deficient C atom of the acetyl goup then undegoes nucleophilic attack by the oxygen atom of a wate molecule. The acetyl enzyme complex is thus hydolyzed and the poducts ae the egeneated enzyme and acetic acid.

12 -9- o= UIý Ic 'ü v d u =d Z4 "' Cu E ZCN dg W 1N o= ý_ \ v v = N U = = W U\,U Z" 1 S2 i i i b tx Ca, -; a d ox o= U. d v I- U, x WI d ". U, 2Z `U z N C W NV" SS U\ /V Nf N C W Ü SZ C= U d /1 1t c, d o? Öz 1 N L 2 ý` N/ Vm I= ü V /V Z u I= n i n Q 'g W d U' a W I_ /Z; N U 46

13 -1- N n -4 9 Q ý/ U ý Q V \ b ý AM,C,,,.,.c ; a u i Z o o ä ö- ä I o 4 1 Z fq Z G _ " s ö ä+ 3 ^' ý+ "-+ d3 E + pco l J 41 N _" E \\ / E ý 4) 2" Co 41N. 2 > ci Co to %D W «o mc)% Z o m a d yw \oý d E ä e ' 95 c ä ö c W ö W V.4 U x V CL. 1.1 cm T Zý W um vi Co MZX.-4 = U/ \V v D+ D. ýöe" 1 o s..2 d p9> ßi = E- ö ö g i w bw c wa,4) i U\V N vý ý. U N y 11 d ul 1.Cp e M a+ º+' u = ( p U of 1 F+ gy / ' p. > '.. a+ p \ _ >4 X. LL.L " w k ()/ Zf 1 W to 1+ \U QV

14 -11- The ate constants in this eaction sequence ae extemely high, thus the time equied fo the complete hydolysis of one ACh molecule (the tunove time), is only 8 mico seconds (Wilson et al., 196). The mechanisms of action of compounds that typify the thee classes of anti-che agents ae shown in Figue 1.1(b, c, d). Simple quatenay compounds, such as tetaetheyfammonium ion, inhibit the enzyme evesibly by combining with it only at the anionic site, and thus blocking attachment of the substate. Much moe potent evesible inhibitos, such as edophonium, combine in addition with the imidazole nitogen atom of the esteatic site by hydogen bonding though the phenolic H goup (Figuet. lb). In all such cases, inhibition is apidly evesible and such dugs have a vey bief duation of action following systemic administation. It was at one time geneally assumed that physostigmine, neostigmine and elated potent inhibitos that possess a cabamyl este linkage o uethane stuctue, in addition to a tetiay amino o quatenay ammonium goup, inhibit the enzyme in the same evesible fashion. Howeve, caeful kinetic studies have since shown that only a negligible amount of an inhibito of this type is eleased evesibly fom the enzyme; actually, physostigmine and neostigmine ae hydolyzed extemely slowly by cholinesteases (Goldstein and Hamlish, 1952; Myes, 1952,1956; Wilson et al., 196). It is likely that inhibitos of this class fom complexes in which the inhibito is attached to the enzyme at both the anionic and esteatic sites, following which thei hydolysis poceeds in a manne

15 -12- analogous to that of ACh (Figuel. lc), the alcoholic moiety is split off, leaving a cabamoylated enzyme that eacts with wate to elease a substituted cabamic acid and egeneated enzyme. The main diffeence between the hydolysis of the natual substate, ACh, and the inhibito, neostigmine, is the velocity of the final step, the half-life of dimethylcabamoyl-ache, fomed by the eaction with neostigmine, is moe than 4 million times that of the acetylated enzyme (3 minutes and 42 micoseconds espectively, Wilson and Haison 1961). Thus, these dugs and newe cabanate insecticides ae called "competitive substates" o "acid tansfeing inhibitos" and can be distinguished fom the simply evesible inhibitos descibed above. The eaction between AChE and most oganophosphoous inhibitos such as di-isopopylphosphoofluoidate (DFP) occus only at the estatic site, but poceeds in a compaable fashion (Figuel. ld). Hee the esultant phosphoylated enzyme is extemely stable; if the attached alkyl goups ae methyl o ethyl, significant egeneation of the enzyme by hydolytic cleavage equies seveal hous; with isopy. opyl goups, as in the example given, vitually no hydolysis occus and the etun of AChE activity is dependent upon synthesis of new enzymes, a pocess equiing days to months. Fom the foegoing account it is appaent that the tems "evesible" and "ievesible" as applied to the cabamoyl este and oganophosphoous anti-che agents espectively, eflect only quantitatively diffeences and that both classes of dugs eact with the enzyme in essentially the same manne as does ACh.

16 -13- THE CHEMISTRY AND STRUCTURE-ACTIVITY RELATINSHIP F SME REVERSIBLE CARBAMATE INHIBITRS. Dugs of this class that ae of theapeutic inteest ae shown in Table 1-1. Afte the stuctue of physostigmine was established, Stedman (1929a, 1929b) undetook a systematic investigation of a numbe of elated synthetic compounds. They concluded that the essential moiety of the physostigmine molecule was the methyl cabamate of a basically substituted simple phenol (Table 1-1). The qua: -tenay ammonium deivative, neostigmine, is a compound of geate stability and equal o geate potency. The simple analogues of neostigmine that lack the cabamoyl goup, such as edophonium, ae less potent and much shote acting anti-che agents, since they poduce a tuly evesible inhibition as descibed above. Retention of the dimethycabamate side chain in the meta position of phenol with the incopoation of the quatenay N atom within the ing to fom a pyidinium nucleus esults in compounds with anti-che and phamalogical popeties simila to those of neostigmine. Pyidostigmine, a dug of this classý)is used in the teatment of myasthenia gavis. A maked incease in anti-che potency and duation of action can esult fom linking of two quatenay ammonium nuclei by a chain of appopiate stuctue and length. one such example is the miotic agent demecaium, which consists of two neostigmine molecules connected at thei cabamate nitogen atoms by a seies of ten methylene goups. It is likely that this compound combines with the anionic and esteatic sites of two adjacent AChE units within the same tetame just as the bis-quatenay

17 CMPUND -14- STRUCTURAL FRMULA (a) TERTIARY AMINE Physostigmine (eseine) CH3 6H3CH3 11 H -C W'ý H3 (b) MNQUATERNARY AMINE Edophonium 'Tensilon' Neostigmine 'Postigmin' F H C2H5-N(CH3)2 CH3 11 N / CH3 N(C Pyidostigmine 'Mestinon' fn CH3 CH3 11 CH3 (c) BISQUATERNARY AMINE Distigmin 'Ubettid' C II (C CH3 2 Demecaium ocý To-oý ýc Jj o 'Rosmilen' fn(chý3 2 TABLE 1.1 Repesentative "evesible" anti-che agents employed clinically.

18 -15- neuomuscula blocking agent, decamethonium, is pesumed to act at two adjacent cholinoeceptos. Anothe class of bis- quatenay compounds is epesented by the dioxamide, ambenenium, used in the teatment of myasthenia gavis. In addition to its potent anti-che activity, this dug has a vaiety of actions at both the pejunctional and postjunctional membanes of the skeletal muscle moto end-plate (Kaczma 1967). The othe bis-quatenay amine, distigmine (Ubetid), is a long acting evesible anti-che agent with a much longe duation of action than neostigmine and pyidostigmine. It is 2.7times moe poweful in the invivo inhibition of non-specific ChE in the whole blood of the at (Chan 198). Clinically, it is used in the pevention and management of post-opeative uinay etention, intestinal atony and paalytic ileus in sugical and gynaecological patients. ccasionally it is also used as an adjuvant in the teatment of myasthenia gavis (Chan 198). PHARMACLGICAL AND TXIC PRPERTIES F ANTI-ChE AGENTS The phamacologic effects of anti-che agents ae due pimaily to the fact that the acetylcholine (ACh) eleased in the couse of nomal physiological activity is not destoyed. The eleased ACh, theefoe, accumulates in the body whee it exets both muscainic and nicotinic actions. The effects of individual anti"che agents may be modified by thei othe popeties; some anti-che agents have an ACh like action in thei own ight, while othes may cause neuomuscula blockade. The effects of some compounds ae complicated by the poduction in vivo of othe toxic substances. Lipid soluble substances, such as oganophosphoous deivatives, penetate easily into the

19 -16- cental nevous system and thei cental effects ae, theefoe, moe ponounced than those poduced by anti-che agents of lowe lipid solubility. Finally, some of the anti-che agents have additional actions which aise, neithe fom thei elationship to ACh o ChE, no to thei metabolic tansfomation into toxic substances. While these possible souces of vaiation in phamacological activity should be bone in mind, the majo actions of all the anti-che can nevetheless be summaized by Table 1-2. All muscainic effects of anti-che can be attenuated o abolished by atopine. The cental stimulation effects ae antagonized by atopine although, not as completely as the muscainic effects at autonomic effecto sites.

20 -17- TABLE 1-2 PHARMACLGIC AND TXIC EFFECTS F ANTI-ChE AGENTS SITES F ACTIN EFFECTS MUSCARINIC EFFECTS Smooth muscles Inceased peistalsis, miosis, elaxed sphinctes, bochoconstiction, vasodilation and cycloplegia. Cadiac muscles Badycadia and vasodilatation, hypotension. Exocine glands Lacimation, salivation and sweating. NICTINIC EFFECTS Skeletal muscles Genealised muscle twitching followed by muscle weakness, muscle camps. Ganglia Stimulation and modification of muscainic effects. CENTRAL EFFECTS Geneal Restlessness, dizziness, temo, insomnia, aphasia, disoientation, hallucination. Poisoning Coma, convulsions and death. * Not all ACNE agents penetate into the cental nevous system.

21 -18- THE EFFECTS F THE UATERNARYANTI-ChE AGENTS N NEURMUSCULAR TRANSMISSIN The quatenay ammonium anti-che dugs such as neostigmine, pyidostigmine and edophonium ae known to have a facilitatoy action on neuomuscula tansmission (Randall and Jampolski 1953). The inceased tension developed in the muscle due to the anti-che agents is thought to esult fom the poduction of a seies of epeated dischages in the muscle fibes in esponse to a single supamaximal neve stimulation (Wescoe and Rike 1951). The pecise mode of action of these dugs has not yet been fully established, but seveal mechanisms have been suggested. It was oiginally poposed that thei facilitatoy action was due to the inhibition of AChE at the end-plate esulting in the pesistance of tansmitte at the junctional egion. Many authos still adhee to this oiginal supposition (Hobbige 1952; Smith, Cohen, Pelikan and Unna, 1954; Katz'and Thesleff 1957; Fey and Mashal 1971; Whittake 1975). Howeve, in-ecent yeas it has been questioned whethe these. anti-che dugs poduce facilitation of neuomuscula tansmission only by the inhibition of AChE at the moto endplate. The possibility that the facilitatoy action of these dugs may be attibuted to the depolaizing action of the quatenay ammonium goup has been poposed on the basis of a diect stimulating action on mammalian skeletal muscle. Fo example, the inte-ateial injection of neostigmine into chonically denevated muscle, o into nomally innevated muscle in which essentially all the AChE has been inactivated by a pio dose of DFP (Di-isopopylphosphoofluoidate), evokes an

22 -19- immediate contaction, wheeas physostigmine does not (Rike and Wescoe 1946). It has also been suggested that the anti-che compounds induce antidomic dischages. in the pe-synaptic neve teminals which ae then disseminated by local axon eflexes causing the additional elease of tansmitte (Rike, Robets, Standet and Fujimot 1957; Wene and Kupeman 1963; Blabe and Chist 1967). Thus, the mode of action of these dugs may be due to one o a combination of any of these suggested mechanisms. THE USE F ANTI-ChE DRUGS IN MYASTHENIA GRAVIS The majo theapeutic uses of the quatenay ammonium anti-che dugs ae in the stimulation of the smooth muscles of the intestinal tact and the uinay bladde, the eduction of high inta-ocula pessue in glaucoma, the evesal of dug induced neuomuscula blockade afte sugical anaesthesia and the diagnosis and teatment of myasthenia gavis. Myasthenia gavis is chaacteised by weakness and apid fatiguability of skeletal muscle. The close esemblance of myasthenia gavis to cuae poisoning epoted by Jolly in 1825, and the success with teatment of myasthenia gavis in the 193's (Walke 1934,1935), have long indicated the involvement of a defect in neuomuscula tansmission. The difficulty has been the exact location of this defect. Thee ae thee majo hypotheses concening the pathogenesis of this disease: Fistly, a pesynaptic defect in the synthesis o elease of ACh, esulting in the failue of an appopiate amount of ACh to each the ecepto sites of the sub-neual appaatus following apid epetitive neve stimulation. Secondly, a ciculating competitive blocking

23 -2- agent intefeing with the nomal inteaction between ACh and the post-synaptic ecepto. Finally, a deficiency in postsynaptic eceptos o a genealised decease in the sensitivity of the eceptos to ACh in consequence of an auto-immune eaction towads the nicotinic eceptos at the moto endplate. The suggestion that myasthenia gavis may be due to impaied elease of ACh fom moto neve endings (Desmedt 1959), esulted fom the esemblance between the post-tetanic exhaustion of myasthenic muscle and of nomal muscle teated with Hemicholinium which inhibits the synthesis of ACh. Elmquist, Hoffman, Kugelbeg and Quastel (1964) found that miniatue end-plate potentials in myasthenic patients wee nomal in fequency, but deceased in amplitude. They tentatively intepeted this obsevation as being due to a decease in the amount of ACh eleased fom the neve teminal. Howeve, this deceased amplitude might equally be due to a deceased numbe o the deceased sensitivity of a nomal numbe of post- synaptic eceptos. Repots have been made of the demonstation in the blood of myastbenic patients of a substance capable of poducing neuomuscula block in expeimental animals (Wilson and Stone 1944). It has been shown that thymopoetin which is the thymic homone, eleased and esponsible fo inducing the diffeentiation of T-cells, in thymic diseases, causes a neostigmine esponsive neuomuscula block in mice which esembles that of myasthenia gavis. Such evidence would lend suppot to the empiically poven teatment in a high popotion of patients

24 -21- with myasthenia gavis by ealy total thymectomy (Keynes 1954). The poo esults of thymectomy in some cases have been attibuted to esidual o ectopic thymictissue (Havey 1948), o to ievesible damage caused by long standing disease (Wolf 1966). Myasthenia gavis may also be due to an auto-immune esponse in which an antibody to the end-plate ecepto potein is poduced by the eticulo-endothelial system (Simpson 196). This suggestion was suppoted by the finding of antibodies to the skeletal muscle which also eact with the myo-epithelial cells of the thymus in the seum of 57. of patients with myasthenia gavis (Downes, Geenwood and Way 1966). Howeve, in patients with obsevable thymoma the incidence was almost 17. (ostehuis, Van De Geld and Feltkamp 1967). In addition, studies utilizing labelled a -bungaotoxin, a snake venom that ievesibly binds to ACh eceptos has shown that the numbe of eceptos at the moto end-plate is consideably educed in myasthenia gavis (Fanboough, Dachman and Satymuti 1973). In myasthenia gavis" thee is, theefoe, evidence of immunological eactions to the ACh ecepto potein which could educe the numbe and possibly the sensitivity of the ACh eceptos at the moto end-plate and theeby poduce the pathological and phamacological symptoms associated with this disease. Noestigmine, pyidostigmine and ambenonium ae the standad anti-che dugs used in the symptomatic teatment of myasthenia gavis. All can incease the esponse of myasthenic muscle to epetitive neve impulses, pobably pimaily by the pesevation of endogenous. ACh and secondaily by thei diect

25 -22- cholinomimetic action. When the diagnosis of myasthenia gavis has been established, teatment can be instituted with any of these dugs, then change if optimal impovement in stength is not achieved initially o at any time subsequently. In the majoity of patients, especially in those with pedominantly bulba involvement, best esults can be obtained with pyidostigmine, which is available only in cetain counties, as time-span tablets containing a total of 18mg of which 6mg is eleased immediately and 12mg ove seveal hous. This pepaation is of paticula value in maintaining patients ovenight. When the muscles of the extemities ae most affected, ambenonium o neostigmine may be the dug of choice. Not infequently bette esults can be obtained by altenating equivalent doses (See Table 1-3) of diffeent anti-che o by combining factional doses of two agents. TABLE 1-3 APPRXIMATE DSES F CMMNLY USED ANTI-ChE DRUGS RAL DSE INTRAVENUS DSE INTRAMUSCULAR DSE Neostigmine "Postigmine" 15mg.5mg mg Pyidostigmine "Mestinon" 6mg 2.mg 2.mg Pyidostigmine "Time-span" 18mg - - Ambenonium "Mytelase" 5-7.5mg none none available available

26 -23- Patients with genealised myasthenia gavis maintained on ambenonium often need supplementation with pyidostigmine 45-6minutes befoe meals to facilitate mastication and deglutition. Paenteal administation of the standad anti- ChE agents is sometimes equested in despaately ill myasthenic patients who do not espond adequately to oal medication, the subcutaneous, intamuscula o intavenous oute may be used. Atopine sulphate.4 -.6mg o moe, intavenously should be given immediately if a sevee eaction (choline4ic cisis) ensues.

27 -24- CHAPTER II PHARMACKINETICS

28 -25- CHAPTER TW Page No. Intoduction. 26 Concepts of Phamacokinetics Modelling. 27 Phamacokinetic Models. 29 ne-compatment Model. 3 Two-Compatment Model. 36 Dug level in cental compatment. 39 Dug level in tissue(peipheal) compatment 44 Some application of phamacokinetic paametes: 46 (a) Plasma o blood levels of dug & plasma t4- (b) The application of aea unde plasma concentation time (AUC) of dugs.

29 -26- PHARMACKINETICS INTRDUCTIN The wod phamacokinetics was intoduced by Dost in 1953 to mean the application of kinetic studies to dugs and poisons (Geek Phamakon). The tem incopoates the kinetic aspects of in vivo absoption, distibution, metabolism and excetion of dugs, poisons and some endogenous substances. Phamacokinetic studies use data obtained fom concentation cuves of dugs o thei metabolites in biological fluids; this data is pocessed by means of mathematical equations. The elationship between phamacological esponse and concentation of dugs o thei metabolites in body fluids is also included within the scope of phamacokinetics. The study of phamacokinetics was oiginated ove 4 yeas ago. Widmak (1922) dealt exhaustively with the phamacokinetics of ethyl alcohol and his mathematical teatment is still used. Equations descibing fist-ode pocesses fo absoption and elimination wee deived by Gehlen (1933) and Beccai (1938). Tasten Teoell (1937 a, b) fist developed the theoy of compatments and this wok laid the foundation fo much of moden phamacokinetics. Teoell assumed that the dug and/o its metabolites wee equitably dispesed in one of seveal tissues of the body and that the dug in these tissues acted as kinetically homogenous. Such a tissue, which acts on isotopicfluid, is temed a compatment. Compatments ae sepaated by baies that inhibit fee diffusion fom one compatment to anothe. The baies ae kinetically definable

30 -27- in that the ate of tanspot of dug (o metabolites) acoss the membane between the compatments is a function of the amounts of dugs in these compatments. Pogess in phamacokinetics was initially slow, due to widespead citicism of the compatment theoy. Howeve, the ideasof Teoell wee ebon in the wok of Duckey and Kupfmulle (1949) and Dost (1953); both goups of wokes utilized data obtained fom blood. The use of data obtained fom uine fo phamacokinetic analysis was extended by Wiegand and Taylo (196). The compatment theoy is now geneally accepted and coupled with the development of moe pecise means of measuing the equied expeimental data phamacokinetics has made damatic advances in the last decade. CNCEPTS F PHARMACKINETIC MDELLING Wagne (1968) descibed easons and advantages of phamacokinetic modelling to intepet quantitatively the fate of a dug in the body. These ae; to summaise obseved data in tems of an equation o equations which may help to descibe bette the fate of a dug than a plasma concentation-time' pofile of the dug; to incease undestanding of the disposition pocess (absoption, distibution and elimination) involved, to be able to pedict dosage egime fo bette quality of theapy, to compae seveal dugs with simila phamacologic action fo sceening of new dugs, and to quantitatively elate biological activity with phamacokinetic data fo bette contol of dug action.

31 -28- The advance of analytical techniques fo the detemination of dugs in biological fluids has allowed moe accuate and pecise analysis to descibe the fate of dugs in the body. The tem 'compatment' should not be esticted to the well-known body fluid compatments o ogans; a phamacokinetic compatment is efeed to as a hypothetical egion in the body whee the dug concentation is eveywhee the same, and is not elated to any paticula anatomical compatment. But it is helpful to conceptualize the cental phamacokinetic compatment as consisting of such physiological units as the extacellula space and well-pefused ogans (e. g., kidney and live), and the peipheal compatment as consisting of pooly pefused ogans and tissues (e. g., muscle o fat). Dug elimination (via enal, biliay and othe outes of excetion and metabolism) is usually assumed to occu fom the cental compatments. It is assumed that the pocesses of absoption, distibution and elimination associated with each model can be descibed by fist-ode ate kinetics, whee the ate of tansfe of a given dug is popotional to the amount o concentation of the dug. Kinetic lineaity was defined by Kuge-Thieme (1968) as diect popotionality of tansfe ates o concentation of diffeences of dugs. Many authos have deived equations to intepet data fo linea phamacokinetics (Benet and Tui 1977; Benet 1979; Thon 1974). A moden view of phamacokinetics was emphasised by Wagne (1973) to include both linea and nonlinea systems. He eviewed ove 16 examples of nonlineaities in phamacokinetics. The cause of nonlinea phamacokinetics is

32 -29- due to a deviation fom fist-ode ate kinetics. Nonlineaities can esult fom incomplete o abnomal dug absoption, in binding eactions to limited numbes of binding sites on plasma poteins o in tissues, in active tanspot pocesses with limited available caie, in active tubula secetion and eabsoption in the kidney, in biliay excetion of dugs and thei metabolites, and in enzymatic tansfomations obeying Michaelis-Menten kinetics which is dose dependent kinetics. Enzyme stimulation and inhibition may also cause nonlineaities. It is impotant to ecognize nonlineaity deiving a phamacokinetic model. Sometimes this deviation becomes less obvious if data ae obtained fom caefully designed expeiments. Beckett, Salmon and Mitchad (1969), by maintaining an acidic uinay PH, demonstated that the uinay excetion ate of amphetamine follows fist-ode kinetics, whilst fomely it was not possible to investigate the phamacokinetics of amphetamine in man (Beckett and Rowland 1964). Although Wagne (1973) emphasised that most dugs show nonlinea phamacokinetics chaactiestics, the concept of linea phamacokinetic theoy can be based just as easily on the simple assumption of lineaity, with no assumptions egading phamacokinetic details. A theoy esting on this foundation is much less vulneable than one based on a possible eoneous specific phamacokinetic model. PHARMACKINETIC MDELS. Afte a dug eaches the geneal ciculation, it can undego a vaiety of pocesses, diffusion and binding in extavascula fluids and tissues, metabolism, nonenzymatic

33 -3- chemical eaction and excetion. Excetion may take place in seveal pathways, the most common one is though the kidneys and bile, othe outes include the skin, lungs and gastointestinal tact. An enteohepatic cycle may develop whee biliay excetion and intestinal absoption continue until most of the dug is eliminated though othe pathways. NE CMPARTMENT MDEL ften the ate of concentation changes fo a dug in blood, plasma, o seum can be descibed as a single fist- ode pocess afte an initial apid distibutive phase (Fig 2.1). That is, afte a apid intavenous injection the logaithm of the plasma dug concentation deceases linealy as a function of time as shown in Figue 2.2. The Figue 2.1 illustates this one compatment ate constant fo elimination K of the dug quantity in the body X, is equal to the sum of the appaent ate constant fo metabolism, Km and excetion Ke: - K-KK+Ke Equation (1) Fo most dugs the ate of elimination is popotional to the amount of dug in the body (X) at any time t: dx -KX Equation (2) The negative sign indicates that dug is being lost fom the body. Integation of equation (2) poduces:

34 -31- FIG 2.1 A typical one compatment model of a dug, whee Ka, Ke, Km ae absobtion, excetion and metabolism ate constants espectively X is amount of dug in the body. dose Ka IV ei 4 a time (hs) FIG 2.2 Log C vesus time, dug plasma level elimination accoding to Model 1.

35 -32- X Xoe-Kt Equation (3) Whee X is the amount injected (i. e., dose) and e epesents the base of the natual logaithm. Taking the natual logaithm of both sides of equation (3) and then conveting to common logaithms (base 1, log), gives: Log X log Kt R o 2.33 Equation (4) The body is obviously not homogenous even if plasma concentation and uinay excetion data can be descibed by epesenting the body as a one-compatment model. Dug concentation in the live, kidneys, heat, muscle, fat and othe tissues will usually diffe fom one anothe as well as fom the concentation in the plasma. If the elative binding of a dug to compatments of these tissues and fluids is essentially independent of a dug concentation, then the atio of dug concentation in the vaious tissues and fluids is constant. Consequently, thee will exist a constant elationship between dug concentation in the plasma, C, and the amount of dug in the body: Xa VdC Equation (5) The popotionality constant Vd in this equation happens to have the units of volume and is known as the appaent volume of distibution. Despite its name, this constant usually has no diect physiologic meaning and does not efe a eal volume.

36 -33- The elationship between plasma concentation and the amount of dug in the body, as expessed by equation (5). enables the convesion of equation (4) fom an amount-time to concentation-time elationship which can be expessed as logc"- logc _ Kt o 2.33 Equation (6) Whee C is the plasma dug concentation immediately afte injection (t - o). Equation (6) shows that a plot of log C vesus t will be linea unde the stated conditions (Fig 2.2). Co may be obtained by extapolation of the log C vesus t plot to time zeo. This intecept, Co, may be used in the calculation of the appaent volume of distibution. Since X equals the amount of dug injected intavenously i. e., the intavenous (I. V. ) dose, Vd may be estimated fom the elationship: VdsI. V= Equation (7) It follows fom equation (6), that if plasma concentations ae plotted on a logaithimic scale against time, a staight line should esult. The slope of such a plot is -K/2.33 and elated to the biological half-life (tb) of dug though equation (8) K-.693 Equation (8) t/

37 -34- Nelson and 'Reilly (196) deived an equation which descibes the amount of unchanged dug exceted at any time duing the post-absoptive phase afte distibution equilibium has been established, equation (9) U-U * (1 - e-kt) KXo (1 - e-kt ý Equation (9) The tems U and U epesent the amount of unchanged dug exceted up to time t and infinite time, espectively. Diffeentiating equation (9) and taking logaithms yield equation (1) log ät - log KU - Kt Equation (1) Kt log KeX It follows fom equation (1), that if the logaithm of the ate of excetion is plotted against time, a staight line should esult. The slope of this line will also be -K/2.33. Equation (8) can be used fo the detemination of the biological half-life of the dug. Data obtained fom uine has been pocessed in the above manne in ode to obtain K and the t fo dugs. such as sulphnamides (Swintosky 1957; Nelson and Schaldemes 1959; Nelson and 'Reilly 196); digitoxin (Swintosky 1957); amphetamines (Chapman et al., 1959; Beckett and Rowland 1965b) paacetamol (Cummings et al., 1967) and chloopopamide (Taylo 1972).

38 -35- Uine data can also be employed to estimate the individual ate constants which ae included in the tem Kel. The excetion ate constant (K can be calculated e) fom equation (11): Re RX U Equation (11) Whee X is the amount of dug absobed. If the dug is completely absobed X will be equivalent to the dose of dug administeed. Substituting the calculated value of Ke in equation (1), m may be detemined. The late value may be a hybid ate constant, since it can combine the ate constants of futhe biotansfomations, as indicated in equation (12). Rý _ (Rml + Kim2 + Km3... ICS ) Equation (12) Plots of log ate of excetion o plasma level against time do not always yield a staight line. The influence of slow distibution of the dug thoughout the body on this elationship has been discussed by Nelson (1964), who also obseved that if the esults of seveal individuals ae aveaged a deviation fom the staight line elationship occus. Plasma potein binding of dugs also has an influence on this cuve (Kuge- Thime 1966c; Kuge-Thime et al., 1969; Dille 1964 and Matin 1965a, b; and Wagne and Notham 1967) have discussed the intoduction of a second body compatment to explain these deviations.

39 -36- In this simplest fom of the one compatment model, afte a apid intavenous (I. V. ) injection, the dug is placed into the single compatment and consequently thee will be no absoption phenomena (see Fig 1.1). Altenatively, an I. V. infusion can be employed and the phamacokinetic paametes can be assessed fom post-infusion blood cuves. TW-CMPARTMENT MDEL Thee is often an initial apid fall of the dug concentation in the blood (and uine) when the dug is administeed intavenously (I. V. ), and then its fist-ode ate of elimination is established (Fig 2.4). Gaett (1964) and Gaett and Alway (1963) have shown that this type of data is best analysed by a two-compatment open model. This model was fist elated to dug kinetics by Teoell (1937a, b). It pesumes that a dug administeed I. V. apidly equilibates between two-compatments, a cental compatment and a peipheal compatment (Fig 2.3). Usually elimination (metabolism and excetion) is consideed to occu at a ate popotional to the dug concentation in the plasma. Howeve, metabolism could occu in the tissues and this possibility was investigated fo acetylalycylic acid / by Rowland et al., (197). If the model shown in Fig 2.3 is assumed to be coect, then thee is a apid initial dop in blood concentation of dug (Fig 2.4), until a pseudo-steady state is established between the dug in the cental (o blood) compatment. Changes in the concentation in the blood can then be elated to changes

40 -37- FIG 2.3 Schematic epesentation of a two-compatment system consisting of a cental compatment and a peipheal (tissue) compatment. Cental K12 Peipheal _Iv Compatment X c K21 X P t K1 whee Xc and Xp ae the amount of dug in the cental and peipheal compatments espectively, K21 and K12 ae the appaent fist-ode intecompatmental ate constants and K1 is the appaent fist-ode elimination ate constant fom the cental compatment.

41 -38- FIG 2.4 A semilog plot of the plasma level of a dug which obeys the kinetics of a two-compatment model of I. V. administation. -d--a E o.. AJ.. t t * 26t - Expeimental values --- Residuals U4 R.. a si 4) u eo.4. TIME (miss). Expeimental values and calculated cuve in man afte I. V. administation of 72.4mg/kg of pyidostigmine bomide. The expeimental values ae the mean of six obsevations (see Chapte IV).

42 -39- in the concentation of the tissue compatment though the equilibium o patition constant (K21/K12). DRUG LEVEL IN THE CENTRAL CMPARTMENT Following apid I. V. injection of a dug that distibutes in the body accoding to a two-compatment model with elimination occuing fom the cental compatment, the ate of change in the amount of dug in the cental compatment Xc can be descibed by the following diffeential equation: dxc dt R -21 Xp K12 Xc - K1 Rc Equation (13) Whee X, is the amount of dug in the peipheal compatment, K21 and K12 ae the appaent fist-ode intecompatmental distibution ate constants, and K1 is the appaent fistode elimination ate constant fom the cental compatment (see Fig 2.3). Taking the Laplace tansfom of equation (13) yields equation (14) and afte patial faction analysis of equations (14) and (15), the amount of dug in the cental compatment Xc is given by equation (16): «+I9 * K12 + K21 + K1 Equation (14) «. 9 - K21K12 Equation (15) Xc Xo (a -K 21) a-, 9 -. t e Xo (K21 - ft > a-, 9 e- Pt Equation (16)

43 -4o- Whee oe and e ae complex constants which simply seve to eplace othe constants as in equations (14) and (15); X is the intavenous dose. Although the cental compatment is obviously not homogeneous by assuming that the atio of dug concentations in the vaious tissues and fluids of the cental compatment is constant (i. e., thee is a vey apid distibution between the plasma and fluids and tissues of the cental compatment), a linea elationship exists between the dug concentation in plasma C and the amount of dug in cental compatment, that is: XV. cc C Equation (17) Whee Vc is the appaent volume of cental compatment. This elationship enables the convesion of equation (16) fom an amount-time to a concentation-time equation, which can be expessed as: C Ro( f-k21) -e+ vc (a- ß) -at Ro(K21-B v (a-ft) )e-, Vt Equation (18) C Ae G(t + Be-. t Equation (19) whee A-x(a-K1) va-e Equation (2)

44 -41- and x (K21-8) Equation (21) A plot of the logaithm of dug plasma concentation vesus time accoding to equation (19) will yield a bioexponential cuve (Fig 2.4). The constant oc is by definition lage than 89 and theefoe at some time the tem Ae at will appoach zeo while Be 'Bt will still have a finite value. Equation (19) will then educe to: C- Be -Bt. Equation (22) which in common logaithms is: log C- log B-2.33 Equation (23) Hence, an estimate of ý9 can be obtained fom the slope - ß/2.33 of the teminal exponential phase, and biological tk can be detemined employing the following elationship:.693 Equation (24) The zeo-time intecept obtained by extapolation of the teminal, linea phase to t-o is B. Application of 'the method of esiduals' yields a second linea segment with a slope equal to - «/2.33 and a zeo-time intecept A. The constants A. B. a and ß may be obtained gaphically as shown in Fig 2.4, o with the aid of a digital compute.

45 -42- The fist appoach is to fit the entie plasma level-time cuve by means of a digital compute pogam which povides a nonlinea egession analysis of the cuve. nce these expeimental constants ae obtained, the phamacokinetic paametes Vc, K12, K21 and K1 can be geneated by consideing the following elationship. At time tao, equation (19) becomes C -A+ B Equation (25) Whee C is the zeo-time plasma concentation, substituting the values of A and B fom equation (2) and equation (21), espectively into equation (25), and then simplify it yields: C= Xo Vc Equation (26) Accoding to equation (25)t yields: Vc = Xo ÄB Equation (27) Whee X is intavenous dose. Reaangement of equation (27) and substitution A+B in equation (21) yields: (A+a)(K21-ý) Ba- Equation (28)

46 -43- K21 =A+B *f Equation (29) A+B K1 =K 21 Äý+ Bä) Equation (3) Detemination of these ate constants pemits an assessment of the elative contibution of distibution and elimination pocesses to the dug concentation vesus time pofile of a dug. It may also aid in elucidating the mechanism of dug inteactions and of effects of disease, age, genetic influence, etc., on dug disposition. ne useful additional equation is the equation fo the total volume of distibution of dug in the body: vd. V _ K Equation (31) Whee Vd can be defined as the appaent volume of distibution of dug in the body (Gibaldi 1975). The paamete Vd will elate the amount of dug in the body to the plasma concentation at any time duing the postdistibutive phase, that is Vd ßf x Cdt Equation (32) which is a useful elationship fo estimating Vd

47 -44- DRUG LEVELS IN THE TISSUE (PERIPHERAL CMPARTMENT) The diffeential equation descibing the ate of change in the amount of dug in the peipheal compatment is: dt - K12Xc - K21XP Equation (33) Whee Xc and Xp ae the amount of dug in the cental and peipheal compatment espectively. Afte the Laplace tansfomation and patial faction analysis of equation (33) yield equation (34) K12Xo Xs e- at K]= p+et -pt Equation (34) o Xs pa X 2X (e- Rt T e- at) Equation (35) Equation 35 descibes the time-couse of the amount of dug in the peipheal compatment following intavenous administation. It is obvious fom equation (35) that afte a sufficiently long peiod of time the tem e- educes to d t appoaches zeo and equation (35) X p R1= a-/4 esgt Equation (36) Hence, the slope of the teminal exponential phase equals to -fß/2.33. Theefoe, in the postdistibutive phase. plasma and peipheal compatment levels decline in paallel.

48 -45- Equation (36) may be useful in detemining the elationship between phamacologic effect and tissue levels of a dug. It must be kept in mind, howeve, that such elationships ae only appoximations. The hypothetical tissue levels may not accuately eflect the concentation of dug at the site of action, even though the site of action may be a pat of the peipheal compatment (Gibaldi 1975).

49 -46- SME APPLICATIN F PHARMACKINETIC PARAMETERS. (a) PLASMA R BLD LEVELS F DRUG AND PLASMA t Measuing plasma o blood concentations of dugs and deducing the plasma half lives, t, fom such data has often been used to monito dug theapy. Plasma level detemination is unnecessay if the patient esponds satisfactoily to a caefully chosen dosage schedule, but can claify the situation when the desied theapeutic effect is not achieved o when toxic effects ae suspected o when the patient has not taken the dug. Howeve the intepetation of dug plasma o blood levels becomes difficult when thee is an intesubject vaiation in the plasma o blood pofile of the dug. Factos which ae esponsible fo thei inte-individual vaiations have been discussed by Vesell (1973). Thee ae genetic vaiations and vaiation in the absoption, distibution and elimination of the dug among individuals which may be due to envionment odisease states. When all these factos ae opeating in seveely ill patients, dug plasma o blood ae difficult to intepet. Theefoe, utilisation of plasma o blood concentation of dug o its plasma tý as a theapeutic guide alone may be limited (Peie and Gibaldi, 1974). (b) THE APPLICATIN F AREA UNDER PLASMA R BLD CNCENTRATIN TIME CURVE (AUC) F DRUGS Fequently, the technique of aea analyais has been applied to assess the absoption and bioavailability of a dug. This is because the aea unde a blood level cuve is popotional to the dug available fom a dose (Wagne and Nelson, 1963; 1964; Loo. and Riegelman, 1968). Howeve, it

50 -k7- is obseved that thee is a eduction in the AUC following oal dug administation as compaed with that obtained afte intavenous dug administation. This obsevation led to the assumption that oally administeed dugs ae eithe incompletely absobed o metabolised in the gasto-intestinal tact. Subsequently, it is demonstated that thee is also a compaable eduction in the AUC afte dug infusion via the potal vein. The total AUC of a dug can be easily calculated using Equation (37), which esults fom integation of equation (19): AUC cc +3 Equation (37) Fom equations(27)9 (3), and (37) Equation (38) can be obtained, which shows that AUC is diectly elated to the dose X and invesely to the elimination constant K1 and the volume of cental compatment Vc. x AUC a V ck1 Equation (38) The AUC of a dug can also be detemined accoding to equation (39): fo o X AUC Xcdt =F(2) Equation (39) Hence, AUC is dependent upon the elimination ate constant, K. the volume of distibution V and the total quantity of available dug FXo. Fo a dug whee the total oally administeed dose

51 -48- is absobed, F=1, in a one compatment model. Similaly, the AUC is identical to that fo the two compatment system except fo the diffeent definitions of distibutive volume (K =, 9 and V= VB). Wagne (1967a) applied equation (4) to twelve sepaate studies with tetacycline and novobiocin F.693V s Ut Xo / Equation (4) combinations fom 167 sets of seum tetacycline cuves. Both childen and adults wee used in the studies. Simila elative tetacycline availability wee evident fom the aveage aea/ dose times half-life atios, which anged fom 1.9 to Absolute availabilities can easily be assessed by a compaison of aeas with the use of equation (4%), whee the AUC pe unit dose by any oute of administation is expessed as a pecent of the intavenous AUC pe unit dose. The total quantities of intact dug exceted afte any administative oute can also be compaed to the intavenous data. Pecent of absolute availability - (AUC) x Dosel. oal V. x 1 AUC I. V. x Dose oal Equation (41) bviously, the pecentage of absoption can only be obtained with equation (41) if no significant metabolism occus befoe the dug eaches the geneal ciculation. This was the case with the compounds studied by Dost (1963) and Gladtke (1964, 1968). Howeve, with oal administation, if the dug is

52 -49- metabolized pio to, o duing absoption, o duing the 'fist pass' though the potal system, then the oal AUC will be less than that obtained afte intavenous administation, even if absoption is completed. This was discussed by Hais and Riegelman (1969) with efeence to a apidly metabolized dug, aspin, using data obtained in dog expeiments afte oal dosage, vena -- cava and hepatic potal vein infusion. Gibaldi et al. (1971) descibed such obsevation as the 'fist-pass' effect, which indicates that dugs ae exposed to metabolism in the live befoe eaching the bloodsteam afte oal administation.

53 -5- THE AIMS F THE THESIS Clinical laboatoies ae in need of a eliable pocedue to analyze quatenay ammonium dugs because this class of compound includes the neuotansmitte acetylcholine as well as the anticholinesteas', neostigmine and pyidostigmine, which ae impotant in the diagnosis and management of myasthenia gavis. Individual diffeences in the dosage equiement of the dugs ae commonly attibuted to vaying seveity in the couse of disease. The ole of inte-individual diffeences in dug metabolism and excetion is a matte of conjectue since analytical methods that can be applied to the outine measuement of pyidostigmine and neostigmine concentations in man have not been developed. The aims of the expeimental wok ae to develop some sensitive and selective analytical methods to measue the concentation of the dug and thei metabolites and also to investigate the phamacokinetics of pyidostigmine and neostigmine afte IV and IM and oal administation. It is hoped that the clinical findings will help to elucidate futhe the mechanisms of action of these dugs, pemit a moe systematic and contolled investigation of a simila natue and ultimately, help to identify the contibuting factos in the difficult management of patients unde going anticholinestease theapy.

54 -51- CHAPTER III PART NE A REVIEW N ANALY'T'ICAL TECHNIQUES AVAILABLE FR STUDIES F ANTI-(ME DRUGS. PART TW DEVFLPMENT F METHDLGY FR THE DETERMINATIN F NIDSTIGMINE, PYRIDSTIGMINE AND THEIR METABLITES IN HUMAN PLASMA AND URINE.

55 CHAPTER THREE Page No. Pat one: A eview on analytical techniques available fo dis- 53 position studies of Anti-ChE dugs. Rational fo monitoing plasma level of Anti-ChE dugs. 57 Conclusion 6 Pat two: Development of methodology fo the detemination of 62 neostigmine, pyidostigmine and thei metabolites in human plasma and uine. Intoduction 63 METHD 1: Appaatus and conditioning 64 Mateials 65 Teatment of glasswae 66 Detemination of neostigmine in plasma 66 Detemination of pyidostigmine in human plasma 67 Detemination of Anti-ChE agents and thei metabolites 68 in human uine. Results 69 Application 74 Discussion 76 METHD 2: The simultaneous detemination of neostigmine 77 and pyidostigmine in human plasma and uine. Synthesis and chaacteization of pyidostigmine analogues. 78 Simultaneous detemination of neostigmine and pyidostigmine. 8 Results 83 Discussion 88

56 -53- A REVIEW N ANALYTICAL TECHNIQUES AVAILABLE FR DISPSITIN STUDIES F ANTI-ChE DRUGS Ealy techniques fo the detemination of quatenay Anti-ChE dugs and thei metabolites in body fluids wee based on spectophotomety. Afte ion-pai extaction of the compounds into oganic solvents, the ion-pai is subsequently dissociated with dilute hydochloic acid and the quatenay amines can then be assayed by standad spectophotometic techniques. These methods have been applied to the measuement of pyidostigmine in plasma (Cope, et al., 1974). The quatenay amine was extacted as an iodide complex into dichloomethane and its absoption was measued at vaious wave lengths. The limit of sensitivity of this assay pocedue is pg/ml, and intefeence by othe dugs and endogeneous substance may occu. Konfeld, et al. (197) and Somani et al. (1972) have had little success with this pocedue which does'not appea to sepaate pyidostigmine fom its main metabolites; appaently, the majo metabolite, 3-hydoxy-N-methylpyidinium, gives maxima at 25,288 and 32nm (Somani et al ). A method fo the estimation of neostigmine and pyidostigmine in uine was descibed using ion exchange teatment and coloimetic estimation of the blue complex poduced when eithe of the dugs was complexed with bomophenol blue (Nowell et al., 1962). Howeve, the pocedue suffes fom lack of sensitivity (lowest limit fo detection is 2 to 3 pg/ml) and selectivity as the assay could not be used to distinguish between. neostigmine and pyidostigmine.

57 Radioisotopic techniques have been used to study the disposition of edophonium in the at using (14C)-edophonium (Back and Calvey 1974). The disposition and metabolism of neostigmine in the hen and at wee studied using 14C-labelled neostigmine (Robets et al., 1965). Similaly, metabolism studies of pyidostigmine in at (Bikt Y et al., 1966) and in man (Konfeld 14C-labelled et al., 197) wee caied out using pyidostigmine. These investigations ae expensive and involve the administation of lage doses of adioactivity and ae not suitable fo human studies. Radioisotopic techniques ae often non-specific and cannot distinguish the paent dug fom its metabolites. Electophoetic and chomatogaphic techniques ae often involved in the isolation and sepaation of paent dugs and metabolites when adioisotopes of the paent dugs ae used. Enzymatic pocedues have been used to assay evesible eta,, anti-che agents, such as edophbnium, in plasma (Babe 1976). These methods have been used to study the coelation between the plasma concentation of the dug and phamacological effects (Calvey et al., 1976). Although these assay pocedues ae sensitive, they ae time-consuming and exacting and can only be used fo tuly evesible anti-che agents. Analyses based on gas-liquid chomatogaphic (GLC) techniques have been used fo the quantification of some quatenay amines. Fo instance, acetylcholine was demethylated into dimethyl-aminoacetate by N-demethylation using benzenethiolate in butanone solution (Jenden at al., 1968); the

58 -55- subsequent dequatenized dimethylaminoacetate was assayed by a GLC pocedue. We have had limited success with this pocedue fo measuing neostigmine and pyidostigmine. Appaently the methylation of the quatenay nitogen by benzenthiolate would also cause the hydolysis of the cabonate este and we did not obtain epoducible demethylation (Chan 198). Methods based on themal dequatenization have been epoted fo a numbe of quatenay compounds; including acetylcholine, acetyl-/-methyl choline, butyylcholine and choline (Szilagyi, 1968 and Schmidt 1975), thiazinolnium (Jonkman et al., 1975), neostigmine and pyidostigmine (Vidie, 1972; Chohan 1976). Vicie(1972) and Chohan (1976) in thei methods used the methyliodide (pat of the dequatenanized poducts, see Chan 198) as a measue of the quatenay amines in the plasma. Howeve, this pocedue suffes fom lack of selectivity as othe quatenay compounds may also be ion-pai extacted and similaly assayed. Recently, Chan et al. (1976) developed a sensitive and selective powiedue based on GLC analysis, fo the detemination of nostigmine and pyidostigmine in human plasma. The pocedue involved peliminay ion-pai extaction of the dugs using potassium iodine-glycine buffe, into dichloomethane, followed by concentation and analysis of the ion-pai complex using GLC system fitted with a nitogen-sensitive detecto. Using peak aea atio techniques, pyidostigmine bomide was used as the intenal make fo the detemination

59 -56- of neostigmine in plasma; neostigmine bomide was used as the intenal standad fo the quantitation of pyidostigmine. The method depends on the dequatenization of the quatenay nitogen to fom the tetiay analogue which is esolved by the GLC system. Chan et al. (1976) epoted that gas chomatogaphy-mass spectomety (GC-MS) of a plasma extact containing neostigmine and pyidostigmine as iodide complex gave a one compound whose mass spectum showed a molecula ion at n/e 166, and second compound with a molecula ion at m/e 28, coesponding to pyidostigmine and neostigmine bomide o the appopiate tetiay analogues of these dugs. Appaently, both Anti-ChE dugs, eithe as the bomide salt o as the iodide complex, wee themally demethylated to thei coesponding tetiay analogues which wee then esolved by the GLC system. the authos have used simila methods to measue quatenay amines in plasma. The plasma and uine level of thiazinamium methylsulphate, an anticholinegic agent fo teatment of bonchial asthma, was detemined by a simila ion pai extaction and GLC pocedue (Jonkman et al., 1975). Acetylcholine and its elated analogues have been measued by an assay based on pyolysis gas chomatogaphy and pyolytic poducts of these compounds ae the coesponding 2-dimethylamine estes and methyl halides (Szilagyi, 1968; Schmidt, 1975). Aquilonius at al., (1979) epoted an identical extaction pocedue using deuteated d6- neostigmine as the intenal standad and mass spectomete as the detecto fo the detemination of neostigmine in plasma. The limit of quantifation of this method. was about lng/ml, but the use of

60 -57- GC-MS system is not always available in evey laboatoy due to the high cost of such a system. Electon captue gas chomatogaphy method fo detemination of quatenay ammonium compounds has been epoted by Pohlman and Cohan (1977). The pinciple of this analytical method is to isolate the quatenay ammonium compound as its iodide fom biological fluids. The iodide extact is then themally degaded to elease methyl iodide upon its injection onto the chomatogaphic column and measued by the electon captue detecto. This method has a few potential poblems, including choline which is found in seum may also be demethylated at the tempeatue employed (Pohlman and Cohan 1977). Although these assay pocedues ae sensitive, they ae time-consuming (ove 48 hous) and non-specific. High pefomance liquid chomatogaphy technique has been epoted fo the quantitation of pyidostigmine in plasma by Yakatan and Tien (1979). Pechloate was used as the cante ion in the extaction of pyidostigmine and measued at a wave length of 269nm using a UV detecto. The limit of sensitivity of this assay pocedue is 2ng/ml by utilizing 1 ml plasma. Mean ecovey fom plasma was (Yakatan and Tien 1979). RATINAL FR MNITRING PLASMA LEVEL F ANTI-ChE DRUGS The influence of plasma concentations of a dug by phamacokinetic pocesses such as disposition and elimination, is often consideed to be esponsible to a geate extent fo the vaiation in individual esponse to the dug than ae the

61 -58- diffeences in taget ogan sensitivity among individuals (Smith et al., 1973). Thus, the monitoing of plasma levels of dugs in phamacotheapy may be of advese side effects o eactions due to the dug o combination of dugs (Mucklow 1973) and povide moe efficient phamacotheapy. At pesent, the egulation of the oal anti-che dugs fo patients with myasthenia gavis is empiical, depending on the patients' subjective esponses and on patients co-opeation when testing moto stength. Edophonium has been used as an* adjunct in egulation dug dosage (sseman et al., 1966), but esults ae difficult to intepet and may not be helpful to the physician in deciding whethe medication is necessay when the patient complains of moto weakness, dyspnea o diplopia. Howeve, if a elationship between the plasma level of these anti- ChE dugs and clinical esponses of the myasthenic patient could be demonstated, it would be possible to egulate the dosage egimen by measuement of the plasma concentation of the dug to detemine if the patient with an unsatisfactoy clinical esponse to the anti-che dug teatment equies moe medication o is manifesting symptoms of cholinegic cisis, due to dug toxicity fom ove medication. Recently, Chan and Calvey (1977) demonstated that the plasma concentation of pyidostigmine was invaiably positively coelated with the impovement in neuomuscula tansmission in patients with typical electomyogaphic decement in the adducto pollices. The plasma concentation to nomal vaied ove a 5-fold ange ( ng/ml), eflecting the vaiable

62 -59- seveity of the disease. In a patient with puely ocula symptoms, thee was a significant coelation between the plasma concentation of the dug and diamete of palpebal fissue. A sepaate study on the plasma level of pyidostigmine in myasthenic patients has shown consideable intesubject vaiation in the bio-availability of the quatenay amine (Calvey and Chan 1977); the six patients on an oal pyidostigmine bomide wee stablized on widely diffeent doses of the dug (6-66mg/day). Theefoe, the ole of individual diffeences in dug disposition may no longe be a matte of conjectue and the monitoing of plasma levels of anti-che dugs should be of paticula value in the clinical assessment of patients with myasthenia gavis. A method to calculate the optimal daily doses of pyidostigmine in individual myasthenic patients is descibed by Chan and Calvey (1977). Fo instance, the optimum plasma level of the dug fo each myasthenic patient can be defined by elating the plasma concentation of pyidostigmine to detemine the volume of distibution in the affected muscle goups. Subsequently, a test dose of oal and intavenous pyidostigmine could be used to detemine the volume of distibution, the elimination half-life and the faction of the dose of the dug equied to maintain the steady-state plasma level can be calculated fo each myasthenic patient, accoding to the following equation: - Dose mg/kg/24h equied steady-state volume of plasma concentation X distibution U&/ml (ml/kg) faction of dose X half-life absobed (min)

63 -6- The phamacokinetics of neostigmine afte intavenous administation to sugical patients duing the evesal of neuomuscula block have ecently been investigated (Aquilonius 1979; Smith 1973). We have done simila IV studies fo pyidostigmine (see chapte 4). It is possible to utilize these phamacokinetic paametes fo the design of a dosage egimen as outlined in the above equation (Chan and Calvey 1977). Such paametes may also help to select which one of the two anti-che dugs is bette fo teatment. Bake et al., (1978) have also studied the plasma cleaance of neostigmine and pyidostigmine in conscious dogs by the use of a coss-ove design. Results suggest that the longe duation of action of pyidostigmine is elated to the diffeential cleaance of the two quatenay amines fom plasma, as the slow disposition half-life of pyidostigmine is appoximately thee times longe than that of neostigmine. CNCLUSIN It is evident that pevious methods fo the study of the metabolism and disposition of anti-che dugs ae not selective and sensitive. ve the last twenty yeas the most impotant pogess obseved in the eseach of fundamental biology has been in the technical means (Deux 1979), such as electonic micoscopy, gas-liquid chomatogaphy, high pefomance liquid chomogaphy, gas chomatogaphy - mass spectoscopy, methods of stuctual detemination fo poteins and the use of immunological methods (enzymatic-multiple immunological

64 -61- techniques and adio-immunoassay). With these techniques it is possible to measue minute quantities of dugs up to the molecula level 1-12g. The detemination of anti-che dugs and thei metabolites offes bette assessment of anti-che theapy in myasthenia gavis and in the evesal of neuomuscula blockade afte sugical opeation.

65 -62- PART TW Development of methodology fo the detemination of neostigmine, pyidostigmine & thei metabolites in human plasma & uine.

66 -63- INTRDUCTIN Sensitive and specific methods fo the analysis of dugs in biological fluids ae essential fo phamacokinetic studies, paticulaly when plasma concentations of dugs which have a low post-absoption concentation o apid elimination ae studied afte administationof small doses. Assay methods epoted fo the detemination of anti-che dugs; as mentioned ealie (Pat ne page 53 ) ae difficult to use, o suffe fom low sensitivity o lead to intefeence by othe compounds. In this pat, the latest development of the analytical methods and thei application to the detemination of anti-che dugs in human plasma and uine is descibed. These developed methods ae based on the peliminay ion-pai extaction of neostigmine, o pyidostigmine and thei majo metabolites as well as appopiate intenal standads into dichloomethane o dichloomethane: acetone(9: 1), followed by concentation and analysis of the ion-pai complex, using a GLC system, These developed pocedues will be descibed in tems of the following section. Method 1 was used fo the isolation and detemination of neostigmine o pyidostigmine and thei metabolites. To measue unchanged dugs, pyidostigmine bomide was used as an intenal make to measue neostigmine; and neostigmine bomide was the intenal make fo the quantititation of pyidostigmine. To measue the metabolites of pyidostigmine and neostigmine in uine, one metabolite was used as the intenal standad fo the othe and vice vesa.

67 -64- Method 2, developed fo use in the simultaneous detemination of the unchanged dugs using a common intenal make. METHD 1. APPARATUS AND CNDITINING A Pekin-Elme model F17 gas chomatogaphy all-glass system, fitted with a phosphous-nitogen detecto and linked to a Pekin-Elme chat pape Model 56 (Pekin-Elme, Beaconsfield, U. K. ) and to an autolab minigato (Specta Physics) was used. Fo the detemination of paent dugs, this instument was opeated with coiled glass columns (2m x 6.5am D) silanized with Hexamethyldisilanize(HMDS) and packed with 17. V-17 o 37 V-17 on chomosob W-AW (1-12mesh); fo the detemination of metabolites, it was opeated with 17. V-17 on chomosob W-AW (1-12 mesh). Gas flow-ates wee: nitogen (caie), 3ml/min; hydogen, 2m1/min; and ai, 1ml/min. The tempeatues wee: 3C fo the injecto and the detecto, and column tempeatue at 2-22 C fo paent dugs; and 19-2C fo metabolites. All columns wee conditioned at 2C below the maximum ecommended tempeatue fo the elevant stationay phases fo 24 hous. Each column was then silanized twice in situ with samples of HMDS (Phase Sepaation LTD, Geat Bitain) befoe use. the appaatus used wee: lml capacity centifuge tubes with well-fitting scew caps (Soviel,, Leval lois-p eet, Fance); 15 ml stoppeed evapoating tubes with finely tapeed bases (Beckett, 1966); ]pl-s. G. E. micosyinge (Chomatogaphy Sevices,

68 -65- Wial, Geat Bitain); Pipetman micosyinges (Anachem, Bedfod U. K. ); thin laye chomatogaphy (TLC), TLC plates coveed with polyamide;.15mm thick (Schleiche and Schull, W. Gemany); Automatic shake (Recto, Denmak). MATERIALS The following mateials wee used. Acetone, dichloomethane, diethylethe, ethanol and methanol, all of anala gade and feshly edistilled; Anala anhydous sodium sulphate; ethe-washed sodium hydoxide solution N and.1n);. 1M glycine buffe which is made up of the following: - Anala glycine 7.55g Anala sodium chloide 5.185g Distilled wate to 1ml Potassiuntodide-glycine buffe which is made up of: - Anala potassium iodide 12.8g. ln Sodium hydoxide solution 4m1.1M glycine buffe 6m1 Potassium tiiodide solution which consists of: - Iodine ig Potassium Iodide 2g Distilled wate 2ml Neostigmine bomide (Koch-Light, Colnbook, U. K. ); pyidostigmine bomide, U. S. P; 3-Hydoxy-pyidine and 3. d imethylaminophenol (Aldich Chemical Company Limited, Gillingham, Doset, U. K); 3-hydoxy-N-methylpyidiniumbomide, 3-HNMP (RocheWelwyn Gaden, U. K); 3-hydoxyphenylti-methylammonium bomide,

69 -66-3-HPTMA (Koch-Light, Colnbook, U. K. ); 14C-3-HNMP and 3H-3-HNMP (The Radiochemical Cente, Amesham, U. K). TREATMENT F GLASSWARES It was found that all glasswae should be cleaned and silanized befoe use. Accodingly the following pocedueýin ode to incease the sensitivity of measuement of the dugs was used. A consideable quantity of the dug might be lost due to adsoption onto the walls of glasswae. Evapoating tubes and scew capped centifuge tubes wee cleaned by soaking in a 2.5% solution of RBS (Chemical concentates -RBS - Limited, London) in wate ovenight, then insed with methanol((mmecial Gade) and hot tap wate, followed by distilled wate. The tubes wee then died at 15 C. Afte cooling at oom tempeatue, all the tubes wee silanized by insing with a 37. solution of HMDS in chloofom. They wee then died at 25 C ovenight. Silanization of tubes should be epeated when necessay, at monthly intevals. DETERMINATIN F NESTIGMINE IN PLASMA Blood samples wee obtained by venous punctue and collected in hepainized polythene tubes. Afte centifugation (5g fo 1mins) the plasma samples ( ml) in a 1-ml glass centifuge tube was made alkaline (ph 1-12) with 2pl 5N sodium hydoxide solution and the make solution (6)jl; equivalent to 5ng base of pyidostigmine bomide in methanol was added. The alkaline solution was extacted with feshly edistilled diethyl ethe (2 x 6m1) by shaking vigoously by hand fo 2min. then centifuged

70 -67- at 3g fo 5min. to beak the emulsion and the etheeal extacts wee discaded. The emaining taces of ethe wee emoved by puging nitogen gas ove the aqueous phase. Potassium iodideglycine buffe (lml) was added to the ethe-washed plasma and the esultant iodide-glycine dug complexes wee extacted into feshly edistilled dichloomethane (2 x 6m1) using an automatic shake. Afte centifugation, the plasma laye was discaded and the bulked dichloomethane extacts wee died by shaking with a quantity of anhydous sodium sulphate (Ca_ 2'9). The wate-fee extact was tansfeed caefully into an evapoating tube and evapoated to dyness by a gentle steam of nitogen gas. Redistilled methanol (2pl) was added, using a Pipetman micosyinge, to edissolvethe died extact by votexing (5secs. ). A sample (2-5pl) of the methanolic solution was injected onto the gas chomatogaphic column with a 1p1 S. G. E. syinge. The concentation of neostigmine pesent in a sample was detemined fom the atio of the integated peak aea fo neostigmine to that fo the intenal make. A calibation gaph was pepaed by adding known amounts of neostigmine bomide to 3m1 of dug-fee plasma, coveing the concentation ange equivalent to 5. to 1ng/ml neostigmine base (Fig 3.1). DETERMINATIN F PYRIDSTIGMINE IN HUMAN PLASMA A pocedue simila to the one descibed above was used fo the detemination of pyidostigmine in human plasma using neosticmine bomide as the intenal make. A calibation gaph was pepaed, by adding known amounts of pyidostigmine bomide to

71 -68-3m1 of dug-fee plasma, coveing the concentation ange equivalent to 5. to 1ng/ml pyidostigmine base. DETERMINATIN F ANTI-ChE AGENTS AND THEIR METABLITES IN HUMAN URINE Neostigmine and its majo metabolite 3-hydoxyphenyltimethyl-ammonium bomide (3-HPTMA) as well as pyidostigmine and its majo metabolite 3-hydoxy-N-methyl-pyidinium (3-HNMP) could be sepaated by a TLC system consisting of a glass TLC plate (polyamide,.15mm thick)& a solvent system of methanol, stong ammonia solution &butan-2-one in the atio, of 15: 2: 6. Visualization of the TLC spots was caied out using Dagendoff eagent (Clake 1969) and unde UV light (Chan and Black unpublished obsevation 1977). This system was used to identify the paent dugs and metabolites extacted fom the uine samples using the pocedue descibed in the scheme (Fig 3.1). Subsequently it was found that the TLC pocedue was not sensitive fo the quantification of the dugs. A GLC system was then developed using a 2m glass column packed with 17. V-17 on chomasob W-AW(1-12 mesh). Extacts fom step 2 of the scheme (Fig 3.1) wee analysed fo the paent dug and the coesponding intenal, standad and extacts fom step 3 of the scheme wee analysed fo the metabolites using GLC system (17. V-17 on chomosob W-AW 1-12mesh). To measue the metabolites, 3-HPTMA., (a majo neostigmine metabolite)was used as the intenal standad fo the quantification of 3-HNMP(a majo pyidostigmine metabolite) and 3-HNMP was used as the intenal standad fo the detemination of 3-HPTMA.

72 -69- RESULTS 1. IN-PAIR EXTRACTIN F PARENT DRUGS AND METABLITES It was demonstated that the mean ecoveies of neostigmine and pyidostigmine fom plasma wee 857. and 897. espectively, using potassium iodide-glycine buffe at ph1 (Chan et al., 1976). In the pesent method the ecoveies wee impoved to 957, and 947. (at 6ng pe ml level) espectively fo neostigmine and pyidostigmine when all elevant glasswae was silanized in the pesent modified pocedue. This pocedue invaiably inceased the sensitivity of detection at a low concentation level. Fig 3.2. shows that the ion-pai extaction of the neostigmine metabolite (3-HPTMA) and the pyidostigmine metabolite (3-HNMP) fom the uine sample spiked with known concentations of the paent dugs(neostigmine and pyidostigmine) and thei coesponding metabolites was poved to be specific. Thee wee no TLC spots coesponding to the metabolites fom concentate of the Step 2 (Fig 3.1) extaction in the geneal scheme no wee thee TLC spots coesponding to the paent dugs extacted by Step 3 in the geneal scheme (Fig 3.1). The mean ecoveies of 3-HPTMA and 3-HNMP, using potassium ti-iodide as counteions at ph4 wee 87. at 1.6nmol level using a pevious pocedue. Simila value of ecoveies wee obtained when adioactive samples wee used (Waeing and Calvey, pesonal communication).

73 -7- STEP 1 1 o 3m1 uine, 5ng intenal makes, 2jil 5NaH (phll), 2x 6m1 diethyyethe Discad diethyl ethe to emove basic dug and lipid soluble substance. STEP 2 Aqueous phase, lml KI glycine buffe (phll), 2x 6m1 dichloomethane. Evapoate oganic extact to dyness, edisolve esidue with methanol (2»l), GLC quantititation fo paent dugs (2p1). STEP 3 Aqueous phase, 6pl 3NHC1 (ph4), 2p1 iodide solution, 2x 6m1 dichloomethane, acetone (9: 1) Evapoate oganic extact to dyness, edissolve esidue with methanol (2»l), GLC quantitation fo metabolites (2p1). FIG 3.1 SCHEME FR THE DETERMINATIN F NESTIGMINE, PYRIDSTIGMINE AND THEIR METABLITES IN URINE.

74 -71- Rf values oq :ý_.58 PYR NE PH NH STEP 1 STEP 2 FIG 3.2 TLC of ion-pai extacts of pyidostigmine (PYR), neostigmine (NE), 3-HNMP(PH) and 3-HPTMA (NH) in uine containing spiked concentation of 5pg each. TLC plates: glassplate (polyamide,.15mm thick), solvent system: stong ammonia solution: butan-2-one: methanol (2: 6: 15). Developing spay: Dagendoff eagent, Tempeatue: C.

75 -72- tin cý w w nwnn =: 3: na: 3 M oko Tw to K to MR CQ p w of W t-n bc fd Q Plh 2 vm o ný wý td «1 Kk Q w CL q* '1 1- W t1 n 1 ft K wto <a 1w v fd o o N R P. - R P" ld I-. b w,.-an ºp,a s pts xmn Cv. p' p ºI pe t+. pw j co M -. Vl ' cs 3 C ap. '. EýÖ "cx m n R Cy I ý K PU ý uelecla esponse, attentuation Ix 128

76 -73- TABLE 3.1 PERFRMANCE F GLC SYSTEMS (17. V-17 N CHRMSRB W-AW, 1-12 MESH)(a) RETENTIN SYMETRTb) RESLUTIN TIME FACTR BETWEEN (MIN) (.95 - MARKER fib) CMPUND IN METHANL 1.5) i>1.) 3-HPTMA bomide HPTMA-iodide complex TLC spot coesponding to 3-HPTMA 3-dimethylamino phenol 3-HNMP bomide HNMP-iodide complex TLC spot coesponding to 3-HNMP 3-hydoxypyidine (a) Tempeatue: injection, 3 C; oven, 2 C; detecto, 3C. Gas flow ates: nitogen (caie), 3m1/min; hydogen 2ml/min ; ai 1m1/min. (b) Bitish Phamacopeia (1968) specification fo GLc analysis.

77 GLC ANALYAIS F PARENT DRUGS AND METABLITES The espective etention times, symmety factos and esolution of the paent dugs (neostigmine and pyidostigmine) wee descibed by Chan et al. (1976). Fig 3.3 shows the chomatogam of neostigmine and pyidostigmine at 5ng/ml level. Table 3.1 and Fig 3.4 summaize the pefomance of GLC system fo the analysis of the metabolites (3-HPTMA and 3-HNMP). The etention times of these compounds indicated that 3-HPTMA and 3-HNMP (eithe as the bomide salts, o as the extact fom TLC spots, o as the iodide ion complexes) wee themally decomposed at the injection pet to thei coesponding tetiay analogues, 3-dimethylaminophenol and 3-hydoxypyidine espectively, which wee esolved by the GLC system. The mechanism of themal dequatenization of the paent quatenay amines has been discussed in detail by Chan et al., (1976) and Chan (198). The epoducibility of dequatenization at the injection pot, whee a glass line was inseted, indicated that the iodide complexes of neostigmine and pyidostigmine yielded tetiay amines which gave linea peak aea atios with thei coesponding intenal makes ove the ange of 5-1ng/ml (Fig 3.5). Similaly, esults wee obtained fo the analysis of the metabolites ove the ange of 5-1äg/ml (Fig 3.6). These gaphs wee found to be epoducible when epeated five times duing the studies. APPLICATIN The pocedue has been used to measue the concentation of pyidostigmine in human plasma afte a single intavenous injection

78 -75- PEAK AREA RATI.. N º+, u, wn ftj W I-ft 1 Qt H 1. - f9 ey C=. - ýt Z ' w x 3öo w ö C '"" ß/ W to _ a! =J" (D ) v 7' º M =' Q C i to v W ". 1 td.7 fd M ty n 'N M X K M Ce * i w CD to Pl b (A p i M 9 t t FA ý+"" m c a IT Wv1. %. D C Y :3. 9 a i eh ý (D i7 AW ö o 9 w!da?i fe t - o Cv (,) 1''- e- =, C W po UI p" WR y K tº g N 6+ fp D MK fp fe ;3 KK C-N Z. IJ1 (a fp l Pf to e e, 94 fö w M A e M" 8 wý. % o Q. -((n C lý E7 ý C e w e C M. =`< w po K X - g Ö fd 4 w X 'N T bw to K l K wo 9 C' 8BC + (A p C "w

79 -76- of the anti-che to twenty-five patients, that is descibed in detail in Chapte 4. DISCUSSIN This method (Method 1) is a selective and sensitive pocedue fo the isolation and subsequent detemination of some clinically useful anti-che and thei majo metabolites. Most peviously published methods based on spectophotometic detection, wee not sensitive enough to measue these quatenay amines. Methods based on themal dequatenization have been epoted fo a numbe of quatenay compounds, including acetylcholine (Szilazyi et al., 1972), thiazinamium (Jonkman et al., 1974) and neostigmine and pyidostigmine (Chan et al., 1976; Pohlmann and Cohan, 1977). Pohlmann and Cohan P (1977) in thei epot used methyiodide (pat of dequatenized poducts) as a measue of quatenay amines in the plasma. Howeve, this method suffes fom the lack of selectivity as othe quatenay compounds may also be ion-pai extacted and similaly assayed. The pocedue descibed above measues appaently, specifically the dequatenized tetiay analogues. Duing the sepaation of majo metabolites fom thei paent compounds, the phenolic H goups of the 3-hydoxy metabolites tend to ionize at alkaline ph. Thus, Step 2 in the geneal Scheme (Fig 3.1) pefeentially extacts the paent dugs. Babe et al., (1972) demonstated that 3-HPTMA, the majo metabolite of neostigmine, was not extacted at phl using tetaphenyl boon sodium as the counte Lou, They wee able to sepaate neostigmine and 3-HPTMA at two diffeent ph anges.

80 -77- The teatment of all glasswae with silylafing agent was found to be essential to incease ecoveies of dug. This is a geneally accepted pocedue fo woking with dugs at low concentation. This method was subsequently published (Chan and Dehghan 1978). METHD 2 THE SIMULTANEUS DETERMINATIN F NESTIGMINE AND PYRIDSTIGMINE IN HUMAN PLASMA AND URINE INTRDUCTIN The anticholinestase agents, neostigmine and pyidostigmine, ae the most widely used compounds in the teatment of myasthenia gavis. A ecent eview has indicated that the monitoing of plasma levels of the anti-che agents may have consideable value in the phamacotheapy of myasthenia patients (Chan 198). Peviously published GLC methods (including Method 1 descibed in page 64 ) fo the detemination of these dugs can only measue one dug at a time while the othe is used as an intenal make (Chan at al., 1976 and Chan and Dehghan 1978). Recently, Aquilonius et al., (1979) epoted an identical extaction pocedue using deuteated (d6) neostigmine as-the intenal standad and mass spectomete (MS) as the detecto fo the detemination of neostigmine in plasma. But the use of a GC-MS system is not always available in evey laboatoy, due to the high cost of such a system. The method (Method 2)

81 --78- descibes the syntheses of a seies of pyidostigmine analogues and the development of one fo use as a common intenal standad fo assaying neostigmine and pyidostigmine in plasma simultaneously. MATERIALS AND METHDS The method and mateials descibed fo the detemination of anticholinestease dugs (Method 1) in plasma was used with the following modification. The injecto and detecto tempeatue was inceased fom 3 to 35 C, and column tempeatue inceased fom 2-22C to 225C. the mateials used wee: TLC plate coated with silica gel.15mm thick (Schleiche and Schull, Dassel, W. G. ) and analogues of pyidostigmine (synthesized in own laboatoy). SYNTHESIS AND CHARACTERIZATIN F PYRIDSTIGMINE ANALGUES Vaious analogues of pyidostigmine bomide wee synthesized in thee stages. 1. The synthesis of acid chloides (CICNR2) was caied out accoding to Wene (1919). Dibutylamine, diethylamine o dipopylamine, equimola with pyidine, was added to phosogene (127. in toluene i. e., in excess) at C. The eaction by poducts wee emoved as follows Pyidine hydochloide by filtation and phosogene, toluene and pyidine by evapoation. The esidual yellow acid was mico-distilled unde vacuum to give the cabonyl chloides (about 67. yield) and the coesponding ueas wee distilled at a high tempeatue.

82 -79- The boiling points at 4mm Hg of diethyl, dipopyl and dibutyl cabonyl chloides wee 55 C, 88 C and 14 C espectively. 2. The cabamates of 3-hydoxypyidine wee synthesized accoding to Wust and Sakel (1951). The 3-hydoxypyidine was dissolved in wamed toluene by stiing. The appopiate acid chloide was added in 1.25fold excess. The eaction was stated by adding tiethylamine, equimola with the acid chloide and the mixtue was stied unde eflex fo fou hous. At the end of the eaction, the tiethylamine hydo chloide was emoved by filtation and the toluene by evapoation and the colouless esidual oil was mico-distilled unde vacuum to yield the appopiate 3-pyidyl-nsn-dialkyl cabamates. The chaacteization of the tetiay analogues ae summaized in Table The quatenization of the tetiay analogues fom Stage 2 (see Table 3.2) was caied out by eacting bomomethane (in excess) to toluene with the 3-pyidyl-n, n- dialkyl cabamates at oom tempeatue. The quatenay bomide was patitioned into wate. Toluene and wate wee initially emoved by evapoation unde vacuum and final poducts wee died ove phosphoous pentoxide. White deliquescent cystals wee poduced, which wee cystallized fom dy ethe/ethanol (1: 1) mixtue and stoed in dissicato befoe use. The poducts at each of the thee synthesis stages wee chaacteized by the measuement of boiling points at 4mmHg infaed spectomety (IR), gas chomatogaphy (GC) o thin-

83 -8- laye chomatogaph (TLC). The chaacteistics of the poducts of Stage 3 ae summaized in Table 3.3. Fom these analogues, NsN-dipopylcabamoyloxy-l-methylpyidinium bomide was consideed the most suitable compound fo use as a common intenal make fo the simultaneous detemination of neostigmine and pyidostigmine in human plasma. SIMULTANEUS DETERMINATIN F NESTIGMINE AND PYRIDSTIGMINE A pocedue simila to the one descibed ealie (detemination of neostigmine in page 66 ) was caied out fo measuing of neostigmine and pyidostigmine simultaneously, using the bomide salt of N, N-dipopylcabamoyloxy-l-methylpyidinium as the intenal make. The concentation of both dugs pesent in a sample was detemined fom the atio of the integated peak aea to that fo the intenal make. A coesponding calibation gaph was pepaed by adding a known amount of the dug to dug-fee plasma (3m1) coveing the ange of concentation between 5.ag to 1. ng/ml base, using 5ng/ml base of the intenal make (Fig. 3.9). The whole pocedue of the assay is summaized in Scheme (Fig 3.7).

84 -81- STEP 1 Plasma (3m1), 15ng intenal ma 2µl 5N NaH solution (ph-11) Discad diethylethe to emove basic dugs and 2x 6ml diethylethe lipid soluble substances STEP 2 Aqueous phase, Lml potassium Evapoate oganic extact iodide glycine, buffe (ph 11) to dyness, edissolve 2x 6m1 dichloomethane esidue with methanol (2p1) GC detemination fo anti-che dugs (2u1) FIG 3.7 A SUMMARY F THE ASSAY PRCEDURE FR NESTIGMINE AND PYRIDSTIGMINE IN PLASMA

85 -82- TABLE 3.2 CHARACTERIZATIN F 3-PYRIDYL-NN-DIALKYL CARBAMATES R II /- -- C-N IýR V CMPUND BILING IR GC RETENTIN TLC Rf* PINT (CARBNYL TIME (AT VALUES 4mmgHg) RESNANCE) (min) A. R-C2H5 136 C B. R-C3H7 144 C C. R-C4H9 168 C * TLC Silica gel plates, developing system consisting of methanol: stong ammonia solution: butan-2-one (15: 2: 6).

86 -83- RESULTS CHARACTERIZATIN F PYRIDSTIGMINE ANALGUES Table 3.2 summaizes the boiling points (at 4mm Hg), the IR chaacteistics (cabonyl esonance), GC etention times and TLC Rf values of the tetiay bases which wee subsequently quatenized to yield the coesponding pyidostigmine analogues. It is inteesting to note that when these tetiay analogues wee un on a non-pola TLC plate (polyamide) thei Rf values (.97) wee indistinguishable. Table 3.3 illustates the GC and TLC chaacteistics of neostigmine (E), pyidostigmine (A) and the thee synthesized pyidostigmine analogues (B, C and D). Thus, Compound C, the dipopylcabamoyloxy analogue was chosen as the common intenal make fo the simultaneous detemination of neostigmine and pyidostigmine. SIMULTANEUS GC ASSAY F NESTIGMINE AND PYRIDSTIGMINE IN PLASMA The espective etention times, symmety factos and esolution of neostigmine, pyidoatigmine and the chosen intenal make ae summaized in Table 3.4 Figue 3.8 shows the chomatogam obtained in the analysis of plasma samples containing 1ng/ml of the dugs. The mean ecoveies of neostigmine and pyidostigmine wee 947. and 937. (at 6ng/ml level) espectively. The epoducibility of dequatenization at the injection pot, whee a glass line was inseted, indicated that bomides o iodide complexes of neostigmine, pyidostigmine and intenal make yielded thei coesponding tetiay amines, which gave linea peak aea atios

87 -84- TABLE 3.3 GC and TLC CHARACTERISTICS F PYRIDSTIGMINE ANALGUES / -- C-N ýr R CH3B CMPUND GC RETENTIN TLC Rf TIME (Minutes) VALUES* A. Rs CH (pyidostigmine) B. R- C2H (diethylcabamoyloxy analogue ) C. R- C3H (dipopylcabamoyloxy analogue ) D. R- C4H (dibutylcabamoyloxy analogue ) E. Neostigmine (3-dimethy icabamoyloxyphenyl timethyl ammonium bomide) * TLC - Polyamide plates, developing solvent system consisting of methanol: stong ammonium solution: butan-2-one (15: 2: 6).

88 -85- TABLE 3.4 PERFRMANCE F THE GC SYSTEM (17. V-17 N CHRMSRB W-AW 1-12 MESH)(a) CMPUND IN METHANL RETENTIN SYMMETRYb) RESLUTIN TIME FACTRY BETWEEN (min. ) (.95- MARKER (b) 1.5) (> 1.) Pyidostigmine bomide o iodide-complex Dipopyl analogue bomide o iodide complex Neostigmine bomide o iodide complex (a) Tempeatue: injection pot 35 C; oven 225; detecto 35 C; gas flow ates: nitogen (caie gas), 3ml/min; hydogen, 2m1/min; ai 1m1/min. (b) Bitish phamacopeia (1973) specification fo GC analysis.

89 -86- LiL bgi{yf. i Vii 1AV m n m ýp K ý B w I v b a!p K K M M n R IV FIG 3.8 Typical chomatogam of plasma extact containing pyidostigmine (P)q intenal make (M) and neostigmine (N).

90 pg IM 9 W a Concentation (ng/m1) FIG 3.9 Calibation gaphs of neostigmine (neo) and pyidostigmine (py) using a common intenal make; (), methanolic solutions of the dugs; (") plasma extacts of the dug-iodide complexes. Each point epesents the mean of six expeiments.

91 -88- ove the concentation ange between 5 to 1ng/ml (Fig 3.9). These gaphs wee found to be epoducible when epeated six times. DISCUSSIN The detemination of plasma concentations of neostigmine and pyidostigmine simultaneously using a common intenal make (N, N-dipopylcabamyloxy-l-meth ylpyidinium bomide) was based on the ion-pai extaction of the quatenay ammonium compounds into dichloomethane. The oganic extact was analyzed by a gas chomatogaphic pocedue. The GC quantitation depends on complete dequateization of the quatenay amines, eithe as the bomide o as the iodide- complexes, to thei coesponding tetiay analogues, which ae then esolved by the GC system. 'This was achieved by applying a high tempeatue (35C) in the injection pot to ensue complete pyolysis of the quatenay moieties. A mechanism fo this themal decomposition of this goup of quatenay compounds was suggested in a eview by Chan (198). The pesent assay pocedue allows the simultaneous monitoing of the plasma levels of neostigmine and pyidostigmine when both of these anti-che agents ae used fo phamacotheapy of myasthenia gavis. The technique can detect as low as lng/ml of the dug in a 3m1 plasma o uine sample, but accuate measuement is achieved within the ange of 5 to 1n&/ml o highe.

92 The evidence of intesubject vaiations in the absoption of neostigmine (Aquilonius et al., 1979) and pyidostigmine (Calvey et al., 1977) suggests that the simultaneous monitoing of both of the anti-che agents in plasma should be impotant, as thee is a lack of infomation concening the effect of combined teatment with neostigmine and pyidostigmine in myasthenia gavis. This pocedue has been used to measue the concentation of neostigmine and pyidostigmine in the plasma of seventeen myasthenic patients, who wee teated with both dugs (combined dose). Fo 119 moe detail see Chapte 6 (page 12, Pat of this wok was pesented at the Bitish Phamacological Society Meeting (1979) and late on published (Davison et al., 198).

93 -9- CHAPTER IV DSE DEPENDENT KINETICS F INThAV NUS PYRIDSTI NE IN SURGICAL PATIFNTS.,

94 -91- CHAPTER IV Page No. Intoduction 92 Patients and methods 92 Calibation gaphs and detemination 95 Results 95 Discussion 15

95 -92- INTRDUCTIN In anaesthetic pactice, anti-che dugs ae widely used to evese non-depolaising neuomuscula blockade. In Geat Bitain, neostigmine methylsulphate (Postigmine) is most commonly employed fo this pupose. ccasionally pyidostigmine bomide is used, since its duation of action is appoximately 3% longe than neostigmine (Mille et al., 1974), and it has been suggested that it is less likely to induce significant bady- cadia Katz(1967). The plasma concentation of pyidostigmine was measued afte thee diffeent doses of the quatenay amine (36.2pg/kg, 72.4pg/kg and 144.8pg/kg) wee given to 23 sugical patients duing geneal anaesthesia. The elation between the plasma concentation of pyidostigmine and time was invaiably expessed as a bi- exponential equation, and the data was intepeted in tems of a two compatment mammillay phamokinetic model (Fig 4.1). PATIENTS AND METHDS Studies wee caied out in twenty-thee patients duing the plastic sugical pocedue. The subjects wee yeas old and thei body weight anged fom kg (see Appendix 4.1)., None of the patients had any clinical evidence of pe-existing hepatic o enal disease and the esults of pe-opeative haematological and biochemical assessment (i. e., haemoglobin and full blood count, plasma electolytes, uea, ceatinine, uic acid, inoganic phosphate, calcium, {on, biliubin, albumin, globulin and aminotansfease) wee usually within nomal limits (Appoximately 1% of the

96 -93- FIGURE 4.1 THE TW-CMPARTMENT PEN MDEL K12 K21 Tissue peipheal o The dug is administeed into the cental compatment by apid intavenous injection. K 12 and K21 ae the fistode distibution ate constants and K1 is the fist-ode elimination ate constant.

97 -9k- esults of biochemical assessment wee slightly outside the nomal ange and the significance of these esults is a matte of conjectue. ) ne subject (Patient 2) was eceiving concuent teatment with anticonvulsant dugs (phenytoin sodium 3mg/day and phenobabitone 9mg/day). All the patients wee pemedicated with nitazepam (1mg oally given on the pevious night). In studies using low o medium doses of pyidostigmine i. e., 36.2 mol/kg) o 72.4p&/kg (4n mol/kg), anaesthesia -}ig/kg(2n was induced by thiopentone sodium (2-4mg); suxametin nium bomide (1mg) with tubocuaine chloide (15mg) was used fo intubation. Anaesthesia was maintained with nitous oxide (6-77. ) and haltane (1-27. ) in oxygen, using intemittent positive pessue ventilation. Duing the couse of the opeation, a polyethylene can la was placed in a supeficial vein, attached to a thee-way tap, and kept patient by the infusion of saline. Pyidostigmine (36.2 pg/kg o 78.4 pg/kg) was injected intavenously as the bomide salt ove a 15 second peiod. Blood samples appoximately 8ml wee emoved at 2,3,4,5,7,1,15,2,3,4,5, 6,7,8,9,1,12 and 14min., and kept in lithium hepain blood tubes. In othe studies, highe doses of pyidostigmine 144.8pg (8n mol/kg) wee used to antagonise non-depolaizing neuomuscula block. In these conditions, anaesthesia was induced by thiopentone sodium (2-4mg), followed by tubncuaine chloide (45mg), and maintained with nitous oxide (6-7%) and halothane (.5%) in oxygen. At the end

98 -95- of the opeation, a polyethylene cannula was placed in a supeficial vein as descibed above, and neuomuscula block was evesed by pyidostigmine (144.8 pg/kg) administeed as the bomide salt and atopine sulphate (1.2mg). Blood samples appoximately 8ml wee usually emoved at 2,3,4,5,7,1,15,2, 4,6,8,1,12,14,16 and 18 min., and kept in lithium hepain. In all expeiments plasma was emoved fom blood samples as soon as possible by centifugation and stoed at -2C befoe analysis. CALIBRATIN GRAPHS AND DETERMINATIN Two calibation gaphs wee pepaed (using method 1 page 61+ ), by adding known amounts of pyidostigmine bomide to 3m1 of dug-fee plasma, coveing the concentation ange 5. to 1. ng/ml base, and 1. to 1. ng/ml base, using neostigmine bomide (5ng/ml base and 2ng/ml base espectively) as the intenal standad (Fig 4.2. a, b). Pyidostigmine was extacted fom plasma of sugical patients as an iodide-'complex, and its concentation fo thee diffeent doses (2,4 and 8nmol/kg o 36.2,72.4 and 144.8pg/kg espectively) was detemined by GLC method 1(page 64 ) and the esults ae summaized in Appendix 4.2; 4.3; and 4.4; espectively. RESULTS Afte intavenous injection of lowe doses of pyidostigmine (36.2p&/kg o 72.4pg/kg), the unchanged dug was apidly

99 -"96- eliminated fom plasma (Fig. 4.3.; 4.4; 4.6). In these studies, only tace amounts of pyidostigmine wee detected afte 9 minutes (patients 8,12 and 16; Appendix 4.2; 4.3). By contast, afte lage doses of pyidostigmine (144.8pg/kg) significant concentations of the dug (16-21ng/ml) wee still detectable in plasma afte thee hous (Fig 4.4; Fig 4.5; Appendix 4.4). In all expeiments, thee was an initial apid fall in the concentation of the unchanged dug, followed by a slowe decline. The elation between the plasma concentation of pyidostigmine and time was invaiably expessed as a biexponential function equation of the fom C Ae- Mt+Be- pt p Equation (19) using non-linea least-squae egession analysis (Appendix 4.5; 4.6; and 4.7) whee Cp is the plasma concentation at time t, and A, B, a and P ae constant. Values fo the fast disposition half-life (T/ a) and the slow disposition half-life (T ) wee deived fom each biexpeiential equation and intepeted in tems of a two compatment mammillay phamacokinetic model (Fig 4.1). The paametes of the model wee detemined by standad method descibed in Chapte II and summaized in Appendix 4.8; 4.9; 4.1. Afte intavenous injection of pyidostigmine (36.2µg/kg), the fast disposition half-life of the dug was min. (mean + SE), and its teminal half-life was min. (Table 4.1). The volume of distibution (Vd) of

100 -97- c 3 ý 3n.. n n P1 - p - "JI " 41 N Peak aea atio ý vi fd C CT fti ä ß' W t Pl eh o Q to fd QQ v- MZ Co cd 1 1 ( '? cd tt IY Vi Ph ' hff to fd td t -< II < a" M t (D In WVW dnd " CD 'l7 to (n W 1 R"? iý WW Mf II 32 IWW In W º'! fd. "y fd W"v x f ºl, N / p " C CD fd M fd W tjý n o po D R S fy W c Z 8 N v N v' pp ID m 1 "R to "1 p 9 C' 9 Co A v m In!AW N Z$ V 9 Peak aea N""" atio wtu, to h+" id CI (A Co mm A '. 7 W ID. In w v, cp t Al fd W fd fd f1 n f t Ko wo 8o n fp / M 7C i D 8

101 -98- log concentation of pyidostigmine (ng/ml) m y cm L.. (D R N G. m ýo ' W y R w* 9 13 g C7 L7 o' to to "t of C -3 V y fd a w CD m C to CD a. i1 C { C y i R Q co x b Co R G n K w p d 'd R fd a ß) y 8 fd t ºi w y R w M w QQ D -i C w fp A W. D cý w -,,, "i a w 3 m 3 w v j: in- 1 ji Ij. II I Ln 1-+ C AII/ ii viv /1 II /M /I If I/ M /1 II /UM /I /1 /fm /I /I/ II N /f II/ f1 n II Ill Ifif I I //111 to / If// to /Iu/ I VI / /N/ I I// II/ I All N/ // IU 1/ /II /N f a f A // //1 " I/ its It //I/ // of 1 // It Il /I / M/1" "/"/1w" //"" I lso I/M I/i It /I It I/ u"/ 4 vn s-fl Q 8 t_t m I- '3 i1 L N A n a b c K «e th b K a ýo n Pº W y M C K Pd. ID R y

102 -99- log concentation of pyidostigmine (ng/ml) K fd b a fd (A b ID (AD "Zi c) A.. tt. v. ÖÖ UI A y a C' fd f, n to ei- A M fd " K K ID n W a. A M 1 w ft a y 7 fp id fa t ft a M Co M M a m " fp t1 M C A C7 a "v K fd a K R7 l< M w a to + w 8 fd ýn w V w iý tt K fp p C to w A A K w v N oý w ti H w K H w 8 w ä v ýý I- b IIs II" 1I 11a 1I // II /It '11 a/ 1 ai 1I AV I "1I `N/ M " of N1I I/ 1N N IR Nn NI so f1 It I 11 N/ II " // I 11 II I 11 I/ / 11 I/ I Jill I/ I 111 / I 11 / If II /1/ t 1I 1NI IN I 1 / *7,. o 3 M n n D L m C, C'1 l) "4 N I C ýt w n

103 -1- M -3 b fd ' M co co { N1 (D CL 9 to ß) fd (A Vn e* fd dp ID fa pa m C') it. iý log concentation of pyidostigmine (ng/ml) Ui Ln ö I CK ED M D (a 1 C D" "' w 4n ED ß) [ º o wc n K `t fd i waw aa n C to w Pl n <I. fo ED v wg Mw A ha ' M ED ºi 8 CA M fc R v ED t f M fa Kw< E ED '%C p to Cy w w n a fo ýq Ph " co (a o < Cý Q w. c! w Da t to " t fd ED < m A 9w G A M v l Q Da 'C p1 /" > f ýº 1 S/ 1' 1 in II$ 1/ y J/ w II 9 1/ /1 I' 11 / 11 - II I s'i j+: /11f /", "1 l11 //f1 II#1 I11 /1au II II 11 /1 II 11 // tl 11 I/ 'll tl II Il ll I1 II 11 I1 Il 1 11 I1H1 11 II II 11 1 IJ /I II 1 il 1 11! 11 / fl /1 11 1n I1 Il 1 1J II ll 1 11 I /J il 1 II f11 It II ) I, - I 11 1 IJ CA I 44 Q Ct iii m Lt d`. T 'i I 1 m Eo K 1

104 -11- log concentation of pyidostigmine (ng/ml) ý w h+" h11 a ' I- to + fc N ßa7 in m m L I-. I- CD u+ o º C?D fd w R ID i MA fo w ' K ID to R Rw AD 'C 4 Ö ý 8 m l i 19 ii ý 3 ID W w 9 h+" ý S3 (D v W zý N e l% fl C» - JQ ý R!D. M Ö v Q Q NW + a JQ N ID 1 ID w " I ti Ip ID 'p fd f1 w 93 v Q n R w C. A %< 9..

105 E ä 5 E U) " v w Ö 1 J. 1 i+ LI 4, V 5 E a Time (min) FIG 4.7 Plasma concentation in one patient (6 o 18) who eceived diffeent doses of pyidostigmine (`1 ' 36.2ug/kg; p, 144.8ug/kg) on sepaate occasions. The dotted lines coespond to the exponential components in each expeiment.

106 _! o3- pyidostigmine ( mi/kg) was geneally geate than pesumptive values fo extacellula fluid volume and the cleaance (C) was ml/min/kg (Table 4.1). In the one subject (Patient 2) studied, these kinetic paametes wee not appaently affected by concuent theapy with phenytoin and phenobabitone. Afte highe doses of pyidostigmine (72.4pg/kg), the fast disposition half-life almost doubled to min. and the cleaance inceased to ml/min/kg. Diffeences between the othe kinetic paametes wee not statistically significant (Table 4.1). By contast, afte the highest dose of pyidostigmine (144.8pg/kg), diffeent esults wee obtained. The fast disposition half-life ( min. ) was simila to the values afte the lowest dose of pyidostigmine, although the teminal halflife ( min. ) was significantly geate (Table 4.1). In two patients (Patients 6 and 1 o 18 and 17 see Appendix 4.1) diffeent doses of pyidostigmine (i. e., 36.2pg/kg and 144.8pg/kg) wee given to the same subject on sepaate occasions. In both instances the slow disposition half-life was clealy polonged afte the lage dose of pyidostigmine (Fig 4.7). In addition, the total body cleaance was significantly deceased (to m1/min/kg) although the othe kinetic paametes wee not significantly affected (Table 4.1).

107 -1.4- Ki pit- ýr n u u n n u u n a J Fi, 8 w 8 S PV Q Q V V d ý 7C º3 w, oý z to v C) x o cn Cn m "3 v, º+ w pi ºt p e ä E ý. w w w ß, M M ', 1 a w w pº R7 p w. n f1 7 'v Co º. gp m- ID 1 Im A M ( Cl fd w to ' `< ºi p. ý d. a w fc fd < i7 w M M-i f M 1 e- w N fc fd to i-n >-+ + tti C ý i W 'G C C' Ö. N ". Ö u L D p N N ýuj "- äi U G f a, ä 1+ D x H "C P I j M f7 ffl I 1? PI F. ºi * M. ö ä N w C (Il N ', 3 h+ý lb M W T - t% N - ý V' N li 4- M `< L! ä g ý a ' fc Vl - h a 8 a i cl ä < {b C cd Co 1-4 ý { N " N N ýp N W W W ý A D V N 7d C+ýJ {b n hfl R K tlb W ý m " " N N ' C p q t %n lt 8 7 n ýp ' v ß1 F+ w K t N ý a a UI ä a ý v p - to ~ h N + V ed ýo w u º -ý v º-. o c. ä % %. o

108 -15- DISCUSSIN If pyidostigmine is distibuted and eliminated by fist-ode pocesses, the half-life of the dug should be independent of the intavenous dose. Howeve, in the pesent study the slow disposition half-life of pyidostigmine was pogessively polonged (fom min, to min) as the dose of the dug was aised fom 36.2pg/kg to 144.8pg/kg. The incease in the teminal half-life of pyidostigmine was also obseved in cossove studies in which diffeent doses of the dug wee administeed to the same patient on sepaate occasions. The explanation fo this phenomenon is obscue. The slow disposition half-life of pyidostigmine is dependent on the volume of distibution and the total plasma cleaance of the dug, and it is difficult to elate the pogessive incease in the half-life of the quatenay amine to coesponding changes in these pimay kinetic paametes. Thus, thee was no significant change in the volume of distibution of pyidostigmine as the dose of the dug was inceased; by contast, the cleaance of the quatenay amine was enhanced at intemediate dose levels (i. e., 72.4pg/kg) but significantly educed at high doses (144.8pg/kg). Connelly at al. (198) have ecently studied the kinetics of a two fold highe dose of pyidostigmine (35pg/kg), administeed ove a two minute peiod in patients with nomal and impaied enal function. Values fo the slow disposition and fast disposition half-lives of the quatenay amine in patients with nomal enal function wee longe than those obseved in the pesent study, although the plasma cleaance was simila. In both studies, the volume of

109 -16- distibution was geate than that pesumptive values fo plasma o extacellula fluid volume. Although the intepetation of the esults is difficult, and thei significance is a matte of conjectue, they may patially account fo the obseved diffeences in the duation of action of neostigmine and pyidostigmine in man (Mille et al., 1974). These diffeences ae difficult to explain on phamacodynamic gounds, since both quatenay amines ae closely elated chemically and inhibit junctional acetylcholinestease in an idential manne (i. e., by cabamylation of the esteatic site on the enzyme). Thus, neostigmine and pyidostigmine poduce the same inhibited enzymes with a halflife of ecovey of appoximately 3minutes (Wilson I. B., et al., 1961; Wilson I. B., 1966; Kitz, 1964). When the kinetics of the lowe dose of pyidostigmine (2nmol/kg) wee compaed with the esults of equimola doses of neostigmine (i. e., nmol/kg) duing the evesal of neuomuscula block (Calvey, et al., 1979), it is clea that thee ae two impotant diffeences in the kinetics of the quatenay amines. In the fist place, the volume of distibution of pyidostigmine ml/kg; mean + SE) is geate than neostigmine ( mlkg mean + SE), eflecting its moe extensive distibution and enhanced tissue up-take. Secondly, the cleaance of pyidostigmine ( ml/min/ kg; mean + SE) is at least five times geate than neostigmine ( ml/min/kg; mean + SE); this diffeence is pesumably elated to the diffeential enal elimination of the two quatenay amines, since pyidostigmine is less extensively

110 -17- metabolised than neostigmine (Sa mani et al., 1972). Simila esults have been obseved in expeimental animals (Bake et al., 1978), and pobably eflect diffeential steic and ionic popeties of the two quatenay amines. In consequence, the elimination half-lives of pyidostigmine ( min; mean + SE) and neostigmine ( min; mean + SE) wee simila and the diffeence between the mean values was not statistically significant (t -.777;.5>P>.4). Howeve, when these dugs ae used in anaesthetic pactice to evese non-depolaizing neuomuscula block, the dose of pyidostigmine is nomally 4 to 5 times geate than neostigmine, due to diffeences in the affinity of thei quatenay goups fo the anionic site on acetylcholinestease. In these conditions, the slow disposition half-life of pyidostigmine may be polonged, allowing significant ecabe mylation of acetylcholineestease to occu, and thus inceasing the duation of action of the dug.

111 -io8- CHAPTER V KINETICS F INTRAMUSCULAR NWSTIGMINE IN MYASTHENIA GRAVIS

112 -19- CHAPTER FIVE Page No. Intoduction 11 Methods AND patients in. Expeimental pocedue iii Results 111 Discussion 116

113 -U o- INTRDUCTIN Neostigmine methylsulphate has been used in the teatment of myasthenia gavis fo moe than foty yeas (see Chapte I page 19 and Chapte 3 page 51 ). In addition, the dug is widely used in clinical anaesthesia to antagonise the effects of muscle elaxants afte opeative sugey. In these conditions, the cleaance of the dug fom plasma has been studied using a novel gas-liquid chomatogaphic technique (see page 68)- Afte apid intavenous injection, neostigmine was eliminated in a biexponential manne, the fast disposition half-life (t/ a) of the dug was invaiably less than one minute and the slow disposition half-life (t anged fom minutes (Calvey at al., 1979). In geneal, these esults have been confimed by the subsequent wok of othes. Fo instance, an identical gasliquid chomatogaphic pocedue (using a mass spectomete as a detecto) has confimed that neostigmine is apidly eliminated fom plasma afte intavenous injection, although slightly diffeent values fo the slow disposition half-life wee obtained (Aquilonius et al., 1979). Both of these studies wee based on the measuement of neostigmine in plasma duing the antagonism of neuomuscula block. In the pesent wok we have studied the kinetics and metabolism of the dug in plasma afte its intamuscula administation to patients with myasthenia gavis. Intamuscula neostigmine methylsulphate is fequently used duing diagnosis o initial theapy of myasthenic patients, and

114 -111- knowledge of its kinetics in these cicumstances may well be of clinical significance. METHDS AND PATIENTS Studies wee caied out in five myasthenic patients who had a classical histoy of voluntay muscle fatiguability fo peiods of two months to two yeas. In most subjects, thee was chaacteistic electomyogaphic decement in esponse to the indiect stimulation of the affected muscle, and a positive esponse to edophonium chloide ('Tensilon') was obtained. None of the patients was concuently taking othe compounds o was on anticholinestease dugs. EXPERIMENTAL PRCEDURE In five patients, neostigmine methylsulphate (2.mg) and atopine sulphate (.6mg) wee given simultaneously by intamuscula injection. Samples of blood wee usually emoved by venepunctue at 15,3,6,9,12,15,18 and 24 min. Plasma was obtained by centifugation and stoed at -2 C befoe analysis. The concentation of neostigmine in plasma was measued by gas-liquid chomatogaphy using a nitogen sensitive detecto and pyidostigmine bomide (5itng/ml base) as the intenal make (see Chapte III page 68 ; and Figue 3.5). RESULTS In two myasthenic patients, neostigmine was detected in plasma within 15 minutes of intamuscula injection (Figue 5.1;

115 -112- Table 5.1). The dug was not pesent until 3D minutes afte administation in the othe thee subjects, and the plasma concentation declined fom ng/ml (mean S. E. M. ) to 9±1 ng/ml mean ± S. E. M. ) between 3 minutes and 12 minutes. In thee patients the neostigmine level was low at 15 minutes; in one of these subjects, the dug was still detectable at 18 minutes. No quatenay amine could be detected in plasma at 24 minutes (Table 5.1). The decline in the plasma concentation of neostigmine afte intamuscula injection was intepeted in tems of a one compatment model (Gibaldi, 1975). Estimates of the half-life (ti) of the dug wee obtained fom a semilogaithmic plot elating the plasma concentation of neostigmine to time between 3 and 15 minutes (The esults obtained at 15 and 18 minutes wee disegaded, since they wee only obtained in a minoity of the patients studied and wee usually inconsistent with the tend of the othe data points. ) In the five patients studied, the half-life of neostigmine anged fom minutes, coesponding to an appaent fist-ode elimina- tion ate constant (K) of.122 min 1 to.69 min-1 (Table 5.2). The extapolated concentation of neostigmine at zeo time (Co) was used to detemine the total appaent volume of distibution V,,,, fom equation (7) in Chapte II (page33). V_ dose (as neostigmine C base) Values fo the total appaent volume of distibution vaied. fom lites. Coesponding values fo total body cleaance as calculated fom the poduct of Vd and K, anged fom

116 " 2 ýýýý.a E 1 6 co,a Aj U) 5.. a 41 4l U) (1) 38.9 (2) 25.7 (3) " 25.7 (4) 22.9 (5) " Time (h) FIGURE 5.1 The plasma concentation of neostigmine in five myasthenic patients afte intamuscula administation of the dug. Each symbol (", o. ". and ) epesents the esults obtained in five diffeent subjects between 3 and 15min. The dotted lines indicate the extapolation to zeotime with Co as concentation in tng/ml.

117 -114- TABLE 5.1 Plasma concentation of neostigmine (ng/ml) in five myasthenic patients following an IM dose of 2mg of neostigmine methylsulphate. TIME SUBJECTS NS 32 NS NS ND 5.2 NS NS NS NS NS NS NS NS NS NS NS ND - non detected NS - non sample

118 TABLE 5.2 Kinetic paamete of neostigmine afte intamuscula administation. PATIENT tk K Vd Vd xk (min) (min-1) (L) (m1/mis) MEAN S. E. M biological half-life K elimination ate constant ýd total appaent volume of distibution Vd xk total body cleaance

119 ml/min (Table 5.2). DISCUSSIN. The pesent expeiments show that neostigmine is apidly absobed and eliminated afte intamuscula administation to patients with myasthenia gavis. In all the subjects studied, maximal concentations (18-32ng/ml) wee pesent in plasma between 15 and 3 minutes, although only small o undetectable concentations wee pesent afte 12 minutes. Thus, the pesence of detectable concentations of the dug in plasma appoximately coesponds to its duation of action in myasthenic patients. In the pesent expeiments as well as in pevious studies of Calvey et al. (1979) and Chan et al. (1976), the analytical method has been shown to detect neostigmine at concentations of 5-7 ng/ml in the plasma, and can be used fo the assessment of anticholinestease theapy in man. the authos (Aquilonius, 1979) suggested that the method only allows accuate measuement of neostigmine at concentations above 5 ng/ml, but this has not been shown in the pesent study. Afte intamuscula administation of neostigmine to five myasthenic patients, thee was a monoexponential decline in the concentation of the dug in plasma between 3 and 12 minutes. When the esults wee intepeted in tems of a one compatment model, the half-life of the dug anged fom minutes, the appaent volume of distibution fom lites. The half-life and intecept of the slow disposition phase was obseved afte intavenous injection of

120 -117- neostigmine (Calvey, 1979). By contast, thee may be a foty-fold diffeence in plasma concentation in patients eceiving oal neostigmine (Aquilonius 1979). Since this vaiability is not obseved afte intamuscula o intavenous neostigmine, it is pesumably elated to inte-individual diffeences in dug absoption and/o vaiation in the fistpass effect. As neostigmine is extensively metabolised in man the fist-pass effect may be of consideable impotance. The volume of distibution of neostigmine was appoximately equal to total body wate. Distibution studies in expeimental animal suggests that the dug is extensively localized in extaceilula fluid, live and kidney, but not in othe tissues (Somani, 1975). The esults of the pesent study ae not inconsistent with this intepetation. Total body cleaance of the dug anged fom ml/min; these values ae geate than the glomeula filtation ate, and suggest the occuance of tubula secetion o extensive metabolism of neostigmine.

121 -118- CHAPTER VI PART NE PLASMA LEVIIß F NFDSTIMWE AND PYRIDS4IGHINE IN MYASTB3VIC PATIENTS AFTER RAL ADMINISTRATIN. PART TW THE RELATINSHIP F PLASMA LEVELS F PYRIDSTIGMINE T CLINICAL EFFECT IN PATIENTS WITH MYAST KIA GRAVIS.

122 -119- CHAPTER SIX Page No. PART NE: Plasma Levels of neostigmine and pyidostigmine 12 in myasthenic patients afte oal administation Intoduction 121 Patients and methods 122 Detemination of plasma concentation of the Anti - ChE dugs 123 Results 123 Plasma neostigmine and pyidostigmine 131 Discussion 131 PART TW 135 Intoductoin 136 Patients and methods 136 Results 138 Discussion 145

123 -12- PAS PLASMA LEVELS F NIDSTIGMINE AND PYRIDSTIM+ANE IN MYSTHFMC PATIP2I AFTER RAL ADMINISTRATIN.

124 -121- INTRDUCTIN ve the past foty yeas, anticholinestease (Anti-ChE) agents such as neostigmine and pyidostigmine have been used as the fist line of teatment fo most patients with myasthenia gavis and pyidostigmine is the most widely used dug. Howeve, only until ecently disposition and phamacokinetic studies of these agents have been caied out in man due to ecent advance of analytical techniques. The metabolism of pyidostigmine in man was studied by Konfeld et al., (1971) using 14_C_ labelled analogue and by Somani and othes (1972) using mass spectomety. Cohen and colleagues (1976) monitoed plasma concentation of pyidostigmine in myasthenic patients and demonstated that thee was a possible elationship between plasma levels of the dug and clinical esponse. Recently, Calvey and Chan (1977) have shown that thee was a consideable vaiation in the bioavailability of pyidostigmine among six myasthenic patients and that the plasma concentation of the dug was invaiably positively coelated with the impovement in neuomuscula tansmission in patients with typical electomyogaphic decement using the adducto pollicis.. The phamacokinetics of neostigmine afte intavenous administation to sugical patients duing the evesal of neuomuscula block have ecently been investigated (Aquilonius et al., 1979; Calvey et al., 1979). Both studies suggested that neostigmine was eliminated fom plasma biexponentially. In a sepaate study on intamuscula

125 -122- neostigmine in myasthenic patients, the kinetics wee best descibed by a one compatment model (Soman& et al., 198). Howeve, thee is no infomation on the kinetics of the two anti-che-agents when both neostigmine and pyidostigmine ae used in sevee cases of myasthenia gavis. Recently, we have developed a gas-liquid chomatogaphic method which can detemine simultaneously the plasma concentations of neostigmine and pyidostigmine using a synthesised pyidostigmine analogue, n, n-dipopylcabamoyloxy-n-methyl pyidinium bomide, as a common intenal make (see Chapte III pat the two). This pat of1wok is concened with the concentation of these two anti-che dugs in the plasma of seventeen myasthenic patients who wee stabilized eithe by pyidostigmine only, o on both neostigmine and pyidostigmine theapy. PATIENTS AND METHDS SUBJECTS Studies wee caied out on eight myasthenic patients who wee stabilized on vaying amounts of oal pyidostigmine bomide only (appendix 6.1), and nine patients who wee maintained by the oal administation of neostigmine and pyido. stigmine (Appendix 6.2 and 6.3). In conjunction with the anti-che teatment, some patients wee also given pedniso]a ne and azathiopine suppoting theapy. PRCEDURE The dosage schedule of each patient was not modified duing the study. Blood samples. (usually 1 ml) wee emoved by

126 -123- venupunctue at two houly intevals afte the initial moning dose (Appendix 6.4), duing the waking day (8.am to 8.pm). The samples wee kept in blood tubes containing lithium hepain and plasma was pomptly obtained by centifugation and stoed at -2 C befoe analysis. DETERMINATIN F PLASMA CNCENTRATIN F THE ANTI-ChE DRUGS. The plasma concentations of neostigmine and pyidostigmine wee detemined by the gas-liquid chomatogaphic technique descibed in Chapte III (page 8) using a co= wn intenal standad n-n-dipopylcabamöloxy-n-methyl pyidinium bomide. A coesponding calibation gaph was pepaed by adding known amounts of the dug to dug-fee plasma (3ml), coveing the ange of concentations between 5. ng to 1. ng/ml base using 5ng/ml base of the intenal make (Fig. 3.9 page 87 ). RESULTS PLASMA PYRIDSTIGMINE Plasma levels of pyidostigmine wee monitoed fom 8.am to 8.pm in eight myasthenic patients, who wee stabilized on intemittent dosage fom 6 to 54 mg oally. In these patients the maximum plasma concentation thoughout the whole waking day anged fom 12.4 to 64.5n&/ml, and the minimum fom 1.3 to 25. ng/ml (Appendix 6.5). Typical plasma pofiles of pyidostigmine fo patient 1 (lowest daily dose) and patient 5 (highest daily dose) ae shown in Fig 6.1. The aea unde plasma concentation-time cuves (AUC) of pyidostigmine in these patients anged fom to 456.ng/ml-1hou, ove the twelve hou peiod (Fig 6.2).

127 E 5 C 3.., FE. n ö 1 -a L T d 7 cv C 5 C V Co E N cu 3 a, 1 FIG 6.1 1ýýb Time (hs) Typical plasma pofile of pyidostigmine fo patient 1 (lowest dug dose 6mg/day) and patient 5 (highest dose 54mg/day) afte oal administation, (b), (+) epesent oal doses of pyidostigmine 6mg and 12mg espectively.

128 o 2 (1) AUC = (2) Auc = 292 6o 4 2 (3) AUC = (4) AUC = 37 6 (5) AUC = 456 (6) Auc = Co 2 6 (7) AUC = 3495 (8) AUC = o- 2 8am pm 8am pm time (hous) Fi. 6.2 The aea unde plasma concentation-time cuve (AUC) of pyidostigmine in 8 patients (anged fom to 456. ng/m1-1 hou) ove 12 hou peiod.

129 C) L s. n C a, L 3. G3 9- CL) "C ca C, FIG Total daily dose of pyidostigmine ( mg ) Diect linea elationship between the aea unde plasma concentation- time cuves (AUC) and total daily dose in the fist goup of myasthenic patients ( ".95. )

130 E v 3 v E N Co ýýb1bbd Time (hs) FIG 6.4 Plasma concentation of neostigmine ( ---- ) and pyidostigmine (S") in a myasthenic patient (Patient 15) on an odinay dose schedule, I epesents oal doses of neostigmine (3mg) plus pyidostigmine (6mg) at indicated intevals, espectively.

131 CD ö - 8. E 6. Cn c CD s.. X 4. C, w D 9 Q Total daily dose of pyidostigmine ( mg ) FIG 6.5 Thee is no linea coelation between AUC of pyidostigmine and total daily dose in this goup of patients who eceived both neostigmine and pyidostigmine theapy ( =-.8).

132 -129- TABLE 6.1 Plasma concentations and bioavailability of pyidostigmine between 8.am and 8.pm afte oal administation of intemittent doses of pyidostigmine only. PATIENT DAILY DSE F MIN. and MAX. AREA UNDER PYRIDSTIGMINE CNCENTRATIN PLASMA CNCENTRATIN- BRMIDE (mg pe ml) TIME CUIVE mg (nit ml h)

133 -13- TAE BL 6.2 Plasma concentations and bioavailability of pyidostigmine between 8.am to 8.pm afte oal administation of intemittent doses of neostigmine and pyidostigmine. PATIENT DAILY DSE F MIN. & MAX. CNCENTRATIN AREA UNDER NESTIG- PYRID- of PLASMA MINE STIGMINE NESTIGMINE PYRIDSTIGMINE CNCENTRATIN- BRMIDE BRMIDE TIME CURVE mg mg (ng (pyidistigmine) (nit pe ml) _ pe ml) (nit/ml h) _ ND ND ND ND ND ND ND ND ND - not detected

134 When the AUC was plotted against the total daily dose of pyidostigmine fo all eight patients a linea elationship was obseved (Fig 6.3). PLASMA PYRIDSTIGMINE AND NESTIGMINE In only one of the nine patients (patient 15) both neostigmine and pyidostigmine wee detected in the plasma (Fig 6.4). In this goup of patients, the plasma concentations of pyidostigmine wee highe and moe divesified; the minimum ange was 5.4 to 6. ng pe ml and the maximum ange was 15.3 to 144. ng pe ml (Table 6.2). No linea coelation between AUC of pyidostigmine and total daily dose was obseved in this goup of patients who eceived both neostigmine and pyidostigmine theapy (Fig 6.5). DISCUSSIN In the pesent investigation, the eight myasthenic patients wee stabilized on widely diffeent daily dose of pyidostigmine (Appendix 6.3 and Table 6.1). Although the concentation of pyidostigmine in plasma, vaied between 12.4 to 64.5ßg/ml (maximum ange) and 1.3 to 25. ng pe ml (minimum ange) thee was a diect elationship between the AUC and total daily dose of the dug in these eight patients ove the twelve hou peiod of investigation. In an investigation on six myasthenic patients, the peak plasma concentation of the dug anged fom 44.2 to 84. ng pe ml; and in most cases the level was maintained between 2 to 6ng pe ml in a peiod of eight hous, when the dose of pyidostigmine was between 6 to 66 mg pe day (Calvey and Chan 1977). These obsevations

135 -132- suggest that the widely diffeent dosage equiement of pyidostigmine in myasthenic patients may be patly elated to intesubject vaiation in dug absoption and disposition and to vaying seveity of the disease. The pesence and significance of intesubject vaiation in the oal absoption of pyidostigmine is difficult to demonstate and assess. In a pevious study, the AUC of pyidostigmine in 3 myasthenic patients was maintained in a naow ange fou hous afte the same oal dose of 6mg (Calvey at al., 1977) suggesting that absoption of the dug fom the gut is elatively constant. In the pesent study, the plasma level of pyidostigmine was monitoed ove a longe peiod of time (up to twelve hous afte the fist oal dose), and thee is a positively diect elationship between the AUC of pyidostigmine and total daily dose duing this peiod, =.95 (Fig. 6.3). These obsevations ae in diect contast to that fo one myasthenic patient, whose bioavailability of the dug was inceased six fold by doubling the dose of pyidostigmine bomide fom 3 to 6mg (Calvey at al., 1977). Pehaps thee is also an intasubject vaiation in the absoption of the dug. In geneal, quatenay amines cannot eadily penetate lipophilic cellula membanes in the gut, due to thei complete ionization at all ph. Some quatenay amines ae known to fom ion-pai complexes with intestinal components such as mucin o bile salt, which ae eadily extacted into oganic solvents (Levine and Clak 1957; Schanke, 1963). Thus, in the pesent expeiments, thee wee fequent fluctuations in

136 -133- the plasma concentation of pyidostigmine which wee not appaently elated to dosing intevals. These fluctuations wee not obseved afte intavenous injection of the dug (see Chapte IV). In patient 1 (Fig. 6.1), thee was a seconday ise in plasma concentation afte a single dose of pyidostigmine bomide (6mg). This obsevations is consistent with pevious obsevations (Calvey et al., 1977). The seconday ise may be elated to the fomation and absoption of lipophilic ion-pai complex although the actual mechanism of absoption of pyidostigmine is unknown. The pocesses of dug absoption and disposition, may be influenced by the pesence of othe dugs. The enal cleaance of pyidostigmine was epoted to be educed by concuent administation of some tetiay amines which ae patially pesent as cations at physiological ph and may comp e, to with pyidostigmine fo enal tubula tanspot (Chan and Calvey, 1978). f the nine myasthenic patients (patients 9-17, Appendix 6.3 and Table 6.2), who wee maintained by both oal neostigmine and pyidostigmine theapy, only in patient 15 both dugs wee detected in the plasma. Two possible explanations may be consideed fo this obsevation. Fistly, the bioavailability of neostigmine was estimated to be 1-2% of the ingested dose and consideable inteindividual diffeences in the absoption of neostigmine afte oal ingestion of the dug wee obseved Aquilonius et al., (1979). The low bioavailability of oal neostigmine is pobably due to its extensive metabolism by the live afte 'fist-pass effect'(somani et al., 1972). The

137 -134- sampling pocedue in the pesent study might have missed the peak concentation o the assay pocedue was not sensitive enough to detect concentation appoaching picogamme ange. Secondly, the possibility of dug-dug intaction between neostigmine and pyidostigmine at the absoption phase should not be uled out. Thee was no diect coelation between the AUC of pyidostigmine and total daily dose in these patients (9-17) who wee teated with both anticholinestease dugs (Fig. 6.5), compaed with pyidostigmine only (Fig. 6.3). The highest and lowest concentation anges of pyidostigmine in patients 9 to 17 wee highe and moe divesified than those of patients 1 to 8 (Table 6.1 and 6.2). When the AUCs of pyidostigmine of all seventeen patients wee plotted against total daily dose of the dug, thee was no coelation ( -.15). Thus, pyidostigmine appeas to compete fo oal absoption with neostigmine. Patient 15 was eceiving the lowest daily dose of pyidostigmine (18mg) and in whom neostigmine was detected in the plasma (Fig 6.4, Table 6.2). These obsevations suggest that if thee is a coelation between clinical esponse and plasma level of anticholinestease dug in myasthenia gavis the concuent administation of oal neostigmine and pyidostigmine may not benefit the patients. A combination of intamuscula and oal outes of these two quatenay amines may be moe advantageous. Howeve, futhe studies should be caied out to substantiate the pesent obsevations of inteaction between neostigmine and pyidostigmine and to elucidate the mechanism of absoption of these dugs in the gut..

138 -135- PART TW The elationship of plasma levels of pyidostigmine to clinical effect in patients with myasthenia gavis. ACKNWLEDGEMENT: The clinical studies of global assessment of Muscle Powe and fatigueability in myasthenic patients and the detemination of antibodies in these patients plasma wee caied out by D. Susan C. Davison in The Radcliffe Infimay, xfod.

139 -136- INTRDUCTIN Pyidostigmine bomide (mestinon) is one of the main anticholinestease dugs used in the teatment of myasthenia gavis (Dachman, 1978). Vey little is known of the effective plasma concentation of the dug in myasthenic patients, although the daily dosage equiement may vay fom 3 to 2 mg pe day (Geene 1969). Thee was a possible elationship between plasma pyidostigmine levels and clinical esponse in myasthenia gavis (Cohen et al., 1976). Recently, Chan and Calvey (1978) showed that in fou patients with typical electomyogaphic decement in the adducto pollicis, the plasma pyidostigmine levels wee diectly elated to the effect on neuomuscula tansmission. The pesent study was undetaken to examine the elation between plasma levels of pyidostigmine and a global clinical evaluation of muscle powe and fatigueability such as could be caied out duing out-patient consultation. The discovey of a positive coelation between a global assessment of muscle powe and plasma levels of pyidostigmine would be a valuable aid in optimising dosage schedules in individual patients. PATIENTS AND METHDS Two male and seven female patients between the ages of 19 and 58 yeas with histoies of myasthenia gavis of two to twenty-two yeas duation wee studied; five of them had had thymectomies and thee wee being teated with pednisolone on altenate days. The total daily dose of

140 -137- pyidostigmine anged fom 6 to 18 mg (Table 6.3). A diagnosis of myasthenia gavis was made when two of the following citeia wee fulfilled: - (a) A chaacteistic electomyogaphic esponse (Geene 1969). (b) A positive edophonium test (Geene and, 1969), (c) An excess of auto antibody diected against human muscle acetylcholine eceptos (Geene 1969)., None of the subjects was seveely disabled by the disease and infomed consent was obtained. Patients wee admitted to hospital fo a peiod of twenty-fou hous and maintained on thei nomal dug egime duing that time. All subjects eheased a outine of timed execises in ode to test a vaiety of diffeent muscle goups fo susceptibility to fatigue befoe stating the expeiment. Details of the outine ae summaized in Table 6.4 togethe with the end points. An individual test took a maximum of twenty minutes to complete, being teminated afte two minutes if no fatigue occued. The test was pefomed immediately befoe each blood sample was taken. Between the hous of 8. and 2.ý(Appendix 6.5) venous blood samples (1ml) wee taken at oughly two hous intevals, the sampling times occuing at one to fou hous afte an oal dose of pyidostigmine. Immediately afte the blood sample was taken it was mixed with lithiumhepain in the blood tube and the plasma was sepaated by centifugation and stoed at -2 C befoe analysis. The concentation of

141 -138- pyidostigmine was subsequently measued in duplicate by gas liquid chomatogaphy (see Chapte VI, Pat ne). A small quantity of plasma set aside to measue the concentation of acetylcholine ecepto antibody using acetylcholine ecepto 1251-bungaotoxin pepaed fom human calf muscle, labelled with as the antigen. RESULTS Fo each subject, each sepaate execise testing a paticula function was evaluated acoss all six tials in two ways; the tials wee anked to 5 fom wost to best pefomance and they wee also ated on a continuous scale with best and wost set 1 and espectively. The global assessment fo each tial was simply expessed as the aveage ank o scale scoe calculated fom the eight diffeent tests. These wee then plotted against plasma concentation of pyidostigmine in ng/ml (Fig. 6.6 and 6.7). Regession analysis demonstated (using a compute pogam, Appendix 6.6) a tend towads a positive coelation between a global assessment of muscle powes and plasma concentation in five of the nine subjects (Fig 6.6), but in only two of them was showed significance at pg.5 level (Patients 8 and 16). f the emaining fou, one showed a negative coelation at p <.1. The findings ae summaized in Table 6.5. No single test of function, e. g., vital capacity, coelated moe closely with plasma dug level than the global assessment. Diunal vaiation in plasma levels of pyidostigmine and global scoes fo muscle stength wee obseved in most subjects,

142 -139- an 1 i) f1 CD Kp DC D' o :3 %9 e +'9 MK Aº KK ý'c cý a ºi K ýg mmw wat º Ko P1 %4 ä º Ko ic Ic a wwt KK gm w ia+ NK owg wn CD D -4 C+ fý 1 a t Z, w N - to ' U ' cn w ö o g "a> c o a o Co ö c Lii f'' W J W V W N v N W' k Gov ha ý!any 1 cn tig o i n wv RW M D fa V md m cs R º1 Cw " Lf CD %< 1 m m b e P-4 m- l" me p 9 fa fa W ^i w H. w ol I. ob ob R C o. b.. ýý D -P, D 1.41, D ý y to N 9- F CA % (4 w C2N -4 cj% vi vý vý xxxxxxxxx ööööööööö tij 65 cýý ýcö - + d Q fýc

143 -14o- TABLE 6.4 THE RUTINE F TIMES EXERCISES AND MEASURES USED T ASSESS SUSCEPTIBILITY T MUSCLE FATIGUE INSTRUCTIN END PINT Look staight ahead and fix on an Widest pat of the palpebal object at eye level fissue measued using a tanspaent ule calibated in millimetes. While facing fowad, fix eyes a point above you head. Diploplia fixation. o loss of Hold ams outstetched, hoizontally fom the shouldes. Gip with each hand in tun the inflated sphigmomanomete cuff as tightly as possible. When ams dop below the hoizontal. When the pessue egisteed on the manomete dops to one half the maximum pessue. Vital capacity Best of thee ties. While lying supine aise each leg in tun and hold it at 45 to the hoizontal fo as long as possible. When the heel touches the bed. While lying supine, aise the head off the bed and hold it thee unsuppoted fo as long as possible. When the occiput bed. touches the Walk a distance of 75 yads, as fast as possible. Cossing the finishing line.

144 -14i- D V ý 1n iý WN C' y CD Ti n H "s m ö p wn" º o. N N N m- e - % 41 u + to ýd x ' N u Hy W w hi w 4> 4 41 p- w Qs W s.. s u' t' -- N ~ \ f7 9 Z Cý ý Z m ßý R w. C1 "C ºh C to "p w 93 ß+ W N m- in fd Wd m ID ºi ' X M ß. w ooo oli Z4 L Z4. v D Vi Vi ýmc, -% : o cs x4l pso (Dz CIf S w C3 N w R b fp i M oä Lfl N co N 1+ A- + " ýt fp! ý7 Ö vý y n tn P1 f f W i b to v p. ý C) a v v v v v v v v b t%) ý ' o o ºd o IV V P P a n P1 to : 7'

145 -142 TABLE 6.6 The diunal vaiation in plasma concentation of pyidostigmine and pefomance in the test outine, the aveage of the thee measues befoe and afte noon ae compaed. SUBJECT BEFRE NN AFTER NN PLASMA SCALE PLASMA SCALE LEVEL SCRE LEVEL SCRE 1 1.7n&/ml ng/ml

146 g 6.EA A A / oý E3 4 / / " Patients Coelation 3 coefficients 1(A) s.8 16( ) =.72 w / 8() -.55 y 7() = (3) -.61 A U) v / Plasma concentation of pyidostigmine (ng/ml) FIG 6.6 Five patients showing tend towads moe actual positive coelation between plasma levels of pyidostigmine and assessment of muscle stength.

147 d W to v y L'. Co PLASMA CNCENTRATIN F PYRIDSTIGMINE (ug/mi) FIG6.7 Fou patients showing low o negative coelation between plasma levels of pyidostigmine and global assessment of muscle stength.

148 -145- both muscle stength and dug level being highe in the moning than in the aftenoon (Table 6.6). DISCUSSIN Studies of plasma levels of anti-che agents in myasthenia gavis ae equied since alteation of doses of oal anti- ChE dugs depend lagely on the patient's own subjective esponse. The edophonium test ( Geen, 1969) to detemine optimal dosage is fequently difficult to intepet because the effect vaies at diffeent times in the same day and on othe occasions it may impove function of some muscles, but eithe does not alte o wosen the stength in othes. It might, theefoe, be pedicted that simila difficulties would be encounteed with the intepetation of seum levels when coelated to clinical signs. In addition, the phamacokinetics of pyidostigmine ae pooly undestood and thee is no good eason to pedict that such a coelation should exist. Nevetheless, some pevious studies suggest that a elationship between seum level and esponse may exist. in one such tial (Cohen, et al., 1976), nine myasthenic patients wee examined at thity minute intevals fo six hous afte dug administation, and impoved moto stength subjectively was appaently paallelled by seum pyidostigmine concentations, but the authos felt that the clinical esponse was not quantifiable. In anothe study (Calvey et al., 1977) wide vaiations in the oal dosage (6 to 66mg/day), nevetheless poduced a elatively naow plasma pyidostigmine ange (2 to 6mg/ml). In an ealie study ( Chan and Calvey, 1977), fou patients with

149 -146- unsatisfactoy contol had lowe seum levels than those who wee well contolled. The stength of these patients impoved when the seum pyidostigmine inceased and they demonstated that the oiginal low levels wee due to malabsoption. A futhe type of coelation was made on fou patients with typical electomyogaphic decements in adducto pollicis consistent with myasthenia gavis ( Chan and Calvey, 1977). Thee was a positive coelation between the concentation of pyidostigmine plasma and the effect of the dug on neuomuscula tansmission. In the pesent seies of nine patients, a wide vaiety of execises each testing paticula functions was thought impotant, due to possible vaiations in the stength of diffeent muscle goups in the same patient. In addition, the clinical esponses wee quantified satisfactoily. In only two subjects was thee a significant coelation between muscle powe and plasma concentation; thee othe patients showed appaent positive coelation. No single test of moto function coelated moe closely with plasma dug level than the global assessment. In addition, the pesence o absence of a possible coelation between muscle powe and plasma concentation was not appaently elated to the duation of the disease, addition pednisolonetheapy o thymectomy.

150 -147- CHAPTER VII FINAL DISCUSSIN

151 -148- Chapte seven Page No. Detemination of Anti-ChE dugs & metabolites in biological fluids. 149 Kinetics of IV pyidostigmine in sugical patients. 151 Kinetics of IM neostigmine. 162 al neostigmine & pyidostigmine, & the 153 coelation of clinical esponse to plasma pyidostigmine in myasthenia gavis.

152 -149- DETERMINATIN F ANTI-ChE DRUGS AND METABLITES IN BILGICAL FLUIDS Quatenay amines in geneal, ae stong bases which have pka values appoaching fouteen and ae completely ionized in all biological fluids. Absoption fom the gastointestinal tact is incomplete and even neglible fo cetain quatenay ammonium compounds such as d-tmbocuaine, pancuonium and elated neuomuscula blocking agents. Distibution in the body is usually esticted to the extacellula fluids as they tavese cell membanes with difficulty (Golstein 1974) and elimination is mainly by excetion of the unchanged dug in the uine o in the bile and also by metabolism in the live (Dal Santo 1969). Thus, the chemical and distibution-elimination chaacteistics of quatenay amines will influence thei extaction pocedue fom biological fluids. In geneal the measuement of dugs in plasma o uine samples afte theapeutic doses involves a peliminay extaction of the dug into a suitable oganic solvent. A subsequent concentation of the oganic extact and the final measuement of the dug in the concentate by a sensitive and selective instumental technique. Most peviously published methods, based on spectophotometic detection, adio isotopic techniques and enzymatic pocedues wee not sensitive enough o selective and these investigations ae expensive o time-consuming. Analysis based on gas-liquid chomatogaphic techniques have been used fo the quantitation of pyidostigmine and neostigmine (Chan 1976). In the development of the method, teatment of all glasswae with silylating agent was found to be essential to incease

153 -15- ecoveies of dug to 957, and 94% (at 6ng/ml level) fo neostigmine and pyidostigmine espectively (see Chapte III). The ion-pai extaction of neostigmine, pyidostigmine and thei metabolites was poved to be specific using TLC and GLC methods. The extaction of paent dugs has been achieved at ph = 11, and the limit of quantitation is ing/ml using 1%V17 on chomosob W-Aki. The extaction of the metabolites of neostigmine and pyidostigmine has been done using potassium ti-iodide as counte-ions at ph=4, and the quantitation is possible down to 5 ng/ml in uine using 3m1 samples. The synthesis of a seies of pyidostigmine analogues and the development of one (N, N-, dipopylcabomoyloxy-l-methyl pyidinium bomide) fo use as a common intenal standad fo assaying neostigmine and pyidostigmine in plasma simultaneously, has also been achieved (Chapte III). The assay involved a peliminay ion-pai extaction of the dugs and the intenal make fom plasma using potassium-iodide glycine buffe. The assay pocedue allows the simultaneous monitoing of the plasma levels of neostigmine and pyidostigmine when both of these anti. ChE agents ae used fo phamacotheapy of myasthenia gavis. The evidence of intesubject vaiations in the absoption of neostigmine (Aquilonius, 1979) and pyidostigmine (Calvey and Chan, 1977) suggests that the simultaneous monitoing of both of the Anti-ChE agents in plasma should be impotant, as thee is a lack of infomation concening the effect of combined teatment with neostigmine and pyidostigmine in myasthenia gavis. The methods developed in the pesent study

154 -151- epesented a significant impovement ove those systems peviously available. KINETICS F IV PYRIDSTIGMINE IN SURGICAL PATIENTS Neostigmine methylsulphate (postigmin ) is one of the Anti-ChE dugs which is widely used to evese non-depolaising neuomuscula blocks in anaesthetic pactice. In the study of IV pyidostigmine (2 nmol/kg) compaed with the kinetics of appoximately equimola doses of neostigmine (i. e., n mol/kg, Calvey et al., 1979), it is clea that thee ae two impotant diffeences in the kinetics of the quatenay amines; fistly, the volume of distibution of pyidostigmine ( mean + SE) is geate than neostigmine ( , mean + SE) eflecting its moe extensive distibution and enhanced tissue uptake. Secondly, the cleaance of pyidostigmine ( , mean + SE) is at least five times geate than neostigmine ( mean + SE); this diffeence pesumably elated to the diffeential enal elimination of the two quatenay amines. In consequence, the elimination halflives of pyidostigmine ( , mean + SE) and neostigmine ( , mean + SE) wee simila. Howeve, when these dugs ae used in anaesthetic pactice to evese nondepolaizing neuomuscula block, the dose of pyidostigmine is nomally 4-5 times geate than neostigmine, pehaps due to diffeences in the affinity of thei quatenay goups fo the anionic site on acetylcholinestease. The slow disposition half-life of pyidostigmine was pogessively polonged (fom min. to

155 min) as the dose of the dug was aised fom 36.2 pg/kg to g/kg. The incease in the teminal half-life of pyidostigmine was also obseved in cossove studies in which diffeent doses of the dug wee administeed to the same patients (see Chapte IV) on sepaate occasions. In addition, the total body cleaance was significantly deceased (to ml/min/kg) although the othe kinetic paametes wee not significantly affected. KINETICS F INTRAMUSCULAR NESTIGMINE Neostigmine was apidly absobed and eliminated afte IM administation, maximal levels wee eached in plasma at 3 minutes but only small o undetectable amounts wee pesent afte 12 minutes (see Chapte V). The kinetic paametes obtained ae based entiely on expeimental ddta duing this peiod. When the esults wee intepeted in tems of a 1-compatment model, the elimination tý anged fom 56.9 to 1.1 minutes and Vd fom 35.2 to 6.5. L., and the cleaance fom 399 to 623 ml/min. In this study the neostigmine t/ ( mean + SE) was 2-3 times as long as the slow disposition t ( mean + SE) afte intavenous injection (Calvey 1979), and the volume of distibution epoted in the IV study was at least thee times that afte intamuscula injection in the pesent study. The volume of distibution of neostigmine was appoximately equal to total body wate. Distibution studies in expeimental animals suggests that the dug is localized in extacellula fluid (Somani, 1975). The esults of IM study ae

156 -153- consistent with this intepetation. Total body cleaance of the dug was geate than glomeula filtation ate, and suggests tubula secetion o extensive metabolism (see also Chapte VI). Studies on the elimination of adioactivity in 14C-neostigmine uine afte intamuscula (Somani, et al., 198) confimed that the dug is patially metabolized in man. The popotion of the dose eliminated as unchanged neostigmine and its metabolites inceased fom appoximately 447. at one hou to 827. at 24 hous. Neostigmine was eliminated pedominantly duing the fist two hous only a futhe 47. of the dose was exceted as unchanged dug between two and twenty-fou hous. Thei esults suggest that metabolism and biliay excetion of the dug may play significant oles in its elimination in man and that neostigmine may be pefeable to pyidostigmine (o othe cholinestease inhibitos) when enal function is impaied. RAL NESTIGMINE AND PYRIDSTIGMINE, AND THE CRRELATIN F CLINICAL RESPNSE T PLASMA PYRIDSTIGMINE IN MYASTHENIA GRAVIS. When plasma levels of pyidostigmine wee monitoed in eight myasthenic patients who wee stabilized on oal pyidostigmine bomide only, thee was a widely diffeent daily "d os age egime (see Chapte VI). Although the concentation of pyidostigmine in plasma vaied, thee was a diect elationship between the AUC and total daily dose of the dug ( -.95). These obsevations ae in diect contast to that fo one myasthenic patients whose bioavailability of the dug was inceased sixfold by doubling the dose of pyidostigmine bomide (Calvey et al., 1977).

157 -154- Pehaps thee is also an intasubject vaiation in the absop- tion of the dug. No such obsevation was noticed in the nine patients who wee teated with both oal pyidostigmine i and neostigmine ( = -648). Neostigmine was only detected in one of the nine patients (second goup). It was suggested that thee might be a dug-dug inteaction between pyidostigmine and neostigmine duing oal absoption, so the concuent administation of oal pyidostigmine and neostigmine may not benefit the patients. A combination of IM and oal outes of these two quatenay amines may be moe advantageous in the teatment of sevee cases of myasthenia gavis. The phamacokinetics of pyidostigmine ae not entiely undestood and thee is no good eason to pedict that a elationship between seum levels and clinical esponses should exist. Pevious studies by Chan and Calvey (1977) indicate that in fou patients with typical electomyogaphic decement in the adducto pollicis, thee was a positive coelation between the concentation of pyidostigmine in plasma and the effect on neuomuscula tansmission. The plasma concentation of pyidostigmine equied to estoe tansmission to nomal vaied ove a five-fold ange, eflecting the vaiable seveity of the disease. In anothe myasthenic patient with puely ocula symptoms they epoted that thee was a significant coelation between the plasma concentation of the dug and the diamete of the palpebal fissue. The pesent studies on plasma pyidostigmine concentation and global assessment of nine patients (Chapte VI) show

158 -155- that thee is a tend towads a positive coelation between global assessment of muscle powe and plasma concentations in five patients, of the emaining fou patients, thee showed coelation appoaching significance at p<.35, while one showed a negative coelation (Chapte Vl'page 35 1). In these studies, a wide vaiety of execises (each testing paticula muscle functions) was used to account fo the possible vaiations in stength of diffeent muscle goups in the patients. Such global assessment, togethe with clinical impovement of the patients could be quantified moe ationally than most of those epoted peviously. Fom the pesent findings it is pobable that the diffeential dosage equiement of pyidostigmine in myasthenia gavis wee elated to both inte-subject vaiation in dug disposition and diffeent seveity of the disease in eight of the nine patients studied. Howeve, it is not possible to explain the negative coelation in the patient (patient six) who was also teated with amitiptyline fo depession. Futhe wok on the kinetics of neostigmine and pyidostigmine should be designed to elucidate whethe the intesubject vaiation in plasma levels and the dugs is due to (a) a diffeence in the actual oal absoption of the Anti-ChE agents o (b) a diffeence in the elimination (i. e., metabolism o excetion) of the dugs. Fom a phamacokinetic point of view, neostigmine may offe no bette advantage ove pyidostigmine in the long tem phamacotheapy of myasthenia gavis. This can be explained in tems of low bioavailability

159 -156- (Chapte VI, Pat I, page 13 )and the shote plasma half- life (ChapteslV and V) of neostigmine. These findings ae also suppoted by othe published wok (Aquilonius et al., 1979 ; 198). A combination theapy of neostigmine and pyidostigmine may not be of advantageous as poven by the findings in Chapte Six (Pat I). Howeve, futhe study on this inteaction between these two Anti-ChE agents at the absoption phase should also be of inteest.

160 -157- APPENDICES

161 -158- APPENDIX 4.1 PATIENT SEX AGE WEIGHT IV DSE F (YR) (KG) PYRIDSTIGMINE BRMIDE (mg) 1 F (36.2pg/kg) 2 M of 3 M " 4 M F of 6 M to 7 F Is 8 M of 9 M " 1 M of THER DRUGS CNCURRENTLY ADMINISTERED phenytoin sodium (3 mg/day) and phenobabitone (9mg/day) 11 M (72.4pg/kg) 12 M F is 14 F to 15 F of 16 M is 17 M (144.8pg/kg) 18 M M " 2 M " 21 M F " 23 F Intavenous injection (IV) doses of pyidostigmine bomide and othe data in 23 myasthenic patients.

162 -159- > % o9 v ' Ui.`wN `A C D V -I ttz- WNA p4 t"w b- :C v tt 3St 7x H - ID N f N to 6 m n ce afä iº vw II ý º Nw. Q D p Q " N '+C w M. w 1-" N. I. pp /ttt vý. fl 1w o'.d VI ýd j, ý Z. ýa JNº - 1ý -NNw Ui Ui I tt Nn t' tº D j, ' Np v, Ll % N 1tt1 U1 WV º+ NW1 NN. Lý - N p+ VW yl V Co % V 1"+ % U % V NWW.. Q' qd VN tt I t1i1t D w %D v v1,.j elf N 1lß º'3 "sj W L NNWp. v ý >v% % C' vl W e% v.d -1 "c. CV t a A. A (D 111w c> e.lw1 vii N Ö a' ", b! a1 m to a t? M Qp 1o m bw H wr lip D n a. to Ph o Ph o w is Nww VI v to 'D w CD W a' o' -", vi " N vi %D ' Nb N fi vi VI v ' viiº 1141Nu ý %V La wwn Z- % NWW % Go. L' ` t 41, oo -wn oo. n w Co % fj NWW '1 X ao w v C% N p" tp" v1. " p xa= to M W In ý VI Go 1n. ý ý hw+ Ö vi... """". """""" Ci7 "' LÖ wnwlým- ()% CD to NZ to to 11 W t 1` is uý to o v Hy 113

163 -16o- APPENDIX 4.3 BLD CNCE NTRATIN F PYRIDSTIGMINE MEAN + SE ýa"'tj NG (n$/ml) of SI X PATIENTS CNCENTRATIN F TIME SIX PATIENTS (min. ) ND* ND ND ND ND ND ND ND ND ND ND ND ND 4 ND ND ND ND ND 3.6 ND ND ND ND 4 ND ND ND ND ND ND ND ND ND ND ND - ND ND *ND non-detectable Plasma concentation of pyidostigmine (ng/ml) in six sugical patients following an IV dose of 72.4pg (4 nmol/kg) as the bomide salt, ove a peiod of time (2 to 14min. ).

164 MISSING PRINT

165 -161- APPENDIX 4.4 BLD CNCENTRATIN F PYRIDSTIGMINE (ug/ml) MEAN + SE SAMPLTNG CNCENTRATIN TIME F SEVEN PATIENTS (min. ) (ng/ml ) ND ND ND ND ND ND ND ND Plasma concentation of pyidostigmine (ng/mi) in seven sugic al patients following an IV dose of }ig/kg (8 nmol/kg) as the bomide salt, ove a peiod of time (2-16mins. ).

166 -162- LX a. % D V % Vi F W ºb y c s m n m x GQ ö (V (o F+ ß7 fd. wg w h" p1 I.» tc fa K ID 'a' t fa a lý jk -i D W " ' ý V V o i' % " V M l- N V 11 " V W.o. " N C' N V v W W lý. " W C' Co. Cy% - % " to. Z- i U' %D ý.. ý N W D U' Vi. - P. " - I- %D Vi % D N Ni S ' + N w ýd vi " W W V % 14 W u " w N ' to fd ßt. 93 ID i C. ý fd v ID K p w ý p M 1 a+ ºi (V º a' m lo ID t7 Ä f º (b C 1 Vý D. v N V S "+ w ýw j, N V 1 S w V t D C) M N S ýn w o C V' V S D w V V' Vs N S ' 1ý D V S N w.o v -4 '. U' S W v w N N l W v oo % ' { S '. D.o (x% '. - v R to ID K fc W m vý ID " fp CD t Q f Co W to w ß/ A/ A P 9" CD C. C \ 7ý' ' 'C 't, M m4. W C) " N a' IV ' V' % VI v " ' N D w '.D " N M ' ' N % '. 11 C) " W '. C ) % 4% D U' Z% " W V1 V D V'. W v GD ' '.. 'p o '. o t ' " N A N hi w N ' V Co CID W N. vp W % QD h+. A 41 N ý w ýg w w 9 w to o C7 n ec 1 ly x " v cc ID x " M f ID f d -. a w 93 ' (b 1 " Ni V C' ' V 1 " Z` N i' U' 1 " W v ' 91 ý 1 " N %D V - u N 1 " N V N ' IJ tj S " p A % D v ' pd 1 " W 1, n % UI 1 " W U % w I " N U Co V ' 1 " N %D N.. -. C) V v V w ID (A ID It ö N b x ýn

167 -163- Z -ý º-ý d % W N 3 Z aý y a z w o X 1 Co C cn Z + mo N 41 w v %. o n i 7 n w t - ýo ya i k oý - md (A w %D +. " w 11 IV Q" 1-+ "D t... v Z, e % La t13 IV Z% w W V Q W Vi s v to w G m G. to G I cb M D D e V e M W 1ý VI t ý` VI -ý W o R t lb ci hl tj 5C W % % - p p ~ V A < W 1I l' VI VI ' o ' '.1 % o m a w o,' fa 'G e ld i ID.G h 'A v i 1-+ 'C W IJ D % p V N fd w N VI F+ % ) \ " " " " " " " ei M p D N Q' '. G V GD to V W Co ee, 1-+ VI U D N tp Z- ' A N Co D W 1+ ID M W V N VI GD W % W o % Z- ul -1 t1 a ' N v UI -i V ' K Xe w ul to Co to o' ul p es» ß. `C ;J N w a 1, 1-6- v to 1 1 c CL eb W N W $ Q x '. F+. F N ib % Z- Co 9- g (IN ci C% W W N '. Co N `-ý µ v' W v N x

168 -164- C to º3 fd X l lb f1 G L7 ßý ', ý lb tp it R 'L7 ßt w to p p1 R ft ww lb.ö C to ý " C7 HR z -4 ýd w W º- N W N N % N N v ý ' % V N v % D t,,, ) N v N Z- w w u, " hi Ln N % Ln a" t" D u %D N Q Q Q Ln V V % N '. to N V N d ^3 z : - PC b ý x C 1o w fd Ka fd m R to w oom 1 a' t i fýd fd Cý pq to a to to RC m a N ý v Co D N V Z % to N % N Co V 1-- %P % N C)% Ln W N v w W 1. % N W N V % SZ ý,,, \ p p, C (ga to ºt to fd (n p- ID Ph M<1. - N fc 4` o ID D to M Co ptp o? \ ºJ D F W N Q ý % N Co C)% V > o N C *Co zý N % Q W o to a D ýe pv wp at m G. w Ro a w al äýw 1 ä tjý u W W. %D : v D N D % pcd A. A " ID 3 " to. ya fp!d "w V vi o N (> T " '. D QW o. Ul 'D Ni % zý V % w 41 W Ut ý -1 ý 1 pd %D ' N Itb (A to (A l fb

169 -165- * mw I+ c, z % Co v C% vi o. Lý w hi ºd * y H i Cý1 : I- PC wc AK pw K N v AW 'C ßl Fß :J CCD AK C fd MM My fd :3 K ºqf K fd t K K ß% cn a ab w Q V N oý 1+ t., 1+ 7C ui V' W W v ui ' 41 1 D w N Ni p' % e v 'C W UI Vi W ýd N N 1.. w V+ %. v ob.. v...c +. t, ý Vi.. a w., hik D ww KW WK M V ߺ fd w :3 %4 K w P$ NN " tj " % v %a 1+ Cý w V' - D i` W N l. W w W tn t N P" oo %D w N N.D w - N o" % W v 9 o p v R k- ºt ow w C. C äy ww o f v.. o pº w ß. m Co C% ýo wv 1+. o " N La + ý " W D % Vý " ý+ %D M % W " pý W " -4 Q% W " C Ni D " o -j '. " W yý " W 1, pý " W D UI e P1 - y V V N C P% V VI '"' v C n Co o% V K 9 ýji N VI Ný N.. V 16 Z., L -4 V1 w % V w %j N w v u, v at N 7 It, w v cl CD ýc ac WM vw "A 7 'Q w R fa UI cý w NQ g uý 1+ 1ý n> uý oý U?% w ný -4 U o" oo g C v V : w cý. U i t- %D w w U m- (» u Co '. to ' % t ý W

170 -166- d CSC Nit- ý W A W "p t, 1 lb º W C º. M B K p" ä B. ob e. " lb eb 1 Co M ý (1) 'C ºi 11 (1) (4 C (Il fu Aý fe W fa (n 3 ) 1+ "!J ý W - 1+ I -, f yy ý u+ W tv y o " N. " W o " N v " W o " W N o " - p "ý,' h+. 4 a o o k t7 f: P W f7 D M ID ߺ fd m op p l M C' A w B ) p C CD A Vi A W 1-h W W Ci B Mh ). ä. ` MM c. W C fy a CD h'a 1+ fa W f/1 e+ º W a1 c º C W K - p q M F: tj' N "V V" tai ý W R 1 W Z, ü VI to +. Av ý o SZ v v ýo w t%) 8 C uo W Q X i W a to W 9 a D.D a a + v W N " N % N V " VN t+ W UI ý Vi. N C D e D D C, W B A v Ö h o W W - W %D v. - 9 v v v v v v p v "" Öý N N N N 6 6 ý 11, ý! o, o1 N tit -j P. VI y1, 93 '9 º 's a M fn 1+ a I H v v v v v W A ' a it W a º N V M Cý Vý AD D co ý+ ' N D J, N u Z- W g w C' co C% *

171 -167- N " fd A ' B fd fd M y cz z 1+ UI co 'i N 1+ NNN- WN - 'D Gý ý.wo iý W ý V' tv -' v v i p - P b - Cý7 t+7 Z ýc >C f'a f CD K u, a CD 9 ew t! w w W t CM MH aq R ww f) W tý V A lb ný w fd º1 Ka cn o to w w L! fd KQ 9 w d fd e - ý+ u+ 1+ N tiý C' D ' N 1+ Go "D Ä %41 1+ N P. Pýý oa N "a R V N s v. N ä ý VI sý V W n W i co Uii D V i % % co co n - w v ti oº oo,ý " X N N W N - ö t \ W 9 CI duo VW IkD C% V D -CS w w - w w o x a o P- - c o V IV V W ti

172 -168- APPENDIX L. 11 /Continued TT W 1ZE-X-1LT -' ICS _.. S! = -=ý - =ýiý: ý-tai(! ' tcla-d=flult', 4XXX) )T.1TT utto- =-= -" -=-- - _ TE; ER _ ý ---I _IJ ý'-t 1 L7-6TDÖI -1 pegt iv 1 -I? --=TpF, F7C'=, -MCil _=A-.-A1, -B1i-XciIFXVj! -V1"ZFR,5UvSIFV'i -`ctc-"ai ` -- -ýs LN-v. C,: x a; 5. C CAICTCPeAtc, so: Dtvti z-i CWTN' -7 TAoS X9 KR -TY2 Vt IsI t- -- -_ _17 ä [Stu -= '=ý f3 I 6fTTDT fib?? ss fan ^'_ 'XST_tT+7LT1T7TiS'TT! sys-! ý-äpt114 2t---_=_ A!_;, Qvft: IT,. TTY-c it -, vi )tx b. j) L7--ýSýTTFT TZ'ý''Z'-p- pt-iivý1rfý1t7äz"l Ä5T"S uaae"cit ( t2ct )' ý) I) t Cýfl. 4(Di(A!. (! 2SS1)'R1('7191)tBE1A'-('19SI7 J4-7ET -Tl- 2C', Tý "-"^ýfs^; : 'TGIT E'(1T;? G ýtstý'rm CP! PLASMÄ, TIME, A, ß, XP Alt, VZFi, 1); -. - jiwy 7 U1. FYD("! S(R). TnMF171 ); C^1'-717s'LAs -71 -c Z), Sý 1)t'ý^ý: `Zlt'iýýc! J"ýATC'TUQý. 55

173 APPEND'-. X 4.11 /Continued... 5::..: = RzýýESSD;: (. "1. PT1A. '?? (1AYZERQ1i)i E V72 na _--- _: 3C 7-! _7a"-ý'"Ctýl: il! t 2, i-7! L. alc-1-z7-s-=y IZFasiXP{=. A 6 Sl e13? TILýEI3: Y2EREXP=(iBS -_- _-ý -c - s2 -- hý-i N (LNýPZULC te LVýZ is*t; ý z2iitz; = ý7_ 3EGINf 7 - -if ET=M. LT. --ý -, aý T 'T FY TC (1 T")t+j+..F Qa-e >=ýxý *TT-t' zt "ný=;. = ý ý. --ý tic "it(äaý.? 3ý AT MSpACE(S) W(TNT('+Ibf/ö, ý)i -=ßi-3= =-=ýis": Ti-ý_ 3i ß' -. _.. = -=1T, 'Ti" RÄ-ii VTM u15 'n1)fv-#'z 2- ö2 -, -(Tß A1- 'ZZ-F'WII... -; may '. GVT i; - _--_ý -. -=1 SD-! d--a Si- = -T 7J- A! ýyi1 ý== ---_. = =_ *-, 717 et' - -ý _ --- VTE - -ßf5 -ý EG-1 ti ? f)jsf, 1-i74TILT!! 11 - ý_ C A G-1 + _ =: Yt=, ew-o(j7; -'IfýPý17=ýTNEýf7GnTQl Gi _,. 12ý. X+TA-1I _XS]xSQ+_T7I1?? ý_ysqt YStALI. 7*2ß_. _ -, ýý - XYSsYV(tjsnI =o. l: --=- _ý i n...: - o: _- acf1)jyzfrc+te'o(5fe): nij.: v7-4 : a=-=- a v/ P. =xia _.: (xy-(x"y/d))/(xso"xt2/aý5i--ý.; _-. =-. :'- --. futºiuý; ---:., "-_ : ý17ftc'4 '" "'('3 C'1'T; <I+EYPýýcNTT'L"LFeST*$ýU1RCscjTýf13C"2ý')'

174 -17- APPENDIX 4.11 /Continued... _ý-=-=3z P -? = ". {_! _l 45=_1_pýT7ýýýl V -1 --E 4, fa. -- -ý' s Lt 11ý ="E!I'C? ýi: t4pta 4x= =i 1 ýxk. o=t ER4J. ý i--)\'ttld"-- - 'c t-4ß-l=" "72 : ate G'. i 1i tt Y T_ '-- -- ' _ "- --- TTü Cs/ T-Eze- ICE = _-_--- is a-. R. "xi -E FITEIME VTZERý"IT; - Es 5? ZfT7 c"1i, LT C CT7 8T, XZ A-A W' lz ZF"-'i T) ý. ---_ T7T-- sc ai i _ x_73==- -. =ý 3 '"' ts is`cý1att337 =i 3dº! II3' ; _- =. =- - - = _= _ _- ýtj 5! TFtf6D V i tisumstv _. ý -_-ý- =. its4t"s-obeýfi? =ý+3ýyt'i-eji %2-; % 8-j =v1-zfr ", MR : 8I-; ' 5 v_zfr! t-- _Tn _1.tý,. _==11TC3 Ts'iT c{ C-' T. TT i= ";; ý itýlt"'-s-l, ýc? ýzxtitý TR-F`A -- ý- _.: _ = =ý1. ý., M i ýsttj'ýstý 'ý ; ý; s'v'7z`r'*')'ýt ý gzl-' ý i"º17 fr1mrefs RT) Mtn sy' 3ER; *ý G A; s Rý. _ q6=--p'v-t U771; 2 7#WT; -Q R TNT'(ý1 iii) ; PST NT Sit xs ýf f8 R) C, -t -Fs-F;- am! 1o? -_ $Z *t n i- =.. 25ý v==? ýa1_i_ý. =ýº. fýsxef ý: EW-+_, _ _. ---ý' 2(A, f.: n- 3IT 'IF. T )T n1svlv I-lýi E CAE 1 ITn`f"Ä 1oU 1- E i l. n ti Lt F C: =.. 17STS-Tfl C'('SCtj! TfIF. u! äp*: "-i7zs+twielpme `=TI ALV(17C+)ý)1)f -? ýý D7ý"jT(ý'/ice:; '47-4L. ý6)1 ýind"cma; L6)"7pZIýTZßEiZE); 7 : T:: T t"! t. Rý. ft RI ý 21 3 ýa<z < 6-).. 1GF, ý. C I R)S 21, 2.: Stsc. 1;

175 -171- APPENDIX 4.11 Digital compute pogame fo bi-ti-exponential analysis of the, plasma pofile of IV pyidostigmine. a 7'I? 7ý 7'3 pst VAY yn 1 iif.: *S to Cn, ''T l. '` ýj IF1Sz L" E '! I' :i 1) T 1ý fý Rai IT)IN'P; G49 IFVýI L171h U^, F! f ;5 17 D1CUIFNT "VQAKR4 ' urf4('vs*kra i LPi N 4/1/ßn AT"11,53 p C'. fsi IINATEI) '±Y XpCK 12X DATE 4/1/8 TIt1E 11/53/25 VR(, Pat VSAK EVTFNNFtl IATA (72AM)- 12 ton^act P 11 UGR. AM (Dit 4) Cw F CLV 4LG)L6 SFG WRITETFXT 1s SFG Eßß :.- SFG D7I9I_T_" 2 SFG A4S-_ SFG NEuLINE 2 SFG ==. L. a SFG AEAf

176 -172- APPENDIX 6.1 The times pyidostigmine (py. ) and othe dugs wee taken. PATIENT NUMBER 1 Py. 6mg 8. pednisolone, 4mg 11. Slow potassium 6mg 8. 6mg Py. 6mg 7.,11.,15.,19.,22.3 Pednisolone 25mg altenate days. 3 Py. 6mg 7.3, noon, Py. 12mg 6. 6mg 1.,15.3,18. 5 Py. 12mg 6.,18.,22. 6mg 1., noon, Py. 12mg 6.,1.15 6mg 14., Py. 12mg 7.5, noon, 17., Py. 6mg 7., noon, 17.,22.

177 -173- APPENDIX 6.2 The times pyidostigmine (py. ), neostigmine (Neo. ), and othe dugs wee taken. PATIENT 9 Py. 6mg 1.,14.,18.,22. Neo. 3mg 7.,1.,14.,18.,22. 1 Py. 6mg 6.3, 12.3, 15.3,18.3,23. Neo. 3mg 6.3, 9.3, 15.3 Neo. 15mg 12.3, 18.3, Py. 12mg 6., noon 6mg 18.,21. Neo. 45mg 6. Neo 3mg 9., noon, 15.15,18. Neo. 15mg Py. 6mg 7.,1., noon, 18.,22. Neo. 15mg 7, noon, 16., Py. 36mg (total daily dose) Neo. 3mg thee houly 14 Py. 36mg (total dose) Neo. 3mg thee houly 15 Py 6mg 7.3,15.,22. Neo. 3mg 7.3,1.3,14.,16.,19., Py. 12mg 1,9.,11.,13.,15., ,21.,23. Neo. 15mg 7. Pednisolone 2mg altenate days 17 Py. 9mg 6.3,9.,11.3,14.,15.45, ,21.3,22.3 Neo. 6mg 6.3, ,15.45,17.45,19.45, 21.3.

178 -174- V + Ný jý WN ý~ ö %D Co V + In -WN ä m b WWNW ' WWNNW to uw V+ W pn W %D 1- % %D > ND C Co wo mm pv = m=mz PI) m ftj pq m psi o X ) (D C %D Cl) w 7ý ý C X a R+ m t+! - p" p"" - Qý N D! -ý IJi WVWVWNV d N M "a7 C eb Vý ti Z V1 ß. -P m A p a 1 o 1R =1 : 21 'WC l< S 1 Ä S fd fd t i A ý V R Fý m IR 'S 1 'Jý ýg K m I ý x cn R m pi v n ä t- 9 ýc C N to +.C '13 W y m a ti ýp MW WNWWWNNW {'I Ww ý iý T oý lý o ý o ý " m gö S 1 4 m n `` 2 1 P--4 9g F1) ý. x ý tig ÖÖÖÖÖÖ I I a+z> ö ' " R dö ýn ä a ýeä ý aý+ " C3 - ti m 1 NJ IV 1 cl% 1 9 pi p 3C t; i 93 ºt o QQ In " zc 1 +ß tn NV l I V CL in 1* ft p - Cs1 d (D G. ' R3 A P, :3 1- W. m go to to x Mwm ý 9

179 -175- APPENDIX 6.4 Blood sample times and concentation of pyidostigmine and neostigmine, thoughout the waking day (8.am to 8.pm). Numbes in backets indicate the patient's numbe. (1) (2) TIME PYR. TIME PYR. (h) n ml) h n tnl) (3) (4) TIME PYR. TIME PYR. (h) (na/ml) (h) ml (5) (6) TIME PYR. TIME PYR. h n ml h n ml)

180 -176- APPENDIX 6.4 /Continued... (7) (8) TIME PYR. TIME PYR. (h) (n ml (h) n ml) , (9) (lo) TIME PYR. NE. TIME PYR. NE. (h) n ml) n ml (h) (n&/ml) (n&/ml) (11) (12) TIME PYR. NE. TIME PYR. NE. (h) ml (na/ml) (h) n ml (nit/ml) (13) (14) TIME PYR. NE. TIME PYR. NE. (h) n ml) (na/ml) (h) (u&/ml) n ml)

181 APPENDIX 6.4 /Continued (15) (16) TIME PYR. NE. TIME PYR. NE. (h) (ng/ml) (nom (h) n ml) (n ml) (17) U 4 $ngl E ý, n (m_1) nq=m

182 -178- APPENDIX 6.5 Plasma concentation of pyidostigmine and pefomance in the test outine. PATIENT TIME PLASMA PYR. RANK SCRE SCALE SCRE h n ml MEAN + SD MEAN + SD MEAN + SD i MEAN + SD

183 -179- APPENDIX 6.5/Continued... PATIENT TIME PLASMA PYR. RANK SCRE SCALE SCRE (h) (ng/ml) MEAN + SD MEAN + SD MEAN + SD MEAN + SD MEAN + SD

184 -18- APPENDIX 6.6 Digital compute pogam fo egession analysis between global assessment of muscle powe and plasma concentation of pyidostigmine, afte oal administation. In ýý/14)3s 1{/f"L/Fn = j: I) i. Y Xni a, uni STATIý'f}JT G 'ýi' ' (I1') C ýsc', D 31 E5. i'cla. "bf: FAU[ I.. 1XWY5 (F1. t. 'egf)f, Au YVSA1G--- it=ffiuttv- P-F ITZACý 7 -- =_- u f EG1N1 _ PEA-LiTßRÄY-bF. MPI 1 ý1ntff, FR'. "J SRCºLý 41 VA S1) S- 3 R1Tj`TP.. 3TZ! ýc -f- -, SUP iijj[jjlmanýt1ý1s1)ý1aolakcetfýticýý}sd G 1(111 1j'cTTe'Ii, tzp. E ý. i '«ß'ßk7; _3! ltfdljalft! T1I161b7i1:. - =- 7" ' F( TNRMFJ - _ n FA II Tt''IT3' Ep 2-RF9'LI CJi'=1s; -TFýQýI"iii? wtip V l7-ý ilktff1cu+7tt1_inýhný i=pmaý ` =' zi ct4.!3 ýi +T=. ý: ±s. 51 = #331= ýa VL1ai57. =CS1t2/ i R-TlT2F=JUio!: i: ':- 7; _- ýi - : mkvtý61=sei: 71i"? 'i. 7ýi3i _ P I '- -i1 ýc: JLid. ti7; SDACwe TiiTAF1Z'TZ1ZiL i1 PIwI S1 /4 LýLI ` ` ;;, ii. I INTp W= fin : 4C111TSC5 1 A'E9 LIN(1)t- 1G - - `= _ -_- '- --" - N1 n 5iiiFTC llfa ýý -- -_ At : 7.

185 -181- APPENDIX 6.6 /Continued Dnf: llnt 'V54wRA, t! V. "l *v5! LD [? +4 I/ 4/ßio AT?h. 1 '4 6 _ 8 CJSI1LIh ati; n?y X3 CK 12 n3tf ýc/f1/, %Ai1 TIFF? )/1ý/ " 1 PP') --4 VCa 12 EYTFVnF nata (22A'-! ) --=-.. =_ CnMPACT PRAP AP4 (s4 1). _..: _. 14 CRE -6 R. =--_. 16 Cld ALG)i_h6 18 ScG.- = WRITFTFJ =_- SFG - -Sch- S RT 22 - ". - SFG dp--..-frf _ =-- -_-ý--_