Reprinted from Veterinary and Human Toxicology, Vol.-39, No. 4, August 1997, pp. 214-219
Potentiation of Pyridostigmine Bromide Toxicity in Mice by Selected Adrenergic Agents and Caffeine _ b
Leslie A Chaney, Robin H Rockhold, James R Mozingo, J and Arthur S Hume ' Department of Pharmacology and Toxicology, -University of Mississippi Medical Center, Jackson, MS
James I Moss V Gainesville, FL
ABSTRACT. Pyridostigmine bromide (PB) is a reversible cholinesterase inhibitor used routinely in the treat- ment of myasthenia gravis and recently by the US Army as a prophylactic agent against potential nerve gas attack in the Persian Gulf War. Pyridostigmine has been implicated as one of several possible causative factors associated with Persian Gulf illnesses. To investigate toxic interactions between PB and other drugs, male ICR mice received contralateral ip injections of either a selected adrenergic drug or caffeine, followed I5 min later by PB. Representative isobolograms plotted for each drug interaction illustrate that a B-adreno— ceptor agonist (isoproterenol), selective B2—adrenoceptor ‘agonists (saibutamol, terbutaline), al-and <12- adrenoceptor antagonists (yohimbine, phentolamine, prazosin), ias well as the stimulant caffeine, strongly potentiate the lethal effect of PB. Agents with agonist activity at both u— and B—adrenoceptors (epinephrine, nbrepinephrine) additively increase PB—induced lethality. The potentiation of toxicity between PB and these agents was counteracted by pretreatment with atropine and atropine methyl nitrate. An u2—adrenoceptor agonist (clonidine) and B-adrenoceptor antagonists (propranolol, nadolol, acebutolol) dicl not increase PB-induced lethalities. These data demonstrate a toxic synergism between PB, several commonly used classes of adrenergic agents and caffeine when exposure occurs in different combinations. Future studies into the mechanism(s) of these interactions may bring into question the usage of PB as a protective agent in combat conditions as well as delineate any possible contributions of the drug to Persian Gulf illnesses.
Pyridostigmine bromide (PB) is a quaternary oncogene and AChE mRNAs following peripheral dimethyl carbamate that has been used histori- administration of “PB _under conditions ,of cally at relatively high: 'd1oses_.(2‘00-1400 ‘mg/d) stress; ‘Recent investigations Fhave linked treat myasthenia.gravisiga'neur0muscular.dis----# the eXp0su’5I*ii3= ,'PB' and the insect order characterized by skeletal muscle weak- repellent‘"N,N—diethyl#m%toluamidee (DEET) to ness band fatigability_ (l); Since yl986, PBi increased ‘lethality 'in “rats_ (10), chickens has been recommended by the US Army for use (ll), and mice-(12).- In studies of inter- as 21 prophylactic agent.against‘organophos- actions between hydrolytic,enzyme_inhibitors phate (OP) nerve gases (2)., Organophosphates andf DEBT in _German. cockroaches, Moss (13) bind irreversibly to “acetylcholinesterase proposed ‘that DEET - may_ Ihave -linherent (AChE) ~in“ thej'centra1_.(CNS)" and. peripheral adrenergic» activity Pthatr could-_play 21 role (PNS) nervous ‘system "to _prevent _hydrolysis in its toxic interaction with PB. , of acetylcholine (ACh) at sites of choliner- gic transmission;- As a result, ACh accumu- Based, on gthe premise that“ activation} of lates at cholinergic receptor sites, produc- the sympathetic nervous system could be in- ing_ excessive ,parasympathetic 'stimulation volved in an adverse_response to PB, we chose leading tfltimately ix) muscle paralysis and to'. investigate? combinations ;Jof» -PB and death. In. contrast, prophylactic doses of se1eeted~adrenergic agents to probe the pos+ PB (30 zmg, po, tid) reversibly inhibit 305 sibility of an acute_interaction between an 40% of AChE_activit'yi~in__.the PNSfto protect- enhanCed* ;cho1inefgiC' state- and altered it from permanent inactivation by OP chemical adrenergic activity. Several of these agents warfare agents. 'Spontaneous dissociation are commonly prescribed, for ‘the treatment over time restores an adequate level of AChE of asthma and for regulation of blood pres- activity to maintain life, -providing “that sure; (l4)l. .Caffeine‘.Was .also included in atropine and oxime treatments are also admin- this study because of its prevalence in com- istered at the time of exposure (3). It is mon 'stimulatory_ beverages tand fits 'ability assumed that PB -does not cross the blood"- to- increase 'levels are circulating catecho— brain barrier to enter the_CNS,.since it is laminesl(l5). _ V v ' a positivelyqfichargedf agent land‘ therefore should(3); not. .._interfere_ _. -,7.with_cognitive_; -,- ,. functiong . “MATERIALS AND METHODS 'The first usage of PB in a combat situation occurred ‘during _the' _Persian >iGulf ”'War. Chemicals" “ ” ' ' - Between August 1990 and April 1991, approxi- Pyridostigmine ~ bromide, ~(i)—epinephrine mately 700,000‘ US 'troops' were deployed, to hydrochloride, (i)-arterenol bitartrate salt they Persian Gulf where -some _were exposed (norepinephrine), (i)-isoproterenol hydro~ simultaneously, to 2i'variety zit potentially chloride,’ sa1butamol,_ terbutaliney hemisul- toxic insults, including "chemical ’Warfare fate, .yohimbine~ hydrochloride, ;phentolamine agents, toxic fumes,” pesticides, infectious hydrochloride§'Yprazosinu hydrochloride, caf- diseases, multiple. immunizations, chemopro- feine,‘,clonidinej hydrochloride; DL-propra— phylactic agents‘ and psychological stress Anololjgjhydrochloride, "nadolol,"” acebutolol (4). Since returning} fromf:theT war, =an - ihydrochlorideQ
- Vet Human Toxicol 39 (4) AU9U$i,,1997 * 215 mg/kg; caffeine, 5 mg/kg; clonidine, 1 mg/kg; nnsmrrs propranolo1;§1i5-cu‘ 3 mg/kg; nadolol, 1~or 5 mg/kg; and acebutolo1,7 1..or <5. mg/kg. The 24 in LD5Os and 95%%CL‘fOI_iDdiVidU&1 Single» ip" injections Iof (these ‘agents 'were drugs are reported in Table 1. (The LD50 of administered at 'the.-abover doses to lgroups prazosin‘waspnotrdeterminedvdue.to;so1ubility of— 6-10 animals, (followed. 15 null later» by difficulties; at rsqgh, high. doses.< Instead, a_ single contralateral ip injection of PB an _LD»50_ value of“6"0 mg/kg‘ with 95% CL be- (1, 2 cu‘ 3’mg/kg).- Mice were returned to tween 47—+*77 mg./kgmwas ‘assu-med for prazosin their cages and .0bserved 'continuously for based (xi a report by Noguchi et a1.(20) for 1 h after dosing. 'Number of deaths was re- ip dosing in male ICR mice; “The above data corded after 24.h.“ <‘ 8 I ." .j were used in conjunction with LDSO data de- rived-from the binary mixtures.0f each drug In a separate study, groups of 6-10 animals with PB to plotiisobolograms. r . .,_ were pretreated_with a musqarinic antagonist prior0t0'severalyof*thelabove,drug*combina- In» the- drug (interaction ystudies, .c0ntrol tions.“ Mice were given simultaneous contra- animals that received the selected doses of lateralliplinjections1ofpeitherfatropiner(10 each adrenergic agent or caffeine alone did mg/kg) or atfbpine methyl nitrate (5.4 mg/kg) not __'e-xhibit_-adver.s‘e" effects, ‘except for epine- and "an ‘adrenergic1.ag6nt_ (epinephrine, 15 Phrine and 'norepinePhrine; both Tof. which mg/kg; norepinephrine, 7.3 mg/kg;‘salbutamol, 0.8 mg/kg; yohimb'i.n;e,‘-,_.‘1,,j-Big/kg.)".or c_.a.ff.einIe’ <5 or 10mg/kg>-r@‘11owed .15 min leer by 5?. Si . PB (3 mg/kg), and lethality was recorded over a 24 h period. I .. " ' ‘ " A A . ,dI~‘.\_‘ _ - _ A - ' =. _I1 . \\\\ I . I. ‘,1 3 I ~\\.. -- 1- * " ' ' , ‘~» ~_ .2- it i" ' 2‘... .. -.\\ 0
Data Analysis -;»~ " ~: - I \ 1 ‘ 1__~ \\\. 1 i LDQT values and 95% confidence limits (CL) .. v - _ 1 I \\ _ . _ -4 _ \ _ . , O_ _ . I , __ _ \ ' I 0 . -T > l - . 1- . e ;_-es, " - I Ito 1 I ~ I ~—I '- t - I for individual drugs. and ‘for PB in the l77'T ' ' '77 '7'?” 7 ~_.0 ‘. 100 ~- 200 300- 0 20-40 60 ~80 100120 .presence of a constant dose of each adrener- in 5_ __ _is0pr_0¢_eren0_l(mg/kg) _ .51 phentolamine (mg/kg) gic drug or [caffeine were calculated; using a computerized version_of the Litchfield Wil- A 4 A 0 . ._ M‘ ._ coxon analysis (18)." Isobolograms were then .; _\ . ___,_\ plotted to -establish whether' combined. drug ,_ 39 0 .\'\ ' _- .'3i,‘ -\ . . g ‘.%¥ “\ . , ii \\ effects were greater, equal‘ or -less ‘than ' 82x. Ir \\< ' _ 2* \ “ would~ be _expected._based on the individual 1, . 1 -_\_ 0, - ¢ . .\- activity of the drugs and the theory of dose _ \ . - \ B _ ' .. . ‘ . \ \ - . . \ . additivity, »as discussed by 'Gessner (19). - _. 0-‘ .' H . O-r__I__ ‘_ L _i__0_ I I Briefly, an iscbologram is 21 graphic repre- gé+ 0 0 xi 40 am so sentation of equieffective doses of 2_drugs, _ I salbutamol‘_(mg/kg) f - B ,' 5_ . Prazosin (mglkg) using rectangular coordinates, such that the coordinate plane is defined by the dose axes . » ' _\ »\ \ of each drug. An isobol is a contour_line, ' \ \ (mg/kg)stigmine \ or line of simple addition,_where all points _ \ \\ _ _ Q~o-,-_' . 2-; - \ represent equieffective quantities of the 0‘ nooz \ 1 . .. \ _\ . 2 drugs. LD§)s and 95% CL for co-adminis- _ . -P l. . .0 _ \ .. 17._y_ V. . \ \ . tered drugs can‘then be plotted on the same pyridfl i \ I . 0 F .1 . t - . 1 , ~ - 0 OJ |- ‘ _ ' i--- -v ”"1' P’ --I graph, and‘drug interactions can be evaluated _ _0 _1fll _Xm’ .10-. _um.- aw. an based on “whe-re"_'the "points fall in rel'ati"on . "5,--0 "terbutaline (mg/kg) . . _‘ _cafféine(mg/kg).‘ '_ to the line of additivity. If a point falls below the line of additivity, without over- lapping' CLs, the interaction ;hs considered to be supra—additive, indicating-potentiation of effects. I 1 1 -Y -4"‘
l\)r--I-La) L4;I‘._-._f4___ " ._._.._,..--' 4-"'-. -;-r“. .__J,_1_+_.| . -|’*->— "f-Tut -0 .10 20 30 40 .50 ' 'yohimbi'ne(mg[kg)f' ' ‘ ‘ - 5, '. _ > . -. _ “ ..*"5< ' Figure I. ~lsobolograms illustrating the combined lethal Table 1. LD50s with 95%‘ CL for individual drugs at 24 hrs. effects (L050) of PB with fixed doses of isoproterenol (3 Ymg/kg), salbutamol (0.0 tn’ 0.8 mg/kg), terbutaline b ' 24 hr LD50 . Lower Upper (5 mg/kg), yohimbine (1 mg/kg), phentolamine (1 mg/kg), pg; _ (mg/kg) 95% CL 0 e5%cL prazosin= (2 mg/kg) or caffeine (5 mg/kg). cThe open circle at the upper left of each isobologram represents caffeine . 251.3 234.3 269.4 the“"LD“5‘0' with 695% confidence limits. (cL)" of -PB. alone, - epinephrine 8 s 12.8 8.5 19.1 while the corresponding open circle at the lower right ., isoproterenol . . 255.3 - 35.0 1860.5 depicts the L050 with 95% CL for the second drug alone. norepinephrine - 30.9 . 23.7 40.-2. Solid and dashed negative slope diagonals represent the phentolamine t 104.3 8.9.2 1-22.0 locus of expected LD50 points for combinations of the PB - 4.3 . ~.3.9 4.9 2 agents, and the lower 95% CL of this line,_respective- ‘ salbutamol .1665 155.0 l78;9 ly,'given.dose additivity. Solid circles represent the terbutaline P. 186.3 168.9 HBA. @: . L050-points.with 95% CL for binary combinations of the Y°himb.i!l°_* 6 - 42.1 7.5 237 4 t d-rugs _i-nfm'-ice. 5 t t .
216 Vet Human -'-l'oxico'l_ 39 (4).-iAu'gu-st1.997 Mice- pretreated with -either- atropine or Table 2. LD50s with 95% CL~_l'or PB following administration of atropine nmthylnitrate exhibited-a-dramati~ ‘adrenergic drugs at fixed doses.‘ - ' " ‘ cally~ reduced incidence ’of lethality for epinephrine, norepinephrine,- salbutamol, Adrener'gic*drug (mg/kg) -24hr LD50 Lower ’ Upper " A yohimbine or~»c-affei-ne combined with PB, as " p_ PB, 7;)’ "(nigi/leg); 95‘{qQL" 9s%cL shown in Table 3. -_' "i - e -
-caffeine (5) " _ V 12.5“ 1.9 3.3 epinephrine (5) 362.6 " 2.2 3.1 ° ‘DISCUSSION 3 epinephrine(l0) ‘:'i"0.9" e 0.3 ' -2.3 - is0protereno1(3) ‘2;0 " 1.5 ' 2.6 Pyridostigmine bromide is zur anticholines- ' norepinephrine (7.3) - 2.2 1.8 2.9‘ " terase _agent that has 'been ‘used. safely for norepinephrine (10) ‘ 2.2 ~ 1.3 " " 3.6 decades _to 'treat' the ~neurological disorder phentolamine-(1) 2.7 2.3 3.2 known as myasthenia gravisf However, the re- prazosin (2) ' -2.4 2.0 " ’ 2.8 cent 'i"1nplicatii_on of "PB as a causative agent salbutamol (0.4)" ' 2.5 2.0‘ 3.1 in Persian Gulf illnesses requires consider- salbuta.mol*(0.8) ' 2.4 ’ ‘ 2.8 ation ”of' they potential“ modification of PB terbuta1ine(5) V 2.6 . 2.1 ' 3.3 toxicity by *concurrently ingested drugs or yohitnbine (1) - j i 1.8 ' 1.4 WW 2.3 ' exposed ‘chemicals, environmental variables and f_underlying pathological ‘"conditions. Previous studies have discussed the potential for adverse interactions between PB and aness thetic agents commonly used in surgery under caused slight tremor, increased respiration and decreased activity ill the animals. Py- ridostigmine administered alone at 1. mg/kg did not produce any adverse reactions; how- ever increasing the dose to-2 mg/kg produced 4i skeletal muscle tremor and inactivity, while \ \ \ 3 mg/kg caused*tremor, lacrymation, salivae \ \ tion and respiratory depression ‘resulting \ 7 \ in approximately 12.9 i 3.8 (SE) % lethality, \ \ with convulsions immediately preceding death. _ \ \ Pretreatment with selected doses (H? the 6- .- \ 2._~ \ adrenoceptor agonist isoproterenol, selective ‘ \ - 1 \ B?-adrenoceptor agonists salbutamol and ter- . \ \ butaline, d@- and cg-adrenoceptor rantagonist \ \ phentolamine, (selective cl-adrenoceptor ,an+ _ \ . \ tagonist prazosin, selective additivity, providing clear-evidence of p0.- _ . .- . ‘I _ tentiation of the drug interaction (19), even in the cases of yohimbine and isoproterenol where large (HJ were noted. The d- and B- 51\ . ‘ l 1 adrenoceptor agonists epinephrine \\ 'l \ 1 \ . . . \ Table 3. Percent lethality at 24 hrs for combinations of adrenergic drugs and PB alone or Ill the presence of O _ _ V atropine or atropine methyl nitrate. - ' | F-— I , . . P ‘*1 ~ . ."*| ~ 1 ~ , - F _.__._.. , . . . , _ + Atropine Methyl. 0 V 10 20 '30 " 40 u , 2_4 hr + Atropine _ Nitrate Drug Ilnteractiofn_ _ (mg/kg) . i _ in Lema lily 24bx%L.gtl1al1ty' _ _ _ __ 24iu%Lethality_ .. p . - _ V Il0l"€PiH€Phl‘iIlB (mg/kg). epinephrine (5) + PB (3) ' 61.2 25.0 12.5 Figure 2. lsobolograms illustrating the combined lethal norepinephrine (7.3) + PB (3) 70.9 0 0 effects (LD50) of PB) withnfixed doses of epinephrine salbutamol (0.4) + PB (3) 62.5 0 ' 0 yohimbine (I) + PB (3) 83.3 5v- (5 or- 10 mg/kg) and norepinephrine (7.3 or 10 mg/kg) caffeine (5) + PB (3) 7 53.0 _@@ ,_ - .0.-_ p in mice. Symbols are the same as in Figel. Vet Human.ToxicoI 39-‘ (4.) AlI9"$l19-97 ' l 217 military conditions (Z1), but until nQw there (tween. the _parasympathetic _and sympathetic have been -no detailed..reports of central toxicity following AChE inhibition Friedman, A, Kaufer ID, Shemer..J et al: A Pyridostigmine brain by‘soman. Our studies support 21 protective penetration under stress enhances neuronal excitability and in- duces “early‘ immediate transcriptional response. Nat- Med‘ 2: role for clonidine"since it failed to increase 1382-1"3.85..1996-1 1 A 1 . s . t » toxicity when administered_prior to PB. -‘. “ McCain WC, Lee R, Johnson MS et al: Lethal interactive effects p. ' . -_ ~. _ -‘I. ‘ of acute oral exposure to pyridostigmine br0mide,.PeFméthPifl, Although blockade of lethal effects by both and DEET in the laboratory rat. ‘Toxicologist 30: 55, 1996. . atropine and atropine methyl‘ nitrate. would Abou-Donia MB, Wiimarth KR, Jensen KF et al:,_Neurotoxicity re- indicate an peripherally* mediated" mechanism sulting -from .coexposure "ua,pyrid0§tigm1fl€_ hP0m1de, DEET, and of lethality in these studies, the possibility permethrin:" implications of_Gu1f War chemical exposures. J for CNS involvement in‘sublethal toxic effects Toxicol Environ Health #8: 35-56, 1996.‘ - L L Chaney L,_ Moss J, Mozingo J et al: Toxic interactions between could still*-exist.. fFriedman; et :11 (9) 're— pyridostigmine (PB), N,N-diethylfm-toluamide (DEET), adrenergic ported Findirect. evidence ‘that' PB can enter agents and caffeine._ Toxicologist 36: 21, 1997. the CNS under conditions of stress. _In the Moss Jl:~ Synergism of toxicity of N,N-diethyl-m-toluamide to brain,‘cholinergic7and"adrenergic'neurons"in- German, cockroaches- (0rthoptera: Blatte1lidae)_’ by hydrolytic teract in the control of essential activities, enzyme inhibitors. J Econ Entomol 89: 1151-1155, 1996: ' such ans cardio-respiratory function and cog- ‘Hoffman“ BB,‘ Lefkowitz. RJ: Catecholamines, sympathomimetic drugs, ¥and- adrenergic *receptor“ antagonists. In Hardman JG, nitive performance. ""‘.-' C " " . "” _ Limhird LE ct al eds: Goodman and Gilman's The Pharmacological Basis of"Therapeutics. 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