( Springer-Verlag 1996

Arch Toxicol (1996) 70: 293—299

OR I G I N A L I NV ES T I G A T I ON

Horst Thiermann · Maria Radtke · Ute Spo¨ hrer Reinhard Klimmek · Peter Eyer

Pharmacokinetics of atropine in dogs after i.m. injection with newly developed dry/wet combination autoinjectors containing HI 6 or HLo¨ 7

Received: 29 May 1995/Accepted: 18 September 1995

Abstract To cope with the rapid onset of the lifethreatening cholinergic crisis after poisoning with organophosphorus compounds, atropine-oxime preparations should be available in autoinjectors allowing i.m. administration also in the absence of a physician. Such a scenario is conceivable in the battlefield, when nerve agents are disseminated, and can no longer be excluded in civilian areas, as demonstrated most recently in Tokyo. In addition, autoinjectors may be of value in agriculture when medical care is remote. The use of second generation oximes with broad antidotal spectrum, e.g., HI 6 (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl)-2-((hydroxyimino)methyl) pyridinium dichloride monohydrate; CAS 34433-31-3) and HLo¨ 7 (1-(((4-(aminocarbonyl)pyridinio)methoxy)methyl) 2,4bis((hydroxyimino)methyl) pyridinium dimethanesulfonate; CAS 145613-73-6) is only possible in dry/wet autoinjectors because their stability is limited in concentrated solution. To detect a possible delay in atropine absorption by the two oximes, the pharmacokinetics of atropine after ‘‘autoinjection’’ in beagle dogs were determined. Commercially available autoinjectors from two manufacturers [STI International Ltd (BJ) and Astra Tech (AT)] were filled with atropine sulfate, either alone (2 mg) or in combination with HI 6 (500 mg) and HLo¨ 7 (200 mg), respectively, and injected according to a complete cross-over design. Atropine concentration was determined as l-hyoscyamine equivalents in a radioreceptor assay (RRA). In the range of 0.1—6.9 ng/ml, atropine sulfate displaced [N-methyl-3H]-scopolamine methyl chloride ([3H]NMS) competitively from rat cerebral cortex membranes. At 200 pmol/l [3H]NMS, IC was 50 1.4$0.1]10~9 M atropine (CV"8.1%). The intraassay deviation was about 6%; day-to-day deviation in

H. Thiermann · M. Radtke · U. Spo¨hrer · R. Klimmek · P. Eyer ( ) Walther-Straub-Institut fu¨r Pharmakologie und Toxikologie, Ludwig-Maximilians-Universita¨t Mu¨nchen, Nubbaumstrasse 26, D-80336 Mu¨nchen, Germany

determination of 1 nM (0.695 ng/ml) atropine was 2.6% (CV"5.2%). AT autoinjectors containing HI 6 delivered only 1.81 mg atropine sulfate while 2.14 mg was released by the other injectors. According to the manufacturer, the reduced delivery was caused by a defective Teflon-coated O-ring as detected later on in the batch used. To allow comparison of the bioavailability of atropine from various autoinjectors, the AUCs were normalized to a constant dose. The atropine absorption half-time (7 min) was not affected either by the autoinjector type or by the combination with oximes. The other pharmacokinetic data likewise did not reveal any differences between the groups. Maximal plasma concentration was 33 ng ml~1, elimination half-life 52 min, V 3.2 l kg~1 and Cl 44 ml min~1 kg~1. The !11 1relatively high clearance of l-hyoscyamine is discussed. Key words Organophosphate antidotes · Oximes · HI 6 · HLo¨ 7 · Autoinjectors · Atropine · Pharmacokinetics

Introduction To cope with the rapid onset of the life-threatening cholinergic crisis after intoxication with organophosphorus compounds, the anticholinergic actions of atropine play the major role in antidotal strategy. Reactivation of the inhibited acetylcholinesterase (AChE) by an oxime is considered an adjunctive, more causally directed therapeutic goal. First generation oximes, pralidoxime and obidoxime, were effective in accidental and suicidal poisoning with a variety of organophosphorus insecticides; however, their value appears rather limited after poisoning with highly toxic organophosphorus nerve agents, particularly soman (for review see Dawson 1994). Second generation oximes, HI 6 (1-(((4(aminocarbonyl) pyridinio)methoxy)methyl)-2-((hydroxyimino)methyl)pyridinium dichloride monohydrate;

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CAS 34433-31-3) and HLo¨ 7 (1-(((4-(aminocarbonyl) pyridinio) methoxy)methyl)-2,4-bis((hydroxy imino) methyl)pyridinium dimethanesulfonate; CAS 14561373-6), being effective in vitro and in animal studies, seem able to fill this therapeutic gap (Oldiges and Schoene 1970; Clement 1981, 1983, 1994; Wolthuis et al. 1981a, b; French et al. 1983; Smith and Wolthuis 1983; Boskovic et al. 1984; Hamilton and Lundy 1989; Alberts 1990; Van Helden et al. 1991, 1992; Melchers et al. 1991, 1994; Clement et al. 1992; Eyer et al. 1992; Lundy et al. 1992; Tattersall 1993; Worek and Szinicz 1993; Adler et al. 1994). For human therapy, single doses of 500 mg HI 6 dichloride (Kusic et al. 1985, 1991) or 200 mg HLo¨ 7 dimethanesulfonate (Eyer et al. 1992) were considered appropriate and should be available for use in a formulation allowing their intramuscular administration also in the absence of a physician. Such a scenario is conceivable in the battlefield, when nerve agents are disseminated, and can no longer be excluded in civilian areas, as demonstrated most recently in Tokyo. In addition, administration by paramedics should be considered in agriculture in those countries where qualified medical care is remote. For this purpose, intramuscular administration by autoinjectors is desirable, requiring dissolution of the oximes in small volumes not exceeding 3 ml (Sidell et al. 1974; Friedl et al. 1989; Schlager et al. 1991). The use of the newer oximes is only possible in dry/wet autoinjectors because their stability is limited in concentrated solution (Eyer and Hell 1985; Eyer et al. 1986, 1988, 1989; Fyhr et al. 1987). The different autoinjector systems have already been evaluated with regard to function (Schlager et al. 1991; Thiermann et al. 1994, 1995) and bioavailability of the oximes (Spo¨hrer et al. 1994). The influence of the oximes on

atropine absorption kinetics, however, has not yet been investigated. In a previous study, a delay in atropine absorption from hyperosmolar solutions containing pralidoxime was reported for humans (Sidell 1974). Such an effect may be detrimental because the early life-threatening effects (broncho constriction, bronchorrhea, central depression of respiration) have to be counteracted as quickly as possible. Hence it was a major objective of our study to investigate possible interactions of oximes on the absorption kinetics of atropine. As a result, the investigation showed that HI 6 and HLo¨ 7 were without effect on the absorption kinetics of atropine, which was determined as l-hyoscyamine equivalents in a radioreceptor assay (RRA).

Materials and methods Autoinjectors Autoinjectors were obtained from STI International Ltd, Frindsbury, Rochester, Kent ME2 4DP/England (Binaject autoinjectors, BJ) and Astra Tech AB, S-43121 Mølndal/Sweden (Astra Tech autoinjectors, AT). For details see Spo¨hrer et al. (1994) and Thiermann et al. (1994).

Experiments with dogs The study was performed in accordance with the current animal welfare regulations, and the protocol was approved by the local Ethical Committee and the local authorities (file number 211-25312-31/92). The dogs were examined by a veterinarian who decided whether the study could be continued after each treatment. Eight male beagles (Hoechst AG, Frankfurt/Main and WILAB, Mu¨nchen, Germany) weighing about 16 kg (Table 1) entered the

Table 1 Pharmacokinetic data of atropine after i.m. injection of atropine alone and of atropine in combination with HI 6 or HLo¨ 7 in dogs. Means$SE, n"8; atropine alone BJ and atropine in combination with HLo¨ 7 BJ, n"7

Weight (kg) Injected dose! (mg kg~1) Absorption t (min) 1@2! c .!9 t .!9 Elimination t (min) "1AUC norm" (ng min ml~1) V (l kg~1) !11 Cl 1(ml min~1 kg~1)

Atropine AT

Atropine BJ

HI 6 AT

HI 6 BJ

HLo¨ 7 AT

HLo¨ 7 BJ

16.19$1.13

16.00$1.03

15.94$1.05

16.05$0.86

15.81$0.95

15.86$0.86

0.137$0.01

0.138$0.01

0.118$0.01

0.136$0.01

0.138$0.01

0.138$0.01

6.37$1.26# 32.16$3.27 22.82$4.01

10.27$2.0 29.27$2.89 28.89$3.31

6.46$2.19 26.94$2.41 17.82$2.05

8.33$2.20 35.96$3.06 20.53$3.64

4.14$0.71 37.88$2.27 14.61$0.72

6.48$0.91 35.13$3.50 19.15$2.60

54.22$3.62

55.81$4.86

50.83$2.34

51.75$3.28

49.08$2.10

50.41$2.54

2928$185 3.36$0.16

2887$261 3.50$0.17

2888$222 3.32$0.14

3332$190 2.81$0.10

2859$232 3.22$0.25

2889$104 3.17$0.19

43.99$2.97

45.43$3.97

45.33$3.90

38.36$2.15

46.12$4.38

43.61$1.63

! The injected amount was calculated from the difference between the amount filled in and the remainder in the system for autoinjectors containing HI 6 and HLo¨ 7; atropine sulfate alone: amount assumed"2.14 mg " AUC was normalized to a dose of 0.125 mg/kg # The absorption in dog 2 was so fast that t and k could not be calculated. Both parameters were excluded from statistics "! !

295 study. It consisted of six different treatments for each dog: atropine alone, atropine in combination with HI 6, and atropine in combination with HLo¨ 7 (both autoinjector types). For details see Spo¨hrer et al. (1994). After ‘‘autoinjection’’, heparinized blood (4 ml, from the foreleg vein) was centrifuged at 10 000 rpm for 2 min. Samples of 300 ll plasma were stored at !78°C until determination. Determination of atropine HP¸C Atropine in concentrated solution as found in autoinjectors was analyzed by HLPC on Li-Chrosphere 60 RP-select-B (5 lM; E. Merck, Darmstadt, Germany) at a flow rate of 1.2 ml/min using an L-6200A pump (Merck-Hitachi, Darmstadt, Germany). The mobile phase consisted of acetonitrile/triethylamine (0.7%)/H O adjusted 2 was apto pH 2.5 with phosphoric acid. An acetonitrile gradient plied, starting with 2% to elute HI 6 completely within 10 min, followed by a linear increase to 10% within 10 min. To clean the column from preservative agents (parabene), acetonitrile was increased to 30% for additional 5 min. Atropine was eluted after 19 min and quantified with a UV/Vis, SPD-6AV detector (Shimadzu, Duisburg, Germany) and a D-2500 Chromato-Integrator (E.Merck, Darmstadt, Germany) calibrated with authentic standards. The detection wavelength was 257 nm. Radioreceptor assay The procedure of Aaltonen (1984) was used with minor modifications. Preparation of receptor material Male Sprague-Dawley rats were decapitated and whole brains (1.51$0.1 g) without cerebellum were rapidly removed and homogenized with a 50 ml glass-Teflon Potter homogenizer (speed control 6 for 1 min; Zell-Homogenisator; Colora Messtechnik GmbH, Lorch, Germany) in 9 vol ice-cold 0.32 M sucrose. The homogenate was centrifuged for 5 min at 2000 g at 4°C, the debris discarded and the supernatant centrifuged twice for 20 min at 10 000 g, with an intermediate rehomogenization in icecold 50 mM sodium phosphate buffer, pH 7.4. The final pellet was suspended in 50 mM sodium phosphate buffer, pH 7.4, to give a protein concentration of about 2 mg/ml and stored at !80°C. The preparation yielded 33 mg receptor protein/rat (Birdsall et al. 1983: 30—35 mg/rat). Protein was determined with the BCA Protein Assay Reagent (Pierce Chemical Company, Rodgau, Germany). Binding of [N-methyl-3H]-scopolamine methyl chloride [N-Methyl3H]-scopolamine methyl chloride ([3H]NMS; specific activity: 85 Ci/mmol) from Amersham Buchler GmbH (Braunschweig, Germany) was diluted automatically (TECAN RSP 5052; Zinsser Analytic, Frankfurt, Germany) to final concentrations from 0.01 to 1.2 nmol/l in 400 ll sodium phosphate buffer (50 mM, pH 7.4) placed in 4 ml polypropylene vials (PR-Ro¨hrchen, Greiner Labortechnik, Nu¨rtingen, Germany). The reaction was started by addition of 100 ll receptor material (400 lg protein/ml) and the assay incubated at 37°C for 60 min. Then the assay was cooled on ice and unbound radioactive material separated by filtration (Zwo¨lffach-Filtrationsgera¨t, Millipore, Eschborn, Germany) through Whatman GF/B glass fiber filters under vacuum (300 mbar), followed by washing the filters five times with 3 ml ice-cold phosphate buffer at 4°C. These steps were executed rapidly to minimize desorption of ligand. The filters were placed in scintillation vials and the receptor membranes solubilized by 100 ll NaOH (1.0 M) for 20 min, followed by addition of 10 ml scintillation cocktail (Aqua Safe, Zinsser Analytic, Frankfurt, Germany). Chemoluminescence was allowed to decay at room temperature for 24 h and liquid scintillation counting (counting time 10 min) was performed in a cooled 1217 Rack Beta Liquid Scintillation Counter (LKB, Turku, Finland). For each ligand concentration, assays were run in triplicates and the arithmetic means used for calculation. To determine non-specific

binding, 10~6 M atropine in phosphate buffer was added to the assays. [3H]NMS bound with high affinity to rat brain membranes (n"4; mean$SD), K being 118$5 pmol/l (Waelbroeck et al. 1986: 120$20 pmol/l) Dand maximal specific binding 1.7 pmol/mg protein (Hulme et al. 1978: 2 pmol/mg). Scatchard analysis confirmed these values (agreeing within 6.3% and 1.9%, respectively) with r2"0.986$0.006. Non-specific binding was linearly related to ligand concentration (r2"0.993$0.005), reaching 2$0.3% at 1.7]K as used in the competition experiments. D Competition assay with atropine The assay consisted of 330 ll 50 mM phosphate buffer, pH 7.4, 50 ll plasma sample, 20 ll [3H]NMS at a final concentration of 200 pmol/l (1.7]K ) and 100 ll receptor preparation (400 lg proD tein/ml). It was incubated and worked up as described above. To keep non-specific binding of atropine (Eckert and Hinderling 1981) and [3H]NMS to plasma proteins constant 50 ll plasma was used throughout (Metcalfe 1981). Plasma was used without any extraction. Atropine concentrations presented in ng/ml refer consistently to racemic atropine sulfate monohydrate (MW 694.8 g). In the range of 0.1—6.9 ng/ml, atropine displaced [3H]NMS competitively from the receptor material (n"7). At 200 pmol/l [3H]NMS IC was 1.37$ 0.1 ]10~9 M atropine (CV"8.1%) 50 !0.94$0.08 (CV"8.6%). After linearization, and the Hill slope the logit-log plot revealed a slope of !0.99$0.05 and an IC of 50 1.44]10~9 M (Fig. 1) without significant difference from parameters obtained by fitting. For atropine determination, an idealized competition curve was used with a Hill coefficient set at 1.0 and an IC of 1.40]10~9 M 50 fitted curve from which lies within the 95% confidence interval of the seven experiments with atropine standards ranging from 1 to 40 ng/ml atropine sulfate in 50 ll plasma. At higher concentrations, up to 70 ng/ml, the measured values deviated by 7.0% from the theoretical ones. The intra-assay deviation was 6.9% for 0.5 nM atropine (0.3475 ng/ml; n"4; CV"7.4%) and 5.1% for 5 nM atropine (3.475 ng/ml; n"4; CV"4.4%). Day-to-day deviation in determination of 1 nM (0.695 ng/ml) atropine was 2.6% (CV"5.2%). Binding and competition analysis (Hulme et al. 1978) The calculations were performed using a computer program (GraphPad INPLOT 4.03, San Diego, Calif., USA). Binding curve: R * K * [¸] #C* [¸] B" t t (1#K * [¸])

(1)

Scatchard transformation: R¸ "R * K!K * R¸ t ¸

(2)

Competition curve: R * K * [¸] t [R¸]" (1#K * [¸]#K @ * [¸@])

(3)

Logit-log transformation: log

A

R¸

B

1

!log [¸@] (4) K@ app B , total binding; R , total amount of receptors; K, association or t affinity constant; ¸, t ligand concentration ([3H] NMS); C, slope of non-specific binding; R¸, receptor-ligand complex concentration; ¸@, competitive ligand concentration (atropine); K@, affinity constant of R¸°!R¸

"log

296

Fig. 1 Competition isotherms of atropine versus [3H]NMS at rat cerebral cortex membranes. Inset panel: linearization in the logit-log plot; ¸@ concentration of competitive ligand; R¸ concentration of receptor-ligand complex; R¸0 specific binding in the presence of L@. n"7; means$SD

¸@; R¸0, specific binding in the presence of ¸@; K@ " app K@/(1#K]¸)"1/IC . 50 binding curve Eq. (1) was used. Linear For fitting the receptor regression by Scatchard analysis followed Eq. (2). The competition curve was fitted by Eq. (3). Linear regression of logit-log transformation was performed using Eq. (4). Pharmacokinetic data were evaluated according to Spo¨hrer et al. (1994).

Statistics The results are usually presented as arithmetic means $SD if not indicated otherwise. Differences between absolute values were estimated for significance (p(0.05, Bonferroni correction) by one-way analysis of variance (ANOVA, Sachs 1978) using the computer program GraphPad Instat 1.0 (San Diego Calif., USA). The coefficients of variation (CV) are given as 100 SD/mean (%).

Results Delivery of atropine from autoinjectors in vitro Both autoinjector types contained approximately 2.35 mg atropine sulfate. BJ autoinjectors delivered 93$5% of the atropine filled in, independent of the ejection back pressure (0—1.2 kg/cm2) (Thiermann et al. 1994). This is about the same magnitude as the release of HI 6 (90$3%). AT autoinjectors delivered 92$4% atropine when ejected without back pressure. However,

when fired against back pressure, delivery of atropine and HI 6 was diminished and scarcely approached 50%, with wide variation. The proportion of released atropine versus released HI 6, however, remained fairly constant.

Delivery of atropine from autoinjectors following injection into beagle dogs The injected amount was calculated as difference between the amount filled in and the remainder determined in the assembly. There was a close correlation between the percentages of remaining atropine and remaining oxime, and the proportion was virtually 1.0. AT autoinjectors containing HI 6 delivered only 1.81 mg atropine sulfate on average, while the other three combination autoinjectors delivered 2.14 mg. A similar delivery was assumed for the autoinjectors containing atropine alone, because in no case (except for one BJ autoinjector, see below) any loss was observed after extracting the cannula (Spo¨hrer et al. 1994; Thiermann et al. 1994). One BJ autoinjector containing HLo¨ 7 delivered only half the declared amount of oxime and atropine. This experiment was considered an outer (Spo¨hrer et al. 1994) and was dropped for statistics. Another BJ autoinjector containing atropine alone showed slow

297

after-dripping when put aside. Inspection of the assembly revealed that the cannula was plugged by a foreign body. This experiment was also excluded from statistics. Pharmacokinetics The pharmacokinetic data as derived from the plasma concentrations are listed in Table 1. A model with first-order absorption and first-order elimination appeared to satisfactorily fit the data. In the case of dog 2 (AT, atropine alone), the absorption of atropine was exceptionally fast and was nearly comparable to i.v. injection so that t could not be determined. Bate1@2! man analysis also was not feasible. Since t was 1@2! normal in the other experiments, this fast absorption may have been caused by injection into blood vessels. The values for t , t and t were not different 1@2! 1@21.!9 between the six groups. AUC and c , after normaliz.!9 ation to a constant dose of 0.125 mg/kg were not different either (p'0.05), whether calculated by Bateman analysis or by residual method, regression analysis and trapezoidal rule (Table 1). To allow comparison of the plasma profile, individual c values were set at 100% .!9 and Bateman analysis was performed. Since no differences were found between the autoinjector types, the data were combined for Fig. 2. The elimination of atropine from plasma was as fast about 52 min) as that of the oximes (t about (t 1@2 1@2 49 min). There were no differences in plasma clearance (Cl 44 ml min~1 kg~1) between the different treat1ments.

Discussion As shown in vitro (Thiermann et al. 1994) and in vivo (Spo¨hrer et al. 1994) AT autoinjectors containing HI 6 delivered their contents incompletely due to defective O-rings, probably resulting in higher friction (Thiermann et al. 1995). To allow comparison of the bioavailability of atropine from the various autoinjectors the AUCs were normalized to an injected dose of 0.125 mg/kg. There was then no difference between the groups, indicating that bioavailability was not influenced by HI 6 or HLo¨ 7 (atropine alone versus atropine combined with HI 6 or HLo¨ 7), and no differences between the two autoinjector types were seen either (AT versus BJ, in each filling mode). The absorption of atropine was not particularly rapid, and t was reached at some 20$5 min with .!9 t "7 min. To our knowledge, no comparable data 1@2! are available for dogs. Studies in man have shown that the velocity of atropine absorption from the musculature varies widely (Berghem et al. 1980; Metcalfe 1981; Saarnivaara et al. 1985; Harrison et al. 1986; Kentala et al. 1989, 1990;

Fig. 2 Time course of atropine plasma concentration after i.m. injection with autoinjectors (both manufacturers) containing atropine alone, atropine in combination with HI 6 and atropine in combination with HLo¨ 7 in beagle dogs. For comparison, the individual curves were normalized to c " 100% derived from max Bateman analysis. The Bateman functions were fitted with r2' 0.999. Means$SE

Ellinwood et al. 1990; Kamimori et al. 1990; Kehe et al. 1992). Also, the site of injection has a significant influence (Kanto et al. 1981; Harrison et al. 1986; Pihlajama¨ki et al. 1986; Kentala et al. 1990). In addition, species differences in absorbance rates have been discussed (Moore et al. 1991). Absorption of atropine may be enhanced when using autoinjectors (Sidell et al. 1974) and can be influenced by the construction of the device (Friedl et al. 1989) or by components mixed to the atropine solution (Sidell 1974). In fact, pralidoxime chloride (300 mg/ml) and hyperosmolar saline (8.5%; 2700 mOsmol/l) did retard atropine absorption in man as measured by the response of heart rate (Sidell et al. 1970). When the concentration of pralidoxime chloride was reduced (190 mg/ml), the effects on atropine absorption were largely reduced (Sidell 1974). They were not observed at all when pralidoxime methanesulfonate (250 mg/ml; 1960 mOsmol/l) was used (Holland and White 1971).

298

The lower osmolarity of the combination autoinjectors used in our study with 1500 and 500 mOsmol/l, respectively, did not affect atropine absorption kinetics, thus supporting the osmolarity hypothesis as put forward by Sidell (1974). Atropine was widely distributed in the tissues of the dogs with V of 3.24 l/kg, which is comparable to data !11 for humans (Adams et al. 1982; Aaltonen et al. 1984; Hinderling et al. 1985; Harrison et al. 1986; Wellstein and Pitschner 1988; Kentala et al. 1989, 1990; Ellinwood et al. 1990; Kamimori et al. 1990; Ali-Melkkila¨ et al. 1993) but at variance with values (2.4 l/kg) found in dogs after i.v. injection of 0.1 mg/kg atropine sulfate (Wurzburger et al. 1977). The unexpected short elimination half-life and the high total plasma clearance of atropine as observed in our study deserve separate comment. Wurzburger et al. (1977) reported an elimination half-life of 2.1 h and a total plasma clearance of 13.5 ml min~1 kg~1. The differences from our results (t 0.9 1@21h; plasma clearance 44 ml min~1 kg~1) are most probably caused by different methodologies of atropine determination. In the study by Wurzburger et al. (1977), an apparently stereoselective radioimmunoassay (RIA) was employed, predominantly resulting in the determination of the pharmacologically inactive d-hyoscyamine. In contrast, the RRA used in our study monitors only biologically active components, principally l-hyoscyamine, but cannot discriminate between the parent drug and possible active metabolites. Human studies with healthy volunteers have repeatedly shown that the best correlation is found between the RRAdetermined concentrations of l-hyoscyamine and the various clinical effects of atropine (Wellstein and Pitschner 1988). In addition, the tissue uptake of the active enantiomer appears to be more intense and hence V D larger in comparison with the inactive enantiomer (for review see Ali-Melkkila¨ et al. 1993). An interesting finding in the studies by Kentala et al. (1989, 1990) should be noted. The authors found that the plasma concentrations of racemic atropine in humans (d,l-hyoscyamine, determined by RIA) were three times higher than those of l-hyoscyamine (determined by RRA), indicating significant differences in the pharmacokinetics of the two enantiomers of atropine. These differences must influence the magnitude of AUC and the metabolic clearance derived therefrom (Hinderling 1985; Kentala 1989, 1990), thus explaining the three times higher clearance of l-hyoscyamine (our study) as compared to d-hyoscyamine (Wurzburger et al. 1977). The elimination rate of atropine appears to be dose dependent. Ellinwood et al. (1990) reported that the elimination half-life of atropine administered i.m. decreased with increasing doses (1.62 h at 4 mg atropine sulfate; assayed by RIA, stereoselectivity not indicated). Since the relative dose of atropine given to our dogs corresponded roughly to that resulting from a human dose of 10 mg atropine sulfate, we feel that an elimina-

tion half-life of approximately 1 h might fit the human data. Moreover, renal clearance referred to kg body weight is expected to be about 1.4 times higher in a 15-kg dog as compared to a 70-kg man (Adolph 1949).

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