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Integrated Pharmacokinetics and Pharmacodynamics of Atropine in Healthy Humans I: Pharmacokinetics
JOURNAL OF PHARMACEUTICAL SCIENCES
July 1985 Volume 74 Number 7
RESEARCH ARTICLES
Integrated Pharmacokinetics and Pharmacodynamics of Atropine in Healthy Humans I: Pharmacokinetics PETERH. HINDERLING*§',URSULA GUNDERT-REMY*~, AND OLGA SCHMIDLIN** Received September 10, 1984,from the *Department of fharmacolo y, University of Basel, 4056 Basel, Switzerland. Accepted for publication March 15, 1985. Present addresses: §F. Hoffmann-La Roche & AG, 4002 Basel, Switzerland, vDepartmentof Clinical Pharmacology, Ruprecht-Karls-University, 6900 Heidelberg, F.R.G., and *Department of Internal Medicine, University of Basel, 4031 Basel, Switzerland.
80.
Abstract 0 The pharrnacokinetics of atropine in three healthy male volunteers after intravenous administration of 1.35 and 2.15 mg of the drug was determined. Pharmacodynamic effects of atropine were measured simultaneously. All the data were fitted to a novel integrated kinetidynamic model. Plasma concentrations of atropine and the amounts of atropine and its primary metabolite, tropine, excreted in the urine were measured by a sensitive gas chromatographic-mass spectrometric assay. The kinetics of elimination of atropine was first order. There was evidence that the kinetics of distribution of the drug was dose dependent. Two phases with apparent half-lives of 1 and 140 min were distinguishablein accordance with a linear two-compartmentdisposition model for atropine. The urinary excretion of unchanged drug was 57%of the dose. The steady-state volume of distribution was 210 L, implying extensive tissue binding and/or partitioning. Renal plasma clearance was 660 mumin, suggesting significant tubular secretion. The renal clearance of atropine depended on urine flow. Urinary excretion of tropine amounted to 29% of the dose. The kinetics of the metabolite was first order.
Atropine, la~,5a~-tropan-3a-ol (&)-tropate,an alkaloid of the belladonna plant, has been employed in medicine for centuries. Today, its therapeutic use is primarily in preanesthetic medication, the treatment of bradycardia and atrioventricular block, and for poisoning with acetylcholine esterase-blocking compounds. Pharmacologically, atropine can be classified as a selective competitive antagonist of acetylcholine at the muscarinic receptors in smooth muscle, cardiac tissue, exocrine glands, and the central nervous system. Atropine is racemic [( -)-(S)/( +)-(R)-hyoscyamine] and the (-)-(,")-form is reportedly primarily responsible for the peripheral effects of the compound.1 Chemically, atropine is an ester of tropic acid and tropine. It is a weak base with a pKa of 9.56.2
Data on the pharmacokinetics of atropine in humans are scarce. Measurements of apparent atropine concentrations in biological fluids of humans have been attempted with bioassays3 as well as measurements of total radioactivity after administration of labeled drug,&' but the data in these studies did not represent true atropine concentrations. A OO22-3549/85/0700-0703$0 1.OO/O 0 1985, American Pharmaceutical Association
sensitive gas chromatographic-mass spectrometric (GC-MS) assay for atropine in plasma and urine was developed in our laboratory. Preliminary pharmacokinetic data obtained with this assay method have been p u b l i ~ h e d .Virtanen ~,~ et aL9 determined the plasma pharmacokinetics of atropine in healthy subjects under general anesthesia after an intravenous bolus injection of 1.4 mg. Adams et a1.l0 reported the plasma pharmacokinetics of atropine in healthy young volunteers after an intravenous bolus injection of 0.83 mg. These two studies used RIA and a radioreceptor assay. Aaltonen et al." measured simultaneously the plasma pharmacokinetics of (-)-(S)- and racemic atropine after an intravenous bolus dose of 1.3 mg of the drug to patients under general anesthesia. These authors applied simultaneously a radioreceptor assay and RIA. The goal of the present study was to determine the plasma and urine pharmacokinetics of atropine simultaneously with the dynamics of the drug in young healthy volunteers at two dosage levels.
Experimental Section Assay-Atropine (in plasma and urine) and its metabolite, tropine (in urine), were measured by the GC-MS method with selected-ion monitoring (SIM), which has been described in detail elsewhere.2 Briefly, the plasma and urine samples were spiked with deuterated atropine which served as a n internal standard. Atropine was then extracted from plasma or urine under alkaline conditions into chloroform. After reextraction into hydrochloric acid, atropine was then hydrolyzed to tropine. Subsequently, tropine was converted to its heptafluorobutyryl derivative, which was measured by GC-MS (SIM). Base peaks a t miz 124 (heptafluorobutyryl derivative of tropine) and m/z 127 (deuterated heptafluorobutyryl derivative of tropine) were simultaneously monitored. The assay measured separately the metabolically and hydrolytically generated tropine; the former was separated from atropine through the back-extraction from chloroform into hydrochloric acid. "he lower level of sensitivity of the assay was 0.5 ngimL. The concentration-independent precision for atropine was 5 and 10% in plasma and urine, respectively, and 15%for tropine in urine. New calibration curves were generated for each of the days in which samples were assayed. Journal of Pharmaceutical Sciences / 703 Vol. 74, No. 7, July 1985
Pharrnacokinetic Procedures-Ampules containing 0.5 mg of atropine sulfate were obtained commercially (Streuli Ltd., Uznach, Switzerland). One hour before administration, the contents of 2.5 or 5 ampules were mixed with 0.9% NaCl to give a total volume of 22.5 mL. The mixture was then taken up in a syringe, and a 21-mL aliquot was injected; the remaining 1.5 mL was used for the determination of the actual dose injected. Three healthy male volunteers (A, B, and C), between 21-24 years old, took part in the study. All experimental protocols were approved by the University of Base1 Committee on Human Research. The volunteers gave their written consent after being informed about the aim and risks of the study. The subjects were considered healthy; their medical histories and status gave no evidence of diseases and the usual laboratory tests were within the normal range. Their creatinine clearance was, on average, 140 (+ 10) mL/min. Subject B was a nonsmoker, subjects A and C were light smokers (0-10 cigarettes/d). The volunteers each received two doses of the drug in a randomized order: 1.35 and 2.15 mg of atropine (base) were given intravenously as a rapid, constant infusion over 3 min. Intervals of at least 3 weeks elapsed between the two studies. All the experiments were started during the same time period in the morning (7-9 a.m.). The volunteers were supine for 2 h prior to and for the duration of the study (22-36 h). Two hours prior to dosing, the volunteers ate a light breakfast (toast, jelly, butter, and milk). To ensure adequate urine flow, the volunteers drank 10 mL/kg of mineral water 1 h prior to drug administration and were encouraged to drink 40 mL/kg of fluid (mineral water or milk) during the initial 12 h of the study. One-half hour before administration, the volunteers were fitted with two catheters (Viggo Venflon; Viggo AB, Halsingborg, Sweden) which were placed into the antecubital veins of both arms. The catheters were used for separate drug injection and blood sampling. Blood samples (10-24 mL) were taken prior to and at appropriate times up to 10 h after drug administration; they were collected into heparinized tubes. After immediate centrifugation, the separated plasma was transferred to fresh tubes containing the internal standard (deuterated atropine). The samples were immediately frozen and kept a t -20°C until needed for assay. Urine specimens were collected at 0.5-h intervals during the initial 3 h of the study and subsequently a t longer intervals up to 22 h after drug administration. In some experiments, urine was sampled up to 36 h after dosing. The volume and pH of the specimens were measured immediately following sampling. Aliquots of 5 mL were transferred into tubes containing the internal standard. After immediate freezing, they were kept at -20°C until needed for assay. Data Analysis-The following procedures were applied: 1. Compartmental topology-dependent methods were employed when the kinetic data (plasma concentrations of atropine and urinary excreted amounts of atropine and tropine) and the dynamic data (heart rate and saliva flow) of each individual were fitted simultaneously t.o the integrated kineticdynamic model of Scheme IA using the nonlinear least-squares estimation program TOPFIT.12 2. Compartmental topology-independent approaches were used when the kinetic data were analyzed separately. Only original, untransformed data with unit weighting were employed in the fittings. The kinetic part of the integrated model consists of a linear intravenous infusion two-compartment model and a peripheral (tissue) compartment ( T ) (Scheme with a central (P) I). It was assumed that exit [renal elimination (U)and metabolism (M)] is froni P only. The data could only be fitted exactly when the formation of a second metabolite M2 was postulated and, consequently, two kinetically equivalent models (Schemes IA and B) had to be Considered: either sequential metabolism of the drug to M1 and subsequently to M,, simultaneously with renal elimination of MI (Scheme IA), or parallel metabolism of the drug to Ml and M,,where M1 is solely eliminated by the kidney (Scheme IB). Biliary routes of elimination for atropine or tropine were not considered, since fecal elimination of total radioactivity after intravenous administration of the labeled drug in humans was reportedly negligible.4 Since the models of Schemes IA and B are kinetically equivalent, only the former model was chosen for the digital computer fittings. The dynamic part of the model comprises two effect compartments (THRand TSF)(Scheme I), which are kinetically indistinguishable from the peripheral compartment (T). The intensity of the heart rate and of the saliva response (decrease), E S F , response (increase),E,, is proportional to the amount of atropine in the peripheral compartment, T , in accordance with the empirical equations: 704 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
where E,,, T(50), and Eo correspond, respectively, to the theoretical maximum effect, the amount of drug in the peripheral compartment evoking a one-half maximum response, and the baseline effect (control values in the absence of drug); y is the Hill coefficient. Equations 1and 2 represent modifications of the Hill equation.13 It was presumed that (a)the whole chain of events starting with the drug-receptor interaction and ending with the measured change in the intensity of the pharmacological endpoints evolves instantaneously14and ( b ) only atropine exerts pharmacological activity. The acceptance of the proposed integrated dynamic-kinetic model was preceded by the evalution of alternative model solutions. Initial fitting attempts, which employed the kinetic data only, showed that only a two-compartment model-but not a one-compartment model-gave random scatter of the data points about the regression line. In other trials, the effect compartments ( T H R and TSF)were set to be kinetically equivalent to the central (P)or peripheral compartment (27;only the latter setting gave satisfactory fits. This was in accordance with the observation that the maximum effects on heart rate and saliva flow occurred consistently later than the maximum concentrations in plasma. In other preliminary attempts, the E-to-T relationship was modeled according to the equations of Clark16 and Hill.16 Only the application of the Hill equation gave satisfactory fits. Pharmacokinetic Calculations-All the pharmacokinetic parameters were referenced to the total concentration of atropine in plasma. It was assumed that the concentration ratio of bound to unbound drug in the plasma of blood flowing through the excretory and circulatory organs was maintained. In addition, the clearance parameters (renal and hepatic clearance) were referenced to the concentration of atropine in whole blood. Experimental evidence obtained in this study indicated that fractional amounts of drug partitioned into the red cells might have been eliminated, together with the total amount of drug in the plasma in the microvessels of the kidney: the renal clearance of atropine referenced to the total concentration of drug in plasma slightly exceeded the renal plasma flow in two of the six experiments. Atropine shows significant red cell distribution in whole blood, and the erythrocyte-to-plasma partition coefficient, K+, amounts to 1.21 at 3TC2An even more effective red cell repartitioning could be anticipated in blood flowing through the hepatic microvessels, since the mean residence time of
(8) Scheme I
blood is reportedly significantly longer in the capillaries of the liver than in the microvessels of the kidney.17.18 For this reason, referencing the hepatic clearance to the concentration of atropine in whole blood seemed appropriate. Atropine-The apparent volume of the central compartment, V,, was estimated from:
Vp = DlC(0) = DIA
+B
(3)
where D corresponds to the dose, C(0) is the drug concentration at time zero, and A and B represent the coefficients of the corresponding intravenous bolus model. The latter were computed in the standard rnanner.l9 The total area under the plasma concentration-time curve, AUC, was calculated from:
The respectively referenced hepatic extraction efficiencies, EH and 4,were obtained in accordance with:
(14)
(15) where UH and U& correspond, respectively, to the hepatic plasma flow (825 mL/min) and hepatic blood flow.21,2z The apparent pseudo-steady-state volume of distribution, V&,, was computed from:Z3
V&,
AUC(tIast,t,) = C(last)/p
(5)
where C(1ast) and p correspond, respectively, to the concentration of the last measured sample and the slope of the terminal logarithmiclinear phase. The total clearance of atropine, CL, was estimated from:
CL
=
DIAUC
(6)
The renal clearance of atropine referenced to the total concentration of drug in plasma, CLR,was computed from linear plots of unchanged drug excreted in urine during a time interval, U(t,tJ, and the partial plasma area during this time interval, AUC(t,,t,), fitted to a straight line in accordance with
U(t&)
=
CLR.AUC(t,,tj)
(7)
Estimates for CLR referenced to the total drug concentration in plasma were also obtained from:
CLR
=
UIAUC
(8)
where U corresponds to the amount of atropine excreted unchanged in urine at infinite time. Renal clearance referenced to the concentration of atropine in whole blood, CL&,was estimated from:2o
where Hc corresponds to the hematocrit measured in the blood from a large vein (Hc = 0.40). The amounts of drug excreted unchanged at infinite time, U , were obtained from:
where U(to,tlaSt) corresponds to the amount of drug excreted during the time interval from dosing up to the time of the last urine sample, and U(tIa8,,tJ represents the cumulative excreted amount in the subsequent interval up to infinite time. Values for U(tlast,tm)were estimated from:
U(t1,,,,tm)
=
dU/dKlast)/P
(11)
where dU/dt(last) corresponds to the urinary excretion rate of atropine at the end of the last sampling interval. The hepatic clearance of atropine referenced to the total concentration of drug in plasma, CLH, was obtained from:
on the assumption that metabolism of the drug occurs exclusively in the liver. Hepatic clearance of atropine referenced to the concentration of drug in whole blood, CL&,was computed from:
(16)
CLIP
The apparent steady-state volume of distribution, Vd,,, was obtained from:24
vd,, where AUC(to,tl) and AUC(tl,tl,,,) correspond, respectively, to the partial area during the time of infusion and the partial area during the time interval after the infusion was stopped up to the last measured sample; AUC(tl,,,t,) is the partial area from the time of the last measured sample to infinite time. AUC(to,tl) and AUC(t,,t,,,,) were obtained by numerical integration (linear trapezoidal rule); AUC(tIast,t,)was determined by algebraic integration in accordance with:
=
=
Vp(1 + kpTlkTp)
(17)
where KPT and kTP correspond to the rate constants of distribution between the central and peripheral compartments. The average fraction of drug residing in the peripheral compartment, fT, was estimated in accordance withz6
fT
=
T ‘ 6t
T * 6t 0
+
lwP
*
6t
= kpT/(kpT
0
+ kTp)
(18)
The extent of absorption of intramuscularly administered atropine, AE, was obtained from:
AE
=
(UID)im/(U/D)iv
(19)
Tropine and Unknown Metabolites-Estimates of the amount of tropine excreted in urine at infinite time, UMl,were obtained in accordance with the procedure outlined above for the parent drug. The amounts of formed unknown metaboliteb) at infinite time after administration of atropine, M2,were obtained from: = 3.46 x lo-’ The conversion factors for molar units were 1 mol for atropine and 1Fg = 7.09 x lo-’ mol for tropine. Paired t test statistics (two sided) were carried out with the pharmacokinetic parameters.
Results and Discussion The plus-and-minus values for mean values given hereafter in the text refer to the standard deviation of such means. The number of values considered is six unless stated otherwise. All the dosages of atropine refer to the base. The plasma areas and the urinary recoveries of atropine were dose proportional (Table I). This implied first-order kinetics of elimination for the drug. In contrast, the dosenormalized plasma concentrations of atropine showed consistent discrepancies during the initial interval between 3 and 90 min after administration, where the concentrations after the higher dose were consistently larger than after the smaller dose (Fig. 1 ) . This indicated that the kinetics of distribution of atropine were possibly dose dependent. Not withstanding, the atropine data were fitted to a linear disposition model (Scheme IA). Based on the available data, a delineation of the type of the suspected nonlinearity of the kinetics (e.g., saturable binding of atropine in the peripheral compartment, pharmacodynamically induced decrease of drug distribution into the peripheral compartment) was not possible. It must be clearly understood that if the transfer of atropine into the peripheral compartment was dose dependent, the distribution parameters in the chosen model were not true constants and represented concentration- and timeaveraged values. The urinary recoveries of atropine and the generated amounts of unknown metabolites, corrected for the dose, were not different a t the two dosage levels (Table I). The tropine data were fitted to a linear one-compartment model (Scheme IA). Journal of Pharmaceutical Sciences / 705 Vol. 74, No. 7, July 1985
8.48 1180 691 489 17.0 243 221 0.92 60.6 5.04 6.91 4.05 2.86 5.08 3.83 1.24 2.16 0.70 5.08 59.5 31.1 9.4
12.60 797 538 259 10.0 207 177 0.94 47.1 2.82 8.89 6.00 2.89 0.39 0.15 0.25 1.09 1.79 0.39 66.8 10.6 22.6
58.3 1.2 0.429 1 62
21 36
8.17 1225 577 648 10.6 261 222 0.95 60.5 3.05 11.90 5.61 6.30 7.20 4.72 2.49 4.12 3.08 7.20 45.3 35.6 19.2
75.0 0.9 0.485 143
1326
1 .o
7.15 1399 790 609 30.8 204 181 0.83 35.2 7.24 5.01 2.83 2.18 5.09 4.55 0.55 1.94 0.23 5.09 58.6 40.5
46.7 1.5 0.776 89
2026
~-
~~
~
8.18 1223 686 537 14.8 259 231 0.87 64.0 4.39 8.88 4.85 4.03 4.26 2.88 1.38 2.27 2.06 4.26 55.7 24.3 20.0 9.:
0.28 42 107 97 3.6 15" 16" 0.06 6.0 1.16 2.66 0.78 1.97 3.43 2.46 1.05 1.80 1.22 3.43
5.9 0.: 0.004 1
r
~
11.1
I15.8
2
-t
t
t
f
5
2
t
t
r
2
t
2
2
t
t
t
2
?
2
76.8 2 0.9 t 0.481 t 144 2
1350
9.39 1126 626 501 23.4 210 189 0.90 47.9 6.73 5.89 3.53 2.36 3.43 2.98 0.45 1.61 0.76 3.43 57.8 33.1 9.05
2.82 305 5 142 t 210 t 11.6 t 7" 5 17" t 0.07 t 13.2 2 3.68 2 2.67 2 2.21 2 0.46 t 2.64 t 2.46 ? 0.18 5 0.45 t 0.90 t 2.64 t 9.3 2 19.9 t 11.8 2
t
_+
60.0 t 14.2 1.2 t 0.3 0.572 0.182 +- 38 121
21 50
Mean
8.78 1175 656 519 19.1 234 210 0.91 56.0 5.57 7.38 4.19 3.19 3.85 2.93 0.92 1.94 1.41 3.85 56.8 28.7 14.6
1.91 202 t 118 r 147 t 9.1 t 29 5 27 f 0.05 t 12.7 t 2.76 t 2.89 2 1.65 t 1.57 & 2.77 zk 2.20 2 0.84 5 1.23 2 1.20 i 2.77 t 8.3 t 16.8 t 11.9 t
-t
68.4 t 13.4 1.0 t 0.2 0.527 t 0.125 t 31 132
Overall
aValuesfor the exponentialswere obtained from the best fits of the kinetic and dynamic data to the integrated kineticdynamic model of Scheme IA by the digital computer program TOPFIT. The pharmacokinetic data were fitted to an intravenous infusion two-compartment modei. bArea under the plasma concentration-time curve was obtained according to eqs. 4 and 5.'Total clearance was computed accordingto eq. 6. Renal clearance referencedto total concentrationof drug in plasma was estimated from eq. 8. Hepatic clearance referenced to total concentration of drug in plasma was calculated in accordance with eq. 12. 'Apparent volume of distribution of the central compartment was obtained from eq. 3.gApparent pseudo-steady-statevolume of distribution was estimated from eq. 16.hApparent steady-state volume of distribution was calculated from eq. 17. 'Average fraction of atropine residing in the peripheral compartment was calculated from eq. 18. 'Microscopic rate constants of distribution and elimination for parent drug, of formation of tropine and unknown metabolite(sj, and of elimination of tropine were obtained from the fittings employing the computer program TOPFIT. Microscopic rate constants obtained when the data were fitted to the model of Scheme IA (superscript "A') with sequential metabolism or of Scheme IB (superscript "B') with parallel metabolism assumed. "Renally excreted amounts of atropine at infinite time in percent of the dose were obtained from eqs. 10 and 11. 'Renally excreted amounts of tropine at infinite time in percent of the dose were calculated from eqs. 10 and 11. mAmounts of formed, unknown metabolite(s) at infinite time after administration of the parent drug, expressed in percent of the dose, were obtained from eq. 20. "Significantly different, p < 0.05.
8.46 1183 549 634 29.4 21 8 208 0.86 61.5 10.10 3.78 1.76 2.02 4.81 4.25 0.57 1.78 0.24 4.81 48.2 48.3 3.6
72.1 I .o 0.483 143
74.9 0.9 0.510 136
83.3 0.8 0.477 145
7.91 1264 791 473 16.7 273 249 0.93 70.9 5.09 7.81 4.89 2.92 0.50 0.09 0.41 0.52 2.40 0.50 62.2 6.3 31.5
1353
2290
1.99
1.86
1.79
1383
C (79,180)
B (72,174)
A (66,173)
Parameters for Atropine, Tropine (Ml), and Unknown Metabolites (&) after Intravenous Administration
Subject, (kg, cm) Surface area, m2 Dose, pg
Table I-Kinetic
r n r Q
.
100
300
500
Q
Y
v v
T
7
. G
700
Minutes
Flgure 1-Plasma concentrations of atropine and amwnts excreted in urine as percent of atropine dose per liter of plasma and percent of dose, respectively, against time after administration of 2150 pg (solid symbols) and 1350 pg [open symbols) to subjects A, B, and C. Key: (0,0 ) plasma levels of atropine; (7,V)urinary excreted amounts of atropine. The plasma data are not superimposable for an initial interval of 3-90 min after administration.The urinary excreted amounts show no consistent discrepanciesat the two dosage levels.
0.2) min ( t l , 2 , J and 136 ( 2 25) min (tl/2,a) could consistently be distinguished (Fig. 3) with considerable intra- and intersubject variability in t1/2,a (Table I). The apparent volume of the central compartment, Vp, was on average 19 (k 9)L. This value is larger than the volume of plasma26 and indicated fast extravascular and erythrocyte distribution. The percentage of the dose renally excreted as unchanged drug averaged 56.8% (f 8.33) (Table I, Fig. l), implying significant nonrenal elimination pathways for atropine. The different methods used for the calculation of CLR,the renal clearance referenced to the concentrations of drug in plasma, gave similar results. The mean value of 656 (2 118) mumin for CLR was close to the renal plasma flow [712 (& 38) mWminl.27 The individual CLR values exceeded the renal plasma flow in two of the six experiments: by 17%in subject A (lower dose) and by 5% in subject C (higher dose). Renal clearance referenced to the concentration of drug in whole blood, CLg, averaged 603 (f 108) mL/min. All these values imply significant tubular secretion of the drug. A large number of basic drugs have been shown to be tubularly secreted.28 The renal clearance of atropine was urine-flow dependent (range 1-16 mL/min) (Fig. 41, which implied tubular reabsorption of the drug.29 There was no pH dependency (range, 5.5-7.5) of CLR. Hepatic clearance referenced to the total concentration of drug in plasma, CLH,amounted to 519 ( 2 147) mL/min (Table I) and can be regarded as mainly due to metabolism, since fecal elimination of radioactivity was reportedly negligible after intravenous administration of the labeled drug.4 Hepatic clearance referenced to the concentration of drug in whole blood CLk,averaged 476 (f136) mL/min. The respectively referenced hepatic extraction efficiencieswere E = 0.63 and E* = 0.32. Based on either of these values, atropine could be classified as an intermediately cleared Mean values of 234 ( f 29) L were obtained for Vd,, (Table I). A slightly smaller average value of 210 ( 2 27) L was
P
'
Satisfactory fits were obtained for the pharmacokinetic data in all cases, when the kinetic and dynamic data were simultaneously fitted to the integrated kinetic-dynamic model of Scheme IA. Figure 2 shows a typical example of a fit of the pharmacokinetic data from an individual. Atropine-Two phases with apparent half-lives of 1 (+
1
Hours
Figure 2-Typical example of a fit of the experimentally measured p/asma (A) and urine (6)data of atropine and urine (C) data of tropine after administration of 1353 pg of atropine to subject B in accordance with a linear intravenous infusion two-compartmentmodel for atropine and a linear one-compartment model for tropine. The pharmacokinetic and pharmacodynamic data were fitted simultaneously to the integrated model of Scheme IA using TOPFIT. Only the fit of the pharmacokinetic data is shown.
L200
2.._--
I
-
L
-
600
_
I
loo0
Minutes
Figure 3-Semilogarithmic plots against time of plasma concentrations of atropine (A) and the amounts not yet excreted in urine of atropine (B) and tropine (C). Data were obtained in subject A after administration of 1383 pg of atropine. The plasma data show two phases with different decays. Plasma and urinary data of atropine give consistent values for the slope p of the slower phase. The terminal, apparently logarithmic, linear decay of tropine is slightly slower than the terminal phase of atropine. The dashed lines represent the best fitting lines. Journal of Pharmaceutical Sciences / 707 Vol. 74, No. 7, July 1985
. al m
u
. :
5
0
oc
7.
.
1
2
In Urine Flow, mL
3
. min-I
Figure 4-Plot of renal clearance of atropine referenced to the total concentration of drug in plasma against the natural logarithm of urine flow. The values for renal clearance in the individuals were determined separafely for each urine sampling interval in accordance with eq. 7. Renal clearance of atropine (CLR, mL min- I) depends on urine flow (v,,, mL . min- ‘) [CLR = 368 (250.7) 187 (233.6). In v”, r = 0.605,(p < O.OOl)].
+
computed for Vd,, (Table I). The relatively small difference between these two values indicated that the rate constants of distribution were equal to or larger than the rate constant of elimination in the assumed two-compartment model (Table I). The peripheral compartment could be considered shallow. Both volumes of distribution were significantly smaller a t the higher dose level than a t the lower dose level (Table I). The large values of the volumes implied significant extraand intracellular binding and partitioning of the drug. The fraction of drug residing in the peripheral compartment a t pseudo steady state, fT, was similar a t both dosage levels, with an average fT of 0.91 ( 4 0.05) (Table I). Tropine and Unknown MetabolitesSemilogarithmic plots of the urinary excretion rates against time showed that the maximum rates of the main metabolite, tropine ( M I ) , were attained within 3 h after administration of the parent drug in all the experiments. Subsequently, there was an apparent monoexponential decay phase observed in plots of the excretion rate and in CT- plots (Fig. 3). This was in accordance with the one-compartment model assumed for M 1 (Scheme I). The decay phase had a n average apparent halflife of 160 ( * 16) min and was slightly longer than the tl12,p of the parent drug [132 ( * 31) min] (Fig. 3). The urinary recovery of M I averaged 28.7%(* 16.8) of the dose with large intra- and intersubject variation (Table I). The remaining 15.1%(t9.9) of the dose was transformed into the unknown metabolite(s), M2, generated either from atropine (Scheme IA) or tropine (Scheme IB). The results obtained in the present study can be compared with the data of the study reported by Adams et al.,1° since these studies were conducted under similar conditions and with healthy male volunteers of a similar age group. Adams et al., using RIA for the determination of the drug in plasma, found on average 530 mL/min for CL and 8.3 h for tllz,p.Both values were at variance with the 1180 mLImin for CL and 2.2 h for tllP,Pobtained in the present study. The values of these parameters varied considerably more in the study of Adams et al. (CL = 250-1100 mL/min, tllz,p= 2.1-28.2 h) than in the present study (CL = 800-1400 mL/min, tl12,p = 1.5-2.7 h). Clearly, the observed discrepancies in the results obtained in the two studies were caused by the use of different assay methodologies and possibly also by an insufficient sampling scheme in the terminal phase in the study by Adams et al. Virtanen et al.9 and Aaltonen et a1.l1 measured the plasma pharmacokinetics of patients under general anesthesia undergoing major surgery. They employed a n identical RIA procedure. The Virtanen and Aaltonen studies used patients of both sexes between 16 and 58 years old and female patients in the age group of 28-71 years, respectively. The average values for CL and tllz,pfound by Virtanen et al. [CL 708 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
3.0 ( 2 0.9) h] and by Aaltonen = 460 ( 2 200) mL/min t et al. [CL = 380 (+ 2 3 6 ) 2 f g i n , t1/2,P[= 4.3 ( ? 1.7) hl were, not surprisingly, similar. In addition, Aaltonen et al. measured the (-)-(S)-enantiomer of atropine on application of a radioreceptor assay and obtained mean values of 990 (+. 660) mL/min for CL and 3.7 (t 2.3) h for tllz,+ Based on the different plasma concentration-time curves obtained with RIA, which measured both enantiomers, and the radioreceptor assay, which measured only the (-1-(S)-enantiomer, they concluded that the disposition of the two enantiomers in the body was not equal. The renal clearance of atropine referenced to the total concentration of drug in plasma was close to renal plasma flow in the present study. Atropine, in dosages equivalent to those used in the present study, was shown to increase cardiac 0 u t p u t 3 ~ 3so~ that ~ even the highest individual CLR value measured in the present study was compatible with renal physiology. On the other hand, CLR approached renal plasma flow, yet was still dependent on urine flow; the red cell partitioning of atropine is significant.2 The latter facts suggested that referencing renal clearance (and other parameters) to the concentration of drug in whole blood may be more appropriate. However, these CL$ values were not physiologically meaningful unless it was possible to indicate a maximum value for CL&of atropine, CL&,,,, i.e., the renal clearance which would be measured if the total amount of drug in whole blood was eliminated during a single passage through the capillaries of the kidney. The value of CL$,,,, for atropine need not necessarily be equal to the renal blood flow. It has been repeatedly reported that, due to “skimming” of blood, the hematocrit in microvessels of various organs is significantly smaller than the hematocrit in large veins.3236 Spleen and liver capillaries appear to be exceptions to this rule.32The extent of “skimming” varies in the capillaries of different organs and tissues, causing hematocrits to range between 0.14 and 0.41.S4 Two mechanisms responsible for lowering the hematocrit of blood in capillaries have been discussed in the literature: (a)rheological differences between plasma and red cells leading to a larger flow velocity of the cells and generating a plasma anulus in the microvess e 1 and ~ ~ (~b ) shunting of red cell-rich blood around the microvessels.~7The CL&,,,, values expected for atropine (and other drugs) are quite different depending on whether mechanism a or b is operative in the kidney. If “skimming” of blood were the result of differences in the flows of red cells and plasma in the capillaries, then CL&,,= would be equal to renal blood flow [1267 (range, 1206-1341) mL/mi111~~ in the present study, so that all the experimentally measured CL& values would be clearly smaller than CLR,,,,. However, if “skimming” of blood occurred by shunting of red cell-rich blood (hematocrit, 0.861, with the assumption that the hematocrit in the renal capillaries is 0.14,37I ~ ‘ L $ , would , ~ ~ be only 709 (range, 675-751) mL/min, so that one of the individual CL$ values of the present study would exceed CL&,,, by 8%. The results on renal clearance of atropine obtained in the present study could best be explained if (a)the amounts of drug in plasma and in red cells were available for elimination in the capillaries of the kidney and ( b ) “skimming” of blood in the renal capillaries was caused by rheological differences between red cells and plasma. However, it must be clearly understood that, based on the experimental evidence obtained in the present study, neither hypothesis a nor b could be postulated. Information was required on the kinetics of red cell partitioning of atropine and on the mechanism responsible for blood “skimming” in the renal capillaries, obtained in separate experiments. In the absence of this knowledge, a definite conclusion regarding renal
elimination of atropine must be restricted to the qualitative statement that glomerular filtration, tubular secretion, and reabsorption are the mechanisms involved. Similarly, in the absence of information on the kinetics of red cell partitioning of atropine, it is not possible to decide whether the prediction of the magnitude of the hepatic first-pass metabolism of orally administered atropine should be based on hepatic clearance referenced to the concentration of drug in whole blood or in plasma.
Appendix: Glossary D
dose of atropine = concentration of atropine in plasma c = concentration of atropine in plasma at C(0) time t = 0 = concentration of atropine in plasma of C(1ast) the last measured sample = amount of atropine in the central comP partment = amount of atropine in the peripheral T (tissue) compartment = respective amounts of atropine excreted in urine unchanged at time t and at infinite time = amount of atropine in the body = respective amounts of tropine excreted in urine a t time t and at infinite time = amount of atropine metabolized to unM2 known metabolite(s) at infinite time = zero-order infusion rate = parameters of the biexponential fit of the atropine data according to an intravenous bolus two-compartment model = amount of atropine excreted in urine dUldt during a time interval = urinary excretion rate of atropine at dU/dt(last) the end of the last sampling interval = generalized form for the microscopic kij first-order rate constant from the ith to the j t h compartment = apparent volume of the central comVP partment = apparent steady-state volume of distriVd.. bution for atropine = apparent pseudo-steady-state volume v4. of distribution for atropine fT = fraction of atropine dose in the periphera1 (tissue) compartment AUC(t,,t,),AUC = partial and total areas under the plasma concentration-time curve of atropine, respectively = total clearance of atropine CL = renal clearance of atropine referenced CLR to the total plasma concentration of drug = renal clearance of atropine referenced CL; to the concentration of drug in whole blood = hepatic (metabolic) clearance of atroCLH pine referenced to the total plasma concentration of drug = hepatic (metabolic) clearance of atroCL;I pine referenced to the concentration of drug in whole blood =
respective hepatic plasma and blood flow = respective hepatic (metabolic) extraction efficiency referenced to the plasma and blood concentration of drug = urineflow = maximum possible renal clearance of atropine, i.e., if the total amount of drug in whole blood was eliminated during a single passage through the capillaries of the kidney = erythrocyte-to-plasma partition coefficient of atropine = extent of absorption = hematocrit = heart rate = saliva flow = respective control values of heart rate and saliva flow obtained in the ahsence of drug = theoretical maximum effect of atropine on heart rate obtained from the fits = Hill coefficient = respective amounts of drug in the peripheral compartment that evoke a one-half maximum effect on heart rate or saliva flow =
AE Hc
HR SF EO,HR, EO,SF Emax.HR
References and Notes 1. Weiner, N. in “The Pharmacological Basis of Therapeutics,” 6th ed.; Gilman Goodman, A.; Goodman, L. S.; Gilman, A., Eds.; 120-128. MacMillan: London, 1980; 2. Eckert, M.; Hinderling, P. Agents Actions 1981, 21, 520. 3. TQnnesen. M. Acta Pharm. 1950. 6. 147. 4. Gosselin, ’R. E.; Gabourel, J . D.; Wills, J . H. Clin. Phurmacol. Ther. 1960, 1, 597. 5. Kalser, S. C. Ann. N . Y . Acad. Sci. 1971, 179, 667. 6. Kalser, S. C.; McLain, P. L. Clin. Pharmacol. Ther. 1970, 1 1 , 214. 7. Beerman, B.; Hellstrom, K.; Rosen, A. Clin. Sci. 1971, 40,95.
ff
8. Gundert-Remy, U.; Schmidlin, 0.; Hinderling, P. H. Naunyn-
Schmiedebergs Arch. Pharmakol. 1980,311 Suppl., 75. 9. Virtanen, R.; Kanto, J.; Iisalo, E.; Iisalo, E. U.; Salo, M.; Sjoval, S. Acta Anaesth. Scand. 1982.26. 297. 10. Adams, R. G.; Verma, P.; Jackson, A. J.; Miller, R. J . Clin. Pharmacol. 1982,22, 417. 11. Aaltonen, L.; Kanto, J.; Iisalo, E.; Pihlajamaki, K. Eur. J . Clin. Pharmacol. 1984.26, 613. 12. Heinzel, G. in “Pharmacokinetics during Drug Development, Data Analysis and Evaluation Techniques”; van Rossum, J.; Bozler, G., Eds.; G. Fischer Verlag: Stuttgart, 1982; p 207. 13. Boeynams, J. M.; Dumont, J. E. “Outlines of Receptor Theory”; Elsevier: Amsterdam, 1980; p 48. 14. Thron, C. D.; Waud, D. R. J . Pharmacol. Exp. Ther. 1968, 160, 91. 15. Bieynams, J . M.; Dumont, J . E. “Outlines of Receptor Theory”; Elsevier: Amsterdam, 1980; p 3. 16. Sheiner. L. B.: Stanski. D. R.: Vozeh. S.: Miller. R. D.: Ham. J . Clin. Pharmacol. Ther.’1979,25,358. ’ 17. Goresky, C. A. Am. J.Physiol. 1963,204,626. 18. Kramer, K.; Thurau, K.; Deetjen, P. Pfluegers Arch. Gesamte Ph siol. Menschen Tiere 1960, 270,251. 19. Gilaldi, M.; Perrier, D. “Pharmacokinetics”; Dekker: New York, 1975; p 73. 20. Hinderling, P. H. J . Pharm. Sci. 1984, 73, 1042. 21. Caesar, J.; Shaldon, S.; Chiadussi, L.; Guevara, L.; Sherlock, S. Clin. Sci. 1961,21, 43. 22. Cohn, J. N.; Khatri, I. M.; Groszmann, R. J.; Kotelanski, B. Am. J . Med. 1972, 53, 704. 23. Gibaldi, M.; Perrier, D. “Pharmacokinetics”;Dekker: New York, 1975; 69 24. Gibali, M:; Perrier, D. “Pharmacokinetics”; Dekker: New York, 1975; p 180. Journal of Pharmaceutical Sciences I’ 709 Vol. 74, No. 7, July 1985
25. Jusko, W.J.; Lewis, G. p.; Ditted, L.w. Chemotherapy 197% 17, 109. 26. “Documenta Geigy, Scientific Tables”; Diem, K.; Lentner, C., Eds.; Geigy Pharmaceuticals: Ardsley, NY, 1975; 555. 27. Smith, H . K . “Principles of Renal Physiology”; Oxgrd University Press: New York, 1956; 32. 28. Weiner, M.; Mudge, G. H. in.J. Med. 1964,36, 743. 29. Tucker. G. T. Br. J . Clin.Pharmacol. 1981. 12. 761. 30. Klotz, U . Eur. J . Drug Metab. Pharmacoki~et.’1976,1, 129. 31. Gorton, R.; Gurmels, J. C . ; Weissler, A. M.; Stead, E. Circ. Res. 1961, 9, 979. 32. Berry, J. N.; Thompson, H. K.; Miller, D. E.; McIntosh, H. D. Am, Heart J . 1959,58, 204. 33. Cohnstein, J.; Zuntz, D. PfluegersArch. GesamtePhysiol. Menschen Tiere 1888,42, 303.
710 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
34. Gibson, J . G., 11; Seligman, A. M.; Peacock, W. C.; Aub, J. C.; Fine, J. J. Clin.Invest. 1946,25, 848. 35. Schmid-Schonbein, G. W.; Zweifach, B. W. Microuasc. Res. 1975, 10, 153. 36. Klitzman, B.; Duling, B. R. Am. J . Physiol. 1979,237, H481. 37. Pappenheimer, R. J.; Kinter, W. B. Am. J. Physiol. 1956, 185, 337.
Acknowledgments The authors acknowledge the valuable comments by Urs Beat Ranalder, Ph.D., Hoffmann-La Roche Inc., Basel, which were helpful in setting up the routine procedure for the GC-MS assay.
July 1985 Volume 74 Number 7
RESEARCH ARTICLES
Integrated Pharmacokinetics and Pharmacodynamics of Atropine in Healthy Humans I: Pharmacokinetics PETERH. HINDERLING*§',URSULA GUNDERT-REMY*~, AND OLGA SCHMIDLIN** Received September 10, 1984,from the *Department of fharmacolo y, University of Basel, 4056 Basel, Switzerland. Accepted for publication March 15, 1985. Present addresses: §F. Hoffmann-La Roche & AG, 4002 Basel, Switzerland, vDepartmentof Clinical Pharmacology, Ruprecht-Karls-University, 6900 Heidelberg, F.R.G., and *Department of Internal Medicine, University of Basel, 4031 Basel, Switzerland.
80.
Abstract 0 The pharrnacokinetics of atropine in three healthy male volunteers after intravenous administration of 1.35 and 2.15 mg of the drug was determined. Pharmacodynamic effects of atropine were measured simultaneously. All the data were fitted to a novel integrated kinetidynamic model. Plasma concentrations of atropine and the amounts of atropine and its primary metabolite, tropine, excreted in the urine were measured by a sensitive gas chromatographic-mass spectrometric assay. The kinetics of elimination of atropine was first order. There was evidence that the kinetics of distribution of the drug was dose dependent. Two phases with apparent half-lives of 1 and 140 min were distinguishablein accordance with a linear two-compartmentdisposition model for atropine. The urinary excretion of unchanged drug was 57%of the dose. The steady-state volume of distribution was 210 L, implying extensive tissue binding and/or partitioning. Renal plasma clearance was 660 mumin, suggesting significant tubular secretion. The renal clearance of atropine depended on urine flow. Urinary excretion of tropine amounted to 29% of the dose. The kinetics of the metabolite was first order.
Atropine, la~,5a~-tropan-3a-ol (&)-tropate,an alkaloid of the belladonna plant, has been employed in medicine for centuries. Today, its therapeutic use is primarily in preanesthetic medication, the treatment of bradycardia and atrioventricular block, and for poisoning with acetylcholine esterase-blocking compounds. Pharmacologically, atropine can be classified as a selective competitive antagonist of acetylcholine at the muscarinic receptors in smooth muscle, cardiac tissue, exocrine glands, and the central nervous system. Atropine is racemic [( -)-(S)/( +)-(R)-hyoscyamine] and the (-)-(,")-form is reportedly primarily responsible for the peripheral effects of the compound.1 Chemically, atropine is an ester of tropic acid and tropine. It is a weak base with a pKa of 9.56.2
Data on the pharmacokinetics of atropine in humans are scarce. Measurements of apparent atropine concentrations in biological fluids of humans have been attempted with bioassays3 as well as measurements of total radioactivity after administration of labeled drug,&' but the data in these studies did not represent true atropine concentrations. A OO22-3549/85/0700-0703$0 1.OO/O 0 1985, American Pharmaceutical Association
sensitive gas chromatographic-mass spectrometric (GC-MS) assay for atropine in plasma and urine was developed in our laboratory. Preliminary pharmacokinetic data obtained with this assay method have been p u b l i ~ h e d .Virtanen ~,~ et aL9 determined the plasma pharmacokinetics of atropine in healthy subjects under general anesthesia after an intravenous bolus injection of 1.4 mg. Adams et a1.l0 reported the plasma pharmacokinetics of atropine in healthy young volunteers after an intravenous bolus injection of 0.83 mg. These two studies used RIA and a radioreceptor assay. Aaltonen et al." measured simultaneously the plasma pharmacokinetics of (-)-(S)- and racemic atropine after an intravenous bolus dose of 1.3 mg of the drug to patients under general anesthesia. These authors applied simultaneously a radioreceptor assay and RIA. The goal of the present study was to determine the plasma and urine pharmacokinetics of atropine simultaneously with the dynamics of the drug in young healthy volunteers at two dosage levels.
Experimental Section Assay-Atropine (in plasma and urine) and its metabolite, tropine (in urine), were measured by the GC-MS method with selected-ion monitoring (SIM), which has been described in detail elsewhere.2 Briefly, the plasma and urine samples were spiked with deuterated atropine which served as a n internal standard. Atropine was then extracted from plasma or urine under alkaline conditions into chloroform. After reextraction into hydrochloric acid, atropine was then hydrolyzed to tropine. Subsequently, tropine was converted to its heptafluorobutyryl derivative, which was measured by GC-MS (SIM). Base peaks a t miz 124 (heptafluorobutyryl derivative of tropine) and m/z 127 (deuterated heptafluorobutyryl derivative of tropine) were simultaneously monitored. The assay measured separately the metabolically and hydrolytically generated tropine; the former was separated from atropine through the back-extraction from chloroform into hydrochloric acid. "he lower level of sensitivity of the assay was 0.5 ngimL. The concentration-independent precision for atropine was 5 and 10% in plasma and urine, respectively, and 15%for tropine in urine. New calibration curves were generated for each of the days in which samples were assayed. Journal of Pharmaceutical Sciences / 703 Vol. 74, No. 7, July 1985
Pharrnacokinetic Procedures-Ampules containing 0.5 mg of atropine sulfate were obtained commercially (Streuli Ltd., Uznach, Switzerland). One hour before administration, the contents of 2.5 or 5 ampules were mixed with 0.9% NaCl to give a total volume of 22.5 mL. The mixture was then taken up in a syringe, and a 21-mL aliquot was injected; the remaining 1.5 mL was used for the determination of the actual dose injected. Three healthy male volunteers (A, B, and C), between 21-24 years old, took part in the study. All experimental protocols were approved by the University of Base1 Committee on Human Research. The volunteers gave their written consent after being informed about the aim and risks of the study. The subjects were considered healthy; their medical histories and status gave no evidence of diseases and the usual laboratory tests were within the normal range. Their creatinine clearance was, on average, 140 (+ 10) mL/min. Subject B was a nonsmoker, subjects A and C were light smokers (0-10 cigarettes/d). The volunteers each received two doses of the drug in a randomized order: 1.35 and 2.15 mg of atropine (base) were given intravenously as a rapid, constant infusion over 3 min. Intervals of at least 3 weeks elapsed between the two studies. All the experiments were started during the same time period in the morning (7-9 a.m.). The volunteers were supine for 2 h prior to and for the duration of the study (22-36 h). Two hours prior to dosing, the volunteers ate a light breakfast (toast, jelly, butter, and milk). To ensure adequate urine flow, the volunteers drank 10 mL/kg of mineral water 1 h prior to drug administration and were encouraged to drink 40 mL/kg of fluid (mineral water or milk) during the initial 12 h of the study. One-half hour before administration, the volunteers were fitted with two catheters (Viggo Venflon; Viggo AB, Halsingborg, Sweden) which were placed into the antecubital veins of both arms. The catheters were used for separate drug injection and blood sampling. Blood samples (10-24 mL) were taken prior to and at appropriate times up to 10 h after drug administration; they were collected into heparinized tubes. After immediate centrifugation, the separated plasma was transferred to fresh tubes containing the internal standard (deuterated atropine). The samples were immediately frozen and kept a t -20°C until needed for assay. Urine specimens were collected at 0.5-h intervals during the initial 3 h of the study and subsequently a t longer intervals up to 22 h after drug administration. In some experiments, urine was sampled up to 36 h after dosing. The volume and pH of the specimens were measured immediately following sampling. Aliquots of 5 mL were transferred into tubes containing the internal standard. After immediate freezing, they were kept at -20°C until needed for assay. Data Analysis-The following procedures were applied: 1. Compartmental topology-dependent methods were employed when the kinetic data (plasma concentrations of atropine and urinary excreted amounts of atropine and tropine) and the dynamic data (heart rate and saliva flow) of each individual were fitted simultaneously t.o the integrated kineticdynamic model of Scheme IA using the nonlinear least-squares estimation program TOPFIT.12 2. Compartmental topology-independent approaches were used when the kinetic data were analyzed separately. Only original, untransformed data with unit weighting were employed in the fittings. The kinetic part of the integrated model consists of a linear intravenous infusion two-compartment model and a peripheral (tissue) compartment ( T ) (Scheme with a central (P) I). It was assumed that exit [renal elimination (U)and metabolism (M)] is froni P only. The data could only be fitted exactly when the formation of a second metabolite M2 was postulated and, consequently, two kinetically equivalent models (Schemes IA and B) had to be Considered: either sequential metabolism of the drug to M1 and subsequently to M,, simultaneously with renal elimination of MI (Scheme IA), or parallel metabolism of the drug to Ml and M,,where M1 is solely eliminated by the kidney (Scheme IB). Biliary routes of elimination for atropine or tropine were not considered, since fecal elimination of total radioactivity after intravenous administration of the labeled drug in humans was reportedly negligible.4 Since the models of Schemes IA and B are kinetically equivalent, only the former model was chosen for the digital computer fittings. The dynamic part of the model comprises two effect compartments (THRand TSF)(Scheme I), which are kinetically indistinguishable from the peripheral compartment (T). The intensity of the heart rate and of the saliva response (decrease), E S F , response (increase),E,, is proportional to the amount of atropine in the peripheral compartment, T , in accordance with the empirical equations: 704 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
where E,,, T(50), and Eo correspond, respectively, to the theoretical maximum effect, the amount of drug in the peripheral compartment evoking a one-half maximum response, and the baseline effect (control values in the absence of drug); y is the Hill coefficient. Equations 1and 2 represent modifications of the Hill equation.13 It was presumed that (a)the whole chain of events starting with the drug-receptor interaction and ending with the measured change in the intensity of the pharmacological endpoints evolves instantaneously14and ( b ) only atropine exerts pharmacological activity. The acceptance of the proposed integrated dynamic-kinetic model was preceded by the evalution of alternative model solutions. Initial fitting attempts, which employed the kinetic data only, showed that only a two-compartment model-but not a one-compartment model-gave random scatter of the data points about the regression line. In other trials, the effect compartments ( T H R and TSF)were set to be kinetically equivalent to the central (P)or peripheral compartment (27;only the latter setting gave satisfactory fits. This was in accordance with the observation that the maximum effects on heart rate and saliva flow occurred consistently later than the maximum concentrations in plasma. In other preliminary attempts, the E-to-T relationship was modeled according to the equations of Clark16 and Hill.16 Only the application of the Hill equation gave satisfactory fits. Pharmacokinetic Calculations-All the pharmacokinetic parameters were referenced to the total concentration of atropine in plasma. It was assumed that the concentration ratio of bound to unbound drug in the plasma of blood flowing through the excretory and circulatory organs was maintained. In addition, the clearance parameters (renal and hepatic clearance) were referenced to the concentration of atropine in whole blood. Experimental evidence obtained in this study indicated that fractional amounts of drug partitioned into the red cells might have been eliminated, together with the total amount of drug in the plasma in the microvessels of the kidney: the renal clearance of atropine referenced to the total concentration of drug in plasma slightly exceeded the renal plasma flow in two of the six experiments. Atropine shows significant red cell distribution in whole blood, and the erythrocyte-to-plasma partition coefficient, K+, amounts to 1.21 at 3TC2An even more effective red cell repartitioning could be anticipated in blood flowing through the hepatic microvessels, since the mean residence time of
(8) Scheme I
blood is reportedly significantly longer in the capillaries of the liver than in the microvessels of the kidney.17.18 For this reason, referencing the hepatic clearance to the concentration of atropine in whole blood seemed appropriate. Atropine-The apparent volume of the central compartment, V,, was estimated from:
Vp = DlC(0) = DIA
+B
(3)
where D corresponds to the dose, C(0) is the drug concentration at time zero, and A and B represent the coefficients of the corresponding intravenous bolus model. The latter were computed in the standard rnanner.l9 The total area under the plasma concentration-time curve, AUC, was calculated from:
The respectively referenced hepatic extraction efficiencies, EH and 4,were obtained in accordance with:
(14)
(15) where UH and U& correspond, respectively, to the hepatic plasma flow (825 mL/min) and hepatic blood flow.21,2z The apparent pseudo-steady-state volume of distribution, V&,, was computed from:Z3
V&,
AUC(tIast,t,) = C(last)/p
(5)
where C(1ast) and p correspond, respectively, to the concentration of the last measured sample and the slope of the terminal logarithmiclinear phase. The total clearance of atropine, CL, was estimated from:
CL
=
DIAUC
(6)
The renal clearance of atropine referenced to the total concentration of drug in plasma, CLR,was computed from linear plots of unchanged drug excreted in urine during a time interval, U(t,tJ, and the partial plasma area during this time interval, AUC(t,,t,), fitted to a straight line in accordance with
U(t&)
=
CLR.AUC(t,,tj)
(7)
Estimates for CLR referenced to the total drug concentration in plasma were also obtained from:
CLR
=
UIAUC
(8)
where U corresponds to the amount of atropine excreted unchanged in urine at infinite time. Renal clearance referenced to the concentration of atropine in whole blood, CL&,was estimated from:2o
where Hc corresponds to the hematocrit measured in the blood from a large vein (Hc = 0.40). The amounts of drug excreted unchanged at infinite time, U , were obtained from:
where U(to,tlaSt) corresponds to the amount of drug excreted during the time interval from dosing up to the time of the last urine sample, and U(tIa8,,tJ represents the cumulative excreted amount in the subsequent interval up to infinite time. Values for U(tlast,tm)were estimated from:
U(t1,,,,tm)
=
dU/dKlast)/P
(11)
where dU/dt(last) corresponds to the urinary excretion rate of atropine at the end of the last sampling interval. The hepatic clearance of atropine referenced to the total concentration of drug in plasma, CLH, was obtained from:
on the assumption that metabolism of the drug occurs exclusively in the liver. Hepatic clearance of atropine referenced to the concentration of drug in whole blood, CL&,was computed from:
(16)
CLIP
The apparent steady-state volume of distribution, Vd,,, was obtained from:24
vd,, where AUC(to,tl) and AUC(tl,tl,,,) correspond, respectively, to the partial area during the time of infusion and the partial area during the time interval after the infusion was stopped up to the last measured sample; AUC(tl,,,t,) is the partial area from the time of the last measured sample to infinite time. AUC(to,tl) and AUC(t,,t,,,,) were obtained by numerical integration (linear trapezoidal rule); AUC(tIast,t,)was determined by algebraic integration in accordance with:
=
=
Vp(1 + kpTlkTp)
(17)
where KPT and kTP correspond to the rate constants of distribution between the central and peripheral compartments. The average fraction of drug residing in the peripheral compartment, fT, was estimated in accordance withz6
fT
=
T ‘ 6t
T * 6t 0
+
lwP
*
6t
= kpT/(kpT
0
+ kTp)
(18)
The extent of absorption of intramuscularly administered atropine, AE, was obtained from:
AE
=
(UID)im/(U/D)iv
(19)
Tropine and Unknown Metabolites-Estimates of the amount of tropine excreted in urine at infinite time, UMl,were obtained in accordance with the procedure outlined above for the parent drug. The amounts of formed unknown metaboliteb) at infinite time after administration of atropine, M2,were obtained from: = 3.46 x lo-’ The conversion factors for molar units were 1 mol for atropine and 1Fg = 7.09 x lo-’ mol for tropine. Paired t test statistics (two sided) were carried out with the pharmacokinetic parameters.
Results and Discussion The plus-and-minus values for mean values given hereafter in the text refer to the standard deviation of such means. The number of values considered is six unless stated otherwise. All the dosages of atropine refer to the base. The plasma areas and the urinary recoveries of atropine were dose proportional (Table I). This implied first-order kinetics of elimination for the drug. In contrast, the dosenormalized plasma concentrations of atropine showed consistent discrepancies during the initial interval between 3 and 90 min after administration, where the concentrations after the higher dose were consistently larger than after the smaller dose (Fig. 1 ) . This indicated that the kinetics of distribution of atropine were possibly dose dependent. Not withstanding, the atropine data were fitted to a linear disposition model (Scheme IA). Based on the available data, a delineation of the type of the suspected nonlinearity of the kinetics (e.g., saturable binding of atropine in the peripheral compartment, pharmacodynamically induced decrease of drug distribution into the peripheral compartment) was not possible. It must be clearly understood that if the transfer of atropine into the peripheral compartment was dose dependent, the distribution parameters in the chosen model were not true constants and represented concentration- and timeaveraged values. The urinary recoveries of atropine and the generated amounts of unknown metabolites, corrected for the dose, were not different a t the two dosage levels (Table I). The tropine data were fitted to a linear one-compartment model (Scheme IA). Journal of Pharmaceutical Sciences / 705 Vol. 74, No. 7, July 1985
8.48 1180 691 489 17.0 243 221 0.92 60.6 5.04 6.91 4.05 2.86 5.08 3.83 1.24 2.16 0.70 5.08 59.5 31.1 9.4
12.60 797 538 259 10.0 207 177 0.94 47.1 2.82 8.89 6.00 2.89 0.39 0.15 0.25 1.09 1.79 0.39 66.8 10.6 22.6
58.3 1.2 0.429 1 62
21 36
8.17 1225 577 648 10.6 261 222 0.95 60.5 3.05 11.90 5.61 6.30 7.20 4.72 2.49 4.12 3.08 7.20 45.3 35.6 19.2
75.0 0.9 0.485 143
1326
1 .o
7.15 1399 790 609 30.8 204 181 0.83 35.2 7.24 5.01 2.83 2.18 5.09 4.55 0.55 1.94 0.23 5.09 58.6 40.5
46.7 1.5 0.776 89
2026
~-
~~
~
8.18 1223 686 537 14.8 259 231 0.87 64.0 4.39 8.88 4.85 4.03 4.26 2.88 1.38 2.27 2.06 4.26 55.7 24.3 20.0 9.:
0.28 42 107 97 3.6 15" 16" 0.06 6.0 1.16 2.66 0.78 1.97 3.43 2.46 1.05 1.80 1.22 3.43
5.9 0.: 0.004 1
r
~
11.1
I15.8
2
-t
t
t
f
5
2
t
t
r
2
t
2
2
t
t
t
2
?
2
76.8 2 0.9 t 0.481 t 144 2
1350
9.39 1126 626 501 23.4 210 189 0.90 47.9 6.73 5.89 3.53 2.36 3.43 2.98 0.45 1.61 0.76 3.43 57.8 33.1 9.05
2.82 305 5 142 t 210 t 11.6 t 7" 5 17" t 0.07 t 13.2 2 3.68 2 2.67 2 2.21 2 0.46 t 2.64 t 2.46 ? 0.18 5 0.45 t 0.90 t 2.64 t 9.3 2 19.9 t 11.8 2
t
_+
60.0 t 14.2 1.2 t 0.3 0.572 0.182 +- 38 121
21 50
Mean
8.78 1175 656 519 19.1 234 210 0.91 56.0 5.57 7.38 4.19 3.19 3.85 2.93 0.92 1.94 1.41 3.85 56.8 28.7 14.6
1.91 202 t 118 r 147 t 9.1 t 29 5 27 f 0.05 t 12.7 t 2.76 t 2.89 2 1.65 t 1.57 & 2.77 zk 2.20 2 0.84 5 1.23 2 1.20 i 2.77 t 8.3 t 16.8 t 11.9 t
-t
68.4 t 13.4 1.0 t 0.2 0.527 t 0.125 t 31 132
Overall
aValuesfor the exponentialswere obtained from the best fits of the kinetic and dynamic data to the integrated kineticdynamic model of Scheme IA by the digital computer program TOPFIT. The pharmacokinetic data were fitted to an intravenous infusion two-compartment modei. bArea under the plasma concentration-time curve was obtained according to eqs. 4 and 5.'Total clearance was computed accordingto eq. 6. Renal clearance referencedto total concentrationof drug in plasma was estimated from eq. 8. Hepatic clearance referenced to total concentration of drug in plasma was calculated in accordance with eq. 12. 'Apparent volume of distribution of the central compartment was obtained from eq. 3.gApparent pseudo-steady-statevolume of distribution was estimated from eq. 16.hApparent steady-state volume of distribution was calculated from eq. 17. 'Average fraction of atropine residing in the peripheral compartment was calculated from eq. 18. 'Microscopic rate constants of distribution and elimination for parent drug, of formation of tropine and unknown metabolite(sj, and of elimination of tropine were obtained from the fittings employing the computer program TOPFIT. Microscopic rate constants obtained when the data were fitted to the model of Scheme IA (superscript "A') with sequential metabolism or of Scheme IB (superscript "B') with parallel metabolism assumed. "Renally excreted amounts of atropine at infinite time in percent of the dose were obtained from eqs. 10 and 11. 'Renally excreted amounts of tropine at infinite time in percent of the dose were calculated from eqs. 10 and 11. mAmounts of formed, unknown metabolite(s) at infinite time after administration of the parent drug, expressed in percent of the dose, were obtained from eq. 20. "Significantly different, p < 0.05.
8.46 1183 549 634 29.4 21 8 208 0.86 61.5 10.10 3.78 1.76 2.02 4.81 4.25 0.57 1.78 0.24 4.81 48.2 48.3 3.6
72.1 I .o 0.483 143
74.9 0.9 0.510 136
83.3 0.8 0.477 145
7.91 1264 791 473 16.7 273 249 0.93 70.9 5.09 7.81 4.89 2.92 0.50 0.09 0.41 0.52 2.40 0.50 62.2 6.3 31.5
1353
2290
1.99
1.86
1.79
1383
C (79,180)
B (72,174)
A (66,173)
Parameters for Atropine, Tropine (Ml), and Unknown Metabolites (&) after Intravenous Administration
Subject, (kg, cm) Surface area, m2 Dose, pg
Table I-Kinetic
r n r Q
.
100
300
500
Q
Y
v v
T
7
. G
700
Minutes
Flgure 1-Plasma concentrations of atropine and amwnts excreted in urine as percent of atropine dose per liter of plasma and percent of dose, respectively, against time after administration of 2150 pg (solid symbols) and 1350 pg [open symbols) to subjects A, B, and C. Key: (0,0 ) plasma levels of atropine; (7,V)urinary excreted amounts of atropine. The plasma data are not superimposable for an initial interval of 3-90 min after administration.The urinary excreted amounts show no consistent discrepanciesat the two dosage levels.
0.2) min ( t l , 2 , J and 136 ( 2 25) min (tl/2,a) could consistently be distinguished (Fig. 3) with considerable intra- and intersubject variability in t1/2,a (Table I). The apparent volume of the central compartment, Vp, was on average 19 (k 9)L. This value is larger than the volume of plasma26 and indicated fast extravascular and erythrocyte distribution. The percentage of the dose renally excreted as unchanged drug averaged 56.8% (f 8.33) (Table I, Fig. l), implying significant nonrenal elimination pathways for atropine. The different methods used for the calculation of CLR,the renal clearance referenced to the concentrations of drug in plasma, gave similar results. The mean value of 656 (2 118) mumin for CLR was close to the renal plasma flow [712 (& 38) mWminl.27 The individual CLR values exceeded the renal plasma flow in two of the six experiments: by 17%in subject A (lower dose) and by 5% in subject C (higher dose). Renal clearance referenced to the concentration of drug in whole blood, CLg, averaged 603 (f 108) mL/min. All these values imply significant tubular secretion of the drug. A large number of basic drugs have been shown to be tubularly secreted.28 The renal clearance of atropine was urine-flow dependent (range 1-16 mL/min) (Fig. 41, which implied tubular reabsorption of the drug.29 There was no pH dependency (range, 5.5-7.5) of CLR. Hepatic clearance referenced to the total concentration of drug in plasma, CLH,amounted to 519 ( 2 147) mL/min (Table I) and can be regarded as mainly due to metabolism, since fecal elimination of radioactivity was reportedly negligible after intravenous administration of the labeled drug.4 Hepatic clearance referenced to the concentration of drug in whole blood CLk,averaged 476 (f136) mL/min. The respectively referenced hepatic extraction efficiencieswere E = 0.63 and E* = 0.32. Based on either of these values, atropine could be classified as an intermediately cleared Mean values of 234 ( f 29) L were obtained for Vd,, (Table I). A slightly smaller average value of 210 ( 2 27) L was
P
'
Satisfactory fits were obtained for the pharmacokinetic data in all cases, when the kinetic and dynamic data were simultaneously fitted to the integrated kinetic-dynamic model of Scheme IA. Figure 2 shows a typical example of a fit of the pharmacokinetic data from an individual. Atropine-Two phases with apparent half-lives of 1 (+
1
Hours
Figure 2-Typical example of a fit of the experimentally measured p/asma (A) and urine (6)data of atropine and urine (C) data of tropine after administration of 1353 pg of atropine to subject B in accordance with a linear intravenous infusion two-compartmentmodel for atropine and a linear one-compartment model for tropine. The pharmacokinetic and pharmacodynamic data were fitted simultaneously to the integrated model of Scheme IA using TOPFIT. Only the fit of the pharmacokinetic data is shown.
L200
2.._--
I
-
L
-
600
_
I
loo0
Minutes
Figure 3-Semilogarithmic plots against time of plasma concentrations of atropine (A) and the amounts not yet excreted in urine of atropine (B) and tropine (C). Data were obtained in subject A after administration of 1383 pg of atropine. The plasma data show two phases with different decays. Plasma and urinary data of atropine give consistent values for the slope p of the slower phase. The terminal, apparently logarithmic, linear decay of tropine is slightly slower than the terminal phase of atropine. The dashed lines represent the best fitting lines. Journal of Pharmaceutical Sciences / 707 Vol. 74, No. 7, July 1985
. al m
u
. :
5
0
oc
7.
.
1
2
In Urine Flow, mL
3
. min-I
Figure 4-Plot of renal clearance of atropine referenced to the total concentration of drug in plasma against the natural logarithm of urine flow. The values for renal clearance in the individuals were determined separafely for each urine sampling interval in accordance with eq. 7. Renal clearance of atropine (CLR, mL min- I) depends on urine flow (v,,, mL . min- ‘) [CLR = 368 (250.7) 187 (233.6). In v”, r = 0.605,(p < O.OOl)].
+
computed for Vd,, (Table I). The relatively small difference between these two values indicated that the rate constants of distribution were equal to or larger than the rate constant of elimination in the assumed two-compartment model (Table I). The peripheral compartment could be considered shallow. Both volumes of distribution were significantly smaller a t the higher dose level than a t the lower dose level (Table I). The large values of the volumes implied significant extraand intracellular binding and partitioning of the drug. The fraction of drug residing in the peripheral compartment a t pseudo steady state, fT, was similar a t both dosage levels, with an average fT of 0.91 ( 4 0.05) (Table I). Tropine and Unknown MetabolitesSemilogarithmic plots of the urinary excretion rates against time showed that the maximum rates of the main metabolite, tropine ( M I ) , were attained within 3 h after administration of the parent drug in all the experiments. Subsequently, there was an apparent monoexponential decay phase observed in plots of the excretion rate and in CT- plots (Fig. 3). This was in accordance with the one-compartment model assumed for M 1 (Scheme I). The decay phase had a n average apparent halflife of 160 ( * 16) min and was slightly longer than the tl12,p of the parent drug [132 ( * 31) min] (Fig. 3). The urinary recovery of M I averaged 28.7%(* 16.8) of the dose with large intra- and intersubject variation (Table I). The remaining 15.1%(t9.9) of the dose was transformed into the unknown metabolite(s), M2, generated either from atropine (Scheme IA) or tropine (Scheme IB). The results obtained in the present study can be compared with the data of the study reported by Adams et al.,1° since these studies were conducted under similar conditions and with healthy male volunteers of a similar age group. Adams et al., using RIA for the determination of the drug in plasma, found on average 530 mL/min for CL and 8.3 h for tllz,p.Both values were at variance with the 1180 mLImin for CL and 2.2 h for tllP,Pobtained in the present study. The values of these parameters varied considerably more in the study of Adams et al. (CL = 250-1100 mL/min, tllz,p= 2.1-28.2 h) than in the present study (CL = 800-1400 mL/min, tl12,p = 1.5-2.7 h). Clearly, the observed discrepancies in the results obtained in the two studies were caused by the use of different assay methodologies and possibly also by an insufficient sampling scheme in the terminal phase in the study by Adams et al. Virtanen et al.9 and Aaltonen et a1.l1 measured the plasma pharmacokinetics of patients under general anesthesia undergoing major surgery. They employed a n identical RIA procedure. The Virtanen and Aaltonen studies used patients of both sexes between 16 and 58 years old and female patients in the age group of 28-71 years, respectively. The average values for CL and tllz,pfound by Virtanen et al. [CL 708 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
3.0 ( 2 0.9) h] and by Aaltonen = 460 ( 2 200) mL/min t et al. [CL = 380 (+ 2 3 6 ) 2 f g i n , t1/2,P[= 4.3 ( ? 1.7) hl were, not surprisingly, similar. In addition, Aaltonen et al. measured the (-)-(S)-enantiomer of atropine on application of a radioreceptor assay and obtained mean values of 990 (+. 660) mL/min for CL and 3.7 (t 2.3) h for tllz,+ Based on the different plasma concentration-time curves obtained with RIA, which measured both enantiomers, and the radioreceptor assay, which measured only the (-1-(S)-enantiomer, they concluded that the disposition of the two enantiomers in the body was not equal. The renal clearance of atropine referenced to the total concentration of drug in plasma was close to renal plasma flow in the present study. Atropine, in dosages equivalent to those used in the present study, was shown to increase cardiac 0 u t p u t 3 ~ 3so~ that ~ even the highest individual CLR value measured in the present study was compatible with renal physiology. On the other hand, CLR approached renal plasma flow, yet was still dependent on urine flow; the red cell partitioning of atropine is significant.2 The latter facts suggested that referencing renal clearance (and other parameters) to the concentration of drug in whole blood may be more appropriate. However, these CL$ values were not physiologically meaningful unless it was possible to indicate a maximum value for CL&of atropine, CL&,,,, i.e., the renal clearance which would be measured if the total amount of drug in whole blood was eliminated during a single passage through the capillaries of the kidney. The value of CL$,,,, for atropine need not necessarily be equal to the renal blood flow. It has been repeatedly reported that, due to “skimming” of blood, the hematocrit in microvessels of various organs is significantly smaller than the hematocrit in large veins.3236 Spleen and liver capillaries appear to be exceptions to this rule.32The extent of “skimming” varies in the capillaries of different organs and tissues, causing hematocrits to range between 0.14 and 0.41.S4 Two mechanisms responsible for lowering the hematocrit of blood in capillaries have been discussed in the literature: (a)rheological differences between plasma and red cells leading to a larger flow velocity of the cells and generating a plasma anulus in the microvess e 1 and ~ ~ (~b ) shunting of red cell-rich blood around the microvessels.~7The CL&,,,, values expected for atropine (and other drugs) are quite different depending on whether mechanism a or b is operative in the kidney. If “skimming” of blood were the result of differences in the flows of red cells and plasma in the capillaries, then CL&,,= would be equal to renal blood flow [1267 (range, 1206-1341) mL/mi111~~ in the present study, so that all the experimentally measured CL& values would be clearly smaller than CLR,,,,. However, if “skimming” of blood occurred by shunting of red cell-rich blood (hematocrit, 0.861, with the assumption that the hematocrit in the renal capillaries is 0.14,37I ~ ‘ L $ , would , ~ ~ be only 709 (range, 675-751) mL/min, so that one of the individual CL$ values of the present study would exceed CL&,,, by 8%. The results on renal clearance of atropine obtained in the present study could best be explained if (a)the amounts of drug in plasma and in red cells were available for elimination in the capillaries of the kidney and ( b ) “skimming” of blood in the renal capillaries was caused by rheological differences between red cells and plasma. However, it must be clearly understood that, based on the experimental evidence obtained in the present study, neither hypothesis a nor b could be postulated. Information was required on the kinetics of red cell partitioning of atropine and on the mechanism responsible for blood “skimming” in the renal capillaries, obtained in separate experiments. In the absence of this knowledge, a definite conclusion regarding renal
elimination of atropine must be restricted to the qualitative statement that glomerular filtration, tubular secretion, and reabsorption are the mechanisms involved. Similarly, in the absence of information on the kinetics of red cell partitioning of atropine, it is not possible to decide whether the prediction of the magnitude of the hepatic first-pass metabolism of orally administered atropine should be based on hepatic clearance referenced to the concentration of drug in whole blood or in plasma.
Appendix: Glossary D
dose of atropine = concentration of atropine in plasma c = concentration of atropine in plasma at C(0) time t = 0 = concentration of atropine in plasma of C(1ast) the last measured sample = amount of atropine in the central comP partment = amount of atropine in the peripheral T (tissue) compartment = respective amounts of atropine excreted in urine unchanged at time t and at infinite time = amount of atropine in the body = respective amounts of tropine excreted in urine a t time t and at infinite time = amount of atropine metabolized to unM2 known metabolite(s) at infinite time = zero-order infusion rate = parameters of the biexponential fit of the atropine data according to an intravenous bolus two-compartment model = amount of atropine excreted in urine dUldt during a time interval = urinary excretion rate of atropine at dU/dt(last) the end of the last sampling interval = generalized form for the microscopic kij first-order rate constant from the ith to the j t h compartment = apparent volume of the central comVP partment = apparent steady-state volume of distriVd.. bution for atropine = apparent pseudo-steady-state volume v4. of distribution for atropine fT = fraction of atropine dose in the periphera1 (tissue) compartment AUC(t,,t,),AUC = partial and total areas under the plasma concentration-time curve of atropine, respectively = total clearance of atropine CL = renal clearance of atropine referenced CLR to the total plasma concentration of drug = renal clearance of atropine referenced CL; to the concentration of drug in whole blood = hepatic (metabolic) clearance of atroCLH pine referenced to the total plasma concentration of drug = hepatic (metabolic) clearance of atroCL;I pine referenced to the concentration of drug in whole blood =
respective hepatic plasma and blood flow = respective hepatic (metabolic) extraction efficiency referenced to the plasma and blood concentration of drug = urineflow = maximum possible renal clearance of atropine, i.e., if the total amount of drug in whole blood was eliminated during a single passage through the capillaries of the kidney = erythrocyte-to-plasma partition coefficient of atropine = extent of absorption = hematocrit = heart rate = saliva flow = respective control values of heart rate and saliva flow obtained in the ahsence of drug = theoretical maximum effect of atropine on heart rate obtained from the fits = Hill coefficient = respective amounts of drug in the peripheral compartment that evoke a one-half maximum effect on heart rate or saliva flow =
AE Hc
HR SF EO,HR, EO,SF Emax.HR
References and Notes 1. Weiner, N. in “The Pharmacological Basis of Therapeutics,” 6th ed.; Gilman Goodman, A.; Goodman, L. S.; Gilman, A., Eds.; 120-128. MacMillan: London, 1980; 2. Eckert, M.; Hinderling, P. Agents Actions 1981, 21, 520. 3. TQnnesen. M. Acta Pharm. 1950. 6. 147. 4. Gosselin, ’R. E.; Gabourel, J . D.; Wills, J . H. Clin. Phurmacol. Ther. 1960, 1, 597. 5. Kalser, S. C. Ann. N . Y . Acad. Sci. 1971, 179, 667. 6. Kalser, S. C.; McLain, P. L. Clin. Pharmacol. Ther. 1970, 1 1 , 214. 7. Beerman, B.; Hellstrom, K.; Rosen, A. Clin. Sci. 1971, 40,95.
ff
8. Gundert-Remy, U.; Schmidlin, 0.; Hinderling, P. H. Naunyn-
Schmiedebergs Arch. Pharmakol. 1980,311 Suppl., 75. 9. Virtanen, R.; Kanto, J.; Iisalo, E.; Iisalo, E. U.; Salo, M.; Sjoval, S. Acta Anaesth. Scand. 1982.26. 297. 10. Adams, R. G.; Verma, P.; Jackson, A. J.; Miller, R. J . Clin. Pharmacol. 1982,22, 417. 11. Aaltonen, L.; Kanto, J.; Iisalo, E.; Pihlajamaki, K. Eur. J . Clin. Pharmacol. 1984.26, 613. 12. Heinzel, G. in “Pharmacokinetics during Drug Development, Data Analysis and Evaluation Techniques”; van Rossum, J.; Bozler, G., Eds.; G. Fischer Verlag: Stuttgart, 1982; p 207. 13. Boeynams, J. M.; Dumont, J. E. “Outlines of Receptor Theory”; Elsevier: Amsterdam, 1980; p 48. 14. Thron, C. D.; Waud, D. R. J . Pharmacol. Exp. Ther. 1968, 160, 91. 15. Bieynams, J . M.; Dumont, J . E. “Outlines of Receptor Theory”; Elsevier: Amsterdam, 1980; p 3. 16. Sheiner. L. B.: Stanski. D. R.: Vozeh. S.: Miller. R. D.: Ham. J . Clin. Pharmacol. Ther.’1979,25,358. ’ 17. Goresky, C. A. Am. J.Physiol. 1963,204,626. 18. Kramer, K.; Thurau, K.; Deetjen, P. Pfluegers Arch. Gesamte Ph siol. Menschen Tiere 1960, 270,251. 19. Gilaldi, M.; Perrier, D. “Pharmacokinetics”; Dekker: New York, 1975; p 73. 20. Hinderling, P. H. J . Pharm. Sci. 1984, 73, 1042. 21. Caesar, J.; Shaldon, S.; Chiadussi, L.; Guevara, L.; Sherlock, S. Clin. Sci. 1961,21, 43. 22. Cohn, J. N.; Khatri, I. M.; Groszmann, R. J.; Kotelanski, B. Am. J . Med. 1972, 53, 704. 23. Gibaldi, M.; Perrier, D. “Pharmacokinetics”;Dekker: New York, 1975; 69 24. Gibali, M:; Perrier, D. “Pharmacokinetics”; Dekker: New York, 1975; p 180. Journal of Pharmaceutical Sciences I’ 709 Vol. 74, No. 7, July 1985
25. Jusko, W.J.; Lewis, G. p.; Ditted, L.w. Chemotherapy 197% 17, 109. 26. “Documenta Geigy, Scientific Tables”; Diem, K.; Lentner, C., Eds.; Geigy Pharmaceuticals: Ardsley, NY, 1975; 555. 27. Smith, H . K . “Principles of Renal Physiology”; Oxgrd University Press: New York, 1956; 32. 28. Weiner, M.; Mudge, G. H. in.J. Med. 1964,36, 743. 29. Tucker. G. T. Br. J . Clin.Pharmacol. 1981. 12. 761. 30. Klotz, U . Eur. J . Drug Metab. Pharmacoki~et.’1976,1, 129. 31. Gorton, R.; Gurmels, J. C . ; Weissler, A. M.; Stead, E. Circ. Res. 1961, 9, 979. 32. Berry, J. N.; Thompson, H. K.; Miller, D. E.; McIntosh, H. D. Am, Heart J . 1959,58, 204. 33. Cohnstein, J.; Zuntz, D. PfluegersArch. GesamtePhysiol. Menschen Tiere 1888,42, 303.
710 / Journal of Pharmaceutical Sciences Vol. 74, No. 7, July 1985
34. Gibson, J . G., 11; Seligman, A. M.; Peacock, W. C.; Aub, J. C.; Fine, J. J. Clin.Invest. 1946,25, 848. 35. Schmid-Schonbein, G. W.; Zweifach, B. W. Microuasc. Res. 1975, 10, 153. 36. Klitzman, B.; Duling, B. R. Am. J . Physiol. 1979,237, H481. 37. Pappenheimer, R. J.; Kinter, W. B. Am. J. Physiol. 1956, 185, 337.
Acknowledgments The authors acknowledge the valuable comments by Urs Beat Ranalder, Ph.D., Hoffmann-La Roche Inc., Basel, which were helpful in setting up the routine procedure for the GC-MS assay.