Previous evidence from laboratory animal studies indicates that R-thiopental has a greater margin of safety than either the more potent S-thiopental or the clinically used rac-thiopental. Although thiopental can cause cardiovascular depression from direct myocardial effects as well as indirect central nervous system and peripheral effects, no studies have yet determined whether its myocardial effects are enantioselective. A lesser direct effect would provide further evidence supporting R-thiopental as a preferred single enantiomer replacement for rac-thiopental.
The direct myocardial effects of the thiopental enantiomers were compared to those of rac-thiopental and propofol, using a crossover design with small incremental doses infused over 3 min, on separate days, into the left coronary arteries of conscious sheep. Hemodynamic and electrocardiographic measurements were acquired, and serial blood samples were collected during the studies for drug analyses.
All three forms of thiopental and propofol produced significant hemodynamic effects consisting of dose-related and rapid-onset decreases in left ventricular dP/dtmax and stroke volume, and increases in left coronary blood flow and heart rate. Cardiac output, mean arterial blood pressure, and central venous pressure remained unaltered. The effects did not differ significantly among rac-thiopental, enantiopure R- or S-thiopental, or propofol. Arterial blood drug concentrations were consistently less than those associated with systemic effects.
Although previous evidence indicates that R-thiopental could make a suitable single-enantiomer replacement for rac-thiopental, the current study did not find a significant difference in direct cardiac effects among the thiopental enantiomers, racemate, or propofol.
THE enantiomers of many racemic drugs differ sufficiently in their potency, side effects, and pharmacokinetics for one enantiomer to be preferred pharmacologically to the other enantiomer or to the racemate. Accordingly, an appreciable number of enantiopure drugs have been introduced to replace racemates originally marketed1; examples in anesthesia include dexmedetomidine for medetomidine and levobupivacaine for bupivacaine.
Thiopental is a chiral drug that is produced and used as a racemate (rac - or RS -thiopental). As judged by studies in laboratory rodents, the thiopental enantiomers have qualitatively similar but quantitatively different central nervous system (CNS) effects.2Whereas S -thiopental is more potent as an anesthetic, R -thiopental has a greater margin of safety.3The greater potency of S -thiopental seems to be a consequence of its higher potency at enhancing the effects of γ-aminobutyric acid (GABA) at the γ-aminobutyric acid type A (GABAA) receptor.4Other evidence suggests that the greater margin of safety of R - than S -thiopental may be due to R -thiopental having a smaller heart:brain distribution ratio, with the heart being the main site of fatal adverse effects.3Other studies have indicated that R -thiopental might also have a small pharmacokinetic advantage in having a somewhat greater mean total body clearance than S -thiopental.5–7
Taken together, the available data suggest that enantiopure R -thiopental could be pharmacologically preferable to rac -thiopental as an intravenous anesthetic, but no studies have yet compared the cardiac depressant effects of racemic thiopentone and its enantiomers. A lesser direct cardiac effect would provide additional evidence supporting R -thiopental as a preferred single enantiomer replacement for rac -thiopental.
Therefore, the aim of this study was to compare the direct cardiac effects of rac -, R -, and S -thiopental; propofol, which is achiral, was used as a comparator. We used a technique of site-directed coronary arterial injection in previously instrumented conscious sheep that we had developed and validated to study direct versus indirect cardiotoxicity of local anesthetic agents.8In this way, we could study the direct cardiac responses from small doses of the drugs that were equivalent to those perfusing the heart from an intravenous anesthetic dose but small enough to preclude indirect responses from interaction of the recirculated drug with the CNS.
Materials and Methods
The study was approved by the joint Animal Care and Ethics Committee of the Royal North Shore Hospital and the University of Technology, Sydney (Sydney, Australia). Using a crossover design, on separate days, small incremental doses of rac -thiopental, R -thiopental, S -thiopental, and propofol were infused for 3 min into the left coronary arteries of previously prepared conscious sheep. During the studies, hemodynamic and electrocardiographic measurements were continuously acquired, and serial blood samples were collected for drug analyses.
The subjects were nonpregnant Merino first-cross ewes (weight, 43–56 kg; n = 10). During general anesthesia, a left thoracotomy was performed to implant an active redirection transit time flow probe (21 mm Triton 200-306-M; Triton Technology Inc., San Diego, CA) around the pulmonary artery for measurement of cardiac output. A transit-time flow probe (4ss; Transonic System Inc., Ithaca, NY) was placed around the left main coronary artery for measurement of coronary arterial blood flow. A spinal catheter (22-gauge Spinocath®; B. Braun Melsungen AG, Melsungen, Germany) to be used for drug infusion was placed retrograde in the left anterior descending coronary artery (paraconal artery in the sheep) and directed into the left main coronary artery to 1 mm proximal to the left coronary artery bifurcation as seen from its exterior. The position of the catheter tip was validated by fluoroscopy. A pressure transducer catheter (Millar 3-French SPR-52; Millar Instruments Inc., Houston, TX) was placed into the left ventricle through the free wall for measurement of left ventricular pressure. Two pairs of stainless steel electrodes (dorsoventral and craniocaudal positions) were sutured onto the pericardium for recording electrocardiographic signal, and the leads exited toward the dorsal midline. The hemiazygos vein, which drains venous blood from the chest of the sheep, was dissected free and ligated at its ventral (proximal) extremity. A catheter (16-gauge, 70-cm polyurethane, Cavafix®; B. Braun Melsungen AG) was inserted into the hemiazygos vein and the tip advanced 5 cm toward the coronary sinus for blood sampling. The chest was closed layer by layer. Two catheters (16-gauge, 70-cm polyurethane cannulae, Cavafix®), one to be used for measurement of mean arterial blood pressure, the other for arterial blood sampling, were placed into the carotid artery and advanced into the aortic arch, and another was placed in the right atrium via the jugular vein for the measurement of central venous pressure. After the neck incision was closed, an intravenous injection of 75 mg carprofen was given. The implanted cannulae were attached to a constant infusion of heparinized saline via minimum volume extension sets connected to high-pressure (300 mmHg), low-flow (3 ml/h) restrictor devices using a multiple port block from a pressurized 1-l bag (0.9% saline) with heparin (10,000 U) and flucloxacillin (1 g) added. The subjects were placed (and thereafter maintained) in metabolic crates and were monitored until conscious and standing. Postoperatively, buprenorphine (0.3 mg intravenous) was administered three times per day for the first 2 days and twice per day for the next 2 days. Ten days were allowed for recovery, during which time body temperature, heart rate, respiration rate, capillary refill time, wound appearance, appetite, demeanor, urine production, and bowel movements were monitored.
Drugs and Dosage Protocol
Thiopental sodium (racemate: Pentothal®; Abbott Laboratories, Sydney, Australia) and propofol (Diprivan®; AstraZeneca, Sydney, Australia) were supplied from standard hospital stock. R - and S -thiopental were prepared and characterized as previously described.9Infusions of 15 ml, containing doses of 20, 40, and 80 mg rac -, R -, and S -thiopental or 7.5, 15, and 30 mg propofol, were given at a constant rate over 3 min. These doses represented approximately 2.5, 5, and 10% of typical anesthetic induction intravenous doses of thiopental (approximately 800 mg) and propofol (approximately 300 mg) in the adult sheep. The doses were calculated as 50, 100, and 200% of the approximate amount expected to reach the heart after intravenous administration of a usual induction dose, given the fraction of cardiac output normally flowing through the left main coronary artery to be approximately 5%.
Because commercial rac -thiopental is prepared as thiopental sodium containing anhydrous sodium carbonate (60 mg/g) to regulate pH for solubility and to maintain chemical stability, each dose of rac -thiopental was prepared with a different concentration of sodium carbonate in saline solution to standardize the conditions to those of the commercial preparation (20 mg dissolved in 24 mg Na2CO3/100 ml saline, 40 mg dissolved in 16 mg Na2CO3/100 ml saline, 80-mg dose dissolved in saline only). All doses of the thiopental enantiomers were prepared with 32 mg Na2CO3/100 ml saline, and the solutions were adjusted, if necessary, to be within pH 10–10.5 as used clinically. The responses to infusion of respective simulated vehicle solutions (thiopental: saline with 32 mg Na2CO3/100 ml at pH 10.5; propofol: 10% Intralipid®[Baxter Healthcare, Sydney, Australia]), designated as 0-mg doses of the respective drugs, were also determined in each subject.
Before each study, the subject was placed in a sling to prevent recumbency and to maintain position relative to transducers but to allow it to rest as required. After settling into the environment of the laboratory, the subject was monitored for 5 min to determine predrug baseline values. Drug was infused over 3 min at a constant rate, and data were acquired for a further 27 min.
Data Acquisition and Processing
Analog signals, consisting of electrocardiogram, mean arterial blood pressure, cardiac output, coronary arterial blood flow, left ventricular pressure, and first derivative of left ventricular pressure (dP/dt), were acquired at 256 Hz by a physiologic monitoring system (System 6; Triton Technology Inc., San Diego, CA), digitally converted (MP100; Biopac Systems Inc., Santa Barbara, CA), and captured using a personal computer. Derived data consisted of heart rate, stroke volume, and maximum positive value of dP/dt (dP/dtmax) on a beat-by-beat basis. The electrocardiogram was analyzed for arrhythmias, and measurements were made of the time between beginning of P wave to beginning of Q wave or R wave if no Q wave was present (P- to R-wave interval), time from onset of Q wave to end of T wave (Q- to T-wave interval), time between consecutive R waves (R- to R-wave interval), and width of the QRS complex. Also, the Q- to T-wave interval was corrected for heart rate by dividing it by the square root of the R- to R-wave interval (QTc). Because the drugs produced tachycardia, the frequency of the various arrhythmias was adjusted for both the duration of observation and the prevailing heart rate.
Predrug baseline data in each individual study were averaged and assigned values of 100% for comparison with the subsequent values from the respective drug/vehicle infusion periods. Hemodynamic data were divided into 20-s epochs, and the averages during each epoch were determined. Five consecutive electrocardiographic complexes were examined every 1 min during the baseline period, every 20 s for the first 5 min after the commencement of drug/vehicle infusions and every 5 min for 15 min. The data were analyzed for peak effects (Emaxor Emin, as appropriate). In addition, the sum of effects differences, which are analogous to the areas under curve, to 5 and 15 min (SED5and SED15) were determined to account for differences in effect time course.8
To measure concentrations of drug recirculated to other tissues, arterial blood samples were collected immediately before drug infusion and then at 1, 2, 3, 4, 5, 10, 15, 20, and 30 min later. Blood samples from a catheter with its tip in the coronary sinus were also collected in a subset of the subjects. Blood drug concentrations were analyzed by high-performance liquid chromatography, chirally for thiopental9and achirally for propofol.10
Statistix for Windows (version 8; Analytical Software, Tallahassee, FL) was used on a personal computer. Staged data analysis was performed. Whether the drug/vehicle infusion produced a significant effect was determined using the Student one-sample t test by comparison of the relevant maximal/minimal values (expressed as percent baseline) to a value of 100%. If a significant effect was found, maximal/minimal effect and SED data were compared using repeated-measures analysis of variance with drug as a between-subject effect, dose as a within-subject effect, and subject as the repeated measure. A finding of significance was investigated further by post hoc testing of differences between mean values using the method of least significant differences and by comparison of mean values for each drug dose to that of the relevant vehicle using the Dunnett procedure. The null hypothesis was equality of the drug effects. A significance criterion of P < 0.05 was used. All tests were two tailed. Baseline values of variables are given in table 1as the pooled mean values and 95% confidence intervals from 22 to 30 individual studies recorded before administration of the three doses of each drug. Changes from the relevant baseline values obtained after drug administration in each individual study are expressed as mean percentage and 95% confidence interval of the respective baseline value before drug administration.
All three forms of thiopental and propofol produced significant hemodynamic effects consisting of decreased dP/dtmaxand stroke volume and increased left coronary blood flow and heart rate, but cardiac output, mean arterial blood pressure, and central venous pressure remained unaltered. Figure 1shows the time course and magnitude of mean effect on dP/dtmaxas a function of dose. Changes to dP/dtmaxand left coronary artery blood flow were extremely rapid in onset, with maximal effects occurring within approximately 1.5 min and with reciprocal maximal changes to heart rate and stroke volume occurring at the end of the 3-min drug infusion period. Figure 2demonstrates the relative temporal changes for the four main variables at the highest doses of thiopental and propofol; this is representative of the relative time courses found with the other doses. It shows the changes in dP/dtmaxand left coronary artery blood flow occurring almost immediately, with the changes in heart rate and stroke volume occurring more slowly. The changes from R -thiopental occurred and regressed a little faster than those from S -thiopental, and changes from all three forms of thiopental consistently regressed faster than those from propofol. Without exception, the changes from all drugs returned to baseline values within 15 min. Although cardiovascular data were collected for 30 min, values beyond 15 min were not included in the subsequent analyses to preclude introduction of experimental noise. There were also small but significant hemodynamic effects of the vehicles for thiopental and propofol. Both maximally decreased dP/dtmaxand left coronary artery blood flow by approximately 10% (P < 0.05), and that for propofol decreased stroke volume and decreased left coronary blood flow by approximately 12% (P < 0.05).
The effects of the drugs were dose related and gave essentially parallel dose–response curves for maximal effect (fig. 3), as well as for SED5and SED15(fig. 4). Not surprisingly, SED5and SED15values were usually similar for thiopental but diverged more for propofol because of its longer duration of effect. Although the maximal effects of propofol tended to be smaller than those of thiopental at the chosen doses, their longer duration produced dose relations as measured by SED5and SED15that were similar for thiopental and propofol.
Using the dose potency ratio preselected for the experiments (rac -thiopental:propofol = 800:300, based upon equianesthetic doses in sheep), no significant differences between drugs were found for maximal effects on dP/dtmax(P = 0.25, not significant) or heart rate (P = 0.18, not significant). However, the effect of propofol was less on stroke volume (P < 0.001) and left coronary artery blood flow (P = 0.049). No significant differences between rac -thiopental and the separate enantiomers were found for any effect.
No systematic significant effects of any of the drugs was found on P wave–R wave interval or width of the QRS complex. However, the vehicle for propofol increased the width of the P– to R–wave interval by 6%, and both vehicles increased the width of the QRS complex by 6%. Propofol/vehicle did not affect the width of the QTc interval, but all three forms of thiopental increased it (P = 0.008: vehicle by 5%, 20 mg by 7%, 40 mg by 11%, 80 mg by 13%).
Cardiac arrhythmias, predominantly premature ventricular and supraventricular ectopic beats, as well as ventricular tachycardia, were observed sporadically. Arrhythmias were observed occasionally in the control period, with vehicle administration, with all of the drugs, in some of the animals, at each dose. There was no obvious pattern for the drugs, nor was a drug dose–response relation observed.
Blood drug concentrations were maximal at the end of the 3-min infusion period and decreased rapidly thereafter (fig. 5). As expected, drug concentrations in coronary sinus blood were at least 10 times those in arterial blood (fig. 6). The arterial drug concentrations, without exception, were far smaller than those associated with anesthesia. Over the time course sampled, there were no significant differences in the blood concentrations of R - and S -thiopental administered separately or as the components of rac -thiopental when scaled to dose.
It is commonly taught that intravenous administration of thiopental for clinical anesthesia causes reductions in mean arterial blood pressure and cardiac output, along with tachycardia; that these effects result from a combination of direct reduction in myocardial contractility, directly induced venodilatation, and indirectly influence hemodynamics through depression of central control mechanisms11; and that propofol also causes these changes, but usually without an increased heart rate.12However, there is considerable debate about the root cause of the cardiovascular depression from these drugs, especially whether it is primarily a direct effect or mediated by effects on the CNS or peripheral vasculature.
Some of the uncertainty over direct versus indirect effects arises from inconsistent results found in different in vivo and ex vivo models used in its investigation. Apart from dose and dose rate, which have clear pharmacokinetic–pharmacodynamic implications,13,14a particular point that we believe leads to discrepancies among in vivo study results is whether and how the subjects are already anesthetized when studied, and whether they have been acutely or chronically prepared surgically for being studied. Anesthesia per se produces a panoply of hemodynamic and pharmacokinetic changes that differ somewhat according to anesthetic agent choice/conditions15—so, too, with the acute stress responses to surgery.16Under such circumstances, it is arguable whether an observed cardiac drug response is true or damped, and even whether effects of test drugs can be satisfactorily differentiated from those of the background. Performing studies in a conscious chronic preparation can avoid this uncertainty when it is appropriate to do so.
This study was designed to avoid the background problem of anesthesia and surgery and to compare dose–response relations for the direct cardiac effects of thiopental enantiomers and propofol. The results provide unequivocal evidence of direct cardiac depression and of qualitatively and quantitatively similar effects of thiopental and propofol, with no material advantage of enantiopure thiopental over rac -thiopental.
To avoid complications caused by the indirect effects of the anesthetic agents on the CNS and peripheral vasculature in an in vivo preparation and of background general anesthesia, we used conscious subjects and a previously developed technique of site-directed left coronary arterial infusions with doses of test drugs believed to be insufficient to cause such extraneous effects. Our drug delivery approach had been validated previously both anatomically by vascular erosion casting and pharmacokinetically by measuring the resultant regional and systemic blood and heart tissue drug concentrations.8Such validation is necessary because of the potential for drug streaming in the affluent blood, thereby delivering an unforeseen pattern of tissue drug deposition. This can be precluded by placing the catheter tip at an appropriate predetermined site, in this case at the bifurcation of the left anterior descending and circumflex arteries, and using retrograde infusion to maximize turbulence. An abbreviated pharmacokinetic validation was repeated in the current studies by demonstrating that the recirculating drug concentrations in aortic blood were at least 10-fold less than those in coronary sinus blood (fig. 6) and were much smaller than those usually associated with anesthesia.11,12,17
Several previous investigations have used intracoronary administration of thiopental, propofol, or both in large animals. One of these reported on cardiac function in previously prepared conscious dogs, but the purpose was to study ischemia–reperfusion injury, and the results were not directly applicable to the current objectives.18The others involved the use of anesthetized, usually paralyzed and ventilated, acutely prepared dogs19–21or sheep.22Although pharmacokinetic principles were recognized in the dosage regimens by estimating the drug concentrations delivered to the heart using dilution principles, only the most recent of these investigations actually measured the propofol concentrations in coronary sinus blood as a measure of myocardial drug exposure.22
Cardiovascular effects of rac -thiopental, sometimes in comparison to propofol, have been previously documented in a variety of rigorous experiments performed with in vivo 23–26and ex vivo drug administration.27–29However, apart from differences in distribution to the heart relative to the CNS,3very little is known regarding the cardiovascular pharmacology of the thiopental enantiomers. A previous study performed in the isolated perfused rat heart with protein-free perfusate found no difference in the wash-in and wash-out kinetics of the thiopental enantiomers when administered as rac -thiopental.30The current study found that the in vivo cardiovascular effects of rac - and separately administered R - and S -thiopental were remarkably similar, but with a tendency of faster wash-in/wash-out of R -thiopental. Moreover, when scaled by dose to equianesthetic potency (in the sheep), the effects were also similar to propofol, which, although generally less in maximum effect, was usually more prolonged in duration.
Inotropic effect or change in inotropic state or both have been used in many studies as a basis for interpreting pharmacokinetic–pharmacodynamic models of anesthetic agents. In studies comparing the myocardial depression of thiopental and propofol ex vivo , thiopental has been found to cause greater depression than propofol, even when their relative anesthetic potencies were taken into account.28,29Various in vivo studies have reported dose-dependent negative inotropic effects of intravenous thiopental24,25and propofol.31,32A recent study found that the magnitude of the negative inotropic effect of propofol was strongly correlated with the measured propofol concentration in coronary sinus blood.22Taken together, these studies support the existence of a direct myocardial depressant effect of these drugs. In the current study, the maximal negative inotropic effect of thiopental was extremely rapid in onset, again consistent with a primary direct effect, and with the high perfusion of the heart and facile drug diffusion from blood to effect sites. A previous study of rac -thiopental administered by intravenous infusion in sheep reported a small apparent volume of distribution in the heart and a calculated effect site equilibration half-time of 0.72 min.24In the current study, the time to maximal negative inotropic effect of propofol was only slightly longer than that of thiopental, but the rate of regression of its effect was much slower than that of thiopental, suggesting that its relative distribution into heart tissue is greater than thiopental. These differences are consistent with a calculated sheep heart tissue:plasma distribution coefficient of 0.36 for thiopental24and a calculated tissue:blood distribution coefficient of 5.94 for propofol.33However, these experiments have not determined the extent to which the regression of effect is prolonged by the triglyceride vehicle that accompanies propofol acting as a local drug depot.
The depressant effect of thiopental on stroke volume is consistent with a previous study in the dog that found direct effects on systolic shortening function by thiopental,20although in that study, propofol had no effect on systolic shortening and, in another study, only supratherapeutic concentrations of propofol caused decreases in segmental shortening.21However, in our study, the time course of onset and regression of decrease in stroke volume was much slower than that of dP/dtmax, bearing a reciprocal relation with increases in heart rate; cardiac output, mean arterial blood pressure, and central venous pressure remained essentially unchanged. Other studies in the closed-chested dog have reported that intravenous propofol infusion decreased myocardial contractility and stroke volume,31but stroke volume was unaltered in open-chested subjects.32The subjects of the current study were closed chested. This is clearly another variable in experimental design and may help to explain why differences in results occur between different studies. Although species differences in sensitivity are recognized in principle, no systematic attempt has been made to examine in vivo cardiac effects of the intravenous anesthetics in different species with consistent methodology, as has been recently reported for propofol in an ex vivo model.34It is clear that different investigators develop particular expertise in specialized models, each claiming particular insights accordingly. However, for there to be more concerted gains, such as in evaluation of principles or new drugs, it seems that developing unifying principles in experimental design for particular aims deserves some urgent attention.
Although differences in the potency of the thiopental enantiomers have been known for some time,35,36potential clinical advantages of the less potent R -thiopental have been recognized only relatively recently. The principal advantages are because of its greater therapeutic index,3more favorable enantioselectivity of distribution between the heart and CNS,3and to its somewhat greater total body clearance,5–7,37although the latter may be mainly because of its higher plasma unbound fraction.6,7,38Even though such evidence has suggested that R -thiopental could make a suitable single enantiomer substitute for rac -thiopental, the current study did not find a significant difference in cardiac effects between enantiomers, although the rate of onset and regression of cardiac effects was consistent with a faster wash-in and wash-out of R -thiopental into cardiac tissues.
The authors thank their colleagues Ray Kearns (Animal House Manager), Sonia Gu, B.Sc. (Research Assistant), Robert Preston, B.Sc. (Research Assistant), and Kylie Wilson, B.Sc. (Technical Officer), all of the Royal North Shore Hospital, St. Leonards, Australia, and Rosemary Einstein, Ph.D. (Associate Professor), of the Department of Pharmacology, The University of Sydney, Sydney, Australia, for their collaboration.