Introduction

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Antipyrine, a marker of total body water (Soberman et al., 1949), including pulmonary extravascular water (Brigham et al., 1971), distributes to a volume as large as total body water in a blood flow-dependent manner in many tissues and is thus a prototype for many lipophilic drugs, including intravenous anesthetics (Renkin, 1952, 1955). Unlike intravenous anesthetics, antipyrine has no systemic cardiovascular effects to affect its own disposition and is thus a useful surrogate for lipophilic drugs in pharmacokinetic studies in which physiologic stability is desirable to meet the assumption of system stationarity (Riggs, 1963).

Factors affecting the early arterial drug concentration versus time profile influence the intensity and timing of the onset of drug effect for rapidly acting drugs, such as intravenous anesthetics (Krejcie and Avram, 1999). We have developed a recirculatory pharmacokinetic model of drug disposition using antipyrine as a surrogate for lipophilic drugs, such as thiopental, to enable studies of factors affecting the initial disposition of drugs with a rapid onset of effect (Krejcie et al., 1996a). This model has been used to study antipyrine disposition in canine studies of various paradigms of altered cardiac output and blood flow distribution, including different levels of halothane (Avram et al., 1997) and isoflurane (Avram et al., 2000) anesthesia, volume loading as well as mild and moderate hypovolemia in awake dogs (Krejcie et al., 1999), and infusions of isoproterenol, nitroprusside, and phenylephrine in awake dogs (Krejcie et al., 2001). These studies have demonstrated that not only cardiac output but also its peripheral distribution affects the early antipyrine concentration history after rapid intravenous administration. Changes in early antipyrine distribution are not proportional to changes in cardiac output because regional blood flow changes depend not only on the altered cardiac output but also on the physiologic circumstances leading to these changes in cardiac output. The purpose of the present study was to test the assumption that antipyrine is a useful surrogate for the flow-dependent tissue distribution of lipophilic drugs, such as thiopental, by comparing the dispositions of antipyrine and thiopental after concomitant administration.

	Materials and Methods

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Experimental Protocol. Five male dogs, weighing 32 to 42.3 kg (36.7 ± 4.6 kg; Table 1), were studied in this Institutional Animal Care and Use Committee-approved study. Approximately 1 month before being studied, a Vascular-Access-Port (Access Technologies, Skokie, IL) was implanted with its catheter tip positioned near the aortic bifurcation via a femoral artery of each dog to facilitate frequent percutaneous arterial blood sampling (Garner and Laks, 1985). Details of the preparation and conduct of the studies have been described in detail previously (Krejcie et al., 1999).

Analytical Methods. Plasma ICG concentrations of all samples obtained up to 20 min were measured on the study day by the HPLC technique of Grasela et al. (1987), as modified in our laboratory (Henthorn et al., 1992). Plasma antipyrine concentrations were measured in all samples using a modification of an HPLC technique developed in our laboratory (Krejcie et al., 1994, 1996a). Plasma thiopental concentrations were measured within 24 h of sample collection using an HPLC technique developed in our laboratory (Avram and Krejcie, 1987).

Pharmacokinetic Model. The pharmacokinetic modeling method (Fig. 1) has been described in detail previously (Krejcie et al., 1996a; Avram et al., 1997). It is based on the approach described by Jacquez (1996) for obtaining information from outflow concentration histories, the so-called inverse problem. Antipyrine and thiopental distributions were analyzed as the convolution of their intravascular behavior, determined by the pharmacokinetics of concomitantly administered ICG, and tissue distribution kinetics (Krejcie et al., 1996a).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   The general model for the recirculatory pharmacokinetics of ICG, antipyrine, and thiopental (Krejcie et al., 1996a). Cardiac output (CO) flows through the central circulation, which is defined by the delay elements (VC). All delay elements are represented generically by rectangles surrounding four compartments, although the number of compartments needed in a delay varied between 2 and 30. The pulmonary tissue volumes (VT-P), subsets of VC, are calculated for antipyrine and thiopental by subtracting the VC of ICG from those of antipyrine and thiopental. Beyond the central circulation, cardiac output distributes to numerous circulatory and tissue pathways which lump, on the basis of their blood volume to flow ratios or tissue volume to distribution clearance ratios (MTTs), into fast (CLND-F, VND-F) and slow (CLND-S, VND-S) peripheral blood circuits (ICG) or the nondistributive peripheral pathway (CLND, VND) and the fast (CLT-F, VT-F) and slow (CLT-S, VT-S) tissue volume groups (antipyrine and thiopental). ICG, which distributes only within the intravascular space, does not have fast and slow tissue volumes. The nondistributive flow for ICG was resolved into fast and slow components; antipyrine and thiopental do not have identifiable (i.e., mathematically distinct) second nondistributive peripheral pathways. The elimination clearances (CLE) are modeled from the arterial sampling site without being associated with any particular peripheral circuit.

	Results

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The pharmacokinetics of ICG and antipyrine in these dogs (Tables 1 and 2) were nearly identical to those described previously in dogs anesthetized with 1.5% halothane but not given thiopental (Avram et al., 1997). Blood ICG, antipyrine, and thiopental concentration versus time relationships were well characterized by the models from the moment of injection (Figs. 2-4). The one-sample runs test confirmed that there were no systematic deviations of observed data from calculated values.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Arterial blood ICG concentration histories for the first 1.5 min (illustrating the first- and second-pass peaks) and for 20 min (inset) following right atrial injection in dog 5. The dots represent drug concentrations, whereas the lines represent concentrations predicted by the model.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Arterial blood antipyrine concentration histories for the first 1.5 min (illustrating the first- and second-pass peaks) and for 600 min (inset) following right atrial injection in dog 5. The dots represent drug concentrations, whereas the lines represent concentrations predicted by the model.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Arterial blood thiopental concentration histories for the first 1.5 min (illustrating the first- and second-pass peaks) and for 600 min (inset) following right atrial injection in dog 5. The dots represent drug concentrations, whereas the solid lines represent the concentrations predicted by the model fit independently to the thiopental data, and the dashed line represents the concentrations predicted by the model fit to the thiopental data with the parameters describing the central circulation and nondistributive peripheral pathway fixed to those of antipyrine.

The total volume of distribution (V_SS) of thiopental was more than 60% larger than that of antipyrine, reflecting differences in the volumes of both the rapidly and the slowly equilibrating tissue compartments, V_T-F and V_T-S, respectively. Nonetheless, the initial distribution kinetics of the two drugs, including the pulmonary tissue volume and the volume of the nondistributive circuit, V_ND, as well as the clearance to it, CL_ND, were nearly identical. As a result, the fraction of cardiac output involved in the distribution of the two drugs to peripheral tissues was similarly identical, although the partitioning of cardiac output between clearance to the rapidly equilibrating tissues, CL_T-F, and clearance to the slowly equilibrating tissues, CL_T-S, differed slightly.

To further test the assumption that antipyrine is a useful surrogate for the flow-dependent tissue distribution of lipophilic drugs, such as thiopental, recirculatory thiopental pharmacokinetics was modeled with several of its parameters fixed to those of the antipyrine model (Table 3). When the parameters describing the central circulation and nondistributive peripheral pathway were fixed to those of antipyrine, the recirculatory thiopental pharmacokinetic model was nearly identical to that of thiopental modeled independently; only V_T-P and CL_T-S differed from those estimated by the independent model by more than 10%. That these differences (a 40-ml distribution volume difference due to fixing V_T-P to the antipyrine volume and a 60 ml/min distributional blood flow difference in CL_T-S) had no practical significance is illustrated by the similarity of the fit of this model to the data to the fit of the independent model to the data (Fig. 4, dashed and solid lines, respectively). When the parameters describing the central circulation, the nondistributive peripheral pathway, and either CL_T-F or CL_T-S were fixed to those of antipyrine, the recirculatory thiopental pharmacokinetic model was quite different from that of thiopental modeled independently; V_SS decreased by more than 13% due largely to a more than 40% decrease in V_T-F, and CL_T-F decreased by more than 10% with a corresponding increase in CL_T-S of more than 69%. The Akaike information criterion and the Schwarz-Bayesian information criterion provided no guidance as to the appropriateness of the choice of model as these parameters differed only in the third significant figure and even then did so inconsistently.

	Discussion

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The physicochemical characteristics of antipyrine and thiopental are quite different. Antipyrine is a base with a pK_a of 1.4, hence its fraction ionized at physiologic pH is less than 0.1% (Wu et al., 1995), whereas thiopental is an acid with a pK_a of 7.6, hence its fraction ionized at physiologic pH is 61.3% (Dundee, 1974). Antipyrine is moderately lipophilic, with an octanol/pH 7.4 Krebs-Henseleit buffer partition coefficient of 1.738 (Wu et al., 1995), whereas thiopental, the prototypical highly lipid-soluble drug (Mühlebach et al., 1985), has an octanol/water partition coefficient of 631 (Steiner et al., 1991). Antipyrine is minimally bound by plasma proteins, with a free fraction of 94% (Wu et al., 1995), whereas thiopental is extensively bound by plasma proteins, with a free fraction of 15% (Burch and Stanski, 1983). Despite the physicochemical differences between antipyrine and thiopental, their pharmacokinetics are remarkably similar.

Transcapillary blood clearance (i.e., diffusion) of both antipyrine and thiopental into tissue is generally reported to be blood flow-limited. The cerebral distribution of thiopental is essentially blood flow-limited (Upton et al., 2000), as is that of antipyrine, which has been used to measure regional cerebral blood flow (Reivich et al., 1969). Renkin (1955) demonstrated blood flow-limited antipyrine distribution in the isolated hindlimb preparation, and similar flow-limited antipyrine distribution has been demonstrated in the isolated perfused liver (Hussein and Rowland, 1992).

Physiologic pharmacokinetic studies of thiopental (Ebling et al., 1994; Wada et al., 1997) and other lipid-soluble drugs, such as fentanyl and alfentanil (Björkman et al., 1993), have found significant diffusion barriers in some tissues. These analyses suggest that the heterogeneity of diffusion barriers may invalidate the simplistic assumption that tissue distribution clearances may be equated with blood flows. However, the limitations to thiopental diffusion into brain, heart, liver, and muscle were low in the report of Ebling et al. (1994) and lower or even absent in their corrected model (Wada et al., 1997). Blood flow to these tissues accounted for 57% of cardiac output (Wada et al., 1997), which compares favorably with the 55% of cardiac output represented by thiopental distributive clearances in the present recirculatory model [i.e., (CL_T-F + CL_T-S)/CL_tot] (Table 2).

The largest parenchymal diffusion barrier reported by Ebling et al. (1994) and Wada et al. (1997) was that of the skin. This corresponds to the observation of Renkin (1955) that blood flow to an isolated hindlimb equaled antipyrine distribution clearance only when the limb was skinned.

The parameters determining the initial distribution of antipyrine and thiopental in the present study were nearly identical (Table 2). The V_C and V_ND of antipyrine and thiopental differed by less than 5% and, like antipyrine, thiopental had minimal first-pass pulmonary uptake, which is consistent with the observations of Roerig et al. (1989). Nondistributive blood flow, CL_ND, of the antipyrine and thiopental models differed by less than 8%. As further evidence of the concordance of the initial distribution kinetics of antipyrine and thiopental, when the parameters describing the central circulation and nondistributive peripheral pathway in the recirculatory thiopental pharmacokinetic model were fixed to those of antipyrine, the thiopental model was minimally affected (Table 3).

An important observation of our work with various paradigms of perturbed physiology is that not only cardiac output but also its distribution affects early drug concentrations, as reflected in the area under the curve in the first minutes after i.v. administration (Avram et al., 1997, 2000; Krejcie et al., 1999, 2001), and suggested by the report of Upton et al. (1999; Krejcie and Avram, 1999). The nondistributive peripheral pathway in the antipyrine model represents blood flow that returns the lipophilic drug to the central circulation after minimal apparent tissue distribution (Krejcie et al., 1996a; Avram et al., 1997). The fraction of cardiac output represented by CL_ND is an important determinant of early drug concentrations. Increased arterial drug concentrations resulting from a larger fractional CL_ND increases drug exposure of the sites of action of drugs with a rapid onset of effect, such as thiopental, and would be expected to produce a more profound and prolonged effect. An important observation of the present study is that the early disposition of antipyrine, including the central circulation, the nondistributive peripheral pathway, and the fraction of cardiac output represented by CL_ND, is nearly identical to that of thiopental. This concordance makes antipyrine a useful physiologically inert surrogate for certain rapidly acting lipophilic drugs in nondestructive studies of the effect of altered cardiac output and blood flow distribution on drug disposition in both animals and humans.

Peripheral distribution of antipyrine and thiopental, on the other hand, differed significantly. Although the total distributive blood flow (CL_T-F + CL_T-S) of antipyrine and thiopental differed by less than 8%, antipyrine CL_T-F was more than 13% less than that of thiopental, whereas antipyrine CL_T-S was 83% more than that of thiopental. Peripheral antipyrine distribution volumes (V_T-F and V_T-S) and V_SS were less than two-thirds those of thiopental. The characteristic distribution pattern of a given drug is dependent on not only blood flow to various tissues but also binding competition among them (Bickel and Gerny, 1980). Antipyrine binds minimally to extracellular and intracellular components (Bickel and Gerny, 1980), whereas thiopental binds weakly to both tissue and plasma (Bickel et al., 1987). Thiopental binds to splanchnic tissues, represented by V_T-F in the recirculatory model (Sedek et al., 1989; Krejcie et al., 1996a), much more extensively than antipyrine does (Bickel et al., 1987) and to muscle, the primary component of V_T-S in the recirculatory model (Krejcie et al., 1996a; Avram et al., 1997), only slightly more than antipyrine does (Bickel et al., 1987). Thus, the much more extensive distribution of thiopental to its V_T-F, which is twice as large as that of antipyrine, delays and prolongs equilibration with its V_T-S, which is only 50% larger than that of antipyrine, relative to that of antipyrine (Upton et al., 1996).

In addition to validating the use of antipyrine as a physiologically inert surrogate for rapidly acting lipophilic drugs, the results of the present study have another practical implication. Interindividual differences in the response to rapidly acting drugs, such as intravenous anesthetics, may have a pharmacokinetic or pharmacodynamic basis. Rapid i.v. drug injection is necessary to describe the pharmacokinetic basis for differences in the dose-response relationship using a recirculatory pharmacokinetic model. In contrast, the pharmacodynamic basis for such differences is best studied when the drug is infused relatively slowly, allowing description of the concentration-effect relationship during the onset and offset of effect. The use of antipyrine as a surrogate for a rapidly acting lipophilic drug like thiopental allows the conduct of pharmacokinetic-pharmacodynamic studies in which antipyrine is administered by rapid i.v. injection to describe early drug disposition, whereas thiopental is administered by continuous infusion to a pharmacodynamic endpoint to enable description of the concentration-response relationships and the tissue distribution and elimination clearance elements of the recirculatory pharmacokinetic model.

This study validates the assumption that antipyrine is a useful surrogate for lipophilic drugs in pharmacokinetic studies in which physiologic stability is desirable to meet the assumption of system stationarity (Riggs, 1963) and to enable accurate description of initial drug distribution in pharmacokinetic-pharmacodynamic studies of rapidly acting lipophilic drugs.