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PBPK MODEL OF NICOTINE Ni?oLn[f-1C.O~:Id~z ~ p N~ CN3 Cotinine f9.1~.,<r t:aus ~- fi3E .- ~ .-~. `-`'N~I ' ' ~ V P. n ~ . 181 "Polar Metabolltes" (PM) In Plasma FIG. 2. Schematic of nicotine clearance on which this PBPK model is based. As illustrated here, nicotine is either metabolized to cotinine and a variety of other "polaPmetabolitea" (shaded rxtangle) or is excreted unchanged in the urine. Based on this scheme, the model indudes a definition of: (I) hepatic metabolism to colinine,(2) hepatic metabolism to "polar metabolites", and (3) renal excretion from the kidney compartment. (Abbreviations used: POXB, y-(3-pyridyi)-y-oxobutyric acid; PMOB, y-(3-pyridyl )-y-oxo-N-melhylbutyramide; PMAB, y-(3-pyridyl)-y-methyaminobutyde acid; and PAA, 3-pyridyiacetic acid). . . . . observation also implied that at the nicotine dose examined (0.1 mg/kg) K,„ 8 CvL• Therefore, the Michaelis-Menten expression was reduced to a )i first-order rate constant, KNc, proportional to V,,,,,,/K,„ The following procedures were used to estimate KNc and KNr. Renal ,iearance (Ct.asrut) was determined experimentally to be 0.2151iters/hr/ kg by measuring the amount of unchanged nicotine excreted into the urine. The volume of distdbution of cotinine VdroT was set to 1.2 liters/kg and total cotinine dearance(CLcoT ^ 0.211 liters/hr/kg) was calculated as the product of Vdcor and Pm. Assuming these constraints, only the rates at which nicotine is metabolized to cotinine (K,c) and PM (K,m) are unknown. These kinetic constants were subsoquentiy estimated by varying these pa- rameters to optimize the model fit to plasma ootinine concentrations. This procedure allowed us to estimate the rate of nicotine metabolism to both cotinine and PM based on the appearance of one metabolite, cotinine. RESULTS Partition Coeicients At the end of a 6-hr infusion of nicotine (0.25 mg/kg/ hr), plasma nicotine reached a steady-state concentration of 85.5 ± 34.9 ng/ml, with a range of 55 to 149 ng/ml. The tissue and plasma concentrations at steady state were used to generate the tissue:plasma partition coefficients listed in Table 2. Nicotine partitioned most notably into kidney and ii%cr, achieving concentrations greater than 24 and 7 times that of plasma, respectively. Although these values are not identical to those reported by Benowitz et al. (1990), also listed in Table 2, they do agree with the relative affinity of nicotine they report for each tissue. Plasma Pharmacokinetics Classical pharmacokinetic parameters were determined in order to assess this study's overall agreement with those pre- viously reported in the literature and to generate kinetic pa- rameters for nicotine and cotinine which were incorporated into the model. Nicotine plasma kinetics followed a simple biexponential decay composed of a rapid distribution phase followed by a single elimination phase (Fig. 3). The ap- pearance of cotinine in the plasma was rapid and reached a maximum of 47.4 ± 5.8 ng/ml at 1.4 ± 0.2 hr, followed by an elimination phase with a ttt2p of 3.9 ± 0.5 hr. Similar to ootinine, PM quickly rose to a maximum of 21.4 ± 3.1 ng/ ml at 0.7 4 0.2 hr. Elimination of PM followed a biexpo- nential decay with ttt='s of 2.4 and 42.9 hr for a and R elim- ination phases, respectively. Pharmacokinetic parameters generated from analyses of these nicotine concentration vs time data are listed in Table 3. Surprisingly, there was little variability within nicotine, cotinine, or PM plasma concen- trations among the six rats as shown in Fig. 3. A comparison of actual nicotine and cotinine plasma•concentrations with model predicted values is illustrated in Fig. 4. The model successfully predicted the plasma kinetics of intraarterially administered nicotine and the appearance, distribution, and clearance ofcotinine. The model was not designed to describe PM disposition, which was included solely to help estimate the metabolic clearance of nicotine. Tissue Kinetics Figure 4 shows the concentration time-course of nicotine for eight tissue compartnients following an intraarterial bolus of 0.1 mg/kg nicotine. The nicotine tissue concentrations predicted by the model were in accordance with experimen- tally determined values for all organs.

t@2 PLOWCHALK, ANDERSEN, ANDDEBETHIZY Kidney tissue had the highest measured nicotine concen- tration, with nicotine concentrations of 969 ng/g tissue fol- lowed by liver (171), brain (49), lung (33), muscle (29), heart (24), skin (21), and fat (8). It should be recognized that these are tissue concentrations measured at 15 min and do not necessarily correspond to the organs which achieved the highest concentrations immediately after dosing. How- ever, the model predicts that the peak tissue concentrations occur between I-15 min and are in the same order. Nicotine was rapidly distributed to most organs within 15-30 min and then was followed by a single elimination phase. 1' Model-Gstimated Physiologic Constants Heart, lung, and brain exhibited a nonlinear nicotine elimination phase. Figure 5 illustrates the elimination of nio- otine from brain, lung, and heart with and without the in- clusion of saturable, high-affinity binding sites. The values of B,„ax (nmol/tissue) and KD (nM) used to obtain these fits were 0.009 and 0. 12 for brain, 0.039 and 2.0 for lung, and TABLE 2 Tissue:Plasma Partition Coefficients for (S)-[5 -3HJNicotine } Determined in Vivo for the Sprague-Dawley (SD) Rat Partition coeffieients • Nlootine • Cotinl,ne •' ~ O Polar Metabolite Is -Model Simulation 10 15 20 25 a0 Hours FIG.3. Plasmaconcentrationsofnicotine,cotinineand"polarmeteb- olite" (PM) following a 0.1 mg/kg intraanerial bolus of (S)-15-rH j nicotine hydrochloride. All data points represent the mean analyle concentration t standard deviation ofsix rats. Model simulations are represented with solid lines. A simulation was not attempted for PM because the model description only aceounted for PM appearance and not distribution or elimination. 0.039 and 0.12 for heart, respectively. These values were estimated by holding all model parameters constant and Benowitz er at. (1990) varying KD and Bm,x to optimize (visually) for either the Our lab• concentration of nicotine in the brain, lung, or heart. Clearly , Organ SDrat° Rat` liabbit~ theadditionofsaturablebindingenhancedtheoverallagree-; 3 16 2 ment between the model prediction and experimental datar Muscle LI t 0 . Skin LImo.3 ND' ND TheoptimizedrateconstantsKHeandKNPwereestimated Fat 0.2 t0.t 0.5 o.s to be 75.8 and 24.3 hr'1, respectively, and are expressed Liver 7.0 m 0.5 3.5 3.7 with respect to the free concentratioit of nicotine in the liver Kidney 24.8 t 9.4 13.3 21,6 (Table 4). Incorporation of these values into the model pre-, "' 7 diets the proportion of the nicotine dose eliminated by, Heart 0 1 1 9 3 6t0 . . . . 2 CLRSNAL, metabolism to cotinine and metabolism to PM Lung 0.9 ± 0.4 2.1 . Slowly pedbsedr 6.4 ND ND to be 8.4, 69.3, and 22.2%, respectively. The metabolicelear Arterial blood' I I I ance of nicotine in the rat has been shown to be linear ove yenousbloodr I I I the dose range of 0.01 to 1.0 mg/kg (Kyerematen et aL; 'Determined after a 6-hr intraarterial nicotine infusion. (4.17 pg/mIN kg). ° Mean and standard deviation for six animals. `Determined after a 72-hr subcutaneous nicotine infusion with osmotic minipumps (2,8 urJmiNkg) (rat strain not stated). °Detcrmined after a 24-hr intravenous nicotine infusion (2.5 pg/min/ kg). `ND, Not determined. - f The partition coefficient for the slowly perfused tissue compartment was estimated by holding all parameters constant and aQiusting the partition coefficient to acheive the best model fit with the initial plasma distribution phase of nicotine. $Not experimentally determined, but equal distribution of nicotine occurs throughout the vascular compartment and plasma protein binding Is less than l0%(Rotenberg, 1978). 1988; Rotenberg, 1978). These data suggest that the tiepatic t metabolic oxidation of nicotine by flavin monooxygenase and cy(ochrome P450 is not saturable up to near lethal do The inability to saturate the metabolism of nicotine in vivo,' makes it impractical to determine accurately the in vivo and V,„x for nicotine metabolism. To circumvent this pro 1em and obtain an estimate of the lower limits of K„, an V,„a, we have used the PBPK model to simulate the nicotin, plasma concentrations from the highest nonlethal dose q nicotine which was used by Kyerematen et al. (1988) (1. mg/kg). In these simulations, K,„ and V,„aK were lowe while maintaining a constant ratio of V,,,/K,,, (0.84) un the plasma elimination of nicotine exhibited nonlinear t r E r 1 52180 8692

186 . PLOWCHALK, ANDERSEN, AND DEBETHIZY CVL Concentration of nicotine in venous blood leav- ing the liver. CLRENAL Renal clearance of nicotine. CLc0T Total plasma clearance of cotinine. Cvt Concentration of nicotine in venous blood leav- ing tissue compartment t. Km Michaelis constant for the metabolism of nicotine to cotinine. KNP First-order rate constant for the metabolism of nicotine to "polar metabolites." KcP First-order rate constant for the metabolism of cotinine to "polar metabolites." Ka Dissociation constant for nicotine in tissue com- partment t. METNc Amount of nicotine metabolized to cotinine. METNP Amount of nicotine metabolized to "polar me- tabolites." Pt 'ilssue:plasma partition coefficient for tissue t. Qt Blood flow through tissue compartment t. QK Blood flow through kidney tissue. QL Blood flow through liver tissue. Vt Volume of tissue compartment t. VL Volume of liver compartment. Vm,x Maximum velocity for the metabolism of nieo- VdcoT tine to cotinine. Volume of distribution of cotinine. (1) Amount ojCottntne in the Body dAcs o dMETNC -(Cf-ooT' Cca) -(Kce • Cca' Vdarr), dt dt where, dMETNC a Vm.x' CvL or (KNC' CvL' VL)• dt Km+CVL (2) Amount oJNicotine in the Liver dAL dMETNC _ dMETNP dt ~ QL (Ce - CvL) - di di where, dMETNP . di KNP' CVL' VL, and where dMETNC/dt is described above. (3) Amount oJNicotine in the Kidney dt K ° 1LK • ( CA - CVK ) - dt p , where, djU ° CLRENAL' CVK• dt (4) Amount oJNicotine In Tissue (1) with Saturable Binding dAt dt _ QXA-Cvt), where the expression At ° Vt' Pt' Cvt +(Bm.xt' Cytl 1K~,+CvJ, is solved for Cvt. This can be done by solving either explicitl as a quadratic in Cvt or implicitly by approximation as done in this study. REFERENCES Adir, J., Miller, P. R„ and Rotenberg, K. S. (1976). Disposition of nicoti in the rat after Intravenous administration. Res. Commun. Chem. Pat PharmacoL 13, 173-182. Andersson, 0., Hansson, E., and Schmiterlow, C. G.(t965). Oastiec oretion of C"-nico8ne. ExpertmenUa 21, 2 t 1-213. . Andersen, M. E., Clewell, H. J., Gargas, M. L, Smith, F. A., and R~ R. H. (1987). Physiologically based pharmacokineties and the risk; sessment process for methylene chloride. ToxlcoL ApOL Pharmaaot. 185-205. Benowitz, N. L., Jacob, P., IIi, Jones, R. T., and Rosenberg, J. (198 Inteuindivldual variability in the metabolism and cardiovascular e6 of nicotine in man. J. Pharmacof. Pxp. Ther. 221(2), 368-372. BenovdK, N. L, and Jacob, P. (1985). Nicotine renal excretion rate i enoea nicotine Intake during cigarette smoking. J. Pharmacol. Exq. 234,153-155. Benowitx, N. L. (1986). The human pharmaeology of nicotine. Res. A Alcohol Drug Prob. 9,1-53. Benowits, N. L, Porchet, H., and Jacob, P. (1990). Pharmacokinetics, tabolism, and pharmacodynamics of nicotine. In Nicotine PsJ+co, matologyr Molecular, Cellular and Behavioral Aspects (S. Wonm M. A. H. Russell, and 1. P. Stolerman, Eds.), pp. 112-157. Oxford versity Press, Oxford. Benowilz, N. L, Jacob, P., Ill, Denaro, C., and Jenkins R. (1991). Ste Isotope studies of nicotine kinetics and bioavailability. C/tn: Pharm Ther. 49, 270-277. Bischo6, K. B., fkdrick, R, L, Zaharko, t). S., and Longstreth, J. A. (19 Methotrexate pharmaookinetics. J. Pharm. Sct. 60,1128-1133. Booth, J., and Boyland, E. (1970). The metabolism of nicotine Into opticxtiyacfive stedoisomers of niootine•t'•oxide by animal tissues In and by cigarette smokers. Biochem. Pharmaml 19, 733-742. I C C C

P6PK MODEL OF NICOTINE 185 for these tissues indicate that this binding is to high-aff nity nicotiitic receptors. Data-based pharmacokinetic parameters determined from these experiments (Table 3) were in excellent agreement with those previously reported by other investigators. The pj2s, !nicotine CiTOratL and Vdp corresponded with those found by Kyerematen el al. (1988 ), Adir et at 1976), and Miller el al. (1977 ). ^:icotine metabolism to cotinine appeared to be linear at the dosages used. Under these conditions, K,„ > CVL and the reaction exhibits first-order kinetics described by the rate t isenstant KNC. Optimizing both KNC and KNV in terms of the ~ppearance of cotinine generated constants that predicted that 91.5% of nicotine clearance is metabolic and 8.5% is renal. This agrees well with our empirical measurement of CLRENAL (i.e., 8% of CI.roTAL; range, 2.4-12.5%) and those determined by Miller el al. (1977), Benowitz et al. (1982), r:nc'• Kyerematen et at (1988). Based on the model, metab- olism of nicotine to cotinine accounts for roughly 70% of CLrorAL, an estimate suggested by several investigators ( Be- nowitz,1986). The remaining 22% of total plasma clearance was attributed to the formation of polar metabolites. AI• though we do not know the detailed kinetics of each metab- olite composing PM, this pathway accounts for a significant portion of the total nicotine dose administered to the rat and literefore cannot be ignored (Benowitz el al., 1990). The constant Kcp, which defines the rate of formation of PM from cotinine appears unimportant to PM formation. In fact, the addition of this constant results in a pattern of metabolite formation inconsistent with our experimental data. Although it is well known that eotinine does indeed form several polar urinary metabolites (Kyerematen et at, 1988), they do not appear to be those measured in the plasma in this study. Many of the metabolites formed from cotininc are quickly excreted in the urine and do not accumulate in the plasma to any great extent (Kyerematen et at, 1988). A true physiologically based pharmacokinetic model for nicotine which possesses t 1 anatomical compartments as well as tissue partitioning, tissue binding, and biotransfor- mation can be a tool for examining both species and indi- vidual differences in the pharmacology and toxicology of nicotine. Large individual differences have been reported in humans for several pharmacokinetic parameters of nicotine including total clearance and volume of distribution (Be- n:)%sitz el at, 1982). There are also reports that smokers have a greater number of nicotine cholinergic receptors in the brain at autopsy (Kellar et at, 1987). Given the large individual differences in nicotine pharmacokinetics and re- ceptor density, it is not surprising that there are also large variations in the smoking habits of individuals (Warburton and Wesnes, 1978). Establishing which individually deter- mined parameters are involved in the varying pharmacolog- ical response should provide insight into the role nicotine plays in smoking. With the recent development of nicotine gum and trans- . dermal patches as adjuncts to smoking cessation programs, this model could be easily expanded to include rate constants for the buccal and percutaneous absorption of nicotine. In addition, recent interest in nicotine as a potential therapy for Alzheimer's disease (Kellar and Wonnacot, 1990) has resulted in the administration of nicotine to elderly Alzhei- mer patients (Sahakian et at, 1989). There are many ques- tions concerning the appropriate route and time course of administration of nicotine to Alzheimer patients (Kellar and Wonnacott, 1990). This model would be useful for address- ing some of these questions prior to clinical studies. For ex- ample, the model can readily determine if changes associated with aging such as altered renal clearance would affect tissue and plasma concentrations of nicotine. In summary, we have described a physiologically based pharmacokinetic model that accurately describes the tissue and plasma kinetics of nicotine in the Sprague-Dawley rat. We found the description of hepatic nicotine metabolism need not include Michaelis-Menten kinetics, since it appears that this pathway is not saturated at the sublethal doses used here. Treatment ofinetabolism in this fashion predicted nia otine elimination by renal and metabolic clearances consis- tent with those demonstrated by other investigators. Some tissues (heart, lung, and brain) have enough nicotinic binding sites to affect the disposition of nicotine in these tissues and were described with saturable nicotine binding. This model should serve as an important first step in the process of de- veloping strategies to assess nicotine tissue kinetics in humans that will be applicable for studying nicotine uptake and dis- position by smokers and nonsmokers alike. ACKNOWLEDGMENTS The authors thank Ms. Robyn Phelps for her expert assistance with animal surgeries, Mr. Jackie Greene for his efforts in developing the HPLC methods for this study, and Dr. William Caldwell forpurification of unlabeled nicotine, APPENDIX Abbrevrations At Amount of nicotine in tissue compartment t. AL Amount of nicotine in the liver compartment. Au Amount of nicotine excreted into the urine. AK Amount of nicotine in the kidney compartment. Aca Amount of cotinine in the body. Qmaxi Maximum binding capacity of nicotine for tissue compartment t. CA Concentration of nicotine in arterial blood. Cca Concentration of cotinine in the body. Cvx Concentration of nicotine in venous blood leav- ing the kidney. ~

188 PLOWCHALK, ANDERSEN, AND DEBETHIZY Shigenaga, M. K„ Jacob, P., III, Trevor, A,, Castagnofi, N., Jr., and Benowitz, N. (1987). Synthesis of specifically labeled (S)-niootinc-5-3H and (S)- cotinine-5-3H by carrier-free tritiolysis of the corresponding 5-bromo de- rivativcs L Labelled Compds. Radiopharm. 24(6), 13-723. Sleight, P., and Widdicombe, J.0. (1965). Aetion potentials in fibers from receptors in the epicardium and myocardium of the dog s left ventricle. J. PhysloL London 181, 235-258. Su, C. (1982 ). Actions of nicotine and smoking on circulation. Pharnracol. 7Ker.17, 129-141. Svensson, C. K. (1987), qinical pharmaookfnetics of nicotine. Clinical Phmmacokiner. 12, 30-40. Tsujimoto, A,, Nakashima, T., Tanino, S., Dohi, T., and Kurogochy~Y= (1975), Toxicol. Appl Pharatacrol 32, 21-31. . Waddell, W. J., and Marlowe, C. (1976). Localization oCnicotino-'4~',, , tinine-"C, and nicotino-1'-N-oxide-"C in lissues of lhe mouse. pr~,y~ lab. Dispos. 4, 530-539. Warbunon, D. M., and Wesnes, K. (1978). Individual difTerenas in smo '` and attentional perfonnance, In Smoking Behavior ( R. E. Thomtoq ~ PP• 19-43. Churchill Livingstone, Edinburgh, 1341 Wonnacott, S. (1990), Characterization of brain nieotipe rxeptor sitrs,s NirorlnePry:»pharmacology.-Molecelar, Celhrlarand BehavioratAsppq (S. Wonnacolt, M. A. H. Russell, and.1. P. Stolerman, Eds.), pp ~ 277. Oxford Univ. Press, Oxford. .. . .~ Cl Skin ~. A LC ,msei fiSSnr ritr0 resic! IPPS L ran uols). CCl dslan a¢rs ( ita'i5i' ;osurn nram nicro: tlild c iions < md Vl athel dgnif c lose ol xteriz :ime a 4indu Aposu auman nodel Nd ars •tatlDn Cher an aris f yesic 'aedcri 52180 8698

POPK MODEL OF NICOTINE . 183 TABLE 3 Pharmaookinetic Parameters for Nicotine and Cotinine De- termined from Six Male Sprague-Dawley Rats Administered 0.1 mg/kg (S)-[5'HJnicotine Intraarterially Pharmacokinetic . parameter Nicotine Cotinine where the model predicted experimental data sets from the literature (Miller et al., 1977). The nicotine partition coefficients agreed fairly closely with the relative tissue affinities within the species previously ex- amined (Benowitz et al., 1990). A notable and potentially important diflcrence was the partition coefficient determined . for the brain. Benowitz reports partition coefficients of 2.8 t,.:, (hr)^ 0.9 ± 0.1 3.9 ± 0.5 and 3 for the rat and rabbit, respectively, whereas we esti- v'C (ng• hr/ml)° 34.9 t 5.5 341.1 153.3 mated a partition coefficient of 1.4 for the Sprague-Dawley ,i tnr ')° 0.8 t 0. 1 0.18 t 0.03 mt. Usingg this twofold greater value in the model would CI.Tw (liters/hr/kg) 2.9 ± 0.4s 0.21 (Ctror) ~lict brain concentrations twice as h' as we determined CI.M,,saK(liters/hr/kg) 2.7 3 0.5` p ~ - .. w (ilers/hr/kg) 0.2 t 0.1 ° - experimentally. It is also worthy to note that Benowitz found t~, (Oters/kg) 3.7 ± 0.6• t,2• different partition coefficients between the rat and rabbit for ~! heart and kidney tissues. Interspecies differences in nicotine • Determined from a biexponential curve fit of plasma nicotine concen- tissue affinity have been reported by other investigators. Loed tration vs time data using RSTRIP, a polyexponential curve stripping and parameter estimation program. and Turner (1977) noted a greater than twofold difference "Ct.r„u = dose/AUCr. CLx.w' Xul,'/AUCg', Xuo° `amount of nicotine excreted in the urine fruntls0- oo. `CLMO,yyk e CLr,w - CLap„1. 'vdi ` CLrou0NiC• . 'CtooT ° VdaoT•dcoT• .. . . I Gabreiisson and Bondesson, 1987; Miller er al., 1977. Imm ~ IWC ~ Ib ~ to netics. This occurred at 9.0 µM and 7.6 pmol/hr for K,„ and 1 respectively. Based on these findings, we know that t: apparent in vivo K„ and Vm. must be greater than these values. Additionally, we can conclude that nicotine metab- olism is linear even at a near lethal iv dose (1.0 mg/kg), and it is not possible to saturate the metabolic clearance of nicotine in vivo to obtain a more precise determination of K,„ and V,,,,x. DISCUSSION Several investigators have previously proposed PBPK models for nicotine. Benowitz et al. (1990) described a per- fusion model constructed of compartments similar to those described in our model. However, no validation of either tissue nicotine concentrations or plasma kinetics of nicotine and cotinine have been published. Similarly, a model by Schwartz et a!. (1987) wastheoretical in nature and described only simulation results. Although it was used to simulate several experimental data sets (personal communication), to •,ur knowledge no validation of this model has been pub- lished. Unlike these previous models, we have described a model and calibrated it by determination of nicotine con- centrations in venous plasma and in eight tissues. Overall, this PBPK model successfully describes nicotine eoncentra- tions in both plasma and tissue compartments, providing good agreement with the experimentally determined data, The model also accounts for the plasma kinetics of cotinine. The general utility of the model is best illustrated in Fig. 5 AI IM to t.un9 aou. IW) to FI 1m .- ~~ Musde 4 . -. , , Q11 J p , 1 1 4 6 1 to 0 a . I , 10 aous F1G.4. Time-course concentrations of nicotinc tn etght otgans following a0.f mg/kginlraartedalbolusof(S)-(5-tH[nicotinehydrochloride.Each data point represents the mean nicotine concentration t standard deviation of three individual rals. Solid lines are simulations without saturable tissue binding. Dashed lines in the brain, heart, and lung panels Illustrate lhe sim- ulated nicotine kinetics with the saturable nicotine binding in lhesa tissues. I H I

1g4 PLOWCHALK, ANDERSEN, AND DEBETHIZY toeo to accumulate nicotine, e.g., salivary glands, adrenals, bone 0 2 4 • Nicotine (Dose. 0.06 mpMg)  NicoOne (Dose . 0.4 mpAcp) 0 eatlnlne (Dose • 0.08 mg/kp) O CotlnIne (Dose .0.4 mg4cg) - Model Prediction marrow, bronchial epithelium, intestine, spleen, and bladder. In part, these organs comprise the slowly perfused tissue compartment which accounts for the remaining body mass not'accounted for by individual organ compartments. It is not unreasonable to speculate that the large partition coe6 ficient for this compartment (6.4) estimated using the model may be a reflection of the affinity of nicotine for these sites When evaluating the in vivo experimental data and model predictions it became obvious that saturable tissue binding was required to describe tissue nicotine kinetics adequately in the heaR, lung, and brain of the rat. These organs exhibited nonlinear elimination of nicotine, and the addition of higti affinity nicotine binding sites permitted a good fit to the ex perimental data at time points greater than 3 hr. These tissues appear to possess nicotine-binding sites which have a signifil 6 e 10 12 icant impact on tissue kinetics, although the Bm,x is lo Hours enough that nicotine binding in these tissues does not affi FIG. 5. Data sets from the literature were also simulated in order to the overall plasma pharma0okinetics at time points mo test the model's predictability with different doses and rat stmin, In a study often examined in pharmaookinetic studies. The authors . by Miller el al.. 1977, Fischer-344 rats were administered (iv) either 0.08 recognize that only three or four data points are present i (circles) or DA (squares) mg niootine/kg. The model accurately predicted . the region of the tissue elimination curves from which th mbols) and cotinine (o en a mbols) lasma icoline (fi11ed s lid ii ) both ~ y p y (so ne p n ,AnMnfmrienc I„rh~, ..Mr;menre binding constants are derived and, therefore, it should be, in kidney and brain affinities for nicotine between Wistar rats, White Carneau pigeons, and cats. Tsujimoto, el a!. (1975) reported that the skeletal muscle of the dog has a greater affinity for nicotine than that in the rhesus monkey. Such differences may become especially critical when trying to establish tissue concentrations of nicotine associated with a pharmacologic effect. Furthermore, this implies that model scale-up to other species, including humans, may require direct determination of partition coefficients or binding in tissues of the species of interest. The greatest tissue:plasma partition coefficient, 24.7, was observed in the kidney. Accumulation and excretion of nic- otine by the kidney appear to be a function of urinary pH. Several investigators (Rosenberg ef al., 1980; Benowitz and Jacob; 1985; and Feyerabend and Russell, 1978) have dem- onstrated that by reducing urinary pH, renal clearance of nicotine is increased. In this study, high variability in both kidney partition coefficients and renal clearance rates (coef- ficients of variation, 40.1 and 47.9%, respectively) may be explained by differences in urinary pH among the experi- mental animals (not determined ). Nicotine, which is a weak base, is ionized at physiologic pH and has been reported to be sequestered by acidic sites within the body (Waddell and Marlowe, 1976; Andersson e1 al., 1965). In whole body autoradiography studies, sub- stantial amounts of nicotine are found in the stomach lumen (Andersson el al., 1965) and melanin-containing tissues (Waddell and Marlowe, 1976). Other organs also were found stressed that the values reported tor lfmu an0 KD are proVl sional. However, because high- and low-affinity nicotinic D ceptors have been reported in mammalian brain and hea (Wonnacott, 1990; Lippiello and Fernandes, 1986; Sleigh and Widdicombe, 1965),theuseofasaturablebindingte in the model is biologically plausible. Recent reports of; second elimination phase for nicotine in human plas (ttl1= 5-6 hr), which is longer lived than generally repo may be explained by these tissue kinetics (R. J. Reynoh 1988; Benowitz et al., 1991). The KD and Bm,a determin TABLE 4 Metabolic Rate Constants and Tissue-Binding Constants Estimated for the Nicotine PBPK Model Metabolic constants Km (µM) a9.0 V.,, (pmol/hr) 0.6' KNC (hr')° . 75.8 ± 1.5 KNP (hr')° 24.3 # 0.5 Kn (hr') <0.001 Nicotine binding constants Bm„ (nmol/heart) 0.039 Bm„ (nmol/brain) 0.009 Bmu (nmol/lung) 0.039 Ku (heart, nM) 0.12 Kp (bmin, nM) . 0.12 Ko (lung, nM) 2.04 • First-order rate constants for the metabolism of nicotine in the li compartment. These values are expressed with respect to free nicotine centrations in the liver. ~

rnxirowov AND APPLIED PxARnfACOwov 116, 177-188 (1992) A Physiologically Based Pharmacokinetic Model for Nicotine Disposition in the Sprague-Dawley Rat DAVID R. PLOWCHALK4`'t MELVIN E. ANDERSEN,t AND J. DONALD DEBETHIZY t" . -• Dnke University Medical Center, Jntegrated Toxicology Program, Box 3005, Davison Building, Durham, Norr4 Carolina 27710; kChemlcal lndustry . Jnslimte ojTa.eirologlt Researclr Triangle Park, North Carolina: and 1R. J. Reynolds Tobacco Compaay, Pdannacology Division, f BuNding630•2,WinstonSalem,NortlrCardina27102 Received September 3, 1991; accepted June 2, 1992 A Physiologically Based Pharmacokinetic Model for Nicotine ni;position in the Sprague-Dawley Rat. Pt.owCHALK, D. R., :\NDERSEN, M. E., AND DEBErHIZY, J. D. (1992). Toxlcol. Appl. Pharnzacol. 116, 177-188. A physiologically based pharmacokinetic (PBPK) model was developed to describe the disposition of nicotine in the Sprague-Dawley (SD) rat. Parameters for the model were either obtained from the literature (blood flows, organ vol- umes) or determined experimentally (partition coefficients). Nicotine metabolism was defined in the liver compartment hy the first-order rate constants KNC and KNP which control tne rate of nicotine metabolism to cotinine and "polar me- tabolites" (PM), respectively. These rate constants were es- timated by optimizing the model fit to pharmacokinetic data obtained by administering an intraarterial (S)-[ 5-'H] nicotine bolus of0.1 mg/kg to 6 rats. Model simulations that optimized for the appearance of cotinine in plasma estimated KNC and KNP to be 75.8 and 24.3 hr'', respectively. Use of these con- stants in the model allowed us to accurately predict nicotine plasma kinetics and the fraction of the dose eliminated by renal (8.5%) and metabolic (91.5%) clearance. To validate the model's ability to predict tissue kinetics of nicotine, 21 male SD rats were administered 0.1 mg/kg (S)-[5- 'H]nicotine intraarterially. At seven time points following treatment, 3 rats were euthanized and tissues were removed and analyzed for nicotine. Model-predicted nicotine tissue kinetics were In agreement with those determined experi- mentally in muscle, liver, skin, fat, and kidney. The brain, heart, and lung exhibited nonlinear nicotine elimination, sug- gesting that saturable nicotinic binding sites may be important in nicotine disposition in these organs. Inclusion of saturable receptor bindi ng expressions in the mathematical description of these compartments resulted In better agreement with the experimental data. The Bm„ and Ko estimated by model sim- ulations for these tissues were brain, 0.009 and 0.12; lung, 0.039 and 2.0; and heart, 0.039 nmol/tissue and 0.12 nm, respectively. This PBPK model can successfully describe the tissue and plasma kinetics of nicotine in the SD rat and i ' To whom correspondence should be addressed. will be a useful tool for pharmacologic studies 1n humans and experimental animals that require insight into the plasma or tissue concentration-effect relationship. 0 1992 Ae.Oem& Prsrs.lne. . ' . Human exposure to nicotine can occur through the con- sumption of tobacco products, exposure to environmental tobacco smoke (ETS), pharmaceuticals such as Nicorette gum, use of nicotine-based insecticides, and dietary sources (Sheen,1988). Tobacco use represents the most substantial exposure to nicotine. Surveys by the Centers for Disease Control indicate that 26.5% of the adult US population are smokers (CDC, 1987). This extensive use of tobacco in our society has generated concern over the potential health risks associated with nicotine (DHHS, 1989). Accurately pre- dicting nicotine uptake and disposition in humans is a nec- essary step in assessing the potential adverse health effects of nicotine. Although plasma pharmacokinetics have been extensivelydocumented in animals (Kyerematen et a1.,1982, 1987, 1988; Adir et al., 1976; Rotenberg et al., 1980) and humans (Rosenberg el aL, 1980; Scherer et at, 1988; Kyer- ematen el al., 1990), the relationship between plasma oon- centrations, tissue concentrations, and pharmacologic or toxicologic effects are not well understood. The principal pharmaoologic actioris of nicotine are me- diated through central and peripheral nicotinic receptori (Su, 1982; Lippiello and Fernandes, 1986). These effects have been reported to be dose dependent and include modulation ofcardiac functions (Rosenberg et at, 1980), brain electro- physiology (Clarke, 1990), and systemic vascular blood flow (Benowitz et al., 1990; Henrich et al., 1984). In addition, recent evidence that chronic nicotine administration results in an increase in the density of nicotinic cholinergic receptors in the mammalian brain has resulted in the experimental administration of nicotine to Alzheimer's patients (New- house et al., 1988; Sahakian et al., 1989). To understand how these pharmacologic effects are related to nicotine ex- (77 0041AOBX/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

178 PLOWCHALK, ANDERSEN, AND DEBETIIIZY posure, the resulting nicotine concentration must be known in the target organ. Early autoradiographic studies indicated that nicotine ao- cum ulates in a variety of tissues (Hansson and Schmiterlow, 1962; Andersson et al., 1965; Waddell and Marlowe, 1976), which may account for its large volume of distribution (I- 4 liters/kg) (Svensson, 1987; Benowitz et al., 1982). Affinity for these tissues may be a function ofthe chemical properties of nicotine in vivo. At physiologic pH, approximately 70- 88% of nicotine is ionized at the pyrrolidine nitrogen (Be• ~ I nowitz, 1986) allowing nicotine to be sequestered into "acidic ~ ~ tissue" compartments (e.g., kidney and stomach ), similarly to other weak bases. Although this nonspecific accumulation has no apparent pharmacologic importance, it may be sig- nificant in the overall,disposition of nicotine. In addition to nonspecific binding, nicotine tissue kinetics may also be af- fected by binding to specific nicotinic receptors in organs such as the brain (neuronal ), heart (peripheral), and muscle (peripheral) (Wonnacott, 1990; Sleight and Widdicombe, 1965). The objective of this study was to develop a physiologically based pharmacokinetic model (PBPK) that describes the tis- sue and plasma pharmacokinetics of nicotine in the Sprague- Dawley I Dawley rat. Although physiologic flow models for nicotine ) have been published (Schwartz et al., 1987; Benowitz ef al., 1990), to our knowledge these models were not tested against experimental data and only the results of simulations were reported. However, a threc*compartment mammillary model that was based on physiologic parameters has been developed for nicotine and was used to assess plasma nicotine kinetics in rabbits (Porchet et al., 1987). When considering the number of humans that are exposed to nicotine from a variety of sources, it is apparent that a model which describes nicotine tissue dosimetry would be a useful tool for relating nicotine exposure with specific phar- macologic etfects. A validated model for nicotine disposition in the rat will be an important first step in the process of developing models that can be used to assess nicotine tissue kinetics in humans. A number of investigators (Ramsey and Andersen, 1984; Andersen et at., 1987) have extrapolated PBPK models developed in rodents to humans using aQo- metric scaling, illustrating the value of such models. More- over, a variety of current issues, such as the selection of ap- propriate dosing regimens for pharmacology studies con- ducted in animals, nicotine pharmacodynamics in humans, and nicotine exposure from ETS and other environmental sources can be examined in greater detail with this type of model. MATERIALS AND METHODS Chemicals. (-)-(Pyrrolidine-2-1'C)cotinine (56 mCi/mmol), DL(pyrrolidine-2'-t"C)nicotine (56 mCi/mmol) and (S)-[5-aH)nicotine hydrochloride (25.8 mCi/mmol ) were purchased from Amersham (Arling- ton Heights. IL). Radiochemical purities determined by high-pressure liquid chromatography were 96.6, 95.8, and 98.6%, respectively. (S)-[5- ..'H)Nicotine hydrochloride was synthesized by Amersham using the pro. cedure described by Shigenaga el al. ( 1987).The starting material for this synthesis, (S)-5-bromonicoline, was obtained from Dr. Peter Crooks (Uni- versity of Kentucky ). Nicotine was purchased from Eastman Kodak (Roch- ester, NY) and distilled from NaOH bt vacuo (bp - 69.0-90D"C at 1-2 Torr) to obtain a purity of 99+%. Unlabeled nicotine was used to dilute labeled nicotine to the desired specific activity (S.A.). Cotinine (99% purity) was purchased from Sigma Chemical (St. Louis, MO). HPLCgrade methanol and acetonitrile were obtained from Burdick & Jackson (Muskegon, MI) and triethylamine (99+% purity) was purchased from Aldrich Chemical Co. (Milwaukee, WI). Animals. Male Sprague-Dawley rals (Crl. CD/BR) weighing between 175 and 200 g were purchased from Charles Rivers Labomtories (Raleigh, NC) and were allowed feed (Purina 5002) and water ad fibfnrm: The rats were kept on a 12-hr light/dark cycle and acclimated to their environment for 2 weeks prior to use. All animals were housed and cared for in accordance with the Animal Wdfare Act or 1970 and amendments (Public Law 91- 579 ) asspecified in CFR Title 9, Part 3 Sub-part E(ILAR,1985 ). Reference was also made to the DHHS document Guide for tlre Care and Use of Labora7oryAafmals (NIH publication 86-23). Surgery. . Rats were anesthetized with a 0.1 mg/kg ip injection of a 1:1 solution of 100 mgfml ketamine HCI (Aveco Co., Fort Dodge, lA) and 20 mg/ml xylazine (Mobay Corp., Shawnee, KS). When the animals reached stage 111(surgical stage) general anesthesia (5-10 min), preparation for sur- gery was initiated. The incision sites(neck and back) were first prepared by shaving and use of a depilatory, followed by disinfection of the exposed skin with Betadine. The rats were restrained on their backs on a heated surgical plalform. An incision was made in the skin I cm on the right and left side of the trachea, and the right jugular vein and left common carotid artery were blunt dissected from surrounding tissues. The right jugular vein was cannulated with OA4-mm OD Micro-renathane( BraintreeScientiflc, Brain- tree, MA) surgical tubing. Similarly, the left carotid artery was cannulated with 0.033-mm OD Micro-renathane tubing. The7rce ends of the cannulas were run subcutaneously around the neck and dorsally to the back where they were exteriorized through a small Incision between the soapula: All Incisions were closed with either wound clips or surgical silk sutures (3-0). Following surgery, the rats were allowed to recover for 48-72 hr before use In an experiment. The cannulae were checked daily and were kept patent with heparinized saline solution (25 Units/ml). . .. . Derermiaarion of parrtrion coo,Q9eieers. Nicotine tissue:plasma partition coefficients were determined by infusing mts with nicotine and measuring nicotine tissue and plasma concentrations at steady-state (Css)• Six male rats weighing between 230 and 280 g were cannulaled in the carotid artery and jugular vot n as described above. The arterial cannula was connected to a Harvard infusion pump (Harvard Apparatus,'1'nc., South Natwick, MA) and the rats were infused with lactated Ringer's solution containing 30 Units/ ml sodium Heparin at a rate of 0.2 ml/hr during the 48- to 72-hr recovery period. Each rat then was Infused inlraarterially for 6 hr (steady-state) whh 0.25 mg/kg/hr (S)-(5-eH)nicotine hydrochloride (SA. = 1.82 kCi/pg) and venous blood samples were collected at I, 2, 3, 4, 5, and 6 hr. Following the last blood sample, the rats were anesthetized with 70% COr In air and a midline incision was made into the chest cavity to expose the hean. The rats were simultaneously exsanguinated and perfused by severing the right atrium and infusing ice-cold 0.9%saline( 15 ml) through the arterial cannula. Following this procedure, tissue samples were removed, rinsed of blood, and frozen to -70'C. The tissues collected included muscle (glutous), fat (epididymal ), brain, skin (back ), heart, liver, kidney, and lung. Plasma and tissue samples were prepared and analyzed for nicotinee and cotinine eon- centmtions as described below. Partition coelficients were calculated for non<liminating and eliminating organs as specified by Chen and Gross (1979). For non-eliminating organs (skir the t bylh (live intrii when 6f th, Cp a respo Nh tweer admii PCt/{ equiv cage a collec (0.25 0.5, 1 collec NJ).: each I Sampl and st Nia betwn piante assignr 8-hr6l hydroc kg). A in air, e samplt Dere sample to an e orousll centrifi pipette at 200( vials (1 Fori goffrr contair Ipyrrol enized i vial wa centrifi centrab Instrun MC 0.4 forskin this tiss Akron, from th exchant All p concent cmX4 52180 8688

180 PLOWCHALK, ANDERSEN, AND DEBETHIZY Ihodel assumes instantaneous mixing or nicotine into the arterial compart- .ment and each subsequent compartment it enters. Partition coefficients are assumed to be concentration and time independent and nicotine distribution is blood flow limited. . - Physiological constants (Table 1), such as organ volumes and blood flows, were obtained from the literature (Gerlowski and Jian, 1983; Gwrhart er at, 1990). Organ and tissue volumes are expressed as a percentage of total body mass and blood flows are expressed as a percentage of cardiac output which was scaled to body weight using the allometric relationship described byAnderseneral.(1987). . Ingeneral,theconcentrationofnicotineinanorganisafunaionoflinear nonspecific binding of nicotine to tissue components, i.e., the product of the partition coefficient and plasma concentration. However, in organs which have capacity-limited nicotinic binding sites ("nicotinic receptors"), total lissuepartitioningwasexpressedasthesumoflincarbindingandsaturable, high-aAmity, binding similar to the models developed for methotrexate (Bis- cho0'er a1., 1971) and 2,3,7,8-TCDD (Leung et a1., 1990). To aecount for saturablebindinginthismodel,abindingexpressionsimilartothatdescribed by Bischofl or ai., (1971) was Included for tissues known to possess large populations of nicotinic receptors (i.e., hean, lung, and brain). Once the overall model structure was formulated, mass-balance differential equations were written to describe the rate of change of nicotine mass in each anatomlcal compartment. These equations are similar to those described by Ramsey and Andersen (1984) except for brain, lung, and heart, which include saturable tissue binding, and kidney, which inctudes a term for renal clearance. A listing of these equations can be found in the Appendix along with equations that describe the formation and elimination ofcotinine and other more polar metabolites. The model equations were "solved" by nu- merical integration using Gear's method for stiff systems with the mathe- matical modeling software ACSL (Advanced Continuous Simulation Lan- guage, Mitchell and Gauthier Associates, Concord, MA ) on a SUN SPARG station I computer. Parameter estimations and final optimizations were performed with Simusolv (Dow Chemical Co., Midland, MI) which employs the maximum likelihood method. Model description ojnicotine metabolism. The metabolism of nicotine has been well established in animals (Kyerematen el at„ 1987; Gorrod and Jenner, 1975) and humans (Kyerematen ei at., 1990; Benowitz, 1986). Nicotine is metabolized to cotinine (Fig. 2) and a variety of other polar metabolites and is also excreted unchanged in the urine. Cotinine is a major metabolite of nicotine and Is formed by a two-step reaction catalyzed by cylochrome P450 (rate-limiting step) and aldehyde oxidase (McCoy et at., 1986; Kyerematen et al.. 1988; Booth and Boyland, 197 t). Although the liver is the primary site for this biotransformation, other organs such as the lung and kidney also can metabolize nicotine, but to a lesser extent (Hansson or al.. 1964). N-oxidation of nicotine to form nicotine-N'-oxide has been reported as an Important metabolic pathway In many species (Booth and Boyland, 1970). Other minor metabolites reported to arise from nicotine include nornicotine, nicotine glucuronide (Byrd er at., 1992), demethyl- cotinine, and y-(-3-pyridyl)-oxybutyric acid. Clearance of cotinine is ac- complished by either renal excretion or metabolism to more polarcompounds such as trans-3'-hydroxycotinine, cotinine glucuronide (Byrd or at, 1992), cotinine-N-oxide, cotinine methonium ion, y-(3-pyridyl)-y-methylami- nobutyric acid, 3-pyridylacetic acid, and y-(3-pyridyl)-y-oxo-N-methyl- butymmide (Kycremakn et al., 1988). The combination of nicotine and cotinine metabolites (excluding cotinine) is defined as PM in this model and represents the unidentified PM peak measured by HPLC. Based on this well-established metabolic scheme, a mathematical descrip tion of nicotine metabolism was formulated for the model. The model as- sumes that nicotine clearance occurs solely by metabolism in the liver and renal excretion from the kidney compartment. Nicotine metabolism is de- scribed as a function of two biotmnsformation pathways: ( I) the conversion of nicotine to colinine and (2) the conversion of nicotine to PM (Fig. 2). The former reaction was addressed in the model by including a saturable TABLE I Physiologic Constants° Used for PBPK Model for Nicotine i the Sprague-Dawley Rat Organ/tissue Organ/tissue volume (Percentage of BW)s Muscle 55 Skin Fat to 7 Liver 4 Kidney 0.75 Brain 0.55 Heart 0.55 Lung Slowly perfused° Arterial blood Is 3 Venous blood 3 °Oerlowski and Jian, 1983; Gearheart el al., 1990; Andersen or a1., 1987. ° Percentage of body weight (0.269 kg). ° Percentage of cardiac output (CO - 5.3 liter/hr). 'Slowly perfused tissue compartment represents the remaining body nusi not accounted for by the other compartments. Michaelis-Menten expression in the liver compartment similar to that de-; scribed by other investigators (Ramsey and Andersen, 1984; Bischofl er aJ; 1971). The rate of polar metabolite formation from nicotine was defineo by the first-order rate constant KNP. g; Cotinine disposition in the model Is controlled by: (1) the rate of nicotine', metabolism to cotinine (Km and V,,, ; or KNc; see below for definition q Kw); (2) cotinine volume of distribution (Vd=) and; (3) total ctearancg rate of cotinine (CLcOT). The metabolism of cotinine to PM was initi separated from the total clearance term. This metabolic pathway was defined by the first-order rate constant Ka. We found through model simulatioas ~~ that when Kcp was set to a value that had an effect on PM kinetics it resul In both PM and cotinine kinetic profiles that were radically different fru the experimental data. Upon this observation, we set Kn to 0.001 so tha it made cotinine clearance via this pathway insignificant and allowed C to account for all clearance mechanisms of eotinine. AUC, ttt=and p wey obtained by first stripping the plasma cotinine concentration vs time da to estimate initial parameters which described a biexponential function a then optimizing the parameters to allow for the best fit to the expedmen data. This analysis was performed with RSTRIP software (MicroMath, Lake City, UT). CLeor and VdceT werablitained from the Iiterature ( brielsson and Bondesson, 1987; Miller et at, 1977). It should be noted tlia' the portion ofthe model which descdbescodnine distribution and eliminati Is based on classical pharmaookinetic techniques. The overall rate of PM formation is described by the first-order rate ooi stants KNP and Kce which specify the rates ofconversion from nicotine e cotinine, respectively. The rate of PM appearance Is of interest because directly affects the amount of nicotine remaining for elimination by otfi pathways (e.g., metabolism to cotinine or renal clearance). However, exact composition and disposition of these metabolites are not inclu since knowledge of the Vd and CL for each component would be requ'. There are no In vlvo data available that allow for calculation of K,R V.„ or KNP directly; therefore these kinetic constants were estimat parameter optimization. Furthermore, we found the ratio of K, and to be more important than their absolute values, an observation simllaa one made by Reltz er a/. (1989) for methylene chloride metabolism.