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Pharmacokinetics of PD123,497, a novel antiarrhythmic drug : early distribution phase in blood and tissues Walker, Maria Louise 1994

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PHARMACOKINETICS OF PDI 23,497, A NOVEL ANTIARRHYTHMIC DRUG:EARLY DISTRIBUTION PHASE IN BLOOD AND TISSUESbyMARIA LOUISE WALKERB.Sc., The University of British Columbia, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Pharmacology and TherapeuticsWe accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1994© Maria Louise Walker, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of tHAOLo&YThe University of British ColumbiaVancouver, CanadaDate Othc ILkDE-6 (2/88)ABSTRACTPDI 23,497 is a novel antiarrhythmic drug and the (+) enantiomer ofPDII7,302, a kappa (ic) opioid agonist. Using a method of liquid-liquid chemicalextraction and high performance liquid chromatography, the pharmacokinetics ofPD123,497 in the rat were determined. Specifically, concentration-vs-time profileswere constructed reflecting the early distribution phase of an intravenous bolus doseof 8 jimol/kg in whole blood, heart, liver, brain, and skeletal muscle. Thepharmacokinetic data for heart and blood correlated well with the time course of heartrate and blood pressure effects as determined in previous pharmacological assays.The data also correlated well with pharmacological screens in dogs and baboonsindicating that the rat model is demonstrative of the situation in other species.Skeletal muscle data indicated a significant capacity for drug accumulation in themuscle beds. Liver data indicated slow uptake and storage of parent drug.Termination of therapeutic effect appeared to be the result of redistribution into othertissues and not metabolism or elimination. Concentrations in the heart were severaltimes higher than that of the blood immediately after administration of drug via the tailvein. This suggests a potential for alpha-phase toxicity. Brain data showed that peakconcentration was achieved after that in the heart indicating redistribution from theheart and suggesting that PD123,497 may be able to produce significant CNS sideeffects.IIITABLE OF CONTENTSAbstract iiTable of ContentsList of Tables ivList of Figures vList of Abbreviations viAcknowledgment viiINTRODUCTION II. The Class I Antiarrhythmics III. Pharmacokinetics 7III. High Performance Liquid Chromatography 16IV. Hypothesis and Objectives 19MATERIALS AND METHODS 20RESULTS 24DISCUSSION 34CONCLUSION 44References 45Bibliography 48Appendix I: Calculations to determine tissue and blood concentration 49ivLIST OF TABLESTable # Title pafleTable 1: Classification Scheme of Antiarrhythmic Drugs 2Table 2: Recovery of PD123,497 and Internal Standardby Chemical Extraction 25VLIST OF FIGURESFigure # Title____Figure 1 Calibration Curves for Blood and Tissues 26a) heart 27b) brain 27c) skeletal muscle 27d) liver 28e) blood 28Figure 2 Concentration-versus-Time Graphs 30a) blood and brain 31b) blood and heart 31c) blood and liver 32d) blood and skeletal muscle 32Figure 3 Pharmacokinetics of PD123,497 33Figure 4 Concentration versus Effect of PD123,497 41a) heart rate 41b) blood pressure 41Figure 5 Effect of PD123,497 on Conduction Velocity in the Dog 42viLIST OF ABBREVIATIONSPVB premature ventricular beatMl myocardial infarctionAA antiarrhythmic drugVmax maximum rate of depolarization(phase 0 of the cardiac action potential)ERP effective refractory periodCNS central nervous systemCAST cardiac arrhythmia suppression trialRSD Rhythm Search Developments Ltd.Ka apparent constant of associationVd apparent volume of distributionK elimination rate constantT112 elimination half-lifeHPLC high performance liquid chromatographyMtB E methyl-tertiary-butyl-etherSEM standard error of the meanVIIACKNOWLEDGMENTSI would like to thank Dr. Michael J.A. Walker and Rhythm Search Developmentswho provided funding and the opportunity for me to do this project. I would also liketo thank my supervisor, Dr. Richard A. Wall, for his invaluable assistance andnever-ending patience.IINTRODUCTIONThis thesis was part of a programme related to the development of newantiarrhythmic agents (the” RSD compounds”). Structure-activity relationship (SAR)studies on the various RSD compounds have been underway for some time. Some ofthese compounds are structural analogues of PDI 17,302, a selective K-opioid agonistwith antiarrhythmic activity. This thesis is concerned with the pharmacokinetics ofPD123,497, the (+) enantiomer of PDI 17,302. We specifically investigated the earlyphase of distribution of PD123,497 in blood and tissue and related this to previouspharmacological studies done with this drug. This introduction provides the readerwith information on Class I antiarrhythmics, the importance of pharmacokineticinformation in the design and presentation of a drug for clinical use, the analyticalmethods used to obtain some of this information, and where our research is likely tolead.I. THE CLASS I ANTIARRHYTHMICS:a) Background information:Sudden cardiac death is a leading cause of death in North America. Over 80%of these deaths are due to ventricular fibrillation. This fatal type of arrhythmia may bepreceded by premature ventricular beats (PVB’s). Patients who have suffered aprevious myocardial infarction (Ml) are at a high risk of fatal arrhythmia and so areoften prescribed antiarrhythmic drugs (AA’s).It was assumed that there was a strong association between PVB’s and2subsequent mortality and so PVB’s were considered to be the prime target ofantiarrhythmic therapy. In consequence, suppression of PVB’s was considered thedefining factor of drug efficacy. Studies conducted so far do not indicate thatsuppression of PVB’s results in decreased mortality for post-MI patients [1,2]. In fact,the Cardiac Arrhythmia Suppression Trial (CAST) of 1989 [3] reported an increasedincidence in mortality for post-MI patients taking encainide and flecainide, a 3.6-folddifference compared to matched post-MI patients receiving placebos. It is now a well-established fact that the Class I drugs have significant pro-arrhythmic effects,although electrophysiologists predicted this many years ago [4,5,6]. The search foran effective antiarrhythmic drug continues.b) Classification of antiarrhythmics:The antiarrhythmic drugs are grouped according to their electrophysiologicaleffects and presumed mechanism of action (see Table 1). This classification schemewas originally described by Vaughan Williams [7]. There are also subclassificationswithin groups based on their effect on Vm (defined as the rate of change of voltagepotential during phase 0 of the action potential).TABLE 1: Classification Scheme of Antiarrhythmic DrugsCLASS subclass action examplesI slow action potential upstrokea moderate 1 Vmax quinidineb minimal 1 V mexijetinec marked ‘1 Vmax encainide2 beta-adrenoceptor blockade propranolol3 prolong repolarization (primarily amiodaronepotassium_channel_blockers)4 calcium channel blockade verapamil3C) Mechanism of action of Class I drugs:An arrhythmia is an alteration in the normal sequence of activation of the atriaand ventricles caused by a disturbance in conduction or an abnormality of rate,regularity, or site of origin of the cardiac impulse. The net effect of someantiarrhythmic drugs (notably the Class lb drugs) is to prolong the effective refractoryperiod (ERP) relative to action potential duration. This reduces the likelihood ofgeneration of abnormal action potentials among the normal frequency of actionpotentials as set by the pacemaker.Class I antiarrhythmics act on the sodium channel responsible for the initialinward current that constitutes the rising phase of the action potential. Much of ourcurrent functional understanding of the sodium channel is based on the work ofHodgkin and Huxley [8] who first demonstrated that a transient inward current ofsodium ions was responsible for action potential propagation in squid giant axons.They characterized three states of the channel: open, inactivated, and closed.Historically, the first drug with antiarrhythmic action discovered was quinidineand related compounds. At concentrations ten to one hundred times theirantiarrhythmic concentrations these drugs are local anaesthetics in nerves. Theywere found to have the common property of “interfering specifically with the processby which depolarizing charge is transferred across the membrane” [9]. This actionwas revealed as a depression of Vmax unless the interstimulus interval was so longthat it permitted full recovery between beats. It was suggested that these AA’sinterfered with the process by which sodium channels were “reactivated in responseto repolarization” [9] and that consequently they “extended the effective refractory4period to a point long after the time at which repolarization was already complete” [9].We now know these conclusions to be substantially correct.The mechanism of action of the class I antiarrhythmics (and possibly other ionchannel blocking antiarrhythmics) is currently best described by the modulatedreceptor hypothesis, postulated by Hille [10] and Hondeghem and Katzung [11] in1977. At the time it was known that the decrease in Vmax due to quinidine wasaccentuated at high frequencies of stimulation and attenuated by slow rates and thatthis effect was observed as a stepwise decrease in Vmax with each action potentialuntil a steady-state level of depression was achieved. The modulated receptorhypothesis accounts for these observations by postulating that the affinity of drug forthe sodium channel is much greater when the channel is in an open or inactivatedstate than when it is in the resting state (closed). This necessitates cycling of thechannel for drug action to occur and thus explains use- or frequency-dependenceseen with some Class I drugs. The higher the rate of stimulation the more channelspass through open or inactivated states, have drug bound to them, and contribute toan overall decrease in sodium conductance. A steady-state level of Vmax depressionis therefore reached sooner (in fewer beats). The modulated receptor hypothesis alsostates that the interaction between drug and channel receptor is voltage-dependent;that is, the drug has a higher affinity for the channel the higher the membranepotential. Thus when membrane potential is increased (depolarized) the drug is morereadily bound and blockade of sodium current is more effective. Furthermore, a drugassociated channel inactivates more readily than drug-free channels and this alsocontributes to an effective decrease in sodium conductance. In cases of ischaemia,5the resting membrane potential is elevated, channel affinity for the drug is increased,and the time to recover from block is thereby increased resulting in a longer effectiverefractory period.Another theory, the guarded receptor hypothesis [12] is a variation on themodulated receptor hypothesis and has not been ruled out at this point. Inherent inthis theory is an explicit acceptance of the Hodgkin-Huxley model, the assumptionthat drugs bind in the sodium channel, and that drug-associated channels do notconduct sodium ions. Where it differs from the modulated receptor theory is aproposal that affinity of the drug for its binding site is constant (i.e. independent ofchannel state) and that in the closed state drug access is restricted. Thus there ismore binding of drug in the open state than in the closed one. Drug binding isproposed to immobilize channel gates thereby blocking sodium influx. The frequency-and voltage-dependent nature of the gates would explain the frequency- and voltage-dependent nature of drug interaction with the sodium channel.The use-dependent block of antiarrhythmics described above requires that theinterval between stimuli be shorter than the time required for the channels toreactivate and recover from channel block. This allows for some specificity since intissues following normal sinus rhythm there is enough time for recovery so that Vmaxshould be only minimally affected, whereas PVB’s result in a shorter diastolic interval.The specificity for arrhythmogenic tissue is also enhanced by the usual relativedepolarization of these tissues (as in the case of ischaemia) which increases theaffinity of the channel binding site for the drug and slows channel recovery time.6d) Goals of our research (rationale):From the results of the CAST study it is evident that current cardiac arrhythmiatherapy with Class I antiarrhythmics is suboptimal and that it is necessary to developchannel blockers for at least three main reasons as put forward by Tamargo et al [13].First, the population of people who need to be treated for supraventricular andventricular arrhythmias averages almost three million patients in the United States[14]. Second, AA’s are likely to remain the mainstay of therapy for the majority ofpatients with cardiac arrhythmias even when electrical (automated implantabledefibrillator, anti-tachycardia pacemakers) and surgical strategies (ablation) canreplace AA’s in selected patients. Third, it is necessary to find better drugs thanthose currently being prescribed with less adverse side effects and higher therapeuticefficacy.Many of the Class I antiarrhythmics in clinical use produce significant sideeffects. For example, quinidine not only blocks sodium channels but also muscariniccholinergic receptors. Dysopyramide also has an anti-muscarinic action and may inaddition block calcium channels. Procainamide can produce a syndrome that issimilar to systemic lupus erythrematosus. Class lb agents such as lidocaine,phenytoin, tocainide, and mexiletine all have CNS side effects and the latter twodrugs can cause clinically significant adverse gastrointestinal effects as well.Therapy with encainide and flecainide, both class Ic agents, was shown in the CASTstudy to increase the risk of sudden cardiac death and cardiac arrest. Another ClassIc drug, propafenone, can cause granulocytopenia.7The RSD antiarrhythmic project is concerned with developing new AA’s withspecificity for ischaemic cardiac tissue, a wide therapeutic margin, and minimal sideeffects. Compounds are synthesized using structural leads from a prototype moleculewith unusual channel blocking actions and antiarrhythmic effects. As part of thisproject, pharmacokinetic determinations of blood and tissue concentration for one ofthe lead compounds provided information which will aid in analyses of in vivo screensby relating pharmacological effects to tissue and blood concentration. Informationwas also obtained with regards to maximum tissue concentrations (possible toxicity),rate of clearance, and time to reach steady state. These data are useful inconsidering drugs as clinical candidates. As quoted from Goodman and Gilman:“optimal therapy of cardiac arrhythmias requires an appreciation of thepharmacokinetic properties of antiarrhythmic drugs”.II. PHARMACOKINETICS:The success or failure of drug therapy is largely dependent on dose regimen.The therapeutic goal is to achieve the optimum concentration of drug at the desiredtarget which produces maximal therapeutic effect with minimal adverse effects.Knowledge of a drug’s pharmacokinetic profile is essential to determine therelationship between concentration at the target tissue and administered dose, and topredict possible interactions with other drugs. Kinetic data also provides informationabout speed of onset and duration of effect. For many drugs a complete8pharmacokinetic profile is unknown or unavailable, and such information is obtainedin the field, sometimes at the expense of the patient.a) History of Pharmacokinetics:Pharmacokinetics is defined as “being concerned with the study andcharacterization of the time course of drug absorption, distribution, metabolism, andexcretion, and with the relationship of these processes to the intensity and timecourse of therapeutic and adverse effects of drugs.” [15]. The term“pharmacokinetics” was first introduced by F.H. Dost in 1953 [16] although the subjectmatter had been discussed previously. A. Buchanen in England in 1847 [17]discussed absorption of ether from arterial blood to the brain, and speed of recoveryas relating to redistribution to other parts of the body. Michaelis and Menten inGermany in 1913 [18] published the classic equation for describing enzyme kinetics,the concepts of which are also used to describe the elimination kinetics of ethanol,salicylate, and several other drugs.From such beginnings pharmacokinetics grew into a distinct scientificdiscipline. With the development of improved analytical methods it has developedbeyond the level of theory and inquiry into physiology and the body’s handling ofdrugs to become a valuable tool in drug development and in the clinical setting toimprove therapeutic uses of drugs [15].9b) The drug in circulation:Absorption from the site of administration and transport into the blood is thefirst major step involved in the processing of drugs by the body. It is dependent onthe drug’s ability to cross various cell membranes. This is irrelevant of course in thecase of intravascular administration. Many small molecule drugs can freely cross thevascular membrane, and since the entire volume of blood in the rat circulates withinone minute, absorption into the blood from other sites of administration is usuallyrapid.In blood the drug partitions between erythrocytes and plasma. While in theplasma fraction it can be protein-bound or free. Drug binding to red blood cellsseldom has a pronounced effect on the body’s handling of drugs, whereas binding toplasma protein does. Different drugs bind to different kinds of plasma protein. If thetested drugs are protein-bound in the plasma, they most likely interact with cL1-acid-glycoprotein. Since the binding of many drugs to plasma protein depends uponhydrophobic interactions it may be regarded as a partitioning phenomenon betweenthe aqueous phase of body water and the hydrophobic part of the plasma protein [191.Binding to plasma macromolecules involves weak interactions; the energies ofdissociation of such bonds differ considerably from those of covalent bonds which aretypically about 150 kcal/mol as opposed to I - 10 kcal/mol for the former. Bindingaffinity is described by the apparent constant of association, Ka, which is the ratio ofthe rate constants k1 and k2 (for binding to and release from the protein, respectively).10The higher Ka, the greater the affinity of a particular drug for its binding site. Suchbinding of drugs to protein is usually reversible and is generally assumed to have veryrapid rates of association and dissociation (milliseconds to microseconds).The ratio of bound to unbound drug is governed by a number of factors whichinclude the drug’s Ka, it’s concentration, and the protein concentration. For mostdrugs used clinically in man, the degree of binding is essentially constant over thetherapeutic concentration range. However, situations do occur where the level ofdrug approaches the molar concentrations of the binding protein and the unboundfraction varies over the plasma concentrations associated with therapy. An exampleis salicylic acid when used for the treatment of arthritis (or in cases of overdose). Inanimal studies, where relatively larger doses are often administered than in humans,concentration-dependent plasma binding is more frequent [20].c) Distribution:From the blood the drug may be distributed to various extravascular“compartments’ of the body such as the extracellular fluid, different organs andtissues. Plasma protein and other binding sites influence tissue distribution of a drug;this is expressed quantitatively as Vd, the apparent volume of distribution. Vd isexpressed in units of volume but is not necessarily significant with respect to realvolumes. A low Vd indicates high plasma concentrations and likely reflects a highdegree of plasma protein binding. A large Vd can be hundreds of litres, and indicateshigh concentrations in tissue with respect to blood; this evidently reflects the degreeof tissue binding. However, the problem of directly assessing tissue binding and11relating it to the overall distribution of a drug has limited investigations of this area. Itis important to remember such factors since there is a possibility that tissue bindingsaturates with increasing dose, especially after large doses and during the distributionphase following intravenous administration of basic drugs [21]. Binding of drugs totissue constituents has not been extensively investigated and the nature of theresponsible macromolecules is largely unknown. Adipose tissue along with solubleand structural proteins and other components (nucleic acids, polypeptides, andpolysaccharides) all have the capacity to reversibly bind a variety of compounds.However, studies of the mechanisms involved, and other characteristics of thephenomenon are hampered by the difficulties in measuring binding in the intactanimal [20].Accumulation of a drug in tissue depends not only on the extent to which it isbound to tissue components, but also on a drug’s lipid solubility. The penetration ofweakly acidic or basic drugs into cells depends on extracellular pH which is quiteconstant in the circulation. The rate of distribution from the circulation to the tissuesof each organ will be determined not only by these characteristics but also by theblood flow to the organ. It is generally believed that blood flow is the rate-limiting stepin distribution. However, distribution depends on the chemical composition of eachtissue, and physicochemical properties of the drug such as PKa. Various types ofdrug may have preferred sites of accumulation in tissues.This partitioning from the blood into the rest of the body is usually reversible,so that eventually equilibrium distribution can be achieved. Although theconcentrations in blood and tissues are rarely equal, they are in equilibrium, and a12change in either will cause a redistribution. After an intravenous bolus dose of a drugit takes time for distribution to occur and because of drug elimination, steady-stateconditions are not maintained. The time required to achieve distribution thendepends not only on plasma protein and tissue binding but on the perfusion of thevarious organs of the body by the blood, the lipophilicity of the drug, and theclearance of drug from the blood. In this case the Vd will change as drug moves fromblood to tissue, finally reaching a “pseudo-distribution equilibrium”. For most drugs,Vd at steady-state and Vd at pseudo-distribution equilibrium are within 10% of eachother. When the difference is large, the more useful value is often the one obtainedat pseudo-distribution equilibrium. This applies to all multicompartment models inwhich elimination occurs from a central compartment [22].d) Elimination of drug from the body:Metabolism and excretion are generally unidirectional processes whichdecrease the amount of available drug in the circulation (as it is either removed orconverted to metabolites). Excretion may occur via urine, bile, and/or milk. As soonas equilibrium distribution is achieved, blood concentrations generally diminish byfirst-order kinetic processes (i.e. linear relationship between blood concentration andrate of elimination).The plasma elimination half life of a drug, T112 (the amount of time required toreduce the concentration of drug in the plasma by half), is arguably the most revealingpharmacokinetic property. This parameter, however, is only secondary and a functionof two primary parameters- Vd and clearance (rate of elimination from the body).13Dividing clearance by Vd gives the elimination rate constant, K. After a bolusintravenous dose, Vd does not remain constant with time and so the elimination rateconstant K also varies with time. As Vd increases, it approaches a limiting value, thusthe K value decreases to a limiting value called the “terminal” rate constant. Its valueand that of its corresponding T112 are the numbers usually referred to in the literature[22].e) Lipid solubility and pharmacokinetics:The pharmacokinetic behavior of a molecule can be well defined in terms ofabsorption, clearance, and volume of distribution. These depend upon thephysicochemical properties of the drug molecule. Lipophilicity at physiological pH isperhaps the most important [23]. The standard measure of lipophilicity is the partitioncoefficient (p) measured as solubility in water versus octanol, and usually expressedas log p. Lipophilicity is increasingly measured by means of indirect methods such asreverse phase liquid chromatography. This method is clean, rapid, and leads touseful structure-activity relationship data especially with in structurally homologousseries’. However, log p does not always reflect relative solubility under physiologicalconditions due to difficulties solubilizing some drugs at certain pH’s and temperatures.A new variable, log D [24], allows for description of effective lipophilicity of a moleculeat a given pH (e.g. physiological pH) and temperature and is probably a more usefulunit of measurement than log p. Log D can be determined using buffered aqueousphases or calculated from p as follows:14for organic bases: log 0 = log p - log[1 + antilog(pKa - pH)]for organic acids: log D = log p - log[1 + antilog(pH - plc)]f) Compartmental systems of pharmacokinetic modeling:Representation of pharmacokinetic data by a compartmental system was firstconsidered in the 1930’s [25,26]. A compartment can be defined as an anatomicalregion in which a drug is uniformly distributed. Usually it does not have an anatomicalor physiological counterpart but may, e.g. plasma. The central compartmentrepresents blood and highly perfused tissues. In a multi-compartmental model theperipheral compartments represent tissue with lower blood flow.In the compartmental model of drug distribution kinetics, the one-compartmentmodel is represented as plasma-time curves with first-order kinetics. For anintravenous dose the amount of drug eliminated from the body will equal the doseadministered; therefore clearance (Cl) = Vd x K. If the plasma-time curve does notshow first-order kinetics (i.e. shows a nonlinear log plasma drug concentration vs.time curve relationship), a multi-compartment model may be used to describe thekinetics of distribution. In the two-compartment model the log concentration-vs-timecurves are biphasic. These are termed the alpha and beta phases, or 2 and ,Each of these “disposition phases” has it’s own T112 value. Generally, T112 valuesreported for drugs refer to the beta phase half-life.15g) Usefulness of pharmacokinetic data:This thesis is primarily concerned with the distribution stage of drug kinetics;specifically tissue distribution. Tissue levels are a valuable piece of information forclinicians since together with the blood concentration they give an accurate method ofcalculating total amount of drug remaining in the body at any time. One can correlatetissue concentration data with pharmacological response and estimate onset andoffset of action. Determining maximum tissue concentration during the alpha phase inorgans where the drug is pharmacologically active may help predict toxicity.For most drugs it is desirable to have specificity of action and this may dependon distribution. This process can involve competition between the target organ andother tissues where affinity may be higher. This could result in less therapeutic effectand possibly unwanted side effects. Conversely, if drug concentration at the targetorgan is too high before steady state levels are reached, toxic effects could manifestthemselves before any therapeutic effects are obtained. This is particularly true fordrugs that affect the heart and have a small therapeutic margin. If the dose is loweredto prevent toxic effects during the alpha phase, the steady state concentration maythen be below the therapeutic level.The rapidity with which a drug is transported to the target organ is the primedeterminant of onset of action. Since the heart is highly perfused and RSDcompounds are lipophilic weak bases we expected no difficulties in achievingtherapeutic concentrations in the target organ. Minimal penetration across the bloodbrain barrier would limit possible central nervous system side effects. However, sincethe tested drugs are i-agonists, significant binding in the brain was expected.16Skeletal muscle data gave insight into accumulation in muscle beds. Storage inmuscle and adipose tissue, if great enough, can be a major cause of decrease inavailability for more perfused tissues and be responsible for maintaining plasmalevels over long periods of time (which could lead to toxicity after chronic use). Inhumans one cannot readily determine tissue levels of drugs by direct sampling butthere are models and mathematical methods for estimating this. Measurement can bedone in animals however, and this can be used to predict distribution in man and becorrelated with mathematical predictions.ifi. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:High performance (or high pressure) liquid chromatography (HPLC) is aquantitative analytical method for measurement of the concentration of compounds insolution. These analyte solutions may be homogenates of tissue or plasma samplesthat have undergone appropriate chemical treatments to reduce interfering substancecontent. Liquid chromatographic techniques complement gas chromatography bytheir application to the separation of non-volatile substances, and are particularlysuitable for separations of compounds that are large, highly polar, thermally unstable,and/or easily ionisable in solution. Optimal rates of solvent flow require conditions ofhigh pressure due to the higher viscosity of liquids as compared to gases.An HPLC system is composed of an optional automated injector system (usefulfor analyzing large sample numbers), a solvent pump (either of constant-pressure or17constant-flow type), the column, a detector (can be ultraviolet, electrochemical,fluorescence or other), and a chart recorder and/or computerized data system.Individual substances show up on the chromatographic record as peaks rising abovethe solvent baseline.A chromatography system is tuned so that peaks are as narrow as possibleand well separated from each other. With HPLC the most important determinants ofpeak profile are the packed bed within the column and the solvent (eluent)composition. The column packing material is usually composed of a chemicallymodified particulate silica gel. The parent silica particles are micrometers in diameter(typically 3-7 ELm) and have on their surfaces silanol groups (Si-OH) that can bereacted with any number of compounds to form a chemically modified surface that canspecifically retain the compound(s) of interest (the analyte). The porous nature of thepacking material may also make it possible to separate on the basis of size andincreases the surface capacity of the column. The size of the gel particles is mostimportant in determining separation efficiency. The smaller the particles, the largerthe plate number (a plate being that fraction of the column length in which an analytemolecule goes through one cycle of attraction and removal). Increasing plate number(within limits set by pressure and time) minimizes peak dispersion. Solventcomposition (organic content, added buffer salts or surfactants) also affects peakprofile and relative retention of analytes.HPLC separations often use several mechanisms of retention with any onecolumn packing chemistry. Molecules of analyte can be retained by reactive silanolgroups, the polar components of the eluent can be adsorbed creating a thin organic18layer on the surface of the gel beads that will allow for separation on the basis ofpolarity (liquid-liquid partition chromatography), charged groups can be attachedeither covalently or by association with the silanols to allow for ion-exchangechromatography, and other non-bonded interactions with alkylated surfaces can alsobe used to modify relative retention. The system used for the studies in this thesiswas primarily operating in the mode known as reverse-phase chromatography.Reverse-phase chromatography is so named because rather than the “normal-phase’ method of using an organic eluent and a polar stationary phase (the reactivegroups on the gel particles), it utilizes aqueous eluents containing a proportion oforganic solvent and a non-polar (hydrocarbon-bonded) stationary phase. The organiccomponent of the eluent is retained by the beads to form an organic-rich layer at theparticle surface which has a high affinity for lipophilic compounds. In this waytransport of the most polar compounds proceeds with little retardation, and the morelipophilic compounds are retained to give extended elution times. This technique isparticularly useful for the separation of non-polar compounds from primarily aqueoussolutions which may have a high level of polar impurities derived from tissues orbiological fluids.The quantitative aspect of the analysis comes from measurement of the peaks.Peak height may be used to estimate analyte amounts on the assumption of constantheight:area ratios but integration of area under the peak is the fundamentalquantitative datum for HPLC quantitation. The area or height values are proportionalto the amount of analyte passed through the column. All samples should contain aninternal standard, which is a substance that is as chemically similar to the analyte as19possible so that both will undergo extraction procedures with equal effectiveness. Aknown concentration of internal standard is used with each sample. Calculations canbe performed using a standard curve of peak area ratio (analyte:standard) vs.concentration of analyte, or using the equation Cx = (PxIPs)Cs where Cs is theconcentration of the internal standard, Ps is peak height (or area) of the standard, andPx is peak height (or area) of the analyte. This latter method is performed following aduplicate run of sample with a standard sample run before and after.IV. HYPOTHESIS AND OBJECTIVES:The hypothesis put forward in this experiment was that drug disposition in theearly distribution phase would mimic the time course of the pharmacodynamics. Toexamine this, a profile of concentration-vs.-time for blood and four different tissueswas constructed using the rat. The objective is to demonstrate that the rise and fall ofdrug concentration in the target organ and the blood would follow the same timecourse as onset and offset of therapeutic effect as observed in the rat in separatestudies using this drug.20MATERIALS AND METHODSMaterials:PD123,497 and the internal standard (a structural analogue of PD123,497)were synthesized by and obtained from Cindy Longley and Anne Morrison of RhythmSearch Developments (Vancouver, B.C., Canada). HPLC grade MeCN, MeOH, andacetone were obtained from Fisher Scientific (Nepean, Ont., Canada). Methyl-t-butylether was obtained from BDH, and various non-organic reagents were used of ACSstandard and obtained from the usual laboratory supply firms.Protocol:Eighteen male, Sprague-Dawley rats weighing from 300 to 450 grams wererandomly assigned to one of six groups (n=3). PD123,497 (8 j.tmol/kg) wasadministered i.v. (tail vein) and rats were sacrificed by decapitation at t=15 sec., 30sec., I mm., 2 mm., 5 mm., or 10 mm. post-injection. Brain, heart, liver and a skeletalmuscle sample taken from the right hind leg were removed, rinsed in cold saline(0.9% wlv), and frozen at -20°C. Blood (8 - 13 mL) was collected from the neck area,mixed with a few drops of heparin (1000 i.u.ImL), and diluted with saline. This mixturewas homogenized and frozen at -20° C.Extraction:There are many methods of chemical extraction used to purify samples foranalysis. Several of these were tried and rejected due to poor recoveries or technical21difficulties. The procedure described below is rather complex, but was necessary formaximum recovery of drugs and reproducibility of results.Tissues were thawed, weighed, and homogenized in saline. Bloodhomogenates were thawed and stirred prior to extraction. Each homogenate wasaliquotted into three 2.5 mL samples. To each sample was added 2.2 nmol/mLinternal standard; samples were lightly vortexed, then 3 mL acetone was added toeach. After vigorous vortexing to a uniform texture, samples were let stand for 15 - 30minutes then centrifuged at 5500 rpm for approximately 15 minutes. A fixed volume ofsupernatant was removed and the pellet discarded. To the supernatant was added0.1 volume of NaH2PO4buffer (0.1 M, pH=9), then 5 mL of MtBE. Tubes were mixedon a Labquake mixer (Lablndustries, Berkeley, California, USA) for 10 minutes. Theorganic fraction was removed and two more washes with 2 mL MtBE were performedon the aqueous fraction. After the last wash the aqueous fractions were discarded. Aback extraction was performed on the organic fractions using 0.4 M sulfuric acid:three washes of 5, 2, and 2 mL respectively. The aqueous fraction was collected thistime and after the final wash the organic fractions were discarded. The aqueousfractions were then neutralized by adding solid sodium carbonate until pH paperindicated a pH between 8 and 9. The MtBE washes were repeated as above and theaqueous fractions discarded. The organic fractions were evaporated under nitrogengas in a Reacti-Therm heating module (Pierce Chemical Co., Rockford, Illinois, USA)set on low heat. Dry samples were reconstituted by first dissolving in 50 L ethanol,sonicating, then adding 450 iL distilled water so that total sample volume = 500 tL.22Recovery of drug during extraction:To determine extraction efficiency blank homogenate solutions from blood andthe four tissues studied were spiked with standard solutions of P0123,497 (0.6, 1.3,2.5, or 5.1 nmoles/mL) and internal standard (2.2 nmoles/mL). Dividing the peak areaof an extracted sample by that of the unextracted standard solution gave percentrecovery of that drug at a particular concentration.Quantitative Analysis:Samples were analyzed using high performance liquid chromatography.Injection volume was 20 jtL. The system was composed of a WISP 71 OB automatedinjector (Waters, Milford, Massachusetts, USA), a twin piston Beckman IOOA pump(Beckman Instruments, CA, USA), a Model 441 Waters Associates 215 nm fixedwavelength UV detector, a Rikadenki model BI 04 chart recorder (Rikadenki KogyoCo., Tokyo, Japan), an SGE cyanophase IOGLC4 precolumn (10mm x 4mm, 5 micronparticle size, 80 angstrom pore size; Fisher Scientific), and an SGE cyanophasecolumn (100mm x 4mm, 80 angstrom pore size, 5 micron particle size; FisherScientific). Data was analyzed with an Apple lIe computer using the Chromatochartprogram (Interactive Microware Inc., State College, PA, USA). The mobile phase wascomposed of 45% MeCN, 16% MeOH, and 8.5% 0.15M ammonium acetate buffer(pH=7) and degassed in situ by helium at ambient temperature. Samples were elutedisocratically.23Calibration curves:Calibration curves were made by plotting peak area ratio (analyte:standard) vs.concentration of P0123,497. A curve was made for unextracted and extractedstandard solutions. A separate graph of the two curves was constructed for eachtissue and for blood.Concentration-vs-time graphs:For each tissue, concentration at each time point was determined. The peakheight ratio of the three aliquots of each sample were averaged and the concentration(nmoles/mL) was determined by extrapolating from the appropriate calibration curve(extracted sample curve). Tissue concentration (nmoles/g tissue) was determined bymultiplying by the appropriate variables (see Appendix I for calculations), then thethree concentrations for each rat were averaged to obtain a mean concentration foreach tissue at each time point.24RESULTSExtractions:The recoveries of PD123,497 and internal standard for blood and the fourtissues are shown in Table 2. The brain and liver homogenates yielded the lowestrecovery of PD123,497 (77 ± 0.7% and 77 ± 3.8% respectively) and the highestrecovery was that of internal standard from brain homogenate (90 ± 0.9%).Calibration curves:Calibration curves for the various tissues and blood and their regressioncoefficients (r) are shown in Figure 1 (a - e). Regression analysis showed allcalibration curves to be linear (of the equation y = mx + b). Concentration in unknownsamples was determined using the extracted sample data on the appropriatecalibration curve.Area under the peak:Variance within days: For each day’s analyses two runs of standard solution wereperformed. The coefficient of variance for these peak areas for any one standardsolution was less than 5%.Variance between days: The same stock solution of standard was used for severaldays. The coefficient of variance for average peak area (n=2) over a series of days(n=5 or 6) was less than 5%.25Mean Recovery(%) ± SEMint.standard PDI 23,497liver 87± 1.1 78±3.8heart 88± 1.5 85±2.1sk.muscle 88±1.7 79±2.6brain 90± 0.9 77±0.7blood 87± 1.4 86±2.0TABLE 2: Recovery of PD123,497 and internal standard by chemical extraction:The % recovery of PD123,497 and internal standard following extraction using themethod described is shown. Blank homogenates were spiked with 0.6, 1.3, 2.5, or5.1 nmol/mL PD123,497 and 2.2 nmol/rnL internal standard before liquid extractionwith MtBE and sulfuric acid. Each number represents the mean and SEM of therecoveries for 12 samples (n=3 at each of four concentrations of PD123,497).26FIGURE 1: Calibration Curves for Blood and Tissues: Blood and tissuehomogenates were spiked with 2.2 nrnoles/mL internal standard and either 0.6, 1.3,2.5, or 5.1 nmoles/mL PD123,497. After extraction using the method described peakarea ratios were plotted against concentration of PD123,497 to give a calibrationcurve (•). These were used to calculate concentration of PD123,497 in unknownsamples by extrapolation using peak area ratio. Curves were also constructed forunextracted standard solutions of PD123,497 and internal standard (A). These wereused to determine extraction efficiency. Regression coefficients (r) are given for allcurves.2704-L.a)I.a)0.2.0001.60c 1.20a)L..L 0.800.400.0.00• standardsHEARTA extracted2.0001.50a,.c 0.50a)0.000.00 0.92 1.83 2.75 3.67 4.58 5.50conc. PD1 23,497 (nmollmL)A0.00 0.92 1.83 2.75 3.67 4.58 5.50BRAIN conc. PD1 23,497 (nmollmL)• standards A extracted2.00OEQ998 B0.00 0.92 1.83 2.75 3.67 4.58 5.50SKELETAL MUSCLEconc. PD1 23,497 (nmollmL)• standards A extractedC281.5001.200.90ci)0.600.3000.000.00• standardsLIVERA extracted2.0001.60(ci 1.200.80.0.400.0.000.00 0.50 1.00 1.50DE2.00conc. of PD123,497 (nmol/mL)BLOOD• standards A extracted0.50 1.00 1.50 2.00conc. of PD1 23,497 (nmol/mL)29Pharmacokinetic data:Figure 2 (a - d) shows concentration-vs-time graphs for brain, heart, skeletalmuscle and liver as compared to blood. Data points are shown with error barsindicating standard error of the mean (SEM). Figure 3 shows all four tissues andblood on one graph so that a complete time profile of the drug’s course through thebody may be observed.The drug distributed rapidly into the well perfused tissues. In fact, theconcentration in the heart after only 15 seconds was several times higher than that inthe blood. The heart and brain exhibited a triphasic distribution profile with aminimum concentration occurring at the 30 second time point and then increasingagain before dropping off to a characteristic beta-phase profile. This coincided withthe apparent transition from alpha-phase to beta-phase in the blood. This triphasictissue profile was also seen in skeletal muscle but the first minimum did not occuruntil around the 60 second time point and the valley was of a broader shape. Theliver exhibited a slow increase in concentration over time and at the 10 minute markwas still increasing.The rise in concentration in the liver mirrored the decrease in concentration inthe heart. Brain concentration was still increasing with liver concentration until the 2minute mark and then it dropped rapidly, leveling off at the 5 minute mark. Skeletalmuscle concentration did not change much after reaching a second maximum ataround 2 minutes.30Figure 2: Concentration vs. time graphs for blood and tissue: The concentrationof PD123,497 in blood (•) and tissue (A) at various times following bolus intravenousinjection of 8 p,mol/kg is shown. Each point is the mean tissue concentration for threeanimals. For each animal, three aliquots of homogenate were analyzed and the peakarea ratios were averaged before extrapolation on a calibration curve to determinedrug concentration. Error bars represent standard error of the mean (SEM). Pointswithout error bars have SEM’s that are too small to be seen on the scale of the graph.A: blood and brain, B: blood and heart, C: blood and liver, D: blood and skeletalmuscle.ABRAIN31• blood ----- brain00.Q-JE0a,C,)U)U,ci>0E000-o00.-jE0a)ci)C’)4-.0,Ci)a)0EC)0020161284080706050403020100B0 60 120 180 240 300 360 420 480 540 600time after injection (seconds)HEART• blood-- heart__ \\\\\-0 60 120 180 24.0 300 360 420 480 540 600time after injection (seconds)32V00-o-JE0a)U)Cl)4-Cl)a)0E000• bloodLIVER---h-- liverV000-JE0G)Cl)Cl)-‘0)U)a)0E000CDI I I I I I I I2016128402016128400 60 120 180 240 300 360 420 480 540 600time after injection (seconds)SKELETAL MUSCLE• blood - sk.musc.A-AI I I I I I0 60 120 180 240 300 360 420 480 540 600time after injection (seconds)33bid. A liver 0 brain + heart—A— sk.m•00.2 8072E 64o 5648Cl)320) 240E 16 - -•-••- AC - ....- - -d 0Co 0 100 200 300 400 500 6000time after injection (seconds)Figure 3: Disposition of PD1 23,497The distribution of P0123,497 in blood and tissues following a bolusintravenous dose of 8 pmol/kg is shown. The peak area ratios ofthree aliquots of a sample homogenate were averaged and this wasextrapolated on a calibration curve to obtain a tissue concentration.Each point on this graph represents the average tissue concentrationfor each of three animals. (for error bars see figure 2)34DISCUSSIONThe distribution of PD123,497 in the blood showed a standard biphasic profileas described by a two-compartment model of pharmacokinetics (see figure 3). Thetwo phases, representing two different rates of removal from the blood, are called thealpha and beta phase respectively. The alpha phase generally representsredistribution into peripheral compartments and the beta phase represents clearancefrom the blood by metabolism and/or elimination mechanisms.Concentration-vs-time profiles for PD123,497 in the brain, heart, and skeletalmuscle were relatively similar with differences mostly in the time course of events(see figure 3). The apparent transient decrease in concentration seen in heart andbrain was lowest at 30 seconds and concentration was rising rapidly at 60 secondswhereas in skeletal muscle concentration was still minimal at this point. This is notsurprising since skeletal muscle is not as well perfused as the other two organs andso one would expect slower rates of drug uptake and elimination. Although drugenters and exits skeletal muscle at a slower rate than the highly perfused tissues, asignificant amount of drug is still getting in. It is necessary to keep in mind that theskeletal muscle data represents a small sample of the total skeletal muscle in thebody of the rat. Although the concentration is lower than that of the other tissues, theactual amount of drug sequestered in the skeletal muscle is considerable given theamount of tissue it represents.Free (unbound) drug on one side of a diffusible membrane will flow to the otherside until concentration is equal on both sides of the membrane. Therefore, any35difference in the total amount of drug between blood and tissue reflects a difference inthe amount of bound drug at steady state (this does not hold for situations where drugis actively transported across the membrane). If the drug concentration in a particulartissue is lower than that of the blood, it means either the drug is bound in the bloodand not in the tissue, or at least bound to a lesser extent than in the blood. If theconcentration is higher in a particular tissue, this reflects a binding site in the tissuewith a higher affinity than binding sites in the blood. The tissue distribution ofPDI 23,497 reflected both such situations. In the heart, the concentration initially wasmuch higher than in the blood. This indicated a readily accessible binding site in theheart with a higher affinity than that of the blood. In the brain, drug concentration wasinitially lower than in the blood, so there may be a low affinity site in the brain or noneat all in which case the total amount of drug measured in the brain immediately afterinjection reflected free drug. Again, this is assuming that the membrane is freelydiffusible and no active transport system is involved in crossing the membrane.The liver is a highly perfused organ and one would expect the drug to obtainmaximum concentrations as rapidly as in other highly perfused organs such as thebrain and the heart. And since the liver is a major organ for metabolism, drugconcentration would likely begin to decrease soon after. The data for PD123,497showed an extremely low concentration in the liver after the first 15 seconds whenconcentrations in the heart and brain were very high. Even if there was no extractionby the liver we would still expect a significant amount of drug due to the high rate ofperfusion. If this low concentration is due to rapid metabolism of drug by the liver, wewould expect the concentration of parent drug to decrease over time as blood36concentration falls; instead, after a short delay, the concentration starts to increase.This does not seem to indicate significant metabolism. Perhaps the time course overwhich data was collected was not sufficient to demonstrate the true profile of drug inthe liver; it is not clear whether a maximum concentration was reached by 10 minutes.However, an explanation for this phenomenon is the presence of a storage depotwithin the liver, possibly the fat globules in hepatocytes. Drug entering the liver is atfirst rapidly extracted and so levels of drug in the tissue remain low. As timeprogresses, the drug molecules eventually make their way deeper into the cell wherethey are sequestered in the fat globules. Thus, concentration of drug in the wholeorgan would rise as drug is trapped in these depots, free from the metabolizingenzymes in the cytoplasm. This process would take time since an intracellular site isnot rapidly accessible from the circulation, but it is saturable and eventually liverconcentration would begin to decrease. This may explain why concentration in theliver is still rising while in other tissues it has reached a minimum.In three of the tissues there was an interesting feature in the kinetics ofPD123,497: a transient decrease in tissue concentration that occurred early in thetime course of distribution. This unusual negative peak” may have been an artifactbut because it appeared in three different tissues (heart, brain, and skeletal muscle) itwas worth examining. Between 15 and 30 seconds after injection, the concentrationof drug in these three tissues decreased with a corresponding decrease in the bloodconcentration, but by 120 seconds the concentration in these tissues rose again eventhough blood concentration was still declining. At this time point we also saw the37concentration in the liver begin to rise rapidly. I have put forward a theory to explainthese events.It is possible that the data above reflects drug binding in a tissue that was notanalyzed in this experiment. This “invisible” binding site competed with binding atother sites and was responsible for the transient decrease seen in the concentration-vs-time profiles. Note that initially most of the drug in tissue will be in the interstitialfluid, a readily accessible, high-capacity compartment. Although our unknown bindingsite is not as accessible to the drug as these extravascular sites in the other tissues,the binding affinity is higher and drug is soon preferentially sequestered here. Thuswe see a rapid drop in concentration for blood, heart, liver, and skeletal muscle asdrug is redistributed to the unknown binding site. This site is soon saturated but thereis a second tissue binding site in the brain, heart, and skeletal muscle locatedintracellularly that is now preferentially sequestering the drug. This high-affinity site isnot as readily accessible, thus the time delay. Drug is redistributed back to the heart,brain, and skeletal muscle. The result of this overall process is concentration-vs-timecurves with two concentration maxima reflecting two separate binding sites: a low-affinity extracellular site, and a high affinity intracellular site. In a compartmentalmodel, the central compartment would represent blood and the extracellular matrix,the unknown site would represent compartment two, and the brain, heart, and skeletalmuscle would represent compartment three. Possible candidates for the unknownsite are the lungs or the kidneys, both highly perfused tissues.It is also possible that the unknown binding site described above is the gut andthat PD123,497 undergoes enterohepatic circulation. In this case drug is rapidly38extracted by the liver into the bile which explains the rapid drop in concentration seenin the blood and other tissues during the first 30 seconds after injection, and the lowconcentration in the liver. The bile is delivered to the gut where it binds to some lowaffinity site. Upon saturation, blood levels stabilize and enough time has passed thatthe drug has penetrated higher affinity intracellular sites in the other tissues and isnow preferentially sequestering here. It is also being stored in the liver and soconcentration in all four tissues goes up. This is one possibility, however, furtherwork is required to clarify the appearance of this transient decrease in concentrationseen early in the time course of tissue distribution of PDI 23,497.After saturation of the second, high-affinity site in the heart, brain, and skeletalmuscle, concentrations declined. This drop occurred early in the heart (peakconcentration at 60 seconds), later in the brain (peak concentration at 120 seconds),and latest in the skeletal muscle (peak concentration at 300 seconds). Thesedifferences reflect affinity of the binding site and of course, rate of perfusion of thetissue. The concentration maxima appear later in other tissues than in the heart andin this way PD123,497 is mimicking thiopental where cessation of therapeutic activityis also due to redistribution into other tissues and not elimination. Concentration ofPD123,497 in the liver was still increasing at 600 seconds reflecting sequestration ofdrug. It is interesting to note that at this time point the liver samples run on the HPLChad an extra peak on the chromatograph that could represent a metabolite.When the actual amount of drug in the five areas is added up, there is still alarge percentage of the administered dose unaccounted for. This is possibly due touptake into body fat. PD123,497 is highly lipophilic and there is no reason to believe39there would be any difficulty crossing lipocyte membranes. Rate of uptake would beslow due to the poor rate of perfusion in fatty tissue, but this site represents a high-capacity depot that should not be overlooked.So how does all of this data relate to pharmacological action? Severalantiarrhythmic screens of PD123,497 have been performed in different animal speciesso far. Many of these tests involved bolus intravenous doses of 8 jimol/kg- the sameprotocol that was used to obtain the pharmacokinetic data presented here. It istherefore possible to directly compare the data.Two dose-response curves were constructed for the effects of PD123,497 onheart rate at 1 minute and 8 minutes after injection in the rat [27]. After 1 minute post-injection (bolus iv.) of 8 tmol/kg PD123,497 there was a drop in heart rate from 415bpm (pre-injection) to 305 bpm. Eight minutes later the heart rate was back up to 385bpm. This correlated well with my data which showed a higher concentration in theheart at 1 minute than at 8 minutes post-injection.The same experiment in the baboon [27] yielded similar results. One minuteafter bolus i.v. injection of 8imol/kg PD123,497 there was a 26% decrease in heartrate from control, whereas at 8 minutes there was only an 8% decrease. The datapresented here for the rat does show that at 8 minutes concentration in the heart islower than at 1 minute post-injection, however the difference is much greater than18%. The results are still appropriate however when one considers that the baboon isa much larger animal than the rat and would therefore be expected to have slowerkinetics of uptake and elimination (due in part to a slower heart rate). The slopes ofthe beta-phase portion of the heart curve may be different for the two species, but the40fact appears to remain that concentration in the heart is declining at this time point inthe rat and the baboon.Another experiment in rats measured the effect of PD123,497 on heart rate andblood pressure at frequent intervals after a bolus intravenous dose of 8 j.tmol/kg [27].These results correlated very well with the heart and blood concentration datapresented here (see Figure 4).In two of three dogs (see figure 5), bolus i.v. doses of 1.0, 2.0, and 4.0 Imol/kgP0123,497 produced a greater percent decrease in conduction velocity 2.5 minutesafter injection than at 5 minutes after injection [27] (the exception being dog #1 at thetwo lower doses where there was no significant change). Although the doses werelower than those used to obtain the rat pharmacokinetic data, it still correlated well.We might expect the time course of elimination from the heart to be slightly longer inthe dog than in the rat, but it would seem that concentration is decreasing in the dogheart by five minutes and thus following the same profile as seen in the rat.Besides relating to pharmacological response, concentration-vs. -time curvescan also provide useful information with regards to potential toxicity. Therapeuticconcentrations must be maintained in the beta phase of distribution, yet the alphaphase concentrations are often much higher. If concentrations in the alpha phase arehigh enough in certain tissues, toxicity may result before any therapeutic benefit isachieved. For example, PD123,497 exerts its primary therapeutic activity in the heartby slowing conduction velocity and increasing the refractory period. Thisantiarrhythmic effect can easily become pro-arrhythmic and the therapeutic margin ofAA’s is notoriously low. The pharmacokinetic data for P0123,497 shows a very high41A heart conc. 0 heart ratea)75 5500) -U)44°Cl) 60a)45- 330Cl) Cl)0)- 0,220EU)110C)15-______C.) 0 0a .1a)0 60 120 180 240 300 360 420 480 540 600 ..time after injection (seconds)B blood conc. 0 blood press.___40 120____J________110 —E °° C =100 EaE30 ° E807060a Cl)500)40.—30a) -vC) 20 a010 —0C.) •. -0 00 60 120 180 240 300 360 420 480 540 600time after injection (seconds)Figure 4: Concentration vs. Effect: The relationshipbetween concentration and pharmacological effect isdemonstrated in the following graphs. Heart and bloodconcentration data were obtained by the methods described.Heart rate and blood pressure data are from pharmacologicalscreens performed by other researchers at Rhythm SearchDevelopments. The data are presented together todemonstrate the strong correlation between the results ofthese different studies. A: heart rate, B: blood pressure.42PD1 23,497 and Conduction VelocityCanine left ventricleDog #1 - Dog #2 Dog #3Ec Effect ci P0123.497 on Conduction Vodty in the caine heart.Ths data, from wxk done by fdlaw researchers at Rh,khm SearchDevopments (unpublished), shc the &fed ci three bdus intra’ienousdoses ci P0123,497 on conduction vocity at 2.5 aid 5.0 ninutes atter eachdose. This data corrates wth the findings presented here thatconcentration ci P0123,497 in the heart is greater a5 ninutes atter a bdusi.v. dose thai at 5.0 ninutes ter.>0CC.C0C0I.U-10090807060501 .Opmolelkg 2.OpmolelkgI4.OimoIe!kg0I2.5 5 7.5 10 12.5 15Time (minutes)43initial concentration in the heart, higher even than in the blood. Not only does thissuggest possible alpha-phase toxicity but it is another good example of why tissuedistribution studies are so important. A clinician relying only on plasma or wholeblood concentration to monitor dosage would have to be aware of the fact that thesefigures underestimate the concentration achieved in the target organ and do notindicate the dangerously high initial concentrations of the alpha phase following i.v.administration.Tissue distribution data is also helpful in drug development. Along with variouspharmacological screens for activity and toxicity, pharmacokinetic profiles can provideuseful information for evaluating the clinical potential of test compounds. The kineticsof PD123,497 indicate that after reaching peak concentration in the heart at 1 minute,a peak brain concentration is achieved at 2 minutes. This implies that ascardiovascular effects are wearing off, significant CNS effects may appear, a likelyevent considering these drugs were developed originally as kappa-opioid agonists.This is an important consideration when evaluating this drug as a potential prototypefor future research. Cardiovascular effects are warranted in the treatment of cardiacarrhythmias; CNS effects are not. The slow buildup of PD123,497 in the liver mayalso be of some concern if this drug proves to be hepatotoxic.44CONCLUSIONThe method presented here for extraction and quantitative analysis ofPD123,497, a novel kappa-opioid agonist, was demonstrated to be effective in termsof recovery and reproducibility. Furthermore, as a tool for the pharmacokineticanalysis of PD123,497 distribution in the rat, it produced results that painted a distinctpicture of the time course of the drug’s travels through the whole animal. The resultsalso correlated well with previous pharmacological screens using the same drug invarious animal models.The data showed that PDI 23,497 was rapidly distributed into the heart and hasa potential for alpha-phase toxicity. Termination of therapeutic effect was due toredistribution to other tissues and not metabolism or elimination. An unusual triphasiccurve observed in three of the tissues suggests another significant compartment forPD123,497 distribution not represented in the samples analyzed here. There may bea significant capacity for storage in muscle and adipose tissue. Possible storage inthe liver could result in hepatotoxicity. Finally, a significant amount of drug wasdistributed to the brain, suggesting a potential for CNS effects.The hypothesis put forward in the Introduction was supported by this work.Specifically, the onset and offset of therapeutic effects correlated well with the timecourse of rise and fall of concentration in the blood and heart.45REFERENCES1. May, G.S.; Eberlein, K.A.; Furberg, C.D.; DeMets, D.L.; Passamani, E.R.Secondary prevention after myocardial infarction: a review of long-term trials.Prog. Cardiovasc. Dis. 24: 331 (1982)2. Furberg, C.D. Effect of antiarrhythmic drugs on mortality after myocardialinfarction. Am. J. Cardiol. 52: 32C (1983)3. The Cardiac Arrhythmia Suppression Trial (CAST) investigators. Preliminaryreport: effect of encainide and flecainide on mortality in a randomized trial ofarrhythmia suppression after myocardial infarction. N. Engi. J. Med.321: 406 (1989)4. Elharrar, V.; Gaum, W.E.; Zipes, D.P. Effect of drugs on conduction delay andincidence of ventricular arrhythmias induced by acute coronary occlusion indogs. Am. J. Cardiol.; 39: 544 (1977)5. Nattel, S.; Pedersen, D.H.; Zipes, D.P. Alterations in regional myocardialdistribution and arrhythmogenic effects of aprindine produced by coronaryartery occlusion in the dog. Cardiovasc. Res. 15: 80 (1981)6. Hondeghem, L.M. Antiarrhythmic agents: modulated receptor applications.Circulation 75: 514 (1987)7. Vaughan Williams, E.M. Classification of dysrhythmic drugs, inPharmacology of Antiarrhythmic Agents (L. Szekeres ed.),Oxford Pergamon, U.K., 1981, pp.125-I 50468. Hodgkin, A.L.; Huxley, A.F. A quantitative description of membrane current andits application to conduction and excitation in nerve.J. Physiol. 117: 500 (1952)9. Szekeres, L.; Vaughan Williams, E.M. Antifibrillatory action. J. Physiol.160: 470 (1962)10. Hille, B. Local anaesthetics: hydrophilic and hydrophobic pathways for thedrug-receptor reaction. J. Gen. Physiol. 69: 497 (1977)11. Hondeghem, L.M.; Katzung, B.G. Time- and voltage-dependent interactions ofantiarrhythmic drugs with cardiac sodium channels.Biochimica et Biophysica Acta 472: 373 (1977)12. Starmer, C.F.; Grant, A.O.; Strauss, H.C. Mechanisms of use-dependent blockof sodium channels in excitable membranes by local anaesthetics. Biophys. J.46: 15(1984)13. Tamargo, J.; Valenzuela, C.; DelpOn, E. New insights into the pharmacology ofsodium channel blockers. Eur. Heart. J. 13(suppl.F): 2 (1992)14. Ratner, S.J. Changing patterns of antiarrhythmic use in the 1990’s.Drugs News Perspectives 3: 295 (1990)15. Wagner, J.G. History of pharmacokinetics, in Pharmacokinetics: Theory andMethodology (M. Rowland and G. Tucker eds.), Pergamon Press, Ontario,Canada, Chapter 1 (1987)16. Dost, F.H. Der blutspeigel-kinetik der konzentrationsablãute, in derKrieslauffUssigkeit(G. Thieme ed.), Leipzig, p. 244 (1953)4717. Buchanen, A. Physiologic effects of the inhalation of ether. London MedicalGazette 39: 715 (1847)18. Michaelis, L.; Menton, M.L. Die Kinetic der Invertinwirkung. Biochem. Z.49: 333 (1913)19. Rolinson, G.N. Beta-lactam antibiotics. J. Antimicrob. Chemother. 6: 311(1986)20. Wilkinson, G.R. Plasma and tissue binding considerations in drug disposition.Drug Metab. Rev. 14(3): 427 (1983)21. Wagner, J.G. A modern view of pharmacokinetics. J. Pharmacokinet.Biopharm. 1: 363 (1985)22. Tozer, T. N. Concepts basic to pharmacokinetics, in Pharmacokinetics: Theoryand Methodology (M. Rowland and G. Tucker eds.) Pergamon Press, Ontario,Canada, Chapter 2 (1987)23. Jezequel, S.G. Central nervous system penetration of drugs: importance ofphysicochemical properties, in Progress in Drug Metabolism, Volume 13 (J.W.Bridges, l.F. Chasseaud, G.G. Gibson eds.), Taylor and Francis Ltd., U.S.A.,Chapter 4 (1987)24. Scherrer, R.A.; Howard, S.M. Use of distribution coefficients in quantitativestructure-activity relationships. J. Med. Chem. 20: 53 (1977)25. Gehlen, W. Wirkungsstarke intravenös verabreichter arzneimittel alszeitfunktion. Em beitrag zur mathematischen behandlung pharmakologischerprobleme. Arch. Exp. Path. Pharmakol. 171: 541 (1933)4826. Teorell, T. Kinetics of distribution of substances administered to the body. II.The intravascular modes of administration. Arch. mt. Pharmacodyn. Thér.57: 226 (1937)27. Rhythm Search Developments, personal communication. (1994).BIBLIOGRAPHY1. Applied Biopharmaceutics and Pharmacokinetics, 2nd edition (L. Shargel andA.B.C. Yu eds.), Appleton-Century-Crofts, Conecticut, U.S.A.2. Biopharmaceutics and Clinical Pharmacokinetics, 2nd edition (M. Gibaldi ed.)Lea and Febiger, Philadelphia, U.S.A.3. The Pharmacological Basis of Therapeutics, 8th edition (A. Goodman-Gilman,T.W. Rail, A.S. Nies, P. Taylor eds.), Pergamon Press, Canada49APPENDIX ICalculations to determine tissue and blood concentration:1. using peak area ratio (average of three aliquots), concentration of PD123,497was extrapolated from the appropriate graph:concentration of volume of reconstituted volume of one aliquot ofPDI 23,497 (nmol/mL) X sample (0.5 mL) ÷ homogenate (2.5 mL)original volume of weight of tissue sample or volume ofX homogenate (mL) ÷ blood taken from rat (g or mL)concentration of PD123,497 in originalsample (nmol/g tissue or mL blood)

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