Clinical Pharmacokinetics 41

Clin Pharmacokinet 2002; 41 (8): 581-602 CONCEPTS 0312-5963/02/0008-0581/$25.00/0

Pharmacokinetic Imaging A Noninvasive Method for Determining Drug Distribution and Action

Alan J. Fischman,1,2 Nathaniel M. Alpert1 and Robert H. Rubin1,2 1 Division of Nuclear Medicine of the Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA 2 Division of Health Sciences and Technology, Center for Experimental Pharmacology and Therapeutics, Harvard-Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Contents Abstract ...... 581 1. Radionuclide Imaging Techniques ...... 583 1.1 Instrumentation ...... 583 1.2 Tracer Techniques ...... 584 1.3 Radiochemistry ...... 585 1.4 Advantages and Limitations of Positron Emission Tomography Measurements ...... 585 2. Types of Studies with Positron Emission Tomography ...... 586 2.1 Antimicrobial Agents ...... 587 2.1.1 Erythromycin ...... 588 2.1.2 Fluconazole ...... 588 2.1.3 Fluoroquinolones ...... 589 2.2 CNS Drugs ...... 590 3. Pharmacodynamics ...... 594 4. Magnetic Resonance Spectroscopy ...... 597 4.1 Antineoplastic Drugs ...... 597 4.2 Antimicrobial Agents ...... 598 4.3 CNS Drugs ...... 598 5. Conclusions ...... 599

Abstract Advances in positron emission tomography (PET), single photon emission computed tomography (SPECT) and magnetic resonance spectroscopy (MRS), and the ability to label a wide variety of compounds for in vivo use in humans, have created a new technology for making precise physiological and pharmacological measurements. Due to the noninvasive nature of these approaches, repetitive and/or continuous measurements have become possible. Thus far, these techniques have been primarily used for one-time assessments of individuals. However, experience suggests that a major use of this technology will be in the evaluation of new drug therapies. Already, these techniques have been used to measure precisely and noninvasively the pharmacokinetics of a variety of antimicrobial, antineoplastic and CNS agents. In the case of CNS drugs, imaging 582 Fischman et al.

techniques (particularly PET) have been used to define the classes of neuro- receptors with which the drug interacts. The physiological, pharmacological and biochemical measurements that can be performed noninvasively using modern imaging techniques can greatly facilitate the evaluation of new therapies. These measurements are most likely to be useful during drug development in preclinical studies and in phase I/II human studies. Preclinically, new drugs can be precisely compared with standard therapies, or a series of analogues can be screened for further development on the basis of performance in animal models. In Phase I/II, imaging measurements can be combined with classical pharmacokinetic data to establish optimal administration schedules, evaluate the utility of interventions in specific clinical situations, and aid in the design of Phase III trials.

In recent years, the techniques employed for rently performed with a much higher degree of so- drug discovery, design and evaluation have under- phistication. In contrast to the largely empirical gone many changes. Historically, the process of procedures of the past, modern drug development drug discovery involved screening of large num- is usually directed to specific molecular targets and bers of samples of natural products for biological is guided by principles of molecular structure and effects until a compound with the desired proper- dynamics, genomics and proteomics. Laborious ties was identified. After elucidation of the struc- one-by one synthesis and biological evaluation of ture of this potential drug, large numbers of ana- analogues has been supplemented by approaches logues were prepared by standard methods of based on combinatorial chemistry and high- organic chemistry and screened with in vitro and throughput screening methods. Applications of ge- in vivo assays until an agent or agents with optimal netic engineering to drug design (gene therapy) has pharmacology emerged. Lead compounds from fostered a transition from developing drugs for this discovery and design process were further treating symptoms to the design of agents that cor- evaluated to define their pharmacokinetic and rect or modulate pathophysiology of disease pro- pharmacodynamic profiles by studies of normal cesses. animals and animal models of the condition for Recent developments in techniques for non- which the drug was being developed. Tissue distri- invasive imaging of drug distribution and effect bution studies in large numbers of animals yielded have begun to have similar impact on drug evalu- precise pharmacokinetic data, and detailed physio- ation in humans. In numerous situations, imaging logical monitoring produced information about procedures such as positron emission tomography pharmacodynamics. Candidate drugs that contin- (PET), single photon emission computed tomogra- ued to show promise in the animal studies were phy (SPECT) and magnetic resonance spectros- further evaluated in Phase I to III studies in hu- copy (MRS) can provide tissue pharmacokinetic mans. Compared with the results of animal studies, information in humans that is comparable in detail data obtained from human clinical trials were less and quality to the results from invasive studies in extensive. In general, the quality of blood and animals. With PET, it has been possible to obtain plasma pharmacokinetic data obtained from hu- detailed quantitative information about drug phar- man subjects were comparable to those from ani- macokinetics and pharmacodynamics in a variety mal studies; however, information about tissue dis- of tissues, including the CNS. This review will tribution was considerably less precise and usually concentrate on recent applications of PET to phar- limited to body secretions and biopsy samples. macokinetic analysis. Although SPECT and MRS Similarly, pharmacodynamic evaluations were much are less generally applicable to pharmacokinetic less extensive. analysis, several important applications of these Drug discovery, design and evaluation are cur- approaches will also be discussed.

1. Radionuclide Imaging Techniques jections of the distribution of radioactivity in the object are obtained. By acquiring images in multi- 1.1 Instrumentation ple projections, three-dimensional distribution of radioactivity can be estimated. The instrumenta- Radionuclide imaging techniques have the goal tion for SPECT is similar to that used for planar of providing noninvasive measurements of the imaging; however, projections at all angles around three-dimensional distribution of radiolabelled an axis of the object are acquired. These data are [1] compounds in living organisms. In many as- then reconstructed using filtered back-projection pects, radionuclide imaging is similar to conven- or iterative algorithms (similar to x-ray computed tional serial section autoradiography. However, in- tomography) to produce transaxial, coronal and sa- stead of film, an array of scintillation detectors is gittal images of the distribution of radioactivity. used to record images. Imprecise quantification is the major disad- Three different procedures are currently used vantage of both planar and SPECT imaging. This for radionuclide imaging: planar imaging, SPECT and PET. The technical details of the three tech- deficiency is caused by loss of photons by tissue niques vary considerably, however, the general attenuation and by imprecise positioning of disin- principles are similar. tegrations due to scatter within the body. Further- γ With planar imaging, a large sodium iodide more, obtaining a - camera image requires absorp- scintillation detector optically coupled to an array tive collimation of photons emitted by the of photomultiplier tubes is placed close to the ob- radioactive object.[1] These collimators are made ject of interest and emitted photons are measured of lead and contain many parallel-aligned channels (figure 1).[1] By appropriate interconnections of that allow detection of only those photons that pass the photomultiplier tubes, two-dimensional pro- through the channels from the source to the crystal.

Collimator Nal crystal

X-position signal

Y-position signal Computer

PHA Position logic circuits Position

Energy information Patient Image

PM tubes

Fig. 1. Basic components of a single photon imaging device (Anger camera) used for planar or single photon emission computed tomography (SPECT) imaging. PHA = pulse height analyser; PM = photomultiplier.

Photons that travel in other orientations are ab- process of annihilation, coincidence and detection sorbed by the collimator (figure 1) and do not con- defines a volume between the detectors from tribute to the image. This method of collimation which coincidence events originate (figure 2).[3] By imposes significant limits on both detection sensi- positioning rings of detectors around the object, tivity and image resolution. maps of the three-dimensional distribution of posi- The main problems inherent to single photon tron emitting isotope in an object can be recon- imaging are not important issues with PET. Many structed using filtered-back projection or iterative radionuclides with low atomic number and relative algorithms. The process of annihilation, coinci- proton excess decay by emission of positrons (posi- dence and detection is frequently referred to as ‘electronic collimation’ and is the primary reason tive electrons, β+).[2] After a positron is emitted, it why the sensitivity of PET is 10- to 20-fold higher rapidly loses kinetic energy and eventually com- than that of SPECT. Since corrections for attenua- bines with an electron (β–). β+-β– interaction re- tion and scatter are straightforward with PET, the sults in an annihilation event in which the rest mass images are quantitative and precise maps of radio- of the positron-electron pair is converted to energy. pharmaceutical distribution.[2] Furthermore, since 2 The relationship E = mc predicts that this process resolution is limited only by positron range and will result in two 511 KeV photons emitted in op- detector characteristics, PET images have higher posite directions. In PET imaging devices, these resolution than SPECT images, 1 to 6mm versus 7 photons are recorded by detector pairs oriented at to 18mm full width at half maximum (FWHM). 180° to each other (figure 2). The use of appropriate electronics, coincidence circuitry, ensures that 1.2 Tracer Techniques only those photon pairs that occur in a very short time interval (5 to 20 nsec) and are the result of Noninvasive measurement of the tissue phar- individual β+-β– annihilations are recorded. This macokinetics of a drug requires radiolabelling with

Coincidence circuitry

Annihilation event Accepted Rejected

Region of coincidence detection

DETECTOR DETECTOR a

Fig. 2. Basic principles of annihilation-coincidence-detection. For an event to be recorded, both photons have to arrive at opposing detectors within the resolving time of the coincidence circuitry. Events registered by a single detector are rejected, giving ‘electronic collimation’.

an isotope that emits penetrating radiation that can straightforward chemically; however, the short be detected by external imaging. A critical require- half-lives of most PET radionuclides introduce ment of the radiolabelling process is that it must complications.[4] Radiopharmaceutical synthesis not modify the biological or biochemical proper- must be sufficiently rapid so that the drug of inter- ties of the drug.[4] Usually, this requires radio- est can be synthesised, isolated, purified and for- labelling of the compound under investigation mulated as a sterile, pyrogen-free, isotonic solu- with a radioactive form of one of the atoms that tion within 2 to 3 half-lives of the radionuclide. is present in its native structure: carbon, hydrogen, Also, since large amounts of radioactivity must be nitrogen or oxygen. Drugs labelled with 14Cand/or handled, limitation of exposure to personnel is an 3H have been of great value for studying pharma- important consideration. For most drugs, these is- cokinetics in animals; however, the negative elec- sues require the design and synthesis of drug pre- trons that are emitted in their radioactive decay cursors that can be radiolabelled in a single step. have a range of only a few millimetres and thus Over the past several years, these requirements cannot be detected outside the human body. The have fostered the development of many new meth- only radioactive forms of carbon, oxygen and ni- ods of rapid remote-controlled and robotics-based trogen with emissions that can be detected exter- chemistry. nally are positron emitters. Also, many drugs contain one or more fluorine atoms in their native 1.4 Advantages and Limitations of Positron structure and can be radiolabelled with the positron Emission Tomography Measurements emitter 18F. Because there are no isotopes of hydrogen that are suitable for external imaging, posi- Compared with conventional methods of phar- tron emitting halogen isotopes are frequently sub- macokinetic analysis and other noninvasive tech- stituted for hydrogen of hydroxyl groups. These niques, PET has many clear and important advan- analogues are not guaranteed to retain the pharma- tages. (i) PET is noninvasive, requiring at most cokinetic properties of the native drug; however, arterial blood sampling. (ii) Extremely high sensi- the substitutions are highly conservative and in tivity permits measurements at drug concentra- many situations have yielded useful probes. Sev- tions as low as 10–12 mol/L (based on detection eral common positron emitting radionuclides that sensitivity of 1 nCi/cm3 and a drug labelled at a have been used in PET studies are listed in table I. specific activity of 1000 mCi/µmol). (iii) The availability of positron emitting radionuclides of 1.3 Radiochemistry nitrogen, carbon and fluorine makes it possible to prepare a PET tracer for almost any drug. (iv) Due The products of most nuclear reactions that are to the quantitative nature of PET measurements, currently used for preparing PET radionuclides are drug concentrations in tissue determined by imag- 13 11 relatively simple molecules, such as NH3, CO2, ing are nearly identical to the results of direct 18 18 H For F2. In many situations, incorporating measurements of radioactivity in samples of ex- these building blocks into complex drugs is cised tissue (figure 3). (v) The anatomical resolu-

Table I. Physical characteristics of radionuclides used for positron emission tomography Radionuclide Half-life (min) Target reaction Common forms 15 14 15 15 15 15 O2.0N (d,n) O O2,C O2,C O 13 16 13 13 13 N 10.0 O(p,α) N NH3, N2 11 14 11 11 11 11 C 20.4 N(p,α) C CO2, CO, CH3I 18 18 18 18 18 F 110 O (p,n) F F2,H F 76 75 3 76 76 Br 972 As ( He,2n) Br Br2 124I 6048 124Te (d,2n) 124I; 124Te (p,n) 124INa124I

tion of PET permits measurements in small volumes of tissue. (vi) The extremely high biochemical resolution of PET allows differentiation of physiological alterations that occur nanometres apart. For example, PET images of patients with Parkinson’s disease (figure 4) demonstrate reduced presynaptic dopamine metabolism (imaged with [18F]fluorodopa) and normal expression of 11 postsynaptic D2 receptors (imaged with [ C]- raclopride). (vii) The short physical half-lives of most PET tracers results in favourable radiation dosimetry that allows repetitive measurements in a single subject after physiological or pharmaco- Fig. 4. Positron emission tomography transaxial, coronal and logical interventions. (viii) Due to the high specific sagittal images of a patient with Parkinson’s disease demon- strating reduced presynaptic dopamine metabolism (imaged activities of most PET tracers, physiological and 18 with [ F]fluorodopa) and normal expression of postsynaptic D2 pharmacological effects are negligible. (ix) Most receptors (imaged with [11C]raclopride). importantly, after suitable toxicity studies and with rigorous process control procedures, PET radiopharmaceuticals developed for studies in animals The short physical half-lives of most PET tra- can be applied to investigations in healthy volun- cers present several complications for pharmaco- teers and patients. Since PET tracers are chemi- kinetic studies. (i) Most radioactive drugs must be cally and biologically identical to the native un- prepared separately for each subject by a cyclo- tron/radiochemistry facility that is in close proxim- labelled drug, only limited toxicity studies are ity to the imaging laboratory. (ii) The time interval required. over which pharmacokinetic studies can be performed is limited. In general, this time frame is approximately four times the physical half-life of 3000 the tracer; approximately 40 min with 13N, 80 min 11 18 r2 = 0.99 with Cand8hourswith F. Obviously, this is 2500 less of a problem with 76Br- and 124I-labelled tra- ) 3 cers. (iii) The number of chemical manipulations 2000 that can be employed for preparing PET radiopharmaceuticals is limited. (iv) Since only total radio- 1500 activity in tissue is measurable by PET, quantifica-

1000 tion is accurate only if metabolism is minimal. (v)

Well counter (nCi/cm Well Although the anatomical resolution of PET is far 500 superior to that of all other radionuclide methods, it is much lower than that of computed tomogra- 0 phy, MRI or ultrasound. 0 500 1 000 1 500 2 000 2 500 3 000 3 PET camera (nCi/cm ) 2. Types of Studies with Positron Emission Tomography Fig. 3. Relationship between drug concentrations determined by positron emission tomography (PET) and direct ‘well- counter’ measurements of excised tissue samples.[5] As indi- From the perspective of PET studies of tissue cated, the points fall nearly along the line of identity. pharmacokinetics, most therapeutic agents can be

classified into one of two broad categories. The constructing regions of interest (ROIs) on the im- first class of drugs includes pharmaceuticals that ages, the time dependence of drug concentrations interact with and/or neutralise agents that invade in specific organs and tissues can be determined the organism from the environment (toxins, and used to calculate standard pharmacokinetic pa- bacteria, fungi and viruses) or alter normal physi- rameters such as peak concentrations, plateau con- ological function from within (neoplastic cells). centrations and areas under concentration-time Important examples of these types of drugs are curves. antimicrobial and antineoplastic agents (table II). The situation is much different for the second Members of the second group of drugs target spe- class of agents. These drugs usually elicit their cific biomolecules and modulate disease-associ- therapeutic effects by interacting with specific re- ated alterations of cellular processes or homeo- ceptors or binding molecules, and PET studies fo- static control systems. Examples of these drugs cus on the derivation of values for receptor number include antipsychotics, steroids, nonsteroidal anti- (Bmax) and affinity (KD). These investigations re- inflammatory agents, enzyme inhibitors and bio- quire high specific activity radiolabelling, arterial logically active proteins and peptides. blood sampling, measurement of metabolite con- In general, the type of pharmacokinetic infor- centrations in the circulation and detailed kinetic mation that is desired is different for the two modelling of the results of single or multiple injec- classes of drugs and the PET techniques required tion studies (see section 2.2). for the measurements vary considerably. For the In the following sections, we will describe the results of PET studies of the pharmacokinetics of first type of agents, only tissue concentrations of both types of drugs. Antimicrobial agents and drug are usually required. For these studies, the drugs that affect the CNS will be used as examples drug is labelled with a positron emitter, injected of the first and second types of drugs. into volunteers or patients and serial PET images are acquired. These images are quantitative maps 2.1 Antimicrobial Agents of the concentration of radioactivity (nCi/cm3)in tissue. If the tracer is not metabolised in the tissues To be effective, antimicrobial therapy must de- of interest, dividing each pixel in the images by the liver concentrations of active drug to the site of specific activity of the tracer (nCi/µmol) produces infection that are sufficient to inhibit growth of, or a map of regional concentrations of intact drug. By kill, the invading microorganism. Drug concentra-

Table II. Examples of type 1 drugs that have been studied by positron emission tomography Drug Radiolabel Class Reference Cisplatin 13N Antineoplastic 6 Carmustine (BCNU) 11C Antineoplastic 7 SarCNU 11C Antineoplastic 7 Elmustine (HECNU) 11C Antineoplastic 8 Fluorotamoxifen 18F Antineoplastic 9 SN-22995 (DACA) 11C Antineoplastic 10 Temozolomide 11C Antineoplastic 11 Fluorouracil 18F Antineoplastic 12 Erythromycin 11C Antifungal 13 Fluconazole 18F Antimicrobial 14 Fleroxacin 18F Antimicrobial 15 Lomefloxacin 18F Antimicrobial 16 Trovafloxacin 18F Antimicrobial 17 DACA = [(dimethylamino)ethyl]acridine-4-carboxamide; SarCNU = 2-chloroethyl-3-sarcosinamide-1-nitrosourea.

tions can be measured serially in blood and urine; slower. This requires the preparation of new pre- however, other body fluids and most tissues are not cursors and radiolabelling with longer lived radio- easily sampled in humans. Thus, in designing clini- nuclides for meaningful measurements. cal trials in patients with infection, administration schedules are usually based on tissue pharmacoki- 2.1.2 Fluconazole neticstudiesinanimalsandonhumanbloodand Fluconazole is a fungistatic agent[19-21] that elic- urine data. Unfortunately, there is frequently an its its effects by inhibiting fungal cytochrome P450 imprecise relationship between drug concentra- sterol C-14 α-demethylation, which results in de- tions in blood and urine and concentrations pletion of normal fungal sterols and accumulation achieved in tissues such as brain, liver and prostate. of 14α-methyl sterols.[22-25] The combination of Clearly, approaches in which traditional blood and broad spectrum fungistatic action, low systemic urine measurements can be correlated with tissue toxicity[26] and a favourable pharmacokinetic pro- concentrations determined by PET hold great file make this drug an attractive therapeutic agent. promise for defining appropriate dosage schedules Use of fluconazole in the treatment of invasive and in accelerating drug development. fungal infections caused by organisms such as Cryptococcus neoformans, Candida species,Coc- 2.1.1 Erythromycin cidioides immitis, Histoplasma capsulatum and Erythromycin was the first antimicrobial agent Blastomyces dermatitidis has been a major ad- for which tissue pharmacokinetic measurements vance in antimicrobial chemotherapy.[27] How- were made with PET. In these studies, N-demethyl- ever, use of fluconazole as the primary treatment erythromycin was radiolabelled with [11C]formal- for disseminated cryptococcal infection in AIDS dehyde by reductive methylation[13] to yield a patients and in the primary treatment of acute life- product that was chromatographically identical to threatening fungal infections in immunocompro- authentic erythromycin. mised hosts has remained somewhat controver- After intravenous injection of this radiopharma- sial.[28] Important aspects of this controversy have ceutical, the time course of drug accumulation was been questions about optimal dosage schedules measured by PET in normal and infected lung of and actual concentrations of drug achieved in dif- patients with pneumonia.[18] During the first hour ferent tissues, particularly sites of infection. Al- after injection, extravascular concentration of drug though tissue distribution of fluconazole has been was similar in normal and infected tissue (6.6 ± 2.2 measured in laboratory animals[29,30] and concen- vs 5.5 ± 2.2 µg/g). These concentrations were trations in body fluids were measured in hu- achieved within 10 min after radiopharmaceutical mans,[31-35] detailed studies of tissue distribution administration and remained relatively constant and concentrations in humans were lacking. for 60 min. The presence of two fluorine atoms in the native This study was successful for two reasons. First, structure of fluconazole made it an ideal candidate thepresenceofanN-methyl group in the native for 18F radiolabelling and PET studies. Also, be- structure of erythromycin and the availability of cause fluconazole undergoes minimal metabolism N-demethyl-erythromycin resulted in a simple and in vivo, measurements of tissue and blood radioac- straightforward (single-step) method for radio- tivity accurately reflect concentrations of intact labelling. Secondly, the rapid equilibration of ery- drug.[29,30,34] Unfortunately, the method for com- thromycininpulmonarytissuemadethe20min mercial preparation of fluconazole introduces both physical half-life of 11C appropriate for labelling – fluorine atoms at an early stage of the synthesis and the tracer and the process were well matched. Un- thus is not applicable for producing 18F-labelled fortunately, precursors for radiolabelling most drug. To overcome this problem, a new flucona- antimicrobial agents are less readily available zole precursor into which 18F can be introduced in and, for many agents, tissue equilibration is much a single synthetic step was synthesised. This pro-

cedure yielded pharmaceutical grade [18F]fluconazole that was identical chemically and in terms of antimicrobial activity to the usually prescribed drug.[15] In preliminary studies, this radiopharmaceutical was used to measure the pharmacokinetics of fluconazole by PET in normal rabbits and rabbits with candidal infections.[5] When 18F-labelled drug was coinjected with a pharmacological dose of unlabelled drug, there was rapid equilibration to a relatively uniform distribution in most organs of the rabbit. In contrast, when [18F]fluconazole was injected ‘carrier-free’, blood clearance was accel- erated, spleen, muscle and heart accumulation was Fig. 5. Representative positron emission tomography images of decreased and liver accumulation was increased. healthy human volunteers co-injected with [18F]fluconazole plus These results illustrate the importance of using unlabelled fluconazole 5 mg/kg. The grey scale is calibrated in terms of absolute concentration (µg/cm3).[36] clinically relevant doses of drug for PET pharmacokinetic studies. In subsequent studies, [18F]fluconazole was ditions such as fungal osteomyelitis or the primary used to measure tissue pharmacokinetics in healthy treatment of acutely life-threatening CNS and in- volunteers by PET.[36] Figure 5 illustrates repre- travascular infections. In the case of CNS infec- sentative PET images of brain, heart, lung, liver, tions, increasing the dosage of drug based on the spleen, kidney and skeletal muscle of human sub- results of the PET measurements resulted in a 18 jects at 90 min after injection of [ F]fluconazole marked improvement in efficacy (unpublished re- plus unlabelled fluconazole (5 mg/kg). These im- sults). ages demonstrate significant accumulation of fluconazole in all organs studied. When injected with 2.1.3 Fluoroquinolones a pharmacological dose of unlabelled drug, [18F]- In recent years, fluoroquinolone antimicrobial fluconazole accumulates to a significant extent in agents have achieved an important role in the treat- all of the peripheral organs of humans. In contrast ment of a variety of bacterial infections. Since with the results obtained in rabbits, the pattern of these agents contain one or more fluorine atoms, fluconazole accumulation is much less homo- 18F-labelled drugs are useful for PET measure- geneous in humans. Fluconazole concentrations ments of in vivo pharmacokinetics. Studies with ranged from a low of 1.24 ± 0.27 µg/g in bone to fleroxacin represent the first application of PET to a high of 22.96 ± 0.24 µg/g in spleen; values in the this class of drugs.[37-40] These studies followed other tissues studied exceeded 6 µg/g. These con- similar lines to the measurements with [18F]fluco- centrations compare well with the concentrations nazole; a specific precursor was synthesised and a required to inhibit the growth of a variety of patho- rapid method for 18F labelling was devised. This gens in vitro. Thus, the PET data indicate that the was followed by PET studies in normal animals, usual fluconazole dose of 5 mg/kg should be effec- animals with bacterial infection, and humans. As tive for treatment of hepatosplenic and urinary with fluconazole, these studies provided important tract candidal infection and maintaining remis- new information about tissue pharmacokinetics in sion of cryptococcal meningitis in AIDS patients. animal models and humans. However, the clinical efficacy of fluconazole Although these types of measurements would mightbeimprovedbyincreasingthedoseforcon- be useful for many other fluoroquinolones, the re-

quirement of a specific precursor for each drug is ceptor types that have been studied by this method a major limitation. Fortunately, this problem has include dopaminergic, serotonergic, cholinergic, been largely resolved by the observation that the opioid and benzodiazepine binding sites. Table III fluoroquinolone lomefloxacin can be radiolabelled lists some of the PET ligands that are currently by 18Ffor19F exchange.[16] Recently, this approach available for this type of study. was applied to the preparation of 18F-labelled A variety of kinetic models have been used for trovafloxacin.[17] estimating parameters of ligand-receptor interac- tionbyPET.[59,64-67] All of these models include 2.2 CNS Drugs transport of free ligand from blood to tissue and receptor binding. Binding may be reversible or ir- Measurements of the tissue pharmacokinetics reversible, and a nonspecific binding compartment of drugs that act in the CNS vary considerably in may or may not be required. Figure 6 illustrates one approach and complexity. These studies can be of the most general models for ligand transport and broadly divided into two types: direct and indirect. binding. In this configuration, flux of ligand across For direct studies, the drug is radiolabelled with a the blood-brain barrier, K1,isequaltop•Vr • positron emitter and dynamic imaging is per- Ca(t), where p is the permeability, V is the fraction formed. If drug distribution is the only information r of brain tissue available for ligand-receptor inter- that is required, the studies are performed by the action and Ca(t) is the metabolite-corrected con- methods described for evaluation of antimicrobial centration of radioactivity in plasma. Free ligand, agents. In contrast, when the goal is to derive spe- F, can either bind directly to an unoccupied recep- cific values for the parameters of drug-receptor in- tor, a nonspecific site (NS, k )orescapefromthe teraction, imaging protocols are much more com- 5 tissue (k ). The probability for ligand-receptor plex. In the case of drugs that bind to a single class 2 interaction depends on the rate constant for asso- of receptors, time-activity curves from single or ciation (k ) and the local concentration of unoc- multiple injection studies can be analysed by com- on ′ partmental modelling to estimate receptor density cupied receptors (B max).Therateconstantsfordis- sociation of specific and nonspecific binding are (Bmax), binding affinity (KD) or binding potential koff and k6. Because even high specific activity (Bmax/KD). When the drug is expected to interact with multiple receptors, kinetic parameters for a ligands contain significant amounts of unlabelled specific receptor type can be measured by block- drug, which can affect local concentrations of un- ing the other receptors with unlabelled ligands occupied receptors, kinetics of unlabelled ligand with which they interact. For example, N-methyl- must also be modelled. Thus, this general model for ligand-receptor interaction contains two com- spiperone binds to both dopamine D2 and serotonin 5-HT2 receptors; however, parameters for binding ponents with the same structure and parameters. to 5-HT2 receptors can be estimated from PET The differential equations defined by the model studies in which D2 receptors are blocked with halo- can be solved by nonlinear least squares, to yield peridol. estimates of the kinetic parameters. In indirect studies, the receptor binding profile With this model, seven parameters must be of the drug of interest is derived from coinjection identified from imaging data. However, the PET or displacement studies with radiolabelled test time-activity curves acquired after injection of a ligands that bind to specific receptors. One of the radioligand are relatively simple; a rapidly rising most important examples of this approach was the phase, a plateau and a falling phase. When these evaluation of the dopamine D2 and D1 receptor data are analysed with the model depicted in figure binding characteristics of the atypical antipsy- 7, the number of parameters to be fitted is too large chotic clozapine using [11C]raclopride and [11C]- to yield meaningful results. Fortunately, in some [41-44] SCH-23390 as test ligands for D2 and D1. Re- cases, this problem can be resolved by multiple

Table III. Examples of radioligands that have been used for positron emission tomography imaging Receptor Ligand Specificity References 11 Dopamine [ C]SCH-23390 D1 41,42 11 [ C]SCH-39166 D1 45 11 [ C]Methylspiperone D2,5HT2 46 76 [ Br]Bromospiperone D2,5HT2 47 11 [ C]Raclopride D2 41,42 76 [ Br]Bromolisuride D2 48 [11C]CFT Transporter 49 [11C]Altropane Transporter 50 18 Serotonin [ F]Setoperone 5HT2 51 11 [ C]MBL 5HT2 52 11 [ C]WAY-100635 5HT1A 53 11 Benzodiazepine [ C]Flumazenil BZ1,BZ2 54 [11C]PK-11195 ‘Peripheral’ 55 Opioid [11C]Carafentanyl µ 56 [11C]Diprenorphine µ, κ 57 [18F]Acetylcyclofoxy µ 58 11 Acetylcholine (muscarinic) [ C]MQNB M1,M2 59 11 [ C]Levetimide M1,M2 60 11 [ C]Scopolamine M1,M2 61 11 [ C]Cogentin M1,M2 62 11 Adrenergic [ C]CGP-12177 β1, β2 63 CFT =2β-carbomethoxy-3β-aryltropane; MBL = N1-methyl-2-bromo-lysergic acid diethylamide; MQNB = methylquinuclidinyl benzilate. injection imaging protocols, which results in much ling CNS kinetics of [11C]flumazenil and [76Br]- more detailed time-activity curves. An example of bromolisuride.[54,67] the application of this approach was use of 11C- In some situations, simpler procedures have labelled 2β-carbomethoxy-3β-aryltropane ([11C]- yielded useful information. For measuring dopa- 18 CFT) to determine the kinetics of dopamine trans- mine D2 receptor density with 3-(2′-[ F]- porter sites using a three-injection protocol.[68] At fluoroethyl)spiperone, only two injections (tracer the beginning of the study, high specific activity and displacement) were required.[69] With ligands radioligand was injected and PET data were col- that dissociate rapidly from their receptors, equi- lected for 30 min. An excess of unlabelled CFT librium binding is established and an even simpler plus a small amount of [11C]CFT was then injected approach has been employed.[70,71] In this situa- and data collection was continued for 60 min. At tion, regional time-activity curves are used to es- 90 min, a second injection of high specific activity tablish a time that represents equilibrium of spe- radioligand was administered and imaging was cific binding. A series of PET scans are then continued for 30 min. Figure 7 illustrates a repre- performed at varying specific activity, and KD and 11 sentative time-activity curve for [ C]CFT in B′max are calculated from saturation curves or monkey striatum measured using this protocol. Scatchard plots.[72] The fitted curve and the time dependence of the Although these complex multi-injection proto- concentration of radioactivity in each compart- cols are required for precise estimates of individual ment are also indicated. Values for KD and B′max values of KD and B′max, the ratio of these parame- [73] estimated by this approach were in excellent agree- ters, referred to as binding potential (B′max /KD ) ment with the results of in vitro assay. Modifica- can be determined from single injection studies. tions of this approach have been used for model- Fortunately, in many situations important informa-

where BPBl = binding potential before drug treat- NS ment and BPDrug = binding potential after drug treatment. This type of analysis was used to study "Labelled k5 k6 K1 central 5-HT receptor occupancy of an antipsy- ligand" konB'max 2 P F B chotic drug, ziprasidone, in healthy volunteers at [74] k2 koff various times after administration. In this inves- SA(t) k B' K1 on max tigation, dynamic PET studies with the 5HT2 re- Pc Fc Bc ceptor ligand [18F]setoperone were performed in k k k k off healthy volunteers, in the drug-naive state and at "Unlabelled 2 5 6 ligand" 4, 8, 12 and 18 hours after a single oral dose of NSc ziprasidone. Quantification of cortical binding potential was performed by a modification of the Fig. 6. Model for ligand transport and binding. This formulation [75] illustrates the interaction of labelled and unlabelled ligand. Al- method described by Petit-Taboue et al. Indi- though unlabelled ligand concentrations are not directly mea- vidual values for the parameters K1,k2,k5 and k6 surable by positron emission tomography, the concentration of specifically bound unlabelled ligand affects the local concentra- were estimated by fitting the three-compartment tion of free receptors and thus binding of labelled ligand. B = model shown in figure 8 to cerebellar time-activity ′ boundligandintissue;B max = local concentration of unoccupied curves. To determine binding potential, the para- receptors; F =freeligandintissue;kon = rate constant for specific binding; koff = rate constant for dissociation of specific binding; meters K1/k2,k5 and k6 were fixed at the cerebellar K1 = rate constant for flux of ligand into tissue; k2 = rate constant values, Vb was fixed at 4.5% and K1,k3 and k4 were for escape from tissue; k5 = rate constant for nonspecific bind- estimated by fitting the cortical time-activity ing; k6 = rate constant for dissociation of nonspecific binding; NS = nonspecifically bound ligand; P =freeligandinplasma; curves to the four-compartment model shown in SA(t) = specific activity at time t. figure 8. Binding potential was calculated from the individual values of k3 and k4. tion can be obtained from measurements of binding As expected, the images demonstrate high con- potential. For example, one of the most common centrations of [18F]setoperone in the cerebral cor- questions in drug development is the degree to tex at baseline. In images acquired at 4 hours after which a new drug affects occupancy (%∆RO)ofa administration of ziprasidone, there was a marked class of receptors (equation 1): decrease in cortical accumulation of radioactivity and concentrations were only slightly greater than RO- RO BB’ - " cerebellar activity. In the images acquired at 8, 12 %D=RO Bl Drug ´=100max max ´ 100 ’ and 18 hours after ziprasidone, intermediate levels ROBl B max of cortical radioactivity were observed. Figure 9 shows the effect of time after administration on where ROBl = receptor occupancy before drug receptor occupancy. Since it can be assumed that treatment, RODrug = receptor occupancy after drug K is unaffected by drug treatment, %RO reflects treatment, B′ = concentration of available re- D max changes in B′ . These data indicate that there is ceptors before drug treatment, and B′′ = concen- max max >95% receptor occupancy at 4 hours after admin- tration of available receptors after drug treatment. istration, and that occupancy remains above 50% Since it can be assumed that KD is unaffected by for more than 19 hours. These findings clearly es- drug treatment, changes in binding potential reflect tablish that once daily administration should be ad- ′ changes in B max and (equation 2): equate with this drug. Also, as indicated in figure 9, the relationship between receptor occupancy BP- BP %D=RO Bl Drug ´100 and blood concentrations of drug is quite impre- BPBl cise.

0.9 Free Bound NS Total PET 0.7

0.5 Concentration (pmol/L) Concentration

0.3

0.1

020406080100 120 Time (min)

Fig. 7. Typical least-squares fit of a positron emission tomography (PET) time-activity curve for the average concentration of 11C-labelled 2β-carbomethoxy-3β-aryltropane in monkey striatum based on the kinetic model in figure 6 and the injection protocol outlined in the text. The time-dependence of the model estimates of concentrations of radioactivity in free, specifically bound and nonspecifically bound (NS) compartments is also illustrated.[68]

In another investigation, this type of analysis namic PET imaging was performed in six volun- was used to study the potential novel antidepres- teers. Two studies were performed in each subject. sant BMS-181101.[76] This drug was believed to For the first study, the subjects were injected with influence CNS serotonin neurotransmission by 10 mCi of high specific activity [11C]BMS-181101 several different mechanisms and thus differs from (~4000 mCi/µmol) and serial PET images and ar- specific serotonin reuptake inhibitors, such as terial blood samples were collected over 90 min. fluoxetine, paroxetine and fluvoxamine. BMS- At 30 min after acquiring the final image, each 181101 is a potent inhibitor of serotonin reuptake subject was co-injected with a second dose of 10 by the terminal bouton in vitro, thereby potentiat- mCi of [11C]BMS-181101 plus 3.9mg of un- ing the synaptic actions of serotonin. In addition, labelled drug (final specific activity ~1.5 mCi- it acts as a full agonist at dorsal raphe somato- /µmol) and imaging and blood collection were re- dentritic 5-HT1A autoreceptors and presynaptic peated. The data were analysed by calculating terminal 5-HT1D receptors that regulate the activ- regional tracer accumulation (% injected dose/g) at ity of serotonin neurons. To study its pharmaco- 60 min after injection and compartmental model- kinetics and receptor binding in the brains of ling. Metabolite-corrected arterial input functions healthy volunteers, 11C-labelled BMS-181101 was were used in the calculations and the vascular frac- 11 prepared by methylation with CH3I at the 5- tion of brain tissue (Vb) was fixed at 4%. Measure- methoxy position of the piperazine ring, and dy- ments of % injected dose/g yielded similar values

In some situations data analysis can be further K1 k5 Cerebellum P F NS simplified by the use of graphical methods. The

k2 k6 first application of this technique was described by Patlak et al.[77] for radioligands that bind irre- versibly to their receptors (k4 ≈ 0) on the imaging NS time scale and free ligand is in steady state with the blood. In this analysis, the ratio of tissue to plasma radioactivity is plotted versus ‘normalised time’ k5 k6 Cortex (integral of plasma radioactivity from time 0 to t

K1 konBmax divided by plasma radioactivity at time t). The P F B asymptotic slope and intercept of the linear part of k koff 2 this curve yields estimates of binding potential. [78] Fig. 8. Compartmental models describing the kinetics of Subsequently, Logan et al. extended this ap- [18F]setoperone in human brain. Three- and four-compartment proach to reversible systems. Although estimates models were used to analyse cerebellar and cortical time-activity of binding potential calculated by these methods curves respectively. B = bound ligand in tissue; Bmax =local concentration of unoccupied receptors; F =freeligandintissue; are sometimes less precise than the results of ki- kon = rate constant for specific binding; koff = rate constant for netic modelling, the techniques can be used to pro- dissociation of specific binding; K1 = rate constant for flux of duce parametric images that represent pixel by ligand into tissue; k2 = rate constant for escape from tissue; k5 = rate constant for nonspecific binding; k6 = rate constant for pixel maps of binding potential. Figure 10 illus- dissociation of nonspecific binding; NS = nonspecifically bound trates binding potential images of the brain of a ligand; P =freeligandinplasma. healthy volunteer at baseline and 4 hours after administration of ziprasidone. for all brain regions, independent of specific activity. Kinetic modelling of time activity curves for 3. Pharmacodynamics cerebellum, caudate, putamen, thalamus, pons and temporal, occipital and frontal cortex demon- In addition to their value for noninvasive in vivo strated that tissue distribution could be described studies of pharmacokinetics, radionuclide tech- by a simple two-compartment flow model. Statis- tical comparisons of the apparent distribution volumes for each region failed to reveal significant 120 100 ) differences between the high and low specific ac- 100 • 80 tivity studies. 80 This study clearly demonstrates the usefulness 60 of PET in drug development. Although in vitro 60 studies of the interaction of BMS-181101 with a va- 40 40 riety of serotonin receptor systems demonstrated 20 significant binding, this was not observed in vivo. 20 Receptor occupancy (%, In fact, the results of the PET study in conjunction (µg/L, o) Blood concentration 0 0 with equivocal results of phase II clinical trials re- 4 6 8 101214161820 sulted in discontinuation of the clinical evaluation Time (h) of BMS-181101. Clearly, PET studies at an earlier Fig. 9. Effect of time after administration of [18F]setoperone on phase of drug development (late Phase I) might serotonin 5HT2 receptor occupancy determined by least- squares fitting of positron emission tomography data to the have resulted in considerable savings of time, model illustrated in figure 8. The time dependence of blood con- money and other resources. centrations of drug is also illustrated.[74]

rised in table IV, and several examples of these procedures are discussed in the remainder of this section. In a recent study, the utility of 99mTc- tetrofosmin (99mTc-TF) and thallium-201 (201TlCl) for predicting multidrug resistance (MDR) and ra- dioresistance was investigated in patients with lung cancer.[79] Thirty patients with untreated disease underwent dual isotope SPECT at 10 and 120 min after co-injection of the radiopharmaceuticals, and retention of each tracer was evaluated semi- quantitatively both at baseline and after sequential (n = 12) or concurrent (n = 18) radiation and che-

Fig. 10. Representative parametric images of the brain of a motherapy (cisplatin plus etoposide). In patients healthy volunteer illustrating the distribution of binding poten- treated with sequential therapy, the response to ra- tial before (left) and 4 hours after (right) oral administration of diation was predicted by 99mTc-TF retention, ziprasidone.[74] whereas 201Tl retention was found not to be predic- tive. Regardless of whether the sequential or con- niques also have considerable utility for assessing current protocol was applied, 14/18 tumours with 99m ≥ drug effects at the organ, tissue and cellular level, high Tc-TF retention ( 15%) exhibited a fa- i.e. pharmacodynamic studies. Although PET mea- vourable response to chemoradiotherapy, whereas all 12 tumours with low 99mTc-TF retention surements have superior sensitivity and quantita- (≤15%) did not respond to the therapy. tive reliability for these measurements, many types The in vivo potency of euphorigenic doses of of pharmacodynamic studies can also be per- intravenous cocaine for displacing binding of 2β- formed with SPECT or planar imaging. The limi- carbomethoxy-3β-(4-[123I]iodophenyl)tropane- ted sensitivity and the difficulty of labelling com- ([123I]β-CIT) binding to striatal dopamine trans- pounds without changing their pharmacological porters (DAT) was assessed in human cocaine properties are major obstacles to the application of abusers by SPECT.[80] In this investigation, co- SPECT in drug development; however, these fac- caine abusers (n = 6) were injected with [123I]β- tors are less severe restrictions for pharmaco- CIT and imaged 24 hours later under equilibrium dynamic studies. Two important advantages of conditions. Sequential cocaine infusions (0.28 ± SPECT are: (i) because of the variety of energies 0.03 and 0.56 ± 0.07 mg/kg) produced significant of photons emitted by SPECT radiopharmaceuti- (p < 0.0005) reductions in DAT binding potential. cals, multiple tracers can be studied simulta- Regression analysis of the logit transformed data neously; and (ii) SPECT facilities are available in enabled reliable determination of the Hill coeffi- almost every nuclear medicine department. cient (0.51) and 50% displacement (ED50)doseof Since nearly every PET or SPECT radiophar- cocaine (2.8 mg/kg). These results suggest that co- maceutical can serve as a ‘biomarker’ for pharma- caine produces behavioural effects in humans at codynamic studies, exhaustive evaluation of this measurable levels of DAT occupancy. In a similar topic would require discussion of a vast array of study, the in vivo potency of mazindol for binding imaging procedures, a task that is clearly beyond to DAT was assessed by SPECT with [123I]β- the scope of this review. Specific examples of PET CIT.[81] Cocaine-dependent individuals underwent and SPECT radiopharmaceuticals that can be ap- three SPECT scans before, during and after me- plied for pharmacodynamic studies are summa- dium-term (1 week) administration of mazindol at

Table IV. Examples of radiopharmaceuticals for pharmacodynamic studies by positron emission tomography (PET) and single photon emission computed tomography (SPECT) Measurement SPECT tracers PET tracers 99m 15 Brain perfusion Tc-HMPAO CO2 99m Tc-ECD H215O [11C] or [15O]Butanol 62Cu-PTSM Brain permeability 99mTc-DTPA 68Ga-EDTA Blood pool 99mTc-RBCs 11CO 99mTc-albumin C15O [11C]Albumin Bone function 99mTc-MDP 18F– Hepatobiliary function 99mTc-DISIDA 68Ga-HBED Infection 111In- or 99mTc-WBCs 18F- or 11C-labelled peptides Reticuloendothelial system function 99mTc-colloid [18F]FDG 68Ga-colloids 133 99m 18 Lung function Xe/ Tc-MAA C O2 13 N2 201 13 Myocardial perfusion Tl NH3 99m Tc-isonitriles H215O 99mTc-teboroxime 82Rb [15O]Butanol 62Cu-PTSM Glomerular filtration rate 99mTc-DTPA 68Ga-EDTA 123I-iothalamate 99m Renal perfusion Tc-MAG3 H215O 82Rb Thyroid function 123I– 124I– 99mTc-pertechnetate Tumours 67Ga-citrate [18F]FDG 99mTc-MIBI [11C]Methionine 99mTc- tetrofosmin [11C]Leucine 123I-MIBG [11C]Thymidine 111In-octreotide 124I-MIBG 18F-estradiol Glucose metabolism [18F]FDG Glucose transport [11C]3-O-Methyl-glucose Protein synthesis 77Se-Methionine [11C]Methionine [11C]Leucine DNA synthesis [11C]Thymidine 18F-FLT 123 15 Energy metabolism I-fatty acid analogues O2 [11C]Acetate [11C]Fatty acids

DISIDA = di-isopropyl iminodiacetic acid; DTPA = diethylenetriaminepenta-acetic acid; ECD = ethylcysteinate dimer; EDTA = ethylenediamine-N,N,N′,N′-tetra-acetic acid; FDG = fluorodeoxyglucose; FLT = 3’-deoxy-3’flurothymidine; HBED = N,N′-bis(2-hydroxybenzyl)- ethylenediamine-N,N′-diacetic acid; HMPAO = hexamethylpropylene amine oxime; MAA = macroaggregated albumin; MAG3 = mercaptoacetyltriglycine; MDP = methylene diphosphonate; MIBG = m-iodobenzylguanidine; MIBI = methoxy isobutyl isonitrile; PTSM = pyruvaldehyde-bis(N4-methylthiosemicarbazone); RBC = red blood cell; WBC = white blood cell.

doses of 2 and 4 mg/day. For each scan, subjects structure, isotopes with non-zero spin are available were injected with [123I]β-CIT and imaged 24 for all atoms in drugs, for example 1H, 13C, 15N, hours later under equilibrium conditions. These 17Oand31P. For MRS, 19F has the advantages of measurements demonstrated a significant effect of high intrinsic sensitivity, a large range of chemical mazindol dose (p < 0.001) on reducing DAT bind- shifts and zero natural background. 1Halsohas ing potential. Regression analysis of the logit high sensitivity; however, the range of chemical transformed data enabled estimation of the ED50 shifts is much smaller and natural background is of mazindol (30 mg/day). These data suggest that high. 13Cand15N have much lower intrinsic sen- low doses of mazindol occupy a small percentage sitivities, but they can be useful for MRS studies (<25%) of DAT in human cocaine abusers and that when advanced pulse sequences are employed. much higher, potentially intolerable, doses (≥30 Thirdly, in contrast to PET tracers, drugs for MRS mg/day) may be required to significantly antagon- studies are stable compounds with unlimited shelf ise cocaine binding in vivo. lives and there is no potential danger from exposure to ionising radiation. Although MRS exposes 4. Magnetic Resonance Spectroscopy humans to radiofrequency energy and strong magnetic fields, studies with conventional imaging Conventional magnetic resonance imaging systems have not been associated with significant (MRI) employs magnetic field gradients to pro- health risks.[83] Finally, the time interval over duce images of the distribution and relaxation which pharmacokinetic studies can be performed properties of nuclei with magnetic spins. Since in- is unlimited with MRS – matching the half-life of dividual atoms of all drugs have specific resonance the tracer to the time scale of the process under frequencies, in principle it should be possible to investigation is not an issue. record MR images of any drug or drug metabolite. In the following sections, applications of MRS Unfortunately, due to the intrinsic insensitivity of to studies of antineoplastic drugs and antimicro- magnet resonance detection, MRI has largely been bial agents, and of drugs that affect the CNS, will limited to images of the distribution and spin prop- be discussed as examples of the first and second erties of water (tissue concentration about 55 types of drugs previously described in section 2. mol/L). Because of this sensitivity limitation, in most applications of MRS images are not recorded. 4.1 Antineoplastic Drugs Instead, field gradients are used to select a specific region of tissue and record a spectrum. Even with Although numerous antineoplastic agents have this approach, sensitivity is much lower than with been evaluated by MRS (table V), the most exten- PET, 10–5 to 10–3 mol/L[82] compared with about sive clinical experience has been gained by 19F- 10–12 mol/L. MRS studies of the pharmacokinetics of fluoroura- Despite this low sensitivity, MRS has much to cil (5- FU).[84] This drug, and several of its offer in specific areas of pharmacokinetic analysis. metabolites, have been measured in vivo in ani- First, although PET can provide precise measure- mals and patients using standard clinical MR sys- ments of total drug concentrations present in small tems. For 5-FU, tumoral half-life is the parameter volumes of tissue, MRS can be used to determine that can be measured most readily. This parameter the contributions of different molecular species is of importance because trapping of 5-FU in present. Also, differentiation between intra- and tumours is a necessary, albeit not a sufficient, con- extracellular drug may be possible. Secondly, as dition for response. Patients in whom tumoral half- with PET, the number of tracers that are available life of 5-FU is ≥20 min are designated as ‘trap- for MRS studies is potentially unlimited. Although pers’, whereas individuals with lower values are the majority of MRS studies have been performed ‘non-trappers’. In a study of 75 patients with liver with drugs that contain fluorine (19F) in their native metastases from colorectal carcinoma,[85] 49% of

Table V. Examples of antineoplastic agents that have been studied by magnetic resonance spectroscopy Drug Signal Measurement References Fluorouracil 19F Pharmacokinetics 84,85 Gemcitabine 19F Distribution and metabolism 86 Difluorochlorambucil 19F Tumour hypoxia 87 SR-4554 19F Tumour hypoxia 88 Iproplatin 1H Early distribution 89 Ifosfamide 31P Pharmacokinetics 90 Temozolomide 13C Pharmacokinetics 91 Carboplatin 195Pt Distribution 92 Tirapazamine 31P (phosphates) Tumour energy metabolism 93 Chemotherapy/radiation 1H Lactate concentration 94

37 patients with tumoral half-life of ≥20 min re- >90% of the drug is in the intact form, such a com- sponded to the drug, whereas 51% did not. In con- bination approach is probably not necessary. How- trast, the 38 patients with tumoral half-life shorter ever, for drugs that undergo a greater degree of than 20 min did not respond. These findings sug- metabolism, such complex analysis could be of gest that 19F-MRS may become a useful screening considerable value. procedure for withdrawing 5-FU treatment in non- trappers. In other investigations, the effects of 4.3 CNS Drugs modulators and other agents on tumoral half-life of 19 5-FU have been measured by MRS. These F- Due to the low sensitivity of the technique, most MRS studies have been extended to other fluoro- MRS studies of CNS active drugs have concen- pyrimidines, some of which are prodrugs of 5-FU, trated on measurements of whole-brain pharmaco- and others where the fluorine atom is on the ribose kinetics. However, despite this limitation, a con- ring. siderable amount of important information has been obtained. One of the earliest investigations in 4.2 Antimicrobial Agents this area used 7Li-MRS to measure brain lithium concentrations in healthy volunteers after single The in vivo pharmacokinetics of fleroxacin has and multiple doses of lithium carbonate.[96] The been measured in human liver and muscle by 19F- results of this study demonstrated that relatively MRS.[95] This study demonstrated a pharmacoki- slow accumulation of lithium in the brain may be netic pattern similar to the results of PET studies. responsible for the delay in therapeutic response However, due to the much lower intrinsic sensitiv- that is frequently observed after initiation of ther- ity of MRS, spatial resolution was quite low, the apy. In a later MRS study, an unexpected variabil- measurements were restricted to large volumes of ity in brain versus serum concentrations of lithium tissue, and absolute quantification was difficult. was demonstrated in patients with bipolar dis- Nevertheless, this study clearly pointed out the po- ease.[97] tential of studies in which PET and MRS measure- Several investigations have employed 19F-MRS ments are combined. PET can provide precise to measure brain concentrations of psychiatric measurements of total drug concentrations present drugs. In one study, the technique was used to mea- in small volumes of tissue, whereas MRS can be sure concentrations of fluoxetine and norfluoxet- used to determine contributions of different molec- ine in patients with obsessive compulsive disorder ular species present (intact drug vs metabolites) and major depression.[98] This study demonstrated and possibly to differentiate between intra- and ex- that the brain to plasma ratio of the parent drug tracellular drug. In the case of fleroxacin, in which (plus metabolite) was significantly elevated to 2.6,

which may have implications for understanding ably and a greater variety of drugs will become both the therapeutic and the toxic effects of appropriate for study. fluoxetine. In another study, brain concentrations of paroxetine or fluoxetine were measured before 5. Conclusions and after 3 days of placebo substitution in patients with remitted major depression who were taking Physiological and biochemical measurements that can be performed noninvasively in humans us- 20 mg/day of either drug.[99] With placebo substi- ing modern imaging techniques can greatly facili- tution, a mean of 88% of brain fluorine signal from tate the evaluation of new therapies. Two critical fluoxetine (plus metabolites) remained, compared points in drug development where these measure- with a mean of 38% of the brain fluorine signal ments are likely to be most useful are in preclinical from paroxetine (plus metabolites). In patients ini- studies and in Phase I/II studies. In preclinical tially taking paroxetine, adverse events during studies, a new drug can be precisely compared with placebo substitution were highly correlated with standard therapies, or a series of analogues can be steady-state drug concentrations in the brain. screened for further development on the basis of In another study, 19F-MRS was used to quantify performance in appropriate animal models. In brain concentrations of dexfenfluramine in hu- Phase I/II human studies, classical pharmacokin- mans receiving clinical doses of the drug and to etic measurements can be combined with imaging determine whether human brain dexfenfluramine measurements to define optimal administration concentrations approach those reported to cause schedules and the potential utility of interventions irreversible neurochemical changes in animals.[82] in particular clinical situations, and to design Twelve obese women received dexfenfluramine Phase III studies that are crucial for drug licensure. 15mg twice daily for 90 days; 19F-MRS measure- In general, the types of measurements that are pos- ments were performed at baseline and three times sible can be grouped into the following categories. during treatment. Brain concentrations of dex- (i) In those situations in which the drug can be fenfluramine and its active metabolite dex- radiolabelled, the time course of tissue delivery norfenfluramine reached steady state (about 4 can be determined noninvasively in vivo in health µmol/L) in the brain after approximately 10 days and disease. Such information should be most use- of treatment and did not increase significantly dur- ful for determining administration schedules, es- ing the remainder of the treatment period. These tablishing efficacy and predicting possible toxicity. findings demonstrated that fluorinated drugs can (ii) Ligand-receptor binding can be assessed in be quantified by 19F-MRS at concentrations below vivo in two ways. The ability of the drug to displace 10 µmol/L in the human brain. The time course standard radiolabelled ligands from their receptors data suggested that brain dexfenfluramine con- can be determined or, alternatively, labelled drug centrations parallel plasma pharmacokinetics. can be used to more directly assess distribution and Also, brain dexfenfluramine/dexnorfenfluramine time course of binding. These measurements are of concentrations were well below those previously particular value for studying drugs that act on the found to cause irreversible brain alterations in an- central nervous and cardiovascular systems. imals. (iii) Measurements of tissue metabolism will be These results clearly indicate that MRS has a useful in determining the effects of therapies bright future for pharmacokinetic analysis. Al- aimed at particular metabolic abnormalities. These though MRS will never have the sensitivity of measurements may also be useful in defining the PET, as higher field magnet systems become more viability and function of tissues in such widely dis- widely available and improved pulse sequences parate clinical situations as cancer chemotherapy are developed, sensitivity will improve consider- and cardiology.

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