D3D4 Lead Optimization and Drug Profiling

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By far, most imaging studies in DDD have been carried out in the lead optimization phase. Imaging has been used for phenotyping disease models and to assess the efficacy of drug candidates using structural, functional, metabolic, and more recently also molecular readouts. We will discuss one example to illustrate the potential and also problems associated with imaging approaches in preclinical DDD: the evaluation of anti-ischemic therapy in models of focal cerebral ischemia.

Stroke is a leading cause of death in industrialized nations and the only clinically approved treatment is thrombolysis using recombinant tissue plasminogen activator (rtPA) applicable in approximately 5% of stroke patients. Hence, there is a high medical need to develop novel pharmacological therapies.

Focal cerebral ischemia is caused by transient or permanent occlusion of a major cerebral artery. Cessation of local perfusion initiates a cascade of detrimental effects within minutes such as energy failure due to the shutdown of aerobic ATP synthesis that affects all energy-dependent processes of the cell including membrane pumps required to maintain ion homeostasis, and intracellular signaling cascades via ATP-dependent protein kinases. Failure of membrane pumps causes intracellular Ca2+ to accumulate prompting a cascade of deleterious downstream events for the cell ultimately leading to cell death. Excessive levels of excitatory neurotransmitters such as glutamate are another major reason for tissue exhaustion and damage as the inactivation of glutamate via glial and neuronal uptake is an energy-dependent process. Elevated glutamate levels cause opening of N-methyl-D-aspartate (NMDA) receptors enhancing Ca2+ influx with the consequences already discussed. Glutamate interaction with metabotropic glutamate receptors (mGluR) activates second-messenger-mediated signaling. In addition to these acute effects, delayed infarct growth due to recruitment of penumbral regions has been observed at time points beyond 48 h after infarction, associated with apoptosis and neuroinflammation. Therapeutic strategies in stroke target the individual processes of the pathophysiological cascade.

Long-term tissue survival can only be achieved through restoration of blood supply to the isch-emic lesion, which has to occur within a short time frame of a few hours. In the acute phase, protective effects can be achieved by reducing the energy demand in affected brain areas that show some residual perfusion (ischemic penumbra). This can be achieved by administration of Ca2+ channel blockers, which reduce Ca2+ influx through voltage-gated channels, or by inhibition of receptor-operated channels such as the NMDA receptor. Both strategies have been rather successful in animal models of focal cerebral ischemia. A number of other therapeutic targets have been investigated such as glycine receptors, prevention of excitotoxicity using antagonists of the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor, free-radical scavengers, inhibitors of death protease, or anti-inflammatory treatment. Yet, all these compounds failed upon translation into the clinics, mostly due to lack of efficacy. More recently, tissue repair strategies using neuronal stem or progenitor cells have been proposed, which revealed beneficial effects in a rat stroke model.

Today, imaging techniques, and in particular MRI-based methods, enable the visualization of individual aspects of the pathophysiological cascade both in humans and in animals from the initial vascular occlusion to the infiltration of inflammatory cells during the postacute phase. The same techniques have been applied to evaluate the efficacy of anti-ischemic drugs.

For example, it has been shown that in models of global ischemia pretreatment with cytoprotec-tive Ca2+ inhibitors significantly delayed ATP depletion and tissue acidosis. Yet, most of the pre-clinical stroke studies evaluating drug efficacy using MRI are based on morphometric readouts, i.e., they use infarct volumes as efficacy biomarker. The underlying assumption is that reduction of the structural damage, i.e., reduction of the infarct volume, will necessarily translate into an improved behavioral or correspondingly clinical outcome. The classical MRI method for the assessment of cerebral infarct volume is based on R2 contrast: formation of a vasogenic edema leads to significantly reduced R2 values (and correspondingly increased T2 = 1/R2 values) providing a good demarcation between ischemic and intact tissue (Figure 7.5). A considerable number of drug candidates have been evaluated using this approach. It has to be kept in mind that cerebral tissue displaying a decreased R2-value is already irreversibly damaged; hence the method indicates an endpoint. Earlier indicators that provide a significant contrast for tissue that is still salvageable are the apparent water diffusion coefficients (ADC) in brain parenchyma and local cerebral blood flow (CBF) rates. It has been demonstrated, at least in animal models of global and focal ischemia, that ADC changes are

FIGURE 7.5 Disease phenotyping in rodent model of human embolic stroke. (a) Cerebral ischemia is induced by permanent transient occlusion of a brain artery, for example, the middle cerebral artery (MCA). The occlusion site is indicated by a cross. As a result, an infarct will develop that occupies large areas of the MCA irrigation territory. The MRI angiogram of a rat brain (top right) demonstrates the absence of blood flow in the left MCA of the animal (yellow arrow). Abbreviations indicate external (ECA) and internal carotid artery (ICA), basilar artery (BA), and Circle of Willis (CW). (b) The tissue damage is reflected by changes of various MRI parameters. The perfusion values (CBF) are dramatically decreased in the affected territory leading to oxidative stress, the apparent water diffusion coefficient (ADC) decreases due to cellular swelling caused by failing membrane pumps, and later the R2 relaxation rate decreases due to the formation of a vasogenic edema. All MRI images have been recorded 24 h following MCA occlusion.

FIGURE 7.5 Disease phenotyping in rodent model of human embolic stroke. (a) Cerebral ischemia is induced by permanent transient occlusion of a brain artery, for example, the middle cerebral artery (MCA). The occlusion site is indicated by a cross. As a result, an infarct will develop that occupies large areas of the MCA irrigation territory. The MRI angiogram of a rat brain (top right) demonstrates the absence of blood flow in the left MCA of the animal (yellow arrow). Abbreviations indicate external (ECA) and internal carotid artery (ICA), basilar artery (BA), and Circle of Willis (CW). (b) The tissue damage is reflected by changes of various MRI parameters. The perfusion values (CBF) are dramatically decreased in the affected territory leading to oxidative stress, the apparent water diffusion coefficient (ADC) decreases due to cellular swelling caused by failing membrane pumps, and later the R2 relaxation rate decreases due to the formation of a vasogenic edema. All MRI images have been recorded 24 h following MCA occlusion.

reversible. There is evidence that these early ischemia markers (ADC, CBF) are of predictive value with regard to the final infarct volume.

A word of caution: the identification of structural damage is based on the deviation of MRI parameters from "normality." This procedure is to some extent arbitrary: CNR depends on multiple factors such as the general signal-to-noise ratio of the image and the morphological heterogeneity of the corresponding brain area. This may limit the accuracy of the evaluation.

The primary objective of stroke therapy is not to reduce infarct volume but to improve the clinical outcome for the patient, i.e., to preserve/restore brain functionality. While several cytoprotective therapy strategies have been shown to reduce infarct volume in animal models of focal cerebral ischemia, it remains to be shown that regions spared from becoming infarcted are in fact functional. Studies in rat focal cerebral ischemia revealed functional recovery only in a fraction of the animals, despite the fact that all animals displayed similar locations and volumes of infarction (Figure 7.6). Obviously, structural integrity is a necessary yet not a sufficient prerequisite for functional integrity. The different functional responses have been attributed to differences in CBF values in the cytopro-tected territory. The validity of the biomarker 'infarct volume' has to be questioned and it is strongly recommended to combine structural outcome information with a functional correlate.

In view of the difficulties in treatment of acute stroke, emphasis is currently shifting toward therapy concepts targeting processes in the postacute phase such as inflammation or aimed at inducing neuroregeneration. For example, it has been shown that administration of stem cells improves behavioral outcome in rat stroke models. In this context, imaging has been used to monitor the

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FIGURE 7.6 The assessment of therapy response in rat stroke model using structural and functional readouts. (a) Reduction of infarct volume following treatment with Ca2+ antagonist isradipine in rat model of permanent MCA occlusion. The R2 weighted MR images demonstrate significant reduction of the hyperin-tense lesion area upon treatment with the drug. The graph displays the dose response curve for isradipine when administered immediately following MCA occlusion. A maximal protection has been obtained at a dose of 2.5 mg/kg. (b) Recovery of functional response in the somatosensory cortex (S1) caused by electrical stimulation of both forepaws simultaneously. The image of the drug-treated animal shows that the cortical area that has been spared from becoming infarcted is in fact functional. The graph displays the functional recovery as a function of time following infarction. Filled symbols are for the contralateral (affected) hemisphere, open symbols for the ipsilateral (unaffected) one. Dark symbols indicate drug, lighter symbols placebo-treated rats. Recovery in drug-treated rats is observed only a day following infarction despite the fact that the cortical tissue is structurally intact. In addition, only 50% of the animals displayed recovery in the observation period of 12 days. In placebo-treated animals the respective S1 was always infarcted, no functional response has been observed. In all animals the response of the ipsilateral unaffected S1 region was normal. (Adapted from Sauter, A. et al., Magn. Reson. Med, 47, 759, 2002.)

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FIGURE 7.6 The assessment of therapy response in rat stroke model using structural and functional readouts. (a) Reduction of infarct volume following treatment with Ca2+ antagonist isradipine in rat model of permanent MCA occlusion. The R2 weighted MR images demonstrate significant reduction of the hyperin-tense lesion area upon treatment with the drug. The graph displays the dose response curve for isradipine when administered immediately following MCA occlusion. A maximal protection has been obtained at a dose of 2.5 mg/kg. (b) Recovery of functional response in the somatosensory cortex (S1) caused by electrical stimulation of both forepaws simultaneously. The image of the drug-treated animal shows that the cortical area that has been spared from becoming infarcted is in fact functional. The graph displays the functional recovery as a function of time following infarction. Filled symbols are for the contralateral (affected) hemisphere, open symbols for the ipsilateral (unaffected) one. Dark symbols indicate drug, lighter symbols placebo-treated rats. Recovery in drug-treated rats is observed only a day following infarction despite the fact that the cortical tissue is structurally intact. In addition, only 50% of the animals displayed recovery in the observation period of 12 days. In placebo-treated animals the respective S1 was always infarcted, no functional response has been observed. In all animals the response of the ipsilateral unaffected S1 region was normal. (Adapted from Sauter, A. et al., Magn. Reson. Med, 47, 759, 2002.)

migration of labeled progenitor cells from the contralateral injection site to the site of infarction. MRI cell tracking using superparamagnetic iron oxide nanoparticles for cell labeling reveals excellent anatomical information, yet no direct viability information. This information can be derived from genetically marked cells: expression of the reporter gene product such as luciferase clearly demonstrates that implanted cells are alive.

7.3.3 Translational Studies, Biomarker

A major reason of using imaging methods in DDD is their potential for translational research: similar or identical techniques are being used in animal and in clinical studies. Imaging biomark-ers are supposed to reveal early evidence for demonstrating the validity of the therapeutic principle in man. This is in line with the Critical Path Initiative of the U.S. Food and Drug Administration (FDA) that aims at "translating basic scientific discoveries more rapidly into new and better medical treatment by creating new tools to find answers about how the safety and effectiveness of new medical products can be demonstrated in faster time frames, with more certainty, at lower cost and with better information." The agency is convinced that DDD programs will benefit from the availability of biomarkers, imaging methods being considered key enabling technologies. The FDA sees an important role for biomarkers in providing proof-of-principle of a therapeutic intervention, for stratification patient populations, and for the evaluation of therapy response or eventual side effects. This enthusiasm has to be viewed in relation to the fact that currently only very few biomarkers are accepted by the FDA as efficacy readouts (see below), and the development of novel ones seems to be a major undertaking. Aspects such as specific versus generic biomarkers, validation, standardization, biomarker profile versus individual markers, and quantification have to be addressed.

As an example, we will discuss two biomarkers for studying the efficacy of tumor therapy that are in a fairly advanced stage of development. The measurement of glucose utilization rates via [18F]-2-fluoro-2-deoxyglucose (FDG) PET has evolved as a sensitive diagnostic tool for the characterization of primary tumors and for the detection of metastases. FDG is taken up by cells via the glucose transporter and phosphorylated by hexokinase to yield FDG-6-phosphate (FDG-6P) and trapped in the cell (Figure 7.7). The PET activity, hence, reflects glucose transporter and hexokinase activity and thus glycolytic activity of the tissue of interest. There is evidence from several clinical

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FIGURE 7.7 Measurement of tissue glucose utilization using PET and 2-fluoro-2-deoxyglucose. The primary energy substrate glucose is taken up from the circulation via glucose transporters and is phosphory-lated within the cells by hexokinase to yield glucose-6-phosphate. The next step in the metabolic cascade is the isomerization to fructose-6-phosphate, which after further processing enters the citric acid cycle. By using 2-deoxy-glucose (2DG) as substrate it could be demonstrated that uptake and phosphorylation are identical as for glucose. Yet, isomerization is not possible due to the lack of the hydroxyl group in the 2-position, and 2DG-6-phosphate is trapped in the cell. Hence, the accumulation of 2DG-6-phosphate, which is measured using autoradiographic techniques, is considered a surrogate for glucose utilization by tissue. In the PET version of the 2DG assay the hydrogen at the 2-position is replaced by a fluorine-18 radionuclide (with half-life 110 min) yielding [18F]-2-fluoro-2-deoxyglucose (FDG). As glucose and FDG are different compounds, the rate constants for enzymatic processing of the two substrates differ. This is accounted for by correcting glucose utilization rates derived from FDG measurements using so-called lumped constants.

FIGURE 7.7 Measurement of tissue glucose utilization using PET and 2-fluoro-2-deoxyglucose. The primary energy substrate glucose is taken up from the circulation via glucose transporters and is phosphory-lated within the cells by hexokinase to yield glucose-6-phosphate. The next step in the metabolic cascade is the isomerization to fructose-6-phosphate, which after further processing enters the citric acid cycle. By using 2-deoxy-glucose (2DG) as substrate it could be demonstrated that uptake and phosphorylation are identical as for glucose. Yet, isomerization is not possible due to the lack of the hydroxyl group in the 2-position, and 2DG-6-phosphate is trapped in the cell. Hence, the accumulation of 2DG-6-phosphate, which is measured using autoradiographic techniques, is considered a surrogate for glucose utilization by tissue. In the PET version of the 2DG assay the hydrogen at the 2-position is replaced by a fluorine-18 radionuclide (with half-life 110 min) yielding [18F]-2-fluoro-2-deoxyglucose (FDG). As glucose and FDG are different compounds, the rate constants for enzymatic processing of the two substrates differ. This is accounted for by correcting glucose utilization rates derived from FDG measurements using so-called lumped constants.

drug trials that changes in glycolytic rate precede effects on the tumor volume. For example, in patients suffering from gastrointestinal stromal tumors treated with imatinib glucose utilization was significantly reduced within 24 h after treatment onset, while there was no effect on tumor volume for several months, indicating that glucose utilization rate predicts therapy response.

A common technique to assess tumor angiogenesis is the so-called dynamic contrast-enhanced (DCE) MRI methods, which exploits the fact that newly formed immature vessels are characterized by increased vascular permeability. DCE-MRI measures the leakage of low molecular weight contrast agents such as GdDTPA (Magnevist®) or GdDOTA (Dotarem®) into the extracellular space. This method is currently evaluated as biomarker for evaluating the efficacy of antiangiogenic therapy. Inhibition of VEGF receptor signaling should be reflected by decreased vascular permeability and potentially also reduced tumor blood volume, as demonstrated for the VEGF tyrosine kinase inhibitor vatalanib for several tumor models in mice. Drug effects could be detected within 48 h following onset of treatment. Clinical studies in patients with liver metastases yielded corresponding results indicating that, in fact, vascular permeability measures may serve as a biomarker of efficacy. The method has been used for translational studies with a number of compounds.

Currently used imaging biomarkers are based on structural (e.g., response evaluation criteria for solid tumors [RECIST] for tumors, infarct volume for stroke, and lesion load for MS) or physiological and metabolic readouts (e.g., DCE-MRI and glucose utilization rates for tumors). A next generation of (molecular) imaging approaches will provide specific mechanistic information tightly linked to the therapeutic pharmacological principle; a term coined in this regard is thera-nostics, the merger of therapeutic and diagnostics.

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