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In vivo magnetic resonance imaging and spectroscopy in pharmacological research: applications to drug development and profiling
EUROPEAN
ELSEVIER
European Journal of Pharmaceutical Sciences 2 (1994) 50-52
JOUlllliAL
OF
PHARMACEUTICAL SCIENCES
In vivo magnetic resonance imaging and spectroscopy in pharmacological research" applications to drug development and profiling Markus Rudin Preclinical Research 386/206, Sandoz Pharma Ltd, CH-4002 Basel, Switzerland
In the last ten years magnetic resonance imaging (MRI) has become an established radiological tool which, due to its excellent soft-tissue contrasting capabilities, is superior to X-ray computer tomography for many indications. In addition to conventional morphological information, advanced M R I techniques have been developed, capable of providing valuable insight into physiological processes such as measurement of hemodynamic parameters or uptake/clearance of paramagnetic tracers. An exciting new application of M R I is the visualization of brain activation due to sensory stimuli and/or cognitive tasks [1]. It is obvious that MRI has a large potential for pharmacological research. Due to its noninvasive character, the method is suited for follow-up studies in order to monitor the progression of a pathological process and/or the effects of therapy in the same individual. This is desirable both from a scientific and ethical point of view. Moreover, similar protocols may be applied in drugtesting programs at the preclinical and clinical level.
1. Classical application of MRI: in vivo morphometric measurements
A large number of successful applications of M R I in drug-testing protocols has been reported. Examples are models for focal cerebral ischemia [2,3,4], neurotoxicity [5,6], hypertrophic cardiomyopathy [7,8], hyperplasia and neoplasms [9,10]. MRI was applied to identify morphological changes associated with the disease/disorder and to assess the extent of the lesion volume. A n important aspect is the validation of the M R I method with conventional techniques. In cerebral ischemia for instance, MRI actually images a change in the transverse relaxation time 0928-0987/94/$07.00 O 1994 Elsevier Science B.V. All rights reserved SSDI 0928-0987(94)00032-U
72, which is related to increased water content in the ischemic region due to edema formation. It had to be shown by comparison with histopathological analysis of brain slices, that the MRI measure corresponds to the actually infarcted tissue [3]. Similarly, MRI volume determinations in models of cardiac hypertrophy had to be correlated with post-mortem heart weights in order to analyze the accuracy of the in vivo technique [7,8]. If MRI provides a relevant measure of the pathologic process (e.g. an infarct size) with sufficient reproducibility, it can be applied to assess the efficacy of a pharmacological intervention. A model which has been extensively studied using MRI is the unilateral occlusion of the middle cerebral artery in rats. MR images are usually recorded 24 to 48 h after embolization; in this time interval the contrast between edematous and intact brain tissue is most pronounced in conventional T2-weighted M R images. A wide spectrum of drugs has been studied such as calcium antagonists [2,3], competitive and noncompetitive N-methyl-D-aspartate antagonists [4], and free radical scavengers, to mention only a few. Similarly it has been shown that the angiotensin converting enzyme inhibitor spirapril reduces left ventricular hypertrophy in rats with aortic valve insufficiency without affecting the functional parameters of the heart [11].
2. 'Non-classicar MRI: measurement of functional parameters of tissue
Even more appealing are MRI applications for which there is no conventional alternative. A typical example is the quantitative assessment of functional parameters of the (rodent) heart, where diastolic and systolic ventricular volumes,
M. Rudin / European Journal of Pharmaceutical Sciences 2 (1994) 50-52
stroke volumes and ejection fractions may be determined in a straightforward manner exploiting the intrinsic contrast between moving blood and stationary tissue [8]. Blood flow rates in major vessels may be determined by measuring the flow of blood to/from the imaging plane. Tissue perfusion at a microscopic level can be assessed via the signal change in a tissue induced by the passage of a paramagnetic contrast agent [12,13] or by using suitable spin label techniques [14]. Using Gd(DTPA) as a contrast agent it could be shown that the cytoprotective efficacy of the Ca antagonist isradipine in the rat middle cerebral artery occlusion model may be partly explained by the increased collateral blood supply to the ischemic region [15]. It has been reported recently that the tissue signal intensity depends on the oxygenation state of blood [16]. This intrinsic BOLD (blood oxygen level dependent) contrast forms the~ basis of functional MRI of the brain [1], which iscurrently extensively used f0rbrain mapping in man. To what extent these results will affect the design of novel animal models for human CNS disorders remains to be shown.
3. The role of magnetic resonance spectroscopy MRS
In vivo MRS provides biochemical information from a defined volume-of-interest within the body. The method has an inherently low sensitivity; therefore only metabolite pools in the millimolar concentration range may be tapped within a reasonable measurement time of several minutes from sample volumes of typically 20100/zl in rodent studies to 8-125 ml in clinical applications. MRS studies are, in general, time consuming and the method is therefore less suited for drug-screening programs. Nevertheless, MRS may be applied to study metabolic changes induced by a disease process and/or a pharmacological intervention, offering a window to metabolism. High-energy phosphate, phospholipid, and carbohydrate metabolism have been extensively studied with multinuclear (1H, 13C, 31p) MRS techniques. Using combined 1I-I/31p MRS it has been shown that calcium antagonists reduce the ATP consumption in the rat brain [17], which may be of importance in situations of impaired energy synthesis such as hypoxia or ischemia. An
51
elegant method to study specific metabolic pathways is the use of 13C spectroscopy in combination with specifically 13C-labeled substrates. Due to the high specificity of the technique and also to the fact that there is little background signal from endogenous compounds, the fate of the label can be studied in great detail. A representative exampie is the study of cerebral glucose metabolism in rat brain [18]. The metabolite levels observed are the steadystate levels resulting from the ongoing production/consumption processes. As long as the synthesis rates match the degradation, the levels remain constant. Thus, measurement of levels may be an insensitive tool for assessing the tissue metabolic state. Reaction turnover rates and substrate fluxes might represent a more subtle indicator. In some cases, advanced MRS techniques such as magnetization transfer allow the noninvasive determination of reaction rates. The best known example in in vivo MRS is the creatine kinase reaction, for which the reaction rates have been determined in brain, heart, and skeletal muscle [19]. Using such techniques it has been demonstrated that the creatine kinase forward flux is reduced by the Ca antagonist isradipine in rat brain, corroborating the interpretation that this drug reduces brain energy consumption [17].
4. Use of M R I / M R S in drug testing programs
Several models involving in vivo MRI morphometry have been developed into tools for routine drug testing protocols. The throughput ranges from typically one animal/hour for dynamic heart studies to four animals/hour for infarct size assessment in stroke studies. Compared to conventional histological methods for the latter application, MRI may be considered to be cost-efficient bearing in mind the work-intensive steps of tissue sectioning, staining, and morphometric analysis. Moreover, since the technique is noninvasive, animals may be monitored over prolonged time periods and/or utilized for additional tests. Thus the information content which may be obtained from an individual may be significantly increased at no or very little additional stress for the animal. Similar throughputs may be achieved with functional MRI experiments. As an example, data acquisition in a blood volume measurement
52
M. Rudin / European Journal of Pharmaceutical Sciences 2 (1994) 50-52
using the bolus tracking techniques is of the order of 1-5 min during which typically 100 sequential images are recorded. The bottle-neck determining the throughput in this experiment is efficient data processing in order to obtain a functional MR image (parameter map) from the raw data. MRS, on the other hand, is less suited for routine drug testing due to the reasons already discussed. A typical spectroscopic session lasts up to several hours per animal. Moreover, due to sensitivity problems, the variability of the MRS data is considerable especially when considering the small sample volumes encountered in rodent studies. The role of MRS is rather to provide supporting data for drug profiling. For this the technique may be unique.
5. Future trends: towards functional MRI
Similar to the situation in radiodiagnostics, MRI may be meanwhile considered an established tool in pharmaceutical research. The measurement of morphological parameters is straightforward. However, unlike the situation in human medicine, pharmaceutical applications of MRI have always to compete with conventional techniques such as histopathological analyses or other invasive methods. An exciting novel avenue of MRI application is functional MRI, which, in addition to static tissue structures, images dynamic (physiological) characteristics. This was made possible by a second generation of spectrometers with selfshielded magnetic field gradient systems allowing images to be achieved in subsecond acquisition times. This reduction of imaging time by two orders of magnitude was a key success factor for functional MRI. The combination of the excellent anatomic definition with the mapping of physiological parameters will definitely become a major application of MRI in pharmaceutical research. Similarly, spectroscopic tools such as spectroscopic imaging (low resolution maps of metabolite distribution) are being developed which will significantly increase the potential of in vivo MRS and eventually enlarge its scope for pharmaceutical research.
The MRI/MRS methodology has made enormous progress in the past ten years and novel exciting applications are emerging on a regular basis. The field is still in rapid development and it is beyond doubt that MRI/MRS techniques will have a major impact on pharmaceutical research both at the preclinical and the clinical level.
References [1] Ogawa, S., Tank, D.W., Menon, R., Ellerman, J.M., Kim, S.G., Merkle, M. and Ugurbil, K. (1992) Proc. Natl. Acad. Sci. USA 89, 5951-5955. [2] Sauter, A. and Rudin, M. (1986) Stroke 17, 12281234. [3] Sauter, A., Rudin, M. and Wiederhold, K.H. (1988) Neurochem. Pathol. 9, 211-236. [4] Allegrini, P.R. and Sauer, D. (1993) Magn. Reson. Imag. 10, 773-778. [5] Sauter, A., Rudin, M. and Urwyler, S. (1991) Proc. Soc. Magn. Reson. Med. p. 291. [6] Sauer, D., Thedinga, K.H., Fagg, G.E., Massieu, G.E., Amaeker, H. and Allegrini, P.R. (1992) J. Neurosci. Meth. 42, 69-74. [7] Manning, W.J., Wei, J.Y., Fossel, E.T., Burstein, D. (1990) Am. J. Physiol. 258, H1181-Hl186. [8] Rudin, M., Pedersen, B., Umemura, K. and Zierhut, W. (1991) Bas. Res. Cardiol. 86, 165-174. [9] Rudin, M., Briner, U. and Doepfner, W. (1988) Magn. Reson. Med. 7, 285-291. [10] Siegel, R., Tolcsvai, L. and Rudin, M. (1988) Cancer Res. 48, 4651-4655. [11] Umemura, K., Zierhut, W., Rudin, M., Novosel, D., Robertson, E., Pedersen, B. and Hof, R.P. (1992) J. Cardiovasc. Pharmacol. 19, 375-381. [12] Villringer, A., Rosen, B.R., Belliveau, J.W., Ackerman, J.L., Lauffer, R.B., Buxton, R.B., Chao, Y.S., Wedeen, V.J. and Brady, T.J. (1988) Magn. Reson. Med. 6, 164-174. [13] Rudin, M. and Sauter, A. (1991) Magn. Reson. Med. 22, 32-46. [14] Detre, J.A., Leigh, J.S., Williams, D.S. and Koretsky, A.P. (1992) Magn. Reson. Med. 23, 37-45. [15] Sauter, A. and Rudin, M. (1990) Drugs 40, 44-51. [16] Ogawa, S., Lee, T.M., Kay, A.R. and Tank, D.W. (1990) Proc. Natl. Acad. Sci. USA 87, 9868-9872. [17] Rudin, M. and Sauter, A. (1989) J. Pharm. Exp. Ther. 251,700-706. [18] K/innecke, B., Cerdan, S. and Seelig, J. (1993) NMR Biomed. 6, 264-277. [19] Bittl, J.A., DeLayre, J. and IngwaU, J.S. (1987) Biochemistry 26, 6083-6088.
ELSEVIER
European Journal of Pharmaceutical Sciences 2 (1994) 50-52
JOUlllliAL
OF
PHARMACEUTICAL SCIENCES
In vivo magnetic resonance imaging and spectroscopy in pharmacological research" applications to drug development and profiling Markus Rudin Preclinical Research 386/206, Sandoz Pharma Ltd, CH-4002 Basel, Switzerland
In the last ten years magnetic resonance imaging (MRI) has become an established radiological tool which, due to its excellent soft-tissue contrasting capabilities, is superior to X-ray computer tomography for many indications. In addition to conventional morphological information, advanced M R I techniques have been developed, capable of providing valuable insight into physiological processes such as measurement of hemodynamic parameters or uptake/clearance of paramagnetic tracers. An exciting new application of M R I is the visualization of brain activation due to sensory stimuli and/or cognitive tasks [1]. It is obvious that MRI has a large potential for pharmacological research. Due to its noninvasive character, the method is suited for follow-up studies in order to monitor the progression of a pathological process and/or the effects of therapy in the same individual. This is desirable both from a scientific and ethical point of view. Moreover, similar protocols may be applied in drugtesting programs at the preclinical and clinical level.
1. Classical application of MRI: in vivo morphometric measurements
A large number of successful applications of M R I in drug-testing protocols has been reported. Examples are models for focal cerebral ischemia [2,3,4], neurotoxicity [5,6], hypertrophic cardiomyopathy [7,8], hyperplasia and neoplasms [9,10]. MRI was applied to identify morphological changes associated with the disease/disorder and to assess the extent of the lesion volume. A n important aspect is the validation of the M R I method with conventional techniques. In cerebral ischemia for instance, MRI actually images a change in the transverse relaxation time 0928-0987/94/$07.00 O 1994 Elsevier Science B.V. All rights reserved SSDI 0928-0987(94)00032-U
72, which is related to increased water content in the ischemic region due to edema formation. It had to be shown by comparison with histopathological analysis of brain slices, that the MRI measure corresponds to the actually infarcted tissue [3]. Similarly, MRI volume determinations in models of cardiac hypertrophy had to be correlated with post-mortem heart weights in order to analyze the accuracy of the in vivo technique [7,8]. If MRI provides a relevant measure of the pathologic process (e.g. an infarct size) with sufficient reproducibility, it can be applied to assess the efficacy of a pharmacological intervention. A model which has been extensively studied using MRI is the unilateral occlusion of the middle cerebral artery in rats. MR images are usually recorded 24 to 48 h after embolization; in this time interval the contrast between edematous and intact brain tissue is most pronounced in conventional T2-weighted M R images. A wide spectrum of drugs has been studied such as calcium antagonists [2,3], competitive and noncompetitive N-methyl-D-aspartate antagonists [4], and free radical scavengers, to mention only a few. Similarly it has been shown that the angiotensin converting enzyme inhibitor spirapril reduces left ventricular hypertrophy in rats with aortic valve insufficiency without affecting the functional parameters of the heart [11].
2. 'Non-classicar MRI: measurement of functional parameters of tissue
Even more appealing are MRI applications for which there is no conventional alternative. A typical example is the quantitative assessment of functional parameters of the (rodent) heart, where diastolic and systolic ventricular volumes,
M. Rudin / European Journal of Pharmaceutical Sciences 2 (1994) 50-52
stroke volumes and ejection fractions may be determined in a straightforward manner exploiting the intrinsic contrast between moving blood and stationary tissue [8]. Blood flow rates in major vessels may be determined by measuring the flow of blood to/from the imaging plane. Tissue perfusion at a microscopic level can be assessed via the signal change in a tissue induced by the passage of a paramagnetic contrast agent [12,13] or by using suitable spin label techniques [14]. Using Gd(DTPA) as a contrast agent it could be shown that the cytoprotective efficacy of the Ca antagonist isradipine in the rat middle cerebral artery occlusion model may be partly explained by the increased collateral blood supply to the ischemic region [15]. It has been reported recently that the tissue signal intensity depends on the oxygenation state of blood [16]. This intrinsic BOLD (blood oxygen level dependent) contrast forms the~ basis of functional MRI of the brain [1], which iscurrently extensively used f0rbrain mapping in man. To what extent these results will affect the design of novel animal models for human CNS disorders remains to be shown.
3. The role of magnetic resonance spectroscopy MRS
In vivo MRS provides biochemical information from a defined volume-of-interest within the body. The method has an inherently low sensitivity; therefore only metabolite pools in the millimolar concentration range may be tapped within a reasonable measurement time of several minutes from sample volumes of typically 20100/zl in rodent studies to 8-125 ml in clinical applications. MRS studies are, in general, time consuming and the method is therefore less suited for drug-screening programs. Nevertheless, MRS may be applied to study metabolic changes induced by a disease process and/or a pharmacological intervention, offering a window to metabolism. High-energy phosphate, phospholipid, and carbohydrate metabolism have been extensively studied with multinuclear (1H, 13C, 31p) MRS techniques. Using combined 1I-I/31p MRS it has been shown that calcium antagonists reduce the ATP consumption in the rat brain [17], which may be of importance in situations of impaired energy synthesis such as hypoxia or ischemia. An
51
elegant method to study specific metabolic pathways is the use of 13C spectroscopy in combination with specifically 13C-labeled substrates. Due to the high specificity of the technique and also to the fact that there is little background signal from endogenous compounds, the fate of the label can be studied in great detail. A representative exampie is the study of cerebral glucose metabolism in rat brain [18]. The metabolite levels observed are the steadystate levels resulting from the ongoing production/consumption processes. As long as the synthesis rates match the degradation, the levels remain constant. Thus, measurement of levels may be an insensitive tool for assessing the tissue metabolic state. Reaction turnover rates and substrate fluxes might represent a more subtle indicator. In some cases, advanced MRS techniques such as magnetization transfer allow the noninvasive determination of reaction rates. The best known example in in vivo MRS is the creatine kinase reaction, for which the reaction rates have been determined in brain, heart, and skeletal muscle [19]. Using such techniques it has been demonstrated that the creatine kinase forward flux is reduced by the Ca antagonist isradipine in rat brain, corroborating the interpretation that this drug reduces brain energy consumption [17].
4. Use of M R I / M R S in drug testing programs
Several models involving in vivo MRI morphometry have been developed into tools for routine drug testing protocols. The throughput ranges from typically one animal/hour for dynamic heart studies to four animals/hour for infarct size assessment in stroke studies. Compared to conventional histological methods for the latter application, MRI may be considered to be cost-efficient bearing in mind the work-intensive steps of tissue sectioning, staining, and morphometric analysis. Moreover, since the technique is noninvasive, animals may be monitored over prolonged time periods and/or utilized for additional tests. Thus the information content which may be obtained from an individual may be significantly increased at no or very little additional stress for the animal. Similar throughputs may be achieved with functional MRI experiments. As an example, data acquisition in a blood volume measurement
52
M. Rudin / European Journal of Pharmaceutical Sciences 2 (1994) 50-52
using the bolus tracking techniques is of the order of 1-5 min during which typically 100 sequential images are recorded. The bottle-neck determining the throughput in this experiment is efficient data processing in order to obtain a functional MR image (parameter map) from the raw data. MRS, on the other hand, is less suited for routine drug testing due to the reasons already discussed. A typical spectroscopic session lasts up to several hours per animal. Moreover, due to sensitivity problems, the variability of the MRS data is considerable especially when considering the small sample volumes encountered in rodent studies. The role of MRS is rather to provide supporting data for drug profiling. For this the technique may be unique.
5. Future trends: towards functional MRI
Similar to the situation in radiodiagnostics, MRI may be meanwhile considered an established tool in pharmaceutical research. The measurement of morphological parameters is straightforward. However, unlike the situation in human medicine, pharmaceutical applications of MRI have always to compete with conventional techniques such as histopathological analyses or other invasive methods. An exciting novel avenue of MRI application is functional MRI, which, in addition to static tissue structures, images dynamic (physiological) characteristics. This was made possible by a second generation of spectrometers with selfshielded magnetic field gradient systems allowing images to be achieved in subsecond acquisition times. This reduction of imaging time by two orders of magnitude was a key success factor for functional MRI. The combination of the excellent anatomic definition with the mapping of physiological parameters will definitely become a major application of MRI in pharmaceutical research. Similarly, spectroscopic tools such as spectroscopic imaging (low resolution maps of metabolite distribution) are being developed which will significantly increase the potential of in vivo MRS and eventually enlarge its scope for pharmaceutical research.
The MRI/MRS methodology has made enormous progress in the past ten years and novel exciting applications are emerging on a regular basis. The field is still in rapid development and it is beyond doubt that MRI/MRS techniques will have a major impact on pharmaceutical research both at the preclinical and the clinical level.
References [1] Ogawa, S., Tank, D.W., Menon, R., Ellerman, J.M., Kim, S.G., Merkle, M. and Ugurbil, K. (1992) Proc. Natl. Acad. Sci. USA 89, 5951-5955. [2] Sauter, A. and Rudin, M. (1986) Stroke 17, 12281234. [3] Sauter, A., Rudin, M. and Wiederhold, K.H. (1988) Neurochem. Pathol. 9, 211-236. [4] Allegrini, P.R. and Sauer, D. (1993) Magn. Reson. Imag. 10, 773-778. [5] Sauter, A., Rudin, M. and Urwyler, S. (1991) Proc. Soc. Magn. Reson. Med. p. 291. [6] Sauer, D., Thedinga, K.H., Fagg, G.E., Massieu, G.E., Amaeker, H. and Allegrini, P.R. (1992) J. Neurosci. Meth. 42, 69-74. [7] Manning, W.J., Wei, J.Y., Fossel, E.T., Burstein, D. (1990) Am. J. Physiol. 258, H1181-Hl186. [8] Rudin, M., Pedersen, B., Umemura, K. and Zierhut, W. (1991) Bas. Res. Cardiol. 86, 165-174. [9] Rudin, M., Briner, U. and Doepfner, W. (1988) Magn. Reson. Med. 7, 285-291. [10] Siegel, R., Tolcsvai, L. and Rudin, M. (1988) Cancer Res. 48, 4651-4655. [11] Umemura, K., Zierhut, W., Rudin, M., Novosel, D., Robertson, E., Pedersen, B. and Hof, R.P. (1992) J. Cardiovasc. Pharmacol. 19, 375-381. [12] Villringer, A., Rosen, B.R., Belliveau, J.W., Ackerman, J.L., Lauffer, R.B., Buxton, R.B., Chao, Y.S., Wedeen, V.J. and Brady, T.J. (1988) Magn. Reson. Med. 6, 164-174. [13] Rudin, M. and Sauter, A. (1991) Magn. Reson. Med. 22, 32-46. [14] Detre, J.A., Leigh, J.S., Williams, D.S. and Koretsky, A.P. (1992) Magn. Reson. Med. 23, 37-45. [15] Sauter, A. and Rudin, M. (1990) Drugs 40, 44-51. [16] Ogawa, S., Lee, T.M., Kay, A.R. and Tank, D.W. (1990) Proc. Natl. Acad. Sci. USA 87, 9868-9872. [17] Rudin, M. and Sauter, A. (1989) J. Pharm. Exp. Ther. 251,700-706. [18] K/innecke, B., Cerdan, S. and Seelig, J. (1993) NMR Biomed. 6, 264-277. [19] Bittl, J.A., DeLayre, J. and IngwaU, J.S. (1987) Biochemistry 26, 6083-6088.