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| EDITORIAL |
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| Year : 2022 | Volume
: 54
| Issue : 5 | Page : 309-313 |
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Imaging techniques in drug development
Shreya Gupta, Ajay Prakash, Bikash Medhi
Department of Pharmacology, PGIMER, Chandigarh, India
| Date of Submission | 29-Jul-2022 |
| Date of Decision | 18-Oct-2022 |
| Date of Acceptance | 09-Nov-2022 |
| Date of Web Publication | 13-Dec-2022 |
Correspondence Address:
Bikash Medhi
Department of Pharmacology, PGIMER, Chandigarh - 160 012
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijp.ijp_533_22
How to cite this article:
Gupta S, Prakash A, Medhi B. Imaging techniques in drug development. Indian J Pharmacol 2022;54:309-13 |
| » Introduction | |  |
The process of drug discovery and development is a risky, lengthy, and resource-intensive endeavor; both in terms of the cost and time, it takes for a single new molecule to enter the market. For most new chemical entities (NCEs) to reach the stage of new drug application leading up to regulatory approval it takes almost 10 years, and coupled with high attrition rates, it has been observed that only one in five compounds make it through the entire development and approval process. Furthermore, the number of novel drug molecules that have been introduced into the market in the past 30 years has remained relatively constant despite the increasing investment, with only two or three NCEs eventually making it to markets every year. Novel strategies that could aid in the early selection of viable candidates to progress onto clinical trials or to terminate entities unlikely to be successful, could significantly hasten the overall process. Molecular and functional imaging is one such modality which if applied in the early phases of the drug development process, can provide key evidence relating to target binding, on-target drug effects, and pharmacokinetic–pharmacodynamic (PKPD) parameters, thereby identifying those who are most likely to benefit from the proposed drug entity.[1],[2]
| » Techniques of Molecular Imaging | | |
Molecular imaging basically entails noninvasive imaging that can assess specific cellular and molecular disease processes such as gene expression, synthesis of proteins, metabolite generation, receptor availability and binding, and enzyme activity. All this information helps in identifying quantitative temporal and spatial information for further preclinical and clinical studies. Hence, molecular imaging can be used to identify new drug targets, estimate drug distribution and dosing strategies, initial efficacy testing in appropriate patient populations, and be used as surrogate endpoints in animal and human studies.[2]
There exist various strategies for molecular imaging. The most common strategy is through the use of various contrast agents which have selective binding to the molecules of interest. Different imaging modalities such as ultrasonography (USG), optical imaging, computed tomography (CT), single-photon emission computerized tomography (SPECT), and magnetic resonance imaging (MRI) use contrast agents which are tailored as per the imaging technique and kinetic alterations.[1],[2],[3]
The target binding molecules can either exist in a linear ratio with the radiological agent, as is the case with SPECT or positron emission tomography (PET), or they may be conjugated in a multivalent manner onto the surface of the contrast agent, as those used in USG or MRI. The biodistribution of the contrast agents also plays an important role in choosing the appropriate agent, with respect to its diffusion capacity versus confinement to the vascular compartment. Contrast agents are also designed to leverage biological processes for their uptake and retention. For example, the use of 18F-fluorodeoxyglucose (FDG) in PET to detect foci of enhanced energy requirement such as cancers, ongoing inflammatory conditions, advanced atherosclerosis, and sarcoidosis. The cellular uptake of certain contrast agents can even help characterize specific immune cell subtypes in atherosclerosis.[4],[5],[6]
Another strategy of molecular imaging involves engaging the disease process to activate the contrast agents. This usually involves developing fluorophores that, after interaction with a pathogenic state, can produce a spectral shift or change in the emission of photons which may be detected by optical imaging.[7],[8]
Furthermore, molecular imaging may be used to detect endogenous signals in a molecular environment, without requiring exogenous contrast agents, such as autofluorescence or MRI-based blood-oxygen-level-dependent imaging.[9]
Selection of the most appropriate technique also plays a major role in various stages of drug development. Few technical considerations for the same include spatial and temporal resolution, target specificity, high sensitivity, and kinetic profile of contrast agent. These practical considerations include availability, cost, and safety. Out of the imaging techniques, some are primarily anatomical or morphological such as CT, MRI, and ultrasound, whereas others are primarily molecular imaging modalities such as optical imaging, PET, and SPECT (single photon emission computed tomography). The primarily anatomical techniques are characterized by high spatial resolution but are unable to detect diseases until tissue or structural damage becomes morphologically evident (e.g., tumorous growth), and are large enough to be detected by the imaging technique. Contrary to this, primary molecular imaging techniques have the potential to detect cellular and molecular changes even before the damage becomes large enough to cause structural changes; however, the spatial resolution of these modalities is poor. Thus, clubbing the strengths of anatomical and molecular techniques would provide a high structural resolution and early detection of pathomorphological changes, for example, PET-CT and PET-MRI technologies, and are currently in use by many large pharmaceutical companies for basic and clinical research projects.[10],[11]
Role in early phase discovery
Imaging can be helpful in various stages of the early drug development phase. Molecule distribution studies help to confirm whether the molecule of interest reaches the target tissue and whether or not it accumulates elsewhere in nontarget tissues causing potential toxicity. Further, dose-target occupancy studies help guide pharmacokinetic measurements and the most appropriate dose selection. Then, imaging can also be used to determine pharmacodynamic biomarkers to help establish proof of pharmacology and as measures of safety or toxicity. Overall translational preclinical imaging is pivotal in identifying and validating imaging biomarkers and in providing early differentiation between promising candidates based on target PKPD response.
Target selection
After choosing a specific target, molecular imaging helps in the detection of said target in vivo, particularly in diseased states, along with its spatial and temporal quantification. For example, for an antiangiogenic drug, it is important to establish the presence and the extent to which a specific target is being expressed in new blood vessels supplying tumor cells. Similarly, to develop a drug against vascular endothelial growth factor receptor (VEGFR), one requires an imaging probe that binds directly to VEGFR, like 64Cu-VEGF in PET imaging which can be helpful in target expression assessment.[12]
A large library of well-established imaging probes already exists called the Molecular Imaging and Contrast Agent Database, which are directed against a number of known targets and can be used for confirmation of these targets for the further drug development process. However, a newly discovered target will warrant de novo synthesis of a customized imaging probe, whose binding sensitivity and specificity to the target will have to be characterized separately.[13]
Another approach to this is through reporter gene imaging, which is a more generalizable process in which the coexpression of the reporter gene and therapeutic target gene is driven by the same promoters. The reporter gene expresses by encoding a protein that can interact with the imaging probe and if the therapeutic target is present, it will interact with the probe and can be visualized through imaging using MRI, radionucleotide, and optical techniques.[14],[15]
Further, in such techniques, the reporter gene can either encode an enzyme that can under transformation, for example, phosphorylation, to trap a specific tracer through enzymatic action, or the reporter gene can encode for an intra or extracellular receptor that can bind to a radioactive ligand which would be detected on imaging. In both approaches, the tracer accumulation depends on the reporter gene expression. A drawback to this approach is also that the gene has to be introduced through vectors such as adenovirus, lentivirus, or liposomes. Since there exists a lack of precise technology for the introduction of these vectors into any part of the body, usually reporter genes are introduced ex vivo into these cells.[16]
Compound screening: Hit and lead compound optimization and validation
After target identification, screening of large libraries of chemicals is usually carried out using high-throughput screening to check for their ability to modulate the said target. Molecular imaging is done through cell-based and animal-based assays, and in addition, enables the screening of compounds by monitoring protein–protein interactions in small rodent models and cell cultures. Optical imaging, bioluminescence, and split-protein strategies are some of the imaging modalities used for compound screening in various settings. Optical imaging is a highly sensitive, high-throughput, and low-cost screening modality, which has been used in recent times for in vivo screening for the development of drugs to treat cutaneous leishmaniasis; the combination of fluorescent nanoprobes with optical imaging has been used as a functional biomarker to identify increased vascular leakage in various disease models.[17],[18],[19]
In recent times, split-protein strategies to assess protein–protein interactions using molecular imaging have been developed for cell culture and in vivo models. In this technique, cleavage of a reporter enzyme or protein is performed into carboxy and amino terminal segments and each segment is fused to one out of the two interacting proteins. Such protein–protein interaction restores the functional reporter enzyme activity, generating signals that can be imaged using molecular techniques. Such assays are being used to screen compounds that act by modulation of protein–protein interactions in in vitro or in vivo settings. After compound identification, these assays can also aid in optimizing the drug dosage, frequency, and routes of administration.[20],[21]
Small animal imaging techniques
Small animal models are widely used in preclinical drug development and form an integral part of the drug testing process. Imaging techniques in small animal models are becoming increasingly popular as they provide an advanced and cost-effective means for the preclinical validation of novel drug molecules. It also aids in the understanding of pathological states such as diabetes, cancer, neurodegenerative conditions, and cardiovascular and immunological diseases, by enabling close monitoring at the molecular level over time.
Specially designed miniaturized machines for clinical diagnoses such as micro-PET (micro-PET), microsingle-photon-emission CT (micro-SPECT), microultrasound, micromagnetic resonance tomography, and optical imaging are some of the modalities in use which enables researchers to longitudinally examine various animal models of human pathologies, before extrapolating the same into clinical research. One major benefit of using imaging in animal models is that they can reduce the number of animals used in preclinical studies while providing a more precise and accurate account of the pathology and treatment effects on the same. Furthermore, small-animal molecular imaging enables noninvasive, quantitative estimates of the experimental interventions, thereby allowing more data to be collected from a single animal.[22],[23]
Role in late drug development
Radiological imaging along with molecular assays is a valuable tool in aiding the go-ahead of NCEs in both preclinical and clinical research. In the late stages, data from imaging is used as a robust basis for generating evidence about efficacy and on-target binding and drug action, which provides additional information while seeking marketing approval from the regulatory bodies.
Especially during the oncology drug development process, imaging plays a fundamental role in exploring the primary, secondary, and surrogate endpoints. For this reason, noninvasive techniques such as CT, MRI, and FDG-PET-CT are generally used to provide evidence of treatment efficacy and reduction in tumor size. Advancement in such techniques might also help prognosticate treatment outcomes in the patient population most likely to benefit from the particular treatment. This would further the aim to enable targeted or personalized drug development in the future. Imaging techniques find their place at various stages of the drug development process, from microdosing/phase 0 studies to phase 3, including drug delivery and biodistribution studies.[1],[2]
Imaging in microdosing/phase 0 studies
Microdosing studies involve using about 1% of the estimated therapeutic dose, i.e., not exceeding 100 μg. At such subtherapeutic doses, toxic effects are usually not expected in healthy human volunteers or patients. Among the various imaging modalities, PET microdosing scan is among the most beneficial. It uses minute quantities of drug tracers which are radiolabelled and can be used to determine the PK profile of the drug, including rate (Cmax), half-life, and extent of tissue absorption using serial PET scans. It has immense scope in onco-therapeutics and psychopharmaceuticals, among other fields of pharmaceutical medicine. It can also be used to determine blood–brain barrier penetration, which often goes unpredicted in preclinical models.[1],[24]
However, certain limitations also exist while employing PET for microdosing. First, the parent drug cannot be differentiated through imaging from its radioactive metabolites in vivo because they both produce the same signal. Also for drugs which are extensively metabolized, the PK data of the parent drug and its metabolites may get confounded. Also to acquire certain quantitative data such as rate constants for the transfer of radiolabelled drug from plasma to various tissue compartments, the concentration-time profile of the unmetabolized parent drug in arterial plasma is required.[1],[24]
Receptor occupancy by imaging
Receptor occupancy entails the calculation of the receptor population that gets engaged by a radiolabelled agent. The combination of SPECT and PET with anatomical imaging techniques like MRI or CT helps to relate target drug occupancy with the anatomical or physiological changes occurring at the tissue level. One can obtain information about the reachability of the drug to its target, information about the kinetics of the drug in relation to its target occupancy level, dose estimation for the optimal occupancy threshold needed to attain desired therapeutic effects, and occupancy levels beyond which risks of adverse events may occur.[25]
For instance, in the field of oncology, in vivo competition studies uses minute amounts of radioligand that binds to specific tumor receptor, thus enabling direct assessment of the relationship between target occupancy and drug plasma concentration. Radioligands which are used include small-molecule ligands such as 18F–FDHT and 18F–FES, and peptides such as arginine-glycine-aspartic acid analogs, somatostatin analogs, gastrin or cholecystokinin derivatives, and antibodies or antibody fragments. Receptor imaging to aid in therapy selection is being employed in various cancers including breast cancer, prostate cancer, glioma, melanoma, etc. Other applications in neuropharmacology include PET occupancy of radioligands for antipsychotics such as paliperidone, blonanserin, and aripiprazole, for D2 and D3 receptor occupancy.[1],[25]
Positron emission tomography in drug biodistribution studies
Biodistribution studies are vital during the early phase drug development process because they help ascertain whether the drug is reaching the target tissue and whether it has the tendency to accumulate or cause unwanted effects at the nontarget sites, which can cause potential toxicity. Imaging techniques play a major role in biodistribution and PK studies of drug and/or drug delivery systems in many diseases, especially cancers. PET, SPECT, MRI, and optical imaging may be used to visualize the localization and further quantify the radioisotope or radiolabelled drug delivery systems as part of theranostics.[26]
MRI-based biodistribution studies have been used to determine the rapid uptake of trastuzumab (5 to 9 h) from plasma and tumor localization within 25 to 32 h, following intraperitoneal injection in human epidermal growth factor receptor 2/neu positive ovarian cancer.[27]
PET is one of the most important imaging modalities for biodistribution studies. It involves using radiolabelled drug molecules, using radioligands with sufficiently long half-lives such as 64Cu, 76Br, 89Zr, or 124I, with T1/2 of 13 h, 16 h, 3.2 days, and 4.2 days, respectively. One also has to avoid any alteration in the biochemical profile of the drug by the labeling process. Following this, the radioligand is introduced into the healthy subject and its distribution can be traced over time using dynamic PET scan imaging.[26]
Similar studies can be performed in preclinical settings using micro-PET and micro-SPECT techniques. Serial PET or SPECT scans are taken once the radiotracer is introduced to monitor their biochemical pathways, distribution, target and nontarget accumulation, etc.
Since PET and SPECT are primarily functional imaging modalities, with a lack of anatomical detail, they are usually combined with CT and MRI, to provide a high spatial resolution, thus merging the physiological and anatomical status in the same image of the region of interest.[23],[27]
Quantification of neurotransmitter concentration by positron emission tomography
Radio imaging can be used to assess the changes in neurotransmitter concentrations, by quantifying the decrease in radiotracer binding to a particular neuroreceptor, flowing as an increase in the endogenous neurotransmitter concentration. While PET is commonly used for such quantification, the combination of PET/MRI provides greater potential for understanding the dynamic physiological and chemical neurotransmission that occurs in space and time. Combining these two modalities also reduces confounding factors, chances of intrasubject, and interscan variability, and enables cross-validation of biological processes, by linking the actions at the receptor site, such as target binding of the ligand to hemodynamical changes.[28]
Simultaneous PET/MR scanners have been used to evaluate how dopamine receptor density affects functional cortical networks for working memory formation.[29]
It has also been instrumental in delineating the importance of opioid receptor availability in pain pathways.[30]
| » Conclusion | | |
Molecular imaging techniques are a promising endeavor for not only clinical diagnostics but also for the evaluation of new therapeutic agents, both in preclinical and clinical settings. They have especially found a place in cardiovascular, neurological, and onco-therapeutics, with many new molecules and biomarkers being evaluated using various imaging modalities, which further aid in making early “go or no-go” decisions, along with assessing their efficacy and safety profile.
| » References | | |
| 1. | Nairne J, Iveson PB, Meijer A. Imaging in drug development. Prog Med Chem 2015;54:231-80.  |
| 2. | Lindner JR, Link J. Molecular imaging in drug discovery and development. Circ Cardiovasc Imaging 2018;11:e005355. |
| 3. | Choudhury RP, Fisher EA. Molecular imaging in atherosclerosis, thrombosis, and vascular inflammation. Arterioscler Thromb Vasc Biol 2009;29:983-91. |
| 4. | Tawakol A, Fayad ZA, Mogg R, Alon A, Klimas MT, Dansky H, et al. Intensification of statin therapy results in a rapid reduction in atherosclerotic inflammation: Results of a multicenter fluorodeoxyglucose-positron emission tomography/computed tomography feasibility study. J Am Coll Cardiol 2013;62:909-17. |
| 5. | Quillard T, Libby P. Molecular imaging of atherosclerosis for improving diagnostic and therapeutic development. Circ Res 2012;111:231-44. |
| 6. | Kostakoglu L, Agress H Jr., Goldsmith SJ. Clinical role of FDG PET in evaluation of cancer patients. Radiographics 2003;23:315-40. |
| 7. | Shuhendler AJ, Pu K, Cui L, Uetrecht JP, Rao J. Real-time imaging of oxidative and nitrosative stress in the liver of live animals for drug-toxicity testing. Nat Biotechnol 2014;32:373-80. |
| 8. | Quillard T, Croce K, Jaffer FA, Weissleder R, Libby P. Molecular imaging of macrophage protease activity in cardiovascular inflammation in vivo. Thromb Haemost 2011;105:828-36. |
| 9. | Friedrich MG, Karamitsos TD. Oxygenation-sensitive cardiovascular magnetic resonance. J Cardiovasc Magn Reson 2013;15:43. |
| 10. | Rudin M, Weissleder R. Molecular imaging in drug discovery and development. Nat Rev Drug Discov 2003;2:123-31. |
| 11. | Willmann JK, van Bruggen N, Dinkelborg LM, Gambhir SS. Molecular imaging in drug development. Nat Rev Drug Discov 2008;7:591-607. |
| 12. | Hu K, Shang J, Xie L, Hanyu M, Zhang Y, Yang Z, et al. PET imaging of VEGFR with a novel 64cu-labeled peptide. ACS Omega 2020;5:8508-14. |
| 13. | |
| 14. | Hildebrandt IJ, Gambhir SS. Molecular imaging applications for immunology. Clin Immunol 2004;111:210-24. |
| 15. | Min JJ, Gambhir SS. Gene therapy progress and prospects: Noninvasive imaging of gene therapy in living subjects. Gene Ther 2004;11:115-25. |
| 16. | Gambhir SS, Herschman HR, Cherry SR, Barrio JR, Satyamurthy N, Toyokuni T, et al. Imaging transgene expression with radionuclide imaging technologies. Neoplasia 2000;2:118-38. |
| 17. | Caridha D, Parriot S, Hudson TH, Lang T, Ngundam F, Leed S, et al. Use of optical imaging technology in the validation of a new, rapid, cost-effective drug screen as part of a tiered in vivo screening paradigm for development of drugs to treat cutaneous leishmaniasis. Antimicrob Agents Chemother 2017;61:e02048-16. |
| 18. | Sandanaraj BS, Gremlich HU, Kneuer R, Dawson J, Wacha S. Fluorescent nanoprobes as a biomarker for increased vascular permeability: Implications in diagnosis and treatment of cancer and inflammation. Bioconjug Chem 2010;21:93-101. |
| 19. | Hilger I, Leistner Y, Berndt A, Fritsche C, Haas KM, Kosmehl H, et al. Near-infrared fluorescence imaging of HER-2 protein over-expression in tumour cells. Eur Radiol 2004;14:1124-9. |
| 20. | Massoud TF, Paulmurugan R, Gambhir SS. Molecular imaging of homodimeric protein-protein interactions in living subjects. FASEB J 2004;18:1105-7. |
| 21. | Paulmurugan R, Gambhir SS. Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals. Cancer Res 2005;65:7413-20. |
| 22. | Dufort S, Sancey L, Wenk C, Josserand V, Coll JL. Optical small animal imaging in the drug discovery process. Biochim Biophys Acta 2010;1798:2266-73. |
| 23. | Yao R, Lecomte R, Crawford ES. Small-animal PET: What is it, and why do we need it? J Nucl Med Technol 2012;40:157-65. |
| 24. | Wagner CC, Langer O. Approaches using molecular imaging technology – Use of PET in clinical microdose studies. Adv Drug Deliv Rev 2011;63:539-46. |
| 25. | Burvenich IJ, Parakh S, Parslow AC, Lee ST, Gan HK, Scott AM. Receptor occupancy imaging studies in oncology drug development. AAPS J 2018;20:43. |
| 26. | Ding H, Wu F. Image guided biodistribution and pharmacokinetic studies of theranostics. Theranostics 2012;2:1040-53. |
| 27. | Palm S, Enmon RM Jr., Matei C, Kolbert KS, Xu S, Zanzonico PB, et al. Pharmacokinetics and biodistribution of (86) Y-trastuzumab for (90) Y dosimetry in an ovarian carcinoma model: Correlative MicroPET and MRI. J Nucl Med 2003;44:1148-55. |
| 28. | Ceccarini J, Liu H, Van Laere K, Morris ED, Sander CY. Methods for quantifying neurotransmitter dynamics in the living brain with PET imaging. Front Physiol 2020;11:792. |
| 29. | Roffman JL, Tanner AS, Eryilmaz H, Rodriguez-Thompson A, Silverstein NJ, Ho NF, et al. Dopamine D1 signaling organizes network dynamics underlying working memory. Sci Adv 2016;2:e1501672. |
| 30. | Karjalainen T, Karlsson HK, Lahnakoski JM, Glerean E, Nuutila P, Jääskeläinen IP, et al. Dissociable roles of cerebral μ-opioid and type 2 dopamine receptors in vicarious pain: A combined PET-fMRI study. Cerebr Cortex 2017;27:4257-66. |
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