The 18-kDa Translocator Protein PET Tracers as a Diagnostic Marker for Neuroinflammation: Development and Current Standing.

Translocator protein (TSPO, 18 kDa) is an evolutionary, well-preserved, and tryptophan-rich 169-amino-acid protein which localizes on the contact sites between the outer and inner mitochondrial membranes of steroid-synthesizing cells. This mitochondrial protein is implicated in an extensive range of cellular activities, including steroid synthesis, cholesterol transport, apoptosis, mitochondrial respiration, and cell proliferation. The upregulation of TSPO is well documented in diverse disease conditions including neuroinflammation, cancer, brain injury, and inflammation in peripheral organs. On the basis of these outcomes, TSPO has been assumed to be a fascinating subcellular target for early stage imaging of the diseased state and for therapeutic purposes. The main outline of this Review is to give an update on dealing with the advances made in TSPO PET tracers for neuroinflammation, synchronously emphasizing the approaches applied for the design and advancement of new tracers with reference to their structure-activity relationship (SAR).

Introduction

The translocator protein, 18 kDa (TSPO), originally reported as a peripheral benzodiazepine receptor (PBR), is a mitochondrial membrane protein and was initially characterized as a high affinity binding site for diazepam.[1] TSPO is chiefly localized on the external mitochondrial membrane in close connection with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide transporter (ANT), thus creating the mitochondrial transition permeability pore (MPTP).[2] The MPTPs effectively participate in the harmonization of signaling pathways, dealing with apoptotic and necrotic tissue damage. During brain injury, TSPO upregulation is associated with microglial activation, which can be visualized using positron emission tomography (PET) and single photon emission computed tomography (SPECT).[3] The TSPO is primarily found in the heart, lungs, kidneys, liver, adrenal glands, and steroidogenic tissues, while in the brain it has relatively low expression. The enhanced expression of TSPO is found under various human pathologies, including several neuropsychiatric disorders such as multiple sclerosis (MS), Huntington’s disease (HD), Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic sclerosis (ALS).[4] The continuous increase in these diseases poses major health concerns throughout the globe, requiring more innovative and more selective radioprobes for diagnosis.[5,6] Apart from the neuroinflammation, pathophysiologic participation of TSPO is also briefly reported in cardiovascular situations in peripheral sterile inflammatory disorders (PSIDs).[7,8]

Structure and Functions of 18 kDa TSPO

TSPO is a hydrophobic and tryptophan-rich protein composed of 169 amino acids and an 18 kDa molecular mass. Antkiewicz-Michaluk et al. in 1988[9] identified the monomer of TSPO using various photolabeling studies. According to three-dimensional modeling, TSPO is a five trans-membrane domain protein where 21 residues of α-helices make the whole membrane a double layer with a smaller loop and tail region. The α-helices are connected to the hydrophobic loop with a carboxyl (−COO–) terminal tail situated externally to mitochondria and amino (−NH2–) termini situated internally to mitochondria. Guo et al.[10] explained the association of TSPO in a trimeric complex with adenine nucleotide translocase (ANT) and a voltage-dependent anion channel (VDAC). The ANT is situated at the internal membrane of mitochondria and displays the characteristic participation during exchange of ATP (adenosine triphosphate) and ADP (adenosine diphosphate) in the operation of oxidative phosphorylation, whereas VDAC is positioned on the inner/outer mitochondrial contact side and allows entry of ions into the mitochondria making the MPTP.[11,12]

The key role of this protein is to transfer the cholesterol from the external mitochondrial membrane to internal mitochondrial membrane, and this translocation process is observed as the rate determining factor in the steroid and neurosteroid synthesis.[12] Transport of porphyrin for the biosynthesis of heme is another important function of TSPO.[13] Apart from these key functions, TSPO also participates in the apoptosis, anion transport, and cell proliferation. In the brain, TSPO expresses predominately on microglia and other immune cells and endorses its significance in immune regulation.[12−19] TSPO is also important for a balanced flow of calcium ions (Ca2+) into the cell, mitochondrial oxidation, and cellular respiration as mentioned in Figure .

Figure 1

Schematic representation of various TSPO functions.

The existence of TSPO on the external mitochondrial membrane and its high concentration in steriogenic endocrine tissues such as Leydig cells and adrenocortical cells also suggest the key role of TSPO in steroid biosynthesis. Testicular, adrenal, ovarian, placental, and glial systems are the various systems where TSPO ligands stimulate the operation of steroid biosynthesis. Transformation of cholesterol into intermediate pregnenolone in the presence of CYP11A1 (steroidogenic enzyme) is the initial step during steroid biosynthesis. In this process, electron transferring protein and cytochrome P450 play an essential role in the cleavage of cholesterol side chains.[12,13]Figure represents a schematic diagram demonstrating cholesterol metabolism in steroidogenic cells.

Figure 2

Schematic diagram demonstrating cholesterol metabolism in steroidogenic cells.

TSPO Endogenous Ligands

Different types of endogenous TSPO ligands have been described with different binding affinities and selectivities. A few well-known endogenous TSPO ligands are tetrapyrol protoporphyrin IX (PPIX), cholesterol, anthralin, and the diazepam-binding inhibitor (DBI).[20,21] Tetrapyrol protoporphyrin was initially recognized as a TSPO endogenous ligand in the 20th century (1987). The important biological porphyrins like heme and protoporphyrin (IX) show high binding affinity for the TSPO with a Ki value of 40.6 ± 13.7 nM and 14.5 ± 10.7 nM, respectively (Table ).[21,22] Association of the TSPO with heme and its precursor in both eukaryotic and prokaryotic cells suggests its role as a porphyrin transporter.

Table 1

Binding Affinity of Various Endogenous TSPO Ligands

ligands	binding affinity (Ki), nM
heme	40.6 ± 13.7
protoporphyrin IX (PPIX)	14.5 ± 10.7
deuteroporphyrin IX (DpIX)	31.3 ± 2
cholestrol	2.0
DBI	6.1 ± 0.9

Synthetic TSPO PET Tracers

The visualization of overexpressed microglial cells in the living brain by high affinity and selective PET tracers has been considered as a powerful tool for staging the neuroinflammation.[22,23] The development of PET tracers was initially started in the mid-1980s using positron emitter isotopes 11C and 18F.[24] PET tracers have much potential for in vivo molecular imaging and play a most impressive role for the assessment and staging of neuroinflammation.[25] PET tracers of TSPO have been categorized in three generations, which are presented in Figure .

Figure 3

Sequential categorization of PET Tracers.

First Generation TSPO Tracers

Benzodiazepine Family

[C]Ro5-4864, (7-chloro-5-(4-chlorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one), belongs to the 4-chlorodiazepam family and is distinguished from other diazepams only in terms of chlorine atom substitution at the para site of the 2-phenyl ring. It presents good binding affinity (Ki = 6 nM) for TSPO parallel to the GABA-A sites. The C-11 isotope radiolabeled Ro5-4864 was the first tracer that successfully discriminated the peripheral benzodiazepine receptor (PBR) from the central benzodiazepine receptor (CBR).[26] Various studies with human subjects and rat models confirmed the unique outcomes of the [3H]Ro5-4864 tracer in terms of TSPO binding.[27] Later, [11C]Ro5-4864 was reported for its binding ability with the anxiolytic etifoxine, with continuous residence time representing its impressive neurosteroidogenic functionality.[28] Therefore, on the basis of consideration of physical and residence duration factors of etifoxine at TSPO, the [3H]Ro5-4864 binding area and dose dependent inhibition of [3H]Ro5-4864 with a Ki value of 9.0 ± 0.9 nM has been established. Moreover, a scatchard binding experiment of [3H]Ro5-4864 confirmed the role of etifoxine for reduction in receptor affinity and a non-effect on the density of binding areas.[28−30] Despite all of these applications, employment of [11C]Ro5-4864 was restricted owing to its limitations such as poor in vitro association affinity, high lipophilicity, enhanced nonspecific binding, and lower brain uptake. Consequently, [11C]Ro5-4864 also failed to deliver better results in the brain tumor patients and for the early PET experiment of glioma imaging.[30]

Isoquinoline Carboxamide

[C]PK11195, (1-[2-chlorophenyl]-N-methyl-N-[1-methyl-propyl]-3-isoquinoline carboxamide), was the initial nonbenzodiazepine compound recognized for its superior binding affinity to TSPO.[31] It has voluminous benefits over the benzodiazepine tracer [11C]Ro5-4864 including high selectivity (Figure ), rapid blood–brain barrier (BBB) penetration, and high brain accumulation. Lipid soluble [11C]PK11195 was the initial PET tracer effectively applied for better understanding of microglia enhancement in humans for the in vivo imaging of TSPO.[32−35] Originally, the recemate [11C]PK11195 was used for imaging the TSPO; however, later it was revealed that its R enantiomer displayed superior binding affinity compared to the (S) enantiomer.[31,32] At present, [11C]PK11195 serves as a gold standard for all of the TSPO tracers. Various studies described the successful utilization of [11C](R)-PK11195 for neuropsychiatric problems.[35] PET distribution studies showed that [11C](R)-PK11195 has significant enhanced binding during the quantitative analysis of AD patients in the entorhinal, cingulate cortex and temporoparietal brain region. Recent studies also confirmed the ability of [11C]PK11195 to minimize Aβ plaques in 3xTg-AD mice and provide effective improvement in AD related cognitive and pathological outcomes.[34] This tracer does not exhibit any binding sensitivity to A147T TSPO in both clinical PET scans and human brain tissue.

Figure 4

First generation TSPO tracers.

Later, Cappelli et al.[36] explained the role of the carbonyl dipole in this skeleton toward TSPO binding. Two of the ligands (chloromethyl and 2-fluorophenyl derivatives) in this study demonstrated promising results toward binding affinity for TSPO comparable to [11C]PK11195. Though [11C]PK11195 was the first clinical prototype for PET imaging of TSPO, it showed limitations which include low in vivo specific binding, relatively very high lipophilicity, and a quasi-satisfactory metabolic profile. These limitations motivated researchers to look for new skeletons, which were later considered as second generation TSPO tracers.

Second Generation TSPO Tracers

Second generation TSPO tracers belong to different structural classes with exceptionally better properties such as lower lipophilicity, higher binding affinity, improved signal-to-noise ratio, etc.[37,38] The second generation tracers of TSPO have been divided into seven classes, which are shown in Figure . Representative examples of second generation TSPO tracers have been mentioned in Figure .

Figure 5

Second generation TSPO tracers.

Phenoxy–Phenyl Acetamide

[C]DAA1106, N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxy-phenyl)acetamide, is a 2-phenoxy-5-fluoroanilide derivative which possesses more than 5-fold higher binding affinity and specificity for TSPO compared to the [11C]PK11195.[39] The opening of the diazepine loop of Ro5-4864 led to this novel DAA1106 ligand for TSPO. DAA1106 and N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (DAA1097) were designed by modification in the bicyclic structure of Ro5-4864. Probst et al.[40] explained two robust methods for the radiosynthesis of [11C]DAA1106 which were performed on a nuclear interface module having 25% radiochemical yields. [11C]DAA1106 was considered as a species-independent PET tracer, as proven by its higher specific binding affinity in both rat and monkey brains (Ki = 0.43 nM and 0.188 nM, respectively).[42] Later effects of various substitution arrangements of phenoxy–acetamide derivatives on TSPO binding were also studied.[43,44]

[F]DAA1106 was also reported as an effective tracer for the imaging of target proteins with PET. [18F]DAA1106 has some advantages over [11C]DAA1106 due to the extended half-life of 18F. Initially (phenyl)(mesityl)iodonium salt precursor was used which was not very stable and provides [18F]DAA1106 having lower radiochemical yield (11%). For human clinical studies, Kumata et al. 2018[41] explained the synthetic methodology for [18F]DAA1106 through automated modules in which a spirocyclic iodonium ylide (SCIDY) was used as precursor. This radiosynthesis process was a Cu-free, one-step reaction having a radiochemical yield (6%). In the biodistribution analysis of [18F]DAA1106, minimum accumulation of radioactivity was observed in bone without significant in vivo defluorination. In the metabolite study, a minimum quantity of radiolabeled metabolite was observed in the brain.[41][F]FMDAA1106 is another methyl fluorinated analogue of DAA1106, which possess almost similar binding affinity for TSPO as DAA1106 but showed substantial absorption in bones in mice and monkeys.[45]

[F]FEDAA1106, the ethyl fluorinated analogue of DAA1106, of the phenoxyphenyl-acetamide class, exhibited higher binding affinity and is a more potent tracer for TSPO compared to [11C]DAA1106 and [11C]PK11195.[46] For the preparation of the fluorinated ethyl analogue of DAA1106, 18F-fluoroethyl bromide was formed by treating 18F–F and 2-bromoethyl triflate using a modern automated system.[47]

[C]PBR28, N-(2-methoxybenzyl)-N-(4-phenoxy-pyridin-3-yl)acetamide, is another analogue of [11C]DAA1106 described as a TSPO PET tracer with better properties than [11C]DAA1106 and [11C]PK11195.[48] [11C]PBR28 was prepared by a reaction between the phenolic precursor and [11C]CH3OTf through O-[11C]methylation in the presence of NaH. The radiochemical yield for this reaction was 70–80% at EOB, based on [11C]CO2.[48] The reaction temperature was raised to 80 °C to reduce the reaction time to only 3 min. Besides that, a “vial” method was used in place of the previously used “loop” method.[49] [11C]PBR28 showed superior properties, including a high binding affinity, lower lipophilicity, improved signal-to-noise ratio, more suitable pharmacokinetics, and an 80-fold higher in vivo association affinity in the monkey brain.[50−52] [11C]PBR28 has also been used in human subjects and exhibited different activities for in vivo PET imaging of TSPO. Because of the lack of real reference area for TSPO expression in the brain, there is much importance to an arterial input activity for the correct assessment of [11C]PBR28 association and total volume distribution.[53] For human subjects, the correct and acceptable quantification of [11C]PBR28 binding is of great importance. The result of this quantification included great benefits like the use of the smallest sample size with a high power, the simplest detection of effects, and a reduction in PET study charges. A new and well developed method (SIME) effectively described the [11C]PBR28 specific binding by deriving estimates.[54,55] Recently, [11C]PBR28 was used successfully in alcoholism patients. In the case of rodents, [11C]PBR28 shows similar brain accumulation among alcohol nondependent rats and alcohol dependent rats.[56]

6-[F]PBR28 and [F]FM-PBR28 are the two other fluorinated analogues of PBR28, which were compared with [3H](R)-PK11195 and [11C]PBR28, respectively, in the same inflammatory rat model. Ex vivo autoradiography showed the distribution of 6-[18F]PBR28 to the cerebellum and olfactory bulb.[57,58]

[ The fluoroethoxy derivative of [11C]PBR28, named [18F]FEPPA, is another TSPO tracer mainly used for the in vivo PET imaging in animals and humans. A nucleophilic substitution reaction was performed with a tosylate precursor at 90 °C followed by HPLC purification (semipreparative). This radio-synthesis was carried out within 55 min with a radiochemical outcome of 34 ± 2% and a molar effectiveness of 198 ± 125 GBq/μmol at EOS.[59,60] In other work, high radiochemical yields of [18F]FEPPA having high specific activity were obtained in a one-pot reaction between its precursor and [18F]fluoride.[61]

In the rat mitochondrial region, FEPPA exhibited favorable binding affinity compared to PBR28, DAA1106, and PK11195.[59] The time activity curve of [18F]FEPPA also shows significantly increased cerebral dispensation in LPS mice when compared with regulation in different regions.[60] Except for all of these superior properties, FEPPA also tolerates few limits to its application, such as three types of binding pattern in human subjects and major problems in quantification due to the lack of reference tissue. From various studies of complete distribution in rats and athymic mice, a higher uptake in the peripheral parts like hearts and kidneys was reported when differentiated from the brain.[59−61] Initially, the in vitro consequence of polymorphism of FEPPA was not reported; only in vivo polymorphism has been considered. But recently, in vitro considerations for the polymorphism rs6971 in human colorectal cancer suggested a similar behavior of FEPPA to those of other TSPO PET tracers with respect to polymorphism rs6971.[62−64]

[F]PBR06 and [C]PBR01 were other derivatives of phenoxyphenyl acetamide that also exhibited effective binding affinity and higher brain uptake for the TSPO in animals and clinical studies parallel to PK11195.[65] The complete synthesis and radiolabeling of PBR06 was described previously by Wang et al.[66] Both tracers showed ample selectivity in their blocking studies. [11C]PBR01 was blocked by PBR01 (nonradioactive form), whereas [18F]PBR06 was specifically blocked by DAA1106.[65]

Indole-Acetamide

[C]SSR180575, (7-chloro-N,N-5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyrid-azino[4,5-b]indole-1-acetamide), is one of the most effective tracers of this class that characteristically participates in the encouragement of neuronal survival and repair.[67] In the brain, [11C]SSR180575 enhances the accumulation of pregnenolone, suggesting its therapeutic effects in steroidogenesis. Damont et al.[68] reported different derivatives of SSR180575 and their respective binding affinity against reference compound [3H]PK11195. All derivatives exhibited nanomolar to subnanomolar binding for TSPO. By introducing fluoroalkoxy substitution at the para site of the phenyl ring, binding affinity toward TSPO increases. In addition, a fluorinated side chain with a polyethylene glycol system in the structure lowered the binding affinity due to the steric limit in the structure. Damont et al.[68] also explained that different substitutions at the N-indole position play the most distinctive role in deciding the superior potency of various derivatives where increasing the fluoroalkoxy chain leads to minimal binding (Table S1).

Vinyl Alkaloid

Eburanamenine-14-carboxyic acid ethyl ester is a derivative of the Vinca minor alkaloid, vincamine, which was initially identified in 1960 and introduced to the market in 1978. It exhibited effective pharmacokinetic properties and has been used for the therapy and relaxation of some cerebrovascular diseases such as a neuroprotective agent.[69] For the preparation of [11C]vinpocetin, two methods have been reported where the semisynthetic method was more effective in terms of better yield, low cost, and simple technology.[69] Initially, Monelo’s patent reported a synthetic route in which Changchun alanine was used as a starting material; 2-fluoro nitrobenzene and acetonitrile were used as a catalyst and solvent, respectively. Despite its high yield, the method suffers from some major limitations such as rare availability of the catalyst and toxicity of the solvent. The second method overcomes all of these limitations where Ti(OEt)4 was used as a catalyst and kuge-like vincamine as the raw material. Ti(OEt)4 catalyzed transesterification to the obtained ethyl vancamine.[70]

It has been successfully used in a PET study as [11C]vinpocetin for four multiple sclerosis (MS) patients. [11C]Vinpocetin showed much more binding efficiency in comparison to [11C]PK11195.[72] [11C]vinpocetin pretreated cynomologous monkeys displayed reduced PK11195 uptake, whereas similar pretreatment with PK11195 increased the accumulation of [11C]vinpocetin by peripheral blockage of the TSPO and suggested its clinical neurological properties. In the brain tissue, [11C]vinpocetin showed very low in vitro association affinity (IC50 = 0.2 μM) with TSPO with the heterogeneous and rapid uptake among all other brain regions, including the brain stem, thalamus, cortex, and striatum except the cerebellum.[73] The main restriction with [11C]vinpocetin was its nonspecific binding with other receptors, ensuring less selectivity for the target protein. Due to all of these reasons, the in vivo selectivity of [11C]vinpocetin for TSPO was questionable. For the determination of binding of [11C]vinpocetin with special areas in the human post-mortem brain, Glyan et al.[71] performed autoradiography and suggested its inferior binding toward the human brain transmitter receptors, which clearly explained the difference in [11C]vinpocetin uptake value between the thalamus, basal ganglia, and cortex.

Aryl-Oxodihydropurine

[C]AC-5216, ([11C]-AC-N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2-pheyl-7,8-dihydro-9H-purin-9-yl)acetamide), is an efficient dihydropurine tracer and the first candidate of the aryl-oxodihydropurine class. It has antidepressant- and antianxiety-like properties, which are mediated by TSPO and utilized in a cancer model.[74] AC-5216 was synthesized from benzamidine hydrochloride by Lee et al.[75] This work also studied the effect of substituted fluorine at different positions of the phenyl ring of aryl-oxodihydropurine in terms of binding efficiency and lipophilicity of the TSPO radiotracers. Out of ortho-, meta-, and para-fluoro derivative of oxodihydropurine, meta substituted derivative displayed highest binding affinity toward TSPO.

AC-5216 radiolabeled with C-11 revealed high binding efficacy (Ki = 0.20 nM) in the rat brain and plays a distinctive function in the displacement of [3H]PK11195 from rat C6 and human Hs683 glioma-derived mitochondrial fractions. It showed improved uptake in the thalamus and inferior binding in the striatum, during human PET studies when coadministration with PK11195.[76] Autoradiography on a kainic acid (KA)-lesioned rat brain (rat model of neuroinflammation related to Huntington’s disease) proved the ability of [11C]AC-5216 to visualize TSPO in neuroinflammation.[77] In addition a PET study on the same preclinical subject reported maximum radioactivity of [11C]AC-5216 in the brain, which is 4- to 6-fold higher than that of the reference compound [11C]PK11195.

[N-benzyl-N-[11C]methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide is a novel analogue of [11C]AC-5216 that exhibits impressive binding efficacy relative to [11C]PK11195(Ki = 0.30 nM) and less lipophilicity than [11C]AC-5216. Lower lipophilicity of [11C]DAC suggested its least nonspecific association with other receptors such as the GABA receptor.[78]In vitro autoradiography of DAC in a unilateral kainic acid (KA)-lesioned rat showed an excellent signal-to-noise ratio, effective differentiation among nonlesioned and lesioned striatum, and increased binding affinity with TSPO concluding it as a selective and potent PET tracer of TSPO for preclinical and clinical investigation. Surprisingly, in the mouse brain homogenate, the detection of [11C]DAC was very low, and its radioactive metabolite could not enter into the brain despite its fast metabolism in plasma, suggesting noninfluence of the radioactive metabolite of [11C]DAC on binding with TSPO.[78]

Except these two promising tracers, [F]FEAC, [F]FEDAC, and [F]FAC were the other three most promising TSPO tracers of the aryl-oxodihydropurine family[75] that display good binding and specificity for TSPO. Among all of the oxopurine derivatives, [18F]FAC was the most effective TSPO tracer, having the highest binding affinity with a Ki value of 0.15 nM compared to [11C]AC-5216 (Ki = 0.20 nM) and [11C]DAC (Ki = 0.23 nM).[79] All three tracers were very potent over the previously known [11C]AC-5216 and [11C]DAC because of the long life of the 18-F isotope, opposite of the 11-C isotopes. 18-F labeling was most favorable for longer collection and transportation and has the ability to give perfect visualization with better resolution. Except for this, they also have some other advantages such as a good signal to neuroinflammation and fast kinetics in the brain and high selectivity for TSPO. Complete synthesis of all of these fluoro derivatives of aryl-oxodihydropurine have been explained by Kawamura et al.[80]

Pyrazolopyrimidine

Pyrazolopyrimidine derivatives were obtained by the modifications at the fifth and seventh positions of the alpidem nucleus and exhibited high association affinity (ranging from 0.8 nM to 6.1 nM) and specificity for the target protein (TSPO).[81] Werry et al.[82] reported that the substitution of various groups at the para site of the phenyl ring in the imidazopyridine system showed good binding efficiency with higher specificity for TSPO. Derivatives with a long carbon chain were able to activate apoptosis and show antiproliferative properties in both human fetal astroglial cells (SVG p12) and T98G cells. Introduction of a benzyl group at this site is also favorable for high binding energy toward the target protein.

[C]DPA713, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide, was effectively used in the in vivo PET imaging of healthy human brain and showed increased accumulation in all regions of the brain.[82] Thominiaux et al.[83] in 2006 described an effective and improved method for the radiolabeling of DPA713 using 11-C methyl triflate with a radiochemical yield of 30–38%. [11C]DPA713 showed high distinction among healthy and diseased brain parenchyma in an AMPA induced rat model for neuroinflammation.[84]

[F]DPA714, N,N-diethyl-2-(2-(4-(2-fluoro-ethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide), is a fluoroethoxy analogue of [11C]DPA713 and another tracer of the pyrazolopyrimidine class.[85,86] [18F]DPA714 was obtained from its tosylate precursor (N,N-diethyl-2-(2-(4-(2-toluenesulfonyl- oxyethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) with fluorine-18 through a nucleophilic substitution reaction in 32% yield.[86] [18F]DPA714 exhibits a higher binding affinity (Ki = 7.0 nM) for the TSPO compared to [11C]PK11195 (Ki = 9.3 nM) and lower with respect to [11C]DPA713 (Ki = 4.7 nM).[87] [18F]DPA714 successfully identifies a neuroinflammatory location in the brain with a good target to background signal ratio. Recently, Wang and co-workers stated that [18F]DPA714 has enhanced lesion-to-normal brain ratios in CCI rats.[88,89] These PET tracers have been successfully implemented for the assessment of TSPO in healthy humans during stroke and Alzheimer disease.[90,91] Currently, [18F]DPA-714 participates in the assessment of the consequences of bone marrow stromal cell (BMSC) delivery, in which it shows the ability to display brain inflammation and assess the BMSC therapeutic impact.[92]

[F]F-DPA, N,N-diethyl-2-(2-(4-fluorophenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide, is another fluoro derivative of the pyrazolopyrimidine group that has binding efficacy, Ki = 1.7 nM, and specificity for the TSPO. In [18F]F-DPA, the fluorine atom is directly associated with the aromatic system (phenyl ring) in place of an alkoxy bridge between the fluorine atom and phenyl ring. The complete synthesis and radiolabeling of [18F]F-DPA has been described by Keller et al.[93] At the periphery, a greater uptake of [18F]F-DPA is attained in the adrenal cells, spleen, heart, lungs, kidneys, and small intestine. Recently [18F]F-DPA also participated in microglia enhancement on a mouse model experiment of Alzheimer’s disease.[94] Because of its rapid washout, [18F]-FDPA shows good differentiation results when compared with [18F]DPA714.

[ is a novel derivative of [18F]DPA714 and another pyrazolopyrimidine derivative with unique properties. It is synthesized by some major structural alterations in the [18F]DPA714 to avert the defluorination issues associated with DPA714.[95] The main structural modification is replacement of the fluorinated moiety with high robust linkage and a phenyl ring of the core structure bridged by an oxygen atom. [18F]CfO-DPA-714 exhibits high binding affinity (in the nanomolar range) compared to [11C]DPA713 and [18F]DPA714, which proves it as an ideal candidate for TSPO imaging.

Cacheux et al.[95] studied the effects of numerous different substitutions of pyrazolopyrimidine derivatives for TSPO binding. All analogues of CfO-DPA-714 exhibited nanomolar to subnanomolar range binding affinities. Introduction of an aromatic moiety at a similar site did not much affect the binding toward TSPO. However, increasing the methylene (−CH2) chain length between the phenyl ring and the nitrogen atom of amide moiety decreases the affinity to a greater extent. These outcomes evidently show that cumulative steric hindrance is unfavorable for the binding affinity of CfO-DPA-714 derivatives.

[F]VUIIS1018A, another newly synthesized analogue belonging to pyrazolopyrimidines, displayed high BPND (nondisplaceable binding affinity) and 20-fold and 700-fold high binding affinity compared with the previous tracers [18F]VUIIS1008 and [18F]DPA-714, respectively. [18F]VUIIS1018A has been synthesized by the modifications at the fifth and seventh positions of the pyrazolopyrimidinal scaffold.[96]

This new tracer displayed an exceptional binding affinity (IC50 = 16.2 pm) to TSPO. Taking the benefits of its binding, this tracer has been represnted as a better candidate to image minimal grade tumors or tumors with low TSPO expression.[97]

Imidazopyridine-Acetamide

Trapani et al.[98] compiled the effects of different substitutions on TSPO affinity and specificity of imidazo-pyridine derivatives. Later on, Wermuth and Bourguignon reported another series of imidazopyridine derivatives with some structural modification in the Trapani’s series derivatives. These modifications included the introduction of an ionizable or polar functional moiety at a para site of the 2-phenyl imidazopyridine skeleton. High polarity groups at the para site of the 2-phenyl ring were the most effective in terms of TSPO efficacy.

[C]CLINME, (2-[6-chloro-2-(4-iodophenyl)-Imidazo[1,2-a]pyridin-3-yl]-N-ethyl-N-methyl-acetamide, is a tracer of the imidazopyridine-acetamide family that has effectively been used in acute rodent neuroinflammation.[99] CLINME is (2-[6-chloro-2-(4-iodophenyl)-imidazo[1,2-a]pyridin-3-yl]-N-ethyl-acetamide), which is radiolabeled with C-11.[100] It is also radiolabeled with the 123-I isotope (with 70–80% radiochemical yield) by the electrophilic introduction of a tributyltin precursor using 123/125I-sodium iodide in acetic acid and per acetic acid as the oxidant.[101] It showed high binding for TSPO, but under in vivo conditions the uptake was lower in the noninflammatory brain region compared to [11C]PK11195, but in inflammation regions both tracers display almost similar uptakes.[100] [11C]CLINME is perceived as superior tracer among imidazopyridine tracers as it displays almost 100-fold higher specificity for TSPO against CBR. This skeleton has been used for PET imaging with radiolabeling with either I-123 or C-11 and 99 mTc based SPECT imaging.[101,102]

[C]PBR111 is an additional imidazopyridine–acetamide derivative that exhibits more impressive TSPO binding in the nanomolar range (Ki = 3.7 ± 0.4 nM) in comaparison to [11C]CLINME and [11C]PK11195.[103] [18F]PBR111 is fluoropropoxy-substituted-2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridine-3-yl)-N,N-diethylacetamide, a fluoro derivative of [11C]PBR111, possessing good in vivo/in vitro binding affinity to TSPO. In the two-tissue compartment model, the in vivo/in vitro binding of [18F]PBR111 has been fully explained by the accelerated aging hypothesis.[103]

The other PET tracer which showed similar efficacy and potential to become a TSPO marker was [18F]PBR102. It also exhibited similar binding and in vivo activity as described by [11C]PBR111 and possesses much better BPND with a minimum signal-to-noise ratio compared to [11C]PK11195.[104]

[C]CB184, (N,N-di-n-propyl-2-[2-(4-[11C]methoxyphenyl)-6,8-dichloroimidazol[1,2-a]pyridine-3-yl] acetamide, is a superior alternative to [11C]PK11195 which displays 7-fold more enhanced binding affinity (Ki = 0.54 nM) and reduced lipophilicity compared to those of PK11195. The complete radio-synthesis has been reported by Denora et al.[105] In PET analysis in a rat animal model, the VT of this tracer was greater in the thalamus and then in the cerebellar cortex. The binding pattern of [11C]CB184 was compatible with the TSPO distribution in a normal human brain with small regional variations.[106]

Acetamidobenzoxazolone

Fukaya et al.[107] thoroughly investigated the consequence of numerous substitutions at diverse positions of the benzoxazolone ring on the binding efficacy and specificity for TSPO (Tables S2–S5).

Taking a lead from these positive findings, [C]MBMP, 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenyl-acetamide, a novel third generation TSPO tracer belonging to the acetamidobenzoxazolone family, has been developed for effective PET imaging of microglia at neuroinflammation sites and the ischemic rat brain.[107,108] [11C]MBMP is obtained by its desmethyl precursor, which is synthesized by 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol and bromo compounds in the mediation of a palladium catalyst (Pd(PPh3)4) through the Suzuki coupling reaction. [11C]MBMP may not have important applications over the various second generation radiotracers, but it demonstrates high association affinity (Ki = 0.29 nM), favorable lipophilicity, higher brain uptake within the minimum time, and negligible nonspecific binding with other receptors such as CBR.[107,109] The autoradiographic studies of [11C]MBMP reveal high binding in the ipsilateral site opposite the contralateral areas, and the contrast disappears with [11C]PK11195, which clearly suggests its superior in vivo affinity with the TSPO. The biodistribution study on [11C]MBMP displayed high radioactive accumulation in the protein-rich peripheral (steroid synthesizing) organs like the blood, heart, lungs, and brain.[108] However, roughly 20% of [11C]MBMP input was identified as a metabolite in the mouse brain, which stalled its further application. To solve this matter 18F-labeled analogues of MBMP, [18F]FEBMP, and [18F]FPBMP were developed for PET.[108−110]

[F]-FPBMP, 2-[5-(4-fluoropropoxy-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-methyl-N-phenylacet-amide, another analogue of [11C]MBMP, displayed similar affinities for TSPO.[110] [18F]FEBMP also exhibited some other advantages such as thw full observation of neuroinflammation in the focal ischemia rat model, greater binding efficiency for TSPO, favorable lipophilicity, and higher penetration into the BBB, confirming the superiority of [18F]FEBMP as an encouraging tracer for TSPO PET imaging.

Another benzoxazolone scaffold-based TSPO tracer, [C]NBMP, N-methyl-2-(5-(naphthalen-1-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-phenylacetamide, has been reported recently and demonstrated the strength to resolve the difficulty of intersubject variability on binding studies in its initial in vitro and preclinical studies.[111] The main advantage with NBMP is its facile synthesis by the reaction among 2-(5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-phenylacetamide and napthalene-1-boronic acid through the Suzuki coupling reaction and better bioavailability, plasma clearance, and suitable metabolic strength in human and rat liver S-9 fractions.[111,112] Both radiotracers ([18F]FEBMP and [18F]FPBMP) have lower lipophilicity compared with reference compound [11C]PK11195, suggesting effective initial brain accumulation for [18F]FEBMP and [18F]FPBMP, the opposite of [11C](R)-PK11195.

[C]′-MPB, [11C]N-(2-methoxyoxyphenyl)-N-methyl-2-(5-nitro-2-oxobenzo[d]oxazol3(2H)-yl)acetamide, another newly synthesized acetamidobenzoxazolone-based tracer, showed good binding efficacy toward TSPO with a Ki value of 4.9 nM.[113] [11C]N′-MPB was prepared by treating its precursor with [11C]methyl iodide with good radiochemical yields (27 ± 08%) and 1.5 h of radiochemical stability. In vitro autoradiography on post-mortem human brains with a previously known tracer of this class [18F]FEBMP exhibited low influence by TSPO rs6971. So, [11C]N′-MPB has advantages over other previously known tracers of the same class.[113]

Some Major Drawbacks of Second Generation Tracers

Despite various efforts to prepare new TSPO PET tracers in clinical studies over past 25 years, several major restrictions are still associated with TSPO as a molecular biomarker of neuroinflammation.[114,115]

The major restrictions include reduced binding, amino-acid (A147T) substitution due to the only nucleotide polymorphism in the gene (rs6971) in the target protein, nonaccuracy in the estimation results of plasma concentration, and, last, difficulty in obtaining perfect kinetic correction for the tissue signals. This polymorphism has affected the second generation tracer-binding affinity of TSPO. Three different forms have been found in association with this polymorphism: Ala/Ala is related with HAB (high affinity binding = 66% of the Caucasian population); Ala/Thr is related with MAB (mixed affinity binding = 29% of the population), while Thr/Thr is linked with LAB (low-affinity binding = 5% of the population).[116−119] Due to these three large forms, intersubject variation in binding has been found in comparison to [11C]PK11195. Given the fact that second-generation PET tracers have been recognized for their sensitivity toward rs6971-polymorphism, this was the main region for scientists to develop third-generation tracers with better properties.[118,119]

Another concern in these TSPO tracers is their nonselectivity for enhanced microglia, as it is also expressed by other types of immune cells, which include astrocytes and others. Similarly, these tracers are also unable to detect the phenotype of microglia and astrocytes, which is also altered with disease progression in neuroinflammatory conditions.[114] These two factors significantly limit the data interpretation or correct quantification of microglia in different neurologic disease. Therefore, alternative and improved radiotracers are required to detect activated microglia. Few researchers have also raised concerns about using the reference tracer, which also requires cluster analysis with dynamic PET studies.[115]

Third Generation and Recent PET Tracers for TSPO

The discovery of third generation tracers overcame almost all of the demerits of second generation tracers and delivered better results in preclinical and especially clinical studies. Figure shows some of the representative structures of third generation TSPO tracers and some new tracers which are supposed to overcome the previous generation’s limitations.

Figure 6

Structural view of new generation PET tracers for TSPO.

Tricyclic Tracers

Wadsworth et al.[120] were the first who reported the effect of different substitutions at various positions of the phenyl ring of the tricyclic scaffold on the binding affinity with TSPO.

Tricyclic-indole compound [F]GE180, (N,N-diethyl-9–2-18F-fluoro-ethyl]-5-methoxy2,3,4,9-tetrahydro-1H-carbazole-4-carboxamide, is a third generation PET tracer that exhibited ideal characteristics for TSPO PET imaging.[120−124] The radiosynthesis of [18F]GE180 has been described by Wadsworth et al., where the final precursor was directly radiolabeled with fluorine-18 from its mesylate derivative on the automated platform FASTlab. Radiochemical yield was found to be 25–35% (nondecay corrected) after HPLC purification. Initially, it was evaluated as recemate, but later it was found that the S-enantiomer has better characteristics to become a standard TSPO PET tracer.[122] It has also improved TSPO binding in both rat and human subjects with Ki values of 0.87 nM and 9.2 nM, respectively, than the R enantiomer (Ki = 3.87 nM in rats and 14.1 nM in humans).[122] Though [18F]GE180 did not achieve the same degree of precision as [11C]DPA-713 for microglial activation, it did have enhanced imaging properties compared to both [11C]PK11195 and [18F]DPA-714 in the preclinical model. The signal-to-noise ratio in focal lesions was shown to be high in relapsing–remitting multiple sclerosis patients, with no notable difference in signal strength among HABs, MABs, and LABs.[121−124] But recent investigations showed that the insensitivity of [18F]GE180 to polymorphism is only due to its poor grade images that cannot be accurately quantified. Second, a greater lesion-to-background ratio was found because of the use of BBB split areas.[125−127]

[F]FEBMP, 2-[5-(4-fluoroethoxy-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-methyl-N-phenyl-acetamide, a novel fluoroalkoxy tracer of the acetamidobenzoxazolone family having nanomolar binding with suitable lipophilicity was developed for TSPO visualization.[128] This fluoroalkoxy analogue [18F]FEBMP was synthesized with 59% yield by heating their precursor with the respective tosylate molecule. Complete synthesis has been mentioned in detail by Tiwari et al.[109]

Similar to [11C]MBMP, the biodistribution of [18F]FEMBP in mice reported high radioactive accumulation in TSPO expressed organs. To assess the effectiveness of [18F]FEBMP for predicting neuroinflammation, focal cerebral ischemia (FCI) rat models having activated TSPO expression have been used.[128]

In vitro autoradiography on post-mortem human brains with [18F]FEBMP exhibited low influence by TSPO rs6971 and possessed nearly identical responses in nonbinder and binder brain compartments. In spite of the excellent properties and effective applications of new generation TSPO tracers, they face certain shortcomings such as an incapability to discriminate between pro-inflammatory microglia and anti-inflammatory microglia, which results in many difficulties in the data interpretation and evaluation of the functional character of microglia in neurological and neurodegenerative disease.[129,130]

[C]ER176, 11C-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide, a modern promising quinazoline relative of [11C]PK11195 for protein quantification, possesses very mild in vitro sensitivity to rs6971 and also shows high binding efficacy in the monkey brain.[131,132] The radiosynthesis of [11C]ER176 has been reported with great selective activity (55 GBq/μmol at EOS) by Castellano et al.[133] In a human brain experiment, four tracers, [11C](R)-PK11195, [11C]DPA-713, [11C]PBR28, and [11C]ER176, compared for TSPO quantification based on described specific-to-non displaceable ratios, and the [11C]ER176 showed a second supreme BPND value and specifically gave a time specific value of VT (tissue volume of distribution) in both HABs and LABs after protein siege by XBD173.[134,135]

[F]FTPQ, [18F]-2-(4-fluoro-2-(p-tolyloxy)phenyl)-1,2-dihydroisoquinolin-3(4H)-one, is another newly developed phenoxy-acetamide derivative with a substituted acetamide moiety. The overall synthesis of [18F]FTPQ has been completed in six steps with the use of a copper catalyst with an overall radiochemical yield of 19% (decay corrected). In a Parkinson’s rat model, [18F]FTPQ has been considered to have a lot of potential for the noninvasive detection of brain changes.[136]

(R)-[F]NEBIFQUINIDE, N-(sec-butyl)-1-(2-fluoropyridin-3-yl)-N-methylisoquinoline-3-carboxamide, is a newly synthesized 18F labeled pyridinyl isoquinoline analogue of [11C](R)-PK11195. The 18F-labeling of [18F]NEBIFQUINIDE is believed to proceed via nucleophilic aromatic interchange reaction and resulted in high radiochemical yield. But the 2-halopyridines activation is somewhat weak and requires very drastic conditions (20 min, ≥150 °C) for successful radiofluorination; therefore, low alteration rates (0.1–0.5%) are achieved even at elevated temperatures.[137] For various human TSPO phenotypes, [18F]NEBIFQUINIDE showed improved binding efficacy and better metabolic stability. This newly synthesized PET tracer has the ability to overcome all of the limitations of [11C]PK11195 like enhanced blood–brain barrier penetration, improved specific binding, a long half-life, and high metabolic stability.[137]

[F]F-FETEM, [18F]fluoroethyltemazepam, (3S)-7-chloro-3-[18F]fluoroethyl-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one, is the newest fluorinated benzodiazepine tracer.[138] For the preparation of this radiotracer in one step, an automated synthesizer is used at a 100 °C temperature with DMSO. However, further validation for in vivo studies in neurodegenerative animal models is awaited.

[18F]Fluorovinpocetine

Recently, a fluorinated analogue of vinpocetine, [18F]fluorovinpocetine, has been synthesized to overcome the limitations of [11C]vinpocetine. 11C radiolabeling of vinpocetine was performed by the reaction of 11C-ethyl iodide and apovincaminic acid in an automated module and completed in three steps.[139] PET studies in cynomolgus monkeys showed that both [18F]fluorovinpocetine and [11C]vinpocetine have similar behaviors in terms of high BBB permeability, high regional uptake, and fast clearance from a non-human primate brain. The computational studies of these two compounds showed identical conformations into the binding pocket of human TSPO protein. On the basis of these results, [18F]fluorovinpocetine has been suggested as an alternative to [11C]vinpocetine for TSPO specificity and sensitivity to rs6971 polymorphism.[139]

[F]LW223 is the most recent 18F labeled fluorinated TSPO-PET tracer with requisite characteristics and insensitivity toward rs6971 polymorphism in human tissue. Ex vivo immunofluorescence staining for TSPO and CD68 (a macrophage marker) reveals a similar device as in the in vivo BPTC (transfer corrected binding potential) study.[140] [18F]LW223 can be the lead candidate for clinical translation, which could ultimately control the advancement of a prognostic tool for function in patients following myocardial infarction.

[F]PBR316 is a new tracer which showed low sensitivity to rs6971 polymorphism in which the oxoacetamide (bis-carbonyl) group has been incorporated. This tracer was developed after replacement of the 4′-fluoroalkoxy group with a 4′-fluoroethyl on the 2-phenyl ring of [18F]PBR111/[18F]PBR102. These structural modifications provided high affinity toward TSPO and displayed 1000-fold selectivity over CBR. The LAB/HAB ratio was 1.5, which was comparable to PK11195.[141]

The recently synthesized TSPO tracer [F]GE-387 was introduced by Qiao et al.[142] This chemical scaffold showed a LAB/HAB ratio of 1.2. The precursor was obtained in eight steps having an 8% outcome. The chiral separation of this derivative was obtained by using supercritical fluid chromatography (>95%). Subsequently automation of radiosynthesis were used to get R and S enantiomers having yields in the range of 20–25% and molar activities of 55.8 ± 35.6 and 63.5 ± 39.5 GBq/μmol.

Third generation PET tracers have more of an advantage than previous generations due to minimum susceptibility to human rs6971 polymorphism, but their evaluation still needs further comparisons and clinical experiments to examine.[142,143]

[F]BS224, 2-(-2-(4-[18F]fuorophenyl)-6,8-dichloro-imidazo[1,2-a]pyridin-3-yl)-N,N-dipropylaceta-mide is the newest 18F-labeled imidazo[1,2-a]pyridine analog-based TSPO tracer, which possesses a high binding efficiency (Ki = 0.51 nM) and specificity for TSPO.[144] [18F]BS224 was prepared with the use of boronic acid pinacol ester via aromatic 18F-fuorination (with 39 ± 6.8% radiochemical yield) by using 18-crown-6/CsHCO3 as a phase transfer catalyst.[145]In vitro analysis with membrane proteins displayed minimum binding responsiveness to the rs6971 polymorphism. This value was comparable to PK11195, which is supposed to be not sensitive to the polymorphism.[145] [18F]BS224 was used in LPS-induced inflammatory and ischemic stroke rat experiments to visualize inflammatory lesions having a BPND = 1.43 ± 0.17 and 1.57 ± 0.37.

[F]CB251, 2-(2-(4-(2-[18F]fluoroethoxy)phenyl)-6,8-dichloroimidazo[1,2-a]pyridin-3-yl)-N,N-dipropylacetamide, is another fluorine-substituted imidazopyridine-acetamide modified analogue which has been reported with good binding affinity (Ki = 0.27 ± 0.09 nM) and specificity for TSPO.[146] Recently, [18F]CB251 was found to be specific toward TSPO-expressing inflammatory cells having a lesser impact of TSPO polymorphism. PET imaging with [18F]CB251 detected the areas of neuroinflammation and also permitted assessment of the therapeutic impact of anti-inflammatory therapy.[146−150] This study was performed by competitive inhibition assay by using membrane proteins isolated from 293FT cells. The ratios of IC50 LAB/HAB were found much better than with GE-180 (1.14 vs 3.96).

Conclusion and Future Outlook

Translocator protein 18 kDa dysregulation has been stated in numerous disease conditions, particularly related to the CNS. To date, various TSPO tracers belonging to different classes have been developed and studied in detail in vitro/in vivo for no-otropic, neuroprotection, anxiety, and antidepressant activities and explored for staging/visualizing neuroinflammation by using molecular imaging techniques, exclusively PET. Even though predominant TSPO expression by microglia is seen in the brain, its expression by other cell types such as peripheral tissues, in brain astrocytes, and the vascular endothelium is a huge hitch itself and should be well considered.

Even though the first classical TSPO ligand, [11C](R)-PK11195, disclosed the existence of microglial activation in glioma, stroke, and multiple scelorosis, the pharmacological potential of [11C](R)-PK11195 faced a huge setback because of maximum nonselective binding and a poor ability to cross the BBB, responsible for its poor signal in both preclinical and clinical conditions where the prominent microglial activation feature is existent. The foremost problem linked to TSPO quantification is associated with its pharmacological presence not only in glial cells of the brain but also in the whole neurovascular system itself. The TSPO density in platelets and monocytes alters with the type of disease, but the fraction of bound radiotracer remains stable at equilibrium.

The subsequently discovered TSPO PET tracers, termed “second generation tracers”, were established on the grounds of concerted restrictions of previously established tracers. Despite the fact that second-generation TSPO tracers have enhanced the specific to nonspecific association ratio, they are responsive for a polymorphism (rs6971) in the TSPO gene. Since the conventional PET imaging in rodents or monkeys could not discriminate rs6971 polymorphism, the human derived in vitro TSPO-specific receptor binding assays would aid in the discovery and development of next-generation TSPO PET tracers with low responsiveness or insensitivity to the genotype.

Lately, numerous next generation TSPO PET tracers that are insensitive to single nucleotide polymorphism (SNP)/rs6971 have been documented, such as [18F]FEBMP, (R,S)-[18F]GE387, (R)-[18F]NEBIFQUINIDE, [18F]LW223, and [18F]PBR316. But all of them have some challenge associated with them; namely, the acetamidobenzoxazolone based [18F]FEBMP tracer has shown excellent superiority related to the influence of TSPO rs6971 polymorphism with a LAB/HAB ratio of 0.9 during in vitro autoradiography on post-mortem human brains. However, the metabolic stability of [18F]FEBMP is an issue due to the existence of an augmented radiometabolite in plasma that has the potential to move into the brain. The other reported 18F-labeled TSPO radiotracers such as (R,S)-[18F]GE387, (R)-[18F]NEBIFQUINIDE and [18F]LW223 also overpowered the binding sensitivity of rs6971 of the TSPO gene, but future exploration for brain PET imaging in a neuroinflammatory model needs to be done. [18F]CB251 also depicts excellent potential for the assessment of neuroinflammation and displays greater TSPO affinity as well as selectivity, irrespective of the rs6971 polymorphism, but it shows nonspecific skull uptake due to defluorination of the parent radiotracer and thus hindrance in the exact TSPO quantification in the brain PET images. [18F]BS224, an 18F-labeled imidazo[1,2-a]pyridine analog, is also a next generation TSPO radiotracer that has no effect of rs6971 and also could endure in vivo defluorination due to the presence of the aromatic C–F bond.

Therefore, to tackle the above-mentioned issues, there is an imperative lookout for the development of clinically effective compounds with the necessary requirement for pharmacokinetics and safety. Due to all of these circumstances, other alternative targets such as cannabinoid receptor type 2, purinergic receptor (P2X7), and cyclooxy-genase-2 come into the light for neuroinflammation PET imaging. The latest comparative PET imaging studies using a second-generation TSPO tracer and [11C]JNJ717, a novel PET tracer, for P2X7R in early symptomatic patients with ALS propose the superiority of TSPO to P2X7R imaging. But P2X7 radiotracers also bind to astrocytes with high variability in healthy humans; therefore detection of genotypes related with this alteration might be applied. Another important target, monoamine oxidase B (MAO-B), is found in enhanced astroglial cells, which is also a home site for neurodegenerative diseases. Recently, SL2511.88 has been found to be promising to determine its density in different CNS conditions.

Conversely, all of these alternative targets of TSPO are specified for the pro-inflammatory phenotype of microglia, but for the comprehensive understanding of the neuroinflammatory operation, it is crucial to give consideration to the anti-inflammatory phenotype as well. Advancement in brain-penetrant tracers that targets the anti-inflammatory phenotype of generated microglia would initiate a great development in neuroinflammation imaging. Future analyses are desirable to enhance our perception of the cellular mechanisms that trigger the alterations in PET signals and the contribution of immune sensitivity to the neurological disorder. Additionally, to reach the full potential in CNS applications, further targets and tracers throughout the immune system and other organs must be identified. The present Review is, in particular, based on the sequential advancement made in the progression of TSPO PET tracers during past 44 years for neuroinflammation imaging and also includes the different tactics used for the advancement of new tracers for an improved understanding on the structure–activity relationships. It also discusses the glitches encountered in current TSPO PET imaging tracer discovery efforts and will address these challenges to assist the translation of TSPO in clinical analysis of neuroinflammation related with CNS diseases.