Synthesis, fluorine-18 radiolabeling, and in vitro characterization of 1-iodophenyl-N-methyl-N-fluoroalkyl-3-isoquinoline carboxamide derivatives as potential PET radioligands for imaging peripheral benzodiazepine receptor

The isoquinoline carboxamide derivative 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195) has been shown to bind strongly and selectively to the peripheral benzodiazepine receptor (PBR) binding sites. A series of PK11195 analogues have been synthesized and biologically characterized. The affinities of the analogues for the PBR were determined using in vitro competitive binding assays with [(3)H]PK11195 in rat kidney mitochondrial membranes. The results showed that the 1-(2-iodophenyl)-N-methyl-N-(3-fluoropropyl)-3-isoquinoline carboxamide (9a) was the most potent compound (K(i)=0.26nM) of this series and is an excellent lead ligand for additional studies for labeling with fluorine-18 to determine whether it possesses the desired in vivo performance in non-human primates by PET imaging. Thus, radiolabeling of 9a with fluorine-18 was developed.     

TspO of rhodobacter sphaeroides. A structural and functional model for the mammalian peripheral benzodiazepine receptor

The function and specific structural aspects of the tryptophan-rich sensory protein (TspO) of Rhodobacter sphaeroides 2.4.1 were studied using site-directed mutagenesis involving some 17 different amino acids. The choice of these amino acids changes was dictated from an analysis of the TspO family of proteins derived from the data bases. These studies demonstrated the importance of several highly conserved tryptophan residues in the sensory transduction pathway involving TspO through the proposed binding of an intermediate(s) in the tetrapyrrole biosynthesis pathway. These studies also revealed that the substitution of one or several of the amino acid residues dramatically affected, either directly or indirectly, the levels of TspO in the membranes of R. sphaeroides. Mounting evidence is presented suggesting that TspO normally forms a dimer within the bacterial outer membrane, and the dimer form of TspO may be the active form for TspO function. Because our earlier studies provided us with a functional framework within which to view these amino acid substitutions, we are able to suggest a preliminary model for TspO structure-function. Not only do these studies tell us more about TspO, but they also shed light on the TspO homologue, the drug-binding component of the mitochondrial peripheral benzodiazepine receptor. Mounting evidence draws numerous parallelism between these proteins and supports the significance of using TspO as a model for the structure and function of the mitochondrial protein.     

A monoclonal antibody against peripheral benzodiazepine receptor activities the human neutrophil NADPH-oxidase.

A murine monoclonal antibody, mAb 8523, raised against whole human pro-monocytic U937 cells recognizes an 18 kDa antigen in human neutrophils (PMN), as determined by immunoprecipitation and by immunodetection on Western blots of SDS-PAGE of PMN membrane fractions. That is 18 kDa antigen corresponds to the phagocyte peripheral benzodiazepine receptor (PBZDR) is evidenced by its co-migration with the 18 kDa covalently labeled PBZDR, detected by autoradiography, and their co-modulation upon phorbol-myristate-acetate activation of PMN. Purified mAb 8523 (IgG2b) is able to dose-dependently and specifically stimulate both the basal and the FMLP-induced oxidative burst of intact human PMN, assessed by luminol-amplified chemiluminescence. This property of the first described monoclonal antibody against PBZDR supports the implication of this receptor in NADPH-oxidase activation and consequently in phagocyte-dependent host defense mechanisms.     

Stress, anxiety and peripheral benzodiazepine receptor mRNA levels in human lymphocytes

Peripheral benzodiazepine receptor (PBR) mRNA levels were measured in lymphocytes obtained from a cohort of university students and clinically diagnosed anxious patients. The average level of PBR mRNA was decreased in anxious patients compared to a control group. This data confirms previously published results, but it also indicates that PBR mRNA levels cannot be used as a sole diagnostic measure of anxiety because the range of the individual PBR mRNA levels of the anxious group overlapped the range of the PBR mRNA levels of the control group. PBR mRNA levels in students following academic examinations were increased in some individuals and decreased in others. In the same cohort of students individual levels of cortisol and prolactin were predominantly increased and decreased respectively. There was no correlation between the individual changes in the hormone levels or PBR mRNA, which suggests that each of these parameters is affected by different environmental and physiological factors. Lymphocyte PBR mRNA measurement is a useful additional methodology for studying human stress responses however, its use in clinical studies would require the elucidation of PBR's physiological role.     

Immunohistochemical assessment of the peripheral benzodiazepine receptor in human tissues.

Exhaustive analysis of the location of the peripheral benzodiazepine receptor (PBR) both at the subcellular and the tissue level is warranted to gain a better understanding of its biological roles. To date, many studies have been performed in animal models, such as rat, mouse, and pig, that yielded important information. However, only a few reports were dedicated to the analysis of PBR expression in humans. To enlarge on previous studies, we investigated PBR expression in different human organs using the monoclonal antibody 8D7 that specifically recognized the human PBR. First, we performed electron microscopic analysis that for the first time unambiguously demonstrated the localization of the PBR on the outer mitochondrial membrane. Second, focusing our analysis on human tissues for which information on PBR expression is sparse (lung, stomach, small intestine, colon, thyroid, adrenal gland, pancreas, breast, prostate, ovary), we found that PBR exhibits selective localization. This characterization of PBR localization in human tissues should provide important insights for the understanding of PBR functions.     

PBRL, a putative peripheral benzodiazepine receptor, in primitive erythropoiesis

Benzodiazepines are a class of psychoactive drugs widely used for their anxiolytic, anticonvulsant, muscle relaxant and hypnotic properties. Although the benzodiazepine receptor in the central nervous system has been well studied, the role of peripheral type benzodiazepine receptor, PBR, remains elusive. Here, we show that there are two PBR homologous genes in amniotes, PBR and PBRL, based on phylogenetic analysis. In chickens, PBRL is exclusively expressed during early development in differentiating primitive erythrocytes and this expression is tightly correlated with that of hemoglobin genes. PBR is not expressed in hematopoietic system during this period and is weakly expressed in developing central nervous system. Because one of PBRs' known functions is to regulate heme transport between the mitochondria and cytoplasm, we investigated expression profiles of heme biosynthesis genes. Seven of the eight enzymes involved in heme biosynthesis, with the exception of protoporphyrinogen oxidase, are present in chicken genome. Five of them, delta-aminolevulinate synthase, delta-aminolevulinic acid dehydrogenase, porphobilinogen deaminase, coproporphyrinogen decarboxylase and ferrochelatase, show stage-specific increase in gene expression correlated with primitive hematopoiesis, but not with primitive erythrocyte differentiation. PBRL protein is localized to the mitochondria in culture cells, and pharmacological inhibition of PBRL activity results in a decrease in globin protein levels during primitive erythropoiesis. Our data suggest a developmental role of PBRs in erythropoiesis in chickens, possibly via the regulation of heme availability for the assembly of functional hemoglobins.     

Involvement of steroids in anti-inflammatory effects of peripheral benzodiazepine receptor ligands

Mouse paw oedema induced by carrageenan is used to determine if glucocorticoids are involved in the anti-inflammatory effects of peripheral benzodiazepine receptor ligands. The anti-inflammatory responses elicited by i.p. treatment with 1-(2-chlorophenyl)-N-methyl-N (1-methyl-propyl)-3-isoquinoline carboxamide (PK11195) and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1, 4-benzodiazepin-2 (Ro5-4864) were reversed by aminoglutethimide, an inhibitor of steroidal synthesis. Intraplantar injection into the ipsilateral paw of Ro5-4864, but not PK11195, inhibited the formation of paw oedema and this effect was reversed by aminoglutethimide. These results suggest that glucocorticoids are involved in the systemic and local anti-inflammatory effects of Ro5-4864 and only in the systemic response to PK11195.     

Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand

The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity of K(i)=4.7 nM. In this study, 1 was successfully synthesised and demethylated to form the phenolic derivative 6 as precursor for labelling with carbon-11 (t(1/2) = 20.4 min). [11C]1 was prepared by O-alkylation of 6 with [11C]methyl iodide. The radiochemical yield of [(11)C]1 was 9% (non-decay corrected) with a specific activity of 36 GBq/micromol at the end of synthesis. The average time of synthesis including formulation was 13.2 min with a radiochemical purity >98%. In vivo assessment of [11C]1 was performed in a healthy Papio hamadryas baboon using positron emission tomography (PET). Following iv administration of [11C]1, significant accumulation was observed in the baboon brain and peripheral organs. In the brain, the radioactivity peaked at 20 min and remained constant for the duration of the imaging experiment. Pre-treatment with the PBR-specific ligand, PK 11195 (5 mg/kg), effectively reduced the binding of [11C]1 at 60 min by 70% in the whole brain, whereas pre-treatment with the central benzodiazepine receptor ligand, flumazenil (1mg/kg), had no inhibitory effect on [11C]1 uptake. These results indicate that accumulation of [11C]1 in the baboon represents selective binding to the PBR. These exceptional in vivo binding properties suggest that [11C]1 may be useful for imaging the PBR in disease states. Furthermore, [11C]1 represents the first ligand of its pharmacological class to be labelled for PET studies and therefore has the potential to generate new information on the pathological role of the PBR in vivo.     

Synthesis and structure-activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published procedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmacophoric (delta1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary allylammonium cation 14. The structure-affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and suggests a common mode of interaction at the PBR binding site.     

Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR.     

A 3D model of the peripheral benzodiazepine receptor and its implication in intra mitochondrial cholesterol transport

A three-dimensional (3D) model of the peripheral benzodiazepine receptor (PBR) has been built using molecular dynamics simulations.The transmembrane domain of the receptor has been modeled as five alpha-helices, which are not long enough to cross the entire bilaver membrane hut correspond approximately to only one phospholipid layer. The receptor model has also been tested as a cholesterol carrier, and molecular dynamics simulations have shown that it could indeed accommodate a cholesterol molecule within the five helices. All three known PBR sequences have been modeled, and no significant difference has been found between them.     

A new optical and nuclear dual-labeled imaging agent targeting interleukin 11 receptor alpha-chain

Optical imaging has great potential for studying molecular recognitions both in vivo and in vitro, yet nuclear imaging is the most effective clinical molecular imaging modality. The combination of optical and nuclear imaging modalities may provide complementary information for improving diagnosis and management of diseases. In this study we developed an optical and nuclear dual-labeled imaging agent, 111In-DTPA-Bz-NH-SA-K(IR-783-S-Ph-CO)-c(CGRRAGGSC)NH2, called DLIA-IL11Ralpha. 111In-DTPA-Bz-NH-SA is the radiotracer moiety; a near-infrared dye IR-783-S-Ph-COOH serves as the fluorescent emitter; and the cyclic peptide c(CGRRAGGSC), which is known to target interleukin 11 receptor alpha-chain (IL-11Ralpha), delivers the desired imaging agent to its target. Experiments revealed that the cyclic peptide c(CGRRAGGSC) continued to possess the targeting capability to IL-11Ralpha after the conjugation of the optical and nuclear tracers. Furthermore, the presence of the metal isotope chelator did not cause quenching of fluorescence emission. The cross validation and direct comparison of optical and nuclear imaging of a tumor was achieved using a single injection, and the preliminary results show the conjugate has tumor targeting capabilities in vivo.     

A novel cholic acid-based contrast enhancement agent for targeted MRI

The novel Gd(III) complexes of heptadentate ligands NE3TA and NE3TA-Bn were prepared, and their relaxivities were measured and favorably compared to the commercially available MRI contrast enhancement agent Gd(DOTA). NE3TA was conjugated with cholic acid (CA) to produce CA-NE3TA. TEM images of Gd(CA-NE3TA) indicate that the complex self-assembles forming nano-sized micelles and displays an over threefold increased relaxivity compared to Gd(DOTA). The new cholic acid-conjugated nanoparticle MR contrast enhancement agent, Gd(CA-NE3TA) possesses great promise for use in targeted MRI.     

Regulation of cardiac and renal peripheral benzodiazepine receptor binding in rapid eye movement sleep-deprived rats

This study was designed to examine the effects of rapid eye movement (REM) sleep deprivation and stress on the properties of peripheral benzodiazepine receptors (PBRs). Rats were deprived of REM sleep for 96 hours by a small pedestal (water tank) method. Using radioligand assays with the selective antagonist [3H]PK11195, receptor density (Bmax) and affinity were measured in the heart and kidney in three groups: 1) REMSD group (REM sleep deprived-group), 2) TC group (tank control group; large pedestal), and 3) CC group (cage control group). REMSD group showed an increase in the density of PBRs in the heart (18%; p<0.002), but not in the kidney (-5%; p=0.058) compared to TC group, and IC group showed an increase in the density of these receptors in the heart (17%; p<0.014), and the kidney (12%; p<0.002) compared to CC group. While the cardiac Kd value was 37% (p<0.0004) and 36% (p<0.002) higher in the REMSD group than in the CC and TC groups, respectively, the renal Kd value was lower (-5%; p=0.057 and -16%; p<0.0004, respectively). The changes in PBRs observed herein are tissue- and treatment-specific, and may suggest a close functional relationship between REM sleep and PBRs.     

A comparison of the high-affinity peripheral benzodiazepine receptor ligands DAA1106 and (R)-PK11195 in rat models of neuroinflammation: implications for PET imaging of microglial activation

Activated microglia are an important feature of many neurological diseases and can be imaged in vivo using 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a ligand that binds the peripheral benzodiazepine receptor (PBR). N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106) is a new PBR-specific ligand that has been reported to bind to PBR with higher affinity compared with PK11195. We hypothesized that this high-affinity binding of DAA1106 to PBR will enable better delineation of microglia in vivo using positron emission tomography. [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195 in brain tissue derived from normal rats and the rats injected intrastriatally with 6-hydroxydopamine or lipopolysaccharide at the site of the lesion. Immunohistochemistry combined with autoradiography in brain tissues as well as correlation analyses showed that increased [(3)H]DAA1106 binding corresponded mainly to activated microglia. Finally, ex vivo autoradiography and positron emission tomography imaging in vivo showed greater retention of [(11)C]DAA1106 compared with [(11)C](R)-PK11195 in animals injected with either lipopolysaccaride or 6-hydroxydopamine at the site of lesion. These results indicate that DAA1106 binds with higher affinity to microglia in rat models of neuroinflammation when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a significant improvement over [(11)C](R)-PK11195 for in vivo imaging of activated microglia in human neuroinflammatory disorders.     

Uniform regulation of central and peripheral benzodiazepine binding sites by GABA receptor agonists in rats

Central and peripheral benzodiazepine binding sites were studied in vitro after the administration of GABAA and GABAB agonists. Cerebral cortex and kidney homogenates were used in this study. Muscimol (1.5 mg/kg, intraperitoneally) pretreatment significantly increased the affinity of benzodiazepine binding sites not only in the cerebral cortex but also in the kidneys. Similar changes were obtained with (-) and (+) baclofen (5 mg/kg, intraperitoneally), with the only exception that (-) baclofen in addition to changes in the affinity caused a marked decrease in the number of binding sites in both structures studied. The mechanism of action of GABA agonists on peripheral benzodiazepine binding sites is discussed.     

Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.     

Neuropharmacological profile of peripheral benzodiazepine receptor agonists, DAA1097 and DAA1106.

Receptor binding and behavioral profiles of N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (DAA1097) and N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (DAA1106), novel, selective agonists for the peripheral benzodiazepine receptor (PBR) were examined. DAA1097 and DAA1106 inhibited [3H]PK 11195 binding to crude mitochondrial preparations of rat whole brain, with IC50 values of 0.92 and 0.28 nM. Likewise, DAA1097 and DAA1106 inhibited [3H]Ro 5-4864 binding to the same mitochondrial preparation, with IC50 values of 0.64 and 0.21 nM. In contrast, DAA1097 and DAA1106 did not inhibit [3H]-flunitrazepam, the central benzodiazepine receptor (CBR) ligand, binding to membranes of rat whole brain (IC50>10,000nM). Oral administration of DAA1097 and DAA1106 had anxiolytic effects in the mouse light/dark exploration test and in the rat elevated plus- maze test. Oral administration of DAA1106, diazepam and buspirone but not DAA1097 significantly increased sleeping time in hexobarbital-induced anesthesia in mice. The order of potency of potentiation of hexobarbital anesthesia was diazepam> buspirone> DAA1106> DAA1097. Oral administration of DAA1097 and DAA1106 but not diazepam and buspirone did not affect spontaneous locomotor activity in mice. These findings indicate that DAA1097 and DAA1106 are PBR selective ligands with potent anxiolytic-like properties, in laboratory animals.     

In vivo imaging of peripheral benzodiazepine receptors in mouse lungs: a biomarker of inflammation

The ability to visualize the immune response with radioligands targeted to immune cells will enhance our understanding of cellular responses in inflammatory diseases. Peripheral benzodiazepine receptors (PBR) are present in monocytes and neutrophils as well as in lung tissue. We used lipopolysaccharide (LPS) as a model of inflammation to assess whether the PBR could be used as a noninvasive marker of inflammation in the lungs. Planar imaging of mice administrated 10 or 30 mg/kg LPS showed increased [(123)I]-(R)-PK11195 radioactivity in the thorax 2 days after LPS treatment relative to control. Following imaging, lungs from control and LPS-treated mice were harvested for ex vivo gamma counting and showed significantly increased radioactivity above control levels. The specificity of the PBR response was determined using a blocking dose of nonradioactive PK11195 given 30 min prior to radiotracer injection. Static planar images of the thorax of nonradioactive PK11195 pretreated animals showed a significantly lower level of radiotracer accumulation in control and in LPS-treated animals (p < .05). These data show that LPS induces specific increases in PBR ligand binding in the lungs. We also used in vivo small-animal PET studies to demonstrate increased [(11)C]-(R)-PK11195 accumulation in the lungs of LPS-treated mice. This study suggests that measuring PBR expression using in vivo imaging techniques may be a useful biomarker to image lung inflammation.