RICERCA ITALIANA     

SYNTHESIS AND PHARMACOLOGICAL CHARACTERIZATION OF NOVEL COMPOUNDS WITH SELECTIVITY FOR THE PERIPHERAL BENZODIAZEPINE RECEPTOR USEFUL FOR THE MODULATION OF NEUROSTEROIDOGENESIS, DIAGNOSIS OF NEURODEGENERATIVE DISORDERS, AND ANTITUMOR DRUG DELIVERY

Università degli Studi di Cagliari

Abstract

Recent studies have demonstrated that the peripheral benzodiazepine receptor (PBR) plays a number of fundamental roles a cellular level, and among other, in the regulation of the synthesis of steroids. Distinct from the “central” benzodiazepine receptor (CBR), PBR is expressed on the mitochondrial membrane, where it controls cholesterol translocation inside the mitochondrion, both in brain (glial and neuronal cells) and peripheral endocrine organs. Steroids, derived from the metabolism of progesterone which are defined “neurosteroids”, such as allopregnanolone and THDOC, are known to induce marked antistress and anxiolytic effects, which are due to their membrane action through the facilitation of the fast inhibitory neurotransmission mediated by gamma-aminobutyric acid type A (GABA-A) receptors in the Central Nervous System. Physiological and pathological fluctuations in neurosteroid concentrations are thought to have important consequence in influencing and modulating emotional states as well as can play a role in the homeostatic processes and neuronal plasticity which are activated in order to counteract and regulate an excessive level of neuronal excitability triggered by various stimuli such as acute stress.
Other studies have demonstrated that PBR expression can be greatly enhanced in different physiological and pathological conditions, such as in several neurodegenerative disorders like Alzheimer’s and Huntington’s diseases. Furthermore, high level of PBR expression have been found in neoplastic tissues and in tumor cell lines, suggesting that this characteristic may by used for the diagnostic purposes or for the site-directed delivery of antitumor drugs particularly in the brain.
Based on such considerations, it becomes evident the different clinic implications of the possibility to modulate, through selective ligands endowed with an agonist or antagonist efficacy, the function of PBRs, and consequently to manipulate pharmacologically the synthesis of neuroactive steroids both in brain and in peripheral tissues.
The objective of this research project, which is to be considered a continuation of previous studies carried out by both these research Units, is to synthesize and characterize a novel series of imidazopyridinacetamide derivatives with high affinity and selectivity for the PBR and that are endowed with agonist or antagonist action on the synthesis of neuroactive steroids. Some of these compounds will be used for preparing conjugates with antitumor drugs in order to improve bioavailability and site-directed delivery of drugs such as citarabine (Ara C) and metotrexate. Furthermore, because the expression of the PBR is dramatically increased in neoplastic tissue, another goal will be to synthesize and characterize a series of imidazopyridinacetamides which will be radiolabelled in order to be utilized as probes for the visualization by PET scanning of PBRs.
These novel compounds with high affinity and selectivity for the PBR might thus prove to represent important tools not only in basic research but also in clinical practice for both the diagnosis of tumors or neurodegenerative pathologies and as therapeutic agents in different pathologies which involve alterations in the synthesis of neuroactive steroids. <<<

Principal Investigator

Enrico Sanna Università degli Studi di CAGLIARI

Research Objectives

The peripheral type of benzodiazepine receptors (PBRs) are receptors distinct from the central type of benzodiazepine receptors (CBR) which are associated to GABA-A receptors, and are expressed on the mitochondrial membrane of glial and neuronal cells as well as in cells of peripheral organs such as adrenals, testis, ovary, lungs, liver, heart, and other circulating cells such as platelets, lymphocytes and erythrocytes. PBRs are involved in a number of cellular functions such as regulation of cell proliferation, immune response, cholesterol transport, porphyrin transport and biosynthesis of heme, oxidative processes, and programmed cell death. In addition, another important physiological role played by PBRs is to promote steroidogenesis. In fact, these receptors facilitate the translocation of cholesterol from intracellular sites to the inner mitochondrial membrane, where this precursor is oxidized to pregnenolone. Thus, this process is the rate-limiting step in the synthesis of steroids. Two reduced metabolites of progesterone, allopregnanolone and THDOC, are known to produce, through a positive modulatory action at the GABA-A receptor, pharmacological effects similar to those of benzodiazepines and barbiturates, suggesting that these hormones are among the most important endogenous regulators of emotional states. Furthermore, it is thought that physiological fluctuations of plasma and brain levels of these hormones might determine, through their influence on GABA-A receptor function, changes in behaviours associated to emotions and affectivity. In fact, because GABA is the major inhibitory neurotransmitter in the mammalian Central Nervous System, alterations in the synthesis and central as well as peripheral secretion of these steroids might represent a crucial neurochemical mechanism that could modify the activity of different neuronal pathways involved in the control of emotions and the regulation of mood. It is then evident both the importance and the possible clinical implications of the capability to modulate, through selective ligands for this receptor endowed with agonist or antagonist activity, the function of this receptor and consequently steroidogenesis both in brain and peripheral organs.
Other studies have also demonstrated that PBR expression can be regulated in response to physiological and pathological alterations, as in neurodegenerative disorders such as Alzheimer’s, Huntington’s, and Parkinson’s disease. Microautoradiographic studies have shown that the increased density of PBRs may reflect an activation of microglia which follows neuronal injury, and this, in turn, may represent a marker for the diagnosis of neurodegenerative disorders. Furthermore, increased levels of PBR expression have been observed in tumors as well as in cultured tumor cell lines, suggesting that this up-regulation may be used as diagnostic marker or as molecular target for site-directed delivery of antitumor drugs particularly in brain.
Considering the relatively large experience of the two laboratories involved in this project proposal in the search and characterization of novel imidazopyridine PBR ligands, the aim of the present project is to synthesize and evaluate the biological activity of a set of novel potential agonist and antagonist ligands for the PBR obtained by modification of the imidazopyrimidinacetamide lead structure by introducing polar or ionisable substituents at the phenyl ring in position 2 or 8. These compounds will be characterized for their physical and chemical as well as for their pharmacological properties, for their PBR affinity and selectivity, and for their capacity of modulating steroidogenesis. In addition, by applying patch-clamp recording in mitochondrial membrane preparations, we will study the effect of these compounds on the function of mitochondrial ion channels that are coupled to PBRs. This study might be useful for identifying compounds endowed with agonist and antagonist action at PBR. Those compounds that possess the best pharmacodynamic and pharmacological characteristics may serve, in addition to the pharmacological modulation of steroidogenesis, also for preparing conjugates with antitumor drugs, such as citarabine (Ara C) and methotrexate, which are characterized by a low bioavailability at brain level due to their hydrophilicity and/or because they are substrates of the Glycoprotein P, the active efflux system at the Blood Brain Barrier. These compounds will be also evaluated, in Prof Gallo’s laboratory, their cytotoxicity because of a synergistic effect due to the possible apoptosis induced by the same ligands. Some of these ligands will serve as starting compounds for the radiosynthesis aimed at the preparation of corresponding [C-11]O-methyl-derivatives that may be used for the development of markers for positron emission tomography (PET). The possibility to have these ligands can have a certain importance from a clinical point of view in the diagnosis of tumor and neurodegerative diseases such as Alzheimer’s.
Thus, the aim of this project is to identify novel compounds with high affinity and selectivity for the PBR which, with the different applications, might represent important tools not only in preclinical research, but also in clinic practice, as high affinity and selective markers in the diagnosis of tumors, and as therapeutical agents that can be used in pathologies involving alterations of steroid synthesis. <<<

Timescale

24 months

National and international background

The peripheral benzodiazepine receptor (PBR), discovered originally because it binds the benzodiazepine diazepam with relatively high affinity (1), is structurally and functionally distinct form the central type of benzodiazepine receptor (CBR), which is a site associated to GABAA receptor complex localized mainly in the central nervous system (CNS). Structurally, PBR is a multimeric complex composed by 3 different subunits. A 18-kDa receptor protein, whose aminoacid sequence (169 AA) presents a 80% homology among different species (human, bovine, murine); hydropathic analysis of primary aminoacid sequence reveals the presence of 5 transmembrane domains, an amino-terminal oriented towards the mitochondrial intermembrane cleft, and a carboxy-terminal oriented towards the cell cytoplasm. This subunit (18-kDa) is associated with 2 other proteins, one of which is a 32-kDa protein that forms a voltage-dependent anion channel required for benzodiazepine binding, and the second is a 30-kDa protein transporter of adenine nucleotides with unknown function (2,3). VDAC is an ion channel of high conductance and limited ion selectivity, localized in the external mitochondrial membrane, in particular on the sites of contact between the external and internal membrane. The adenine nucleotide transporter is localized in the internal mitochondrial membrane.
Although PBR is present in most tissues examined (for example, lungs, heart, skeletal muscles, platelets, lymphocytes, red blood cells), it is particularly abundant in steroid producing tissues (adrenals, testicle, ovary) where it found in the outer mitochondrial membrane (2,3), being more abundant in the outer/inner mitochondrial membrane contact sites (4).
PBR differs substantially from the CBR also in terms of its pharmacological sensitivity. There are anxiolytic and hypnotic compounds such as diazepam, flunitrazepam, zolpidem, and alpidem, which, in addition to have high affinity for the CBR, can bind to PBR with relatively good affinity (Ki 10-20 nM). Moreover, there are other drugs such as the benzodiazepine Ro5-4864, the isoquinoline PK11195, the indolacetamide FGIN-1, and several imidazopyridine CB derivatives (synthesized and characterized by the two Units of this project), which bind selectively and with high affinity (Ki 1-5 nM) only to the PBR.
Although the physiological role of PBR is not completely understood, experimental evidence suggests its involvement in important and fundamental cellular functions such as calcium homeostasis, mitochondrial oxidation, lipid metabolism, cell growth and differentiation, proliferation of tumor cells, immune response, porphyrin transport and heme biosynthesis, and regulation of programmed cell death or apoptosis (2,3).
However, in line with the high level of expression in steroid producing tissues, both “in vitro” and “in vivo” studies have demonstrated that PBR is involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, which represents the rate-limiting step in steroid biosynthesis (2). In the inner mitochondrial membrane cholesterol is converted to pregnenolone by the P450 “side chain cleavage” (P450scc) which catalyzes a series of reactions involving the formation of the 22R-OH-cholesterol and 20,22R-OH-cholesterol intermediates, followed by cleavage of the bond between C20 and C22 of colesterol (5). Pregnenolone then leaves the mitochondrion to undergo enzymatic transformation in the endoplasmic reticulum that will give rise to the final steroid products.
“In vitro” reconstitution experiments demonstrate that the 18-kDa PBR protein binds with high affinity both drug ligands, such as Ro5-4864 and PK11195, and cholesterol (6,7). At nanomolar concentrations, PBR ligands enhance the formation of pregnenolone and neuroactive steroids (2). “In situ” and “in vitro” studies have indicated that in steroidogenic cells the steroidogenic acute regulatory protein (StAR)-induced cholesterol import into mitochondria was regulated by PBR in the outer mitochondrial membrane (8), indicating that there is a functional interaction between StAR and PBR required for cholesterol delivery into mitochondria and subsequent steroid formation.
In search of the tissue specificity of PBR expression, it has been demonstrated that the high levels of PBR expression in steroidogenic cells are due, at least in part, to the expression of Sp1/Sp3 transcription factors (9). Thus, expression of specific transcription factors results in the over-expression of PBR and increased cholesterol transport into mitochondria associated with steroidogenesis. In other tissues, PBR expression might be part of the mitochondrial membrane biogenesis process involved in increased cell proliferation (cancer, gliosis) and tissue repair (nerve damage and ischemia-reperfusion injury) where PBR levels have been shown to be elevated (10-13).
It is well known that steroid hormones act by regulating gene expression, inducing cellular processes such as growth and differentiation in steroid-sensitive tissues. The genomic effects of steroids are mediated through proteins members of the superfamily of steroid hormone receptors, a group of intracellular transcription factors (14). Steroids have also rapid, non-genomic effects, particularly in the brain, that were first described in the 1940s (15,16). These non-genomic actions are characterized by extremely rapid effects, lasting from milliseconds to minutes, and do not require interaction with steroid hormone receptors (17). In the CNS, these effects are thought to involve steroid modulation of membrane-bound neurotransmitter receptors (18,19), including the GABAA receptor, the glutamatergic NMDA receptor, and sigma receptors (20,21). In particular, the 3alpha,5alpha-reduced metabolites of progesterone, such as the 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), are among the most potent and efficacious positive modulators of GABAA receptors, which mediates for most part the rapid inhibitory neurotransmission in CNS. GABAA receptors play a crucial role in the control of neuronal excitability and mediate the anxiolytic, sedative-hypnotic, and anticonvulsant actions of drugs such as benzodiazepines, barbiturates, imidazopyridines, cyclopyrrolones, and pyrazolopyrimidines. Systemic or intracerebroventricular administration of allopregnanolone induces behavioral and pharmacological responses (anxiolytic, anticonvulsant, sedative-hypnotic) very similar to those induced by the above mentioned drugs. An anxiolytic effect in humans and in animals can be reached following an increase in brain concentrations of endogenous allopregnanolone elicited by the systemic administration of the precursor progesterone.
Neuroactive steroids have been intensively studied in recent years, due to great appeal as potential drugs for the treatment of a number of neuropathological and clinical conditions (22, 25). Because of their lipophilic structure, steroids can easily diffuse across the blood-brain barrier when administered systemically. Furthermore, the concentration of steroids required to induce changes in neuronal activity is extremely low, usually in the nanomolar range. The term “neurosteroids” was first introduced by Baulieu and Robel (26) to designate the steroid hormone intermediates, dehydroepiandrosterone and pregnenolone and their sulfate forms, which could be measured in brain at concentrations independent from peripheral sources, such as adrenal glands or the gonads (27,28).
It appears thus evident that the possibility to control and modulate pharmacologically the function of PBR and consequently the synthesis of steroid hormones both in CNS and peripheral organs by using selective ligands endowed with agonist or antagonist activity, bears a great potential relevance and might have profound consequences in the clinic practice.
In brain, PBR is mainly expressed in glial cells and in low levels in neurons (29). PBR basal expression is up-regulated in a number of neuropathologies, including gliomas (30,31), neurodegenerative disorders such as Alzheimer’s disease (32-34), and in various forms of brain injury and inflammation (13,35), induced by neurotoxins or cuprizone (35,36-38). A typical feature of neurodegenerative diseases as well as brain injury is activation of microglia and inflammation, leading to gliosis. Considering the high level of expression of PBR in glia as well as the increased proliferation of glia in gliosis, it is evident that PBR could serve as diagnostic marker of the pathological state progression. In fact, in addition to the diseases and injury reported above, increased PBR levels have been observed in multiple sclerosis (39), Parkinson’s (33), and Huntington’s diseases (32,40) as well as in animal models of epilepsy (41-43). Increased PBR levels in astroglia have been also associated with hepatic encephalopathy, a condition where a role of ammonia-induced dysfunction of astrocytes has been proposed (44). PBR has also been implicated in peripheral nerve degeneration and regeneration (11), where PBR density and the levels of the endogenous PBR ligand, the polypeptide “diazepam binding inhibitor” (DBI) and its metabolites, were increased after peripheral nerve injury. Once regeneration of the peripheral nerve was complete, PBR and DBI levels returned to normal. The precise role of PBR in neurodegeneration is not known. However, considering the increased requirement for mitochondrial biogenesis needed to support the accelerated cell proliferation seen in gliomas and during gliosis, part of the above mentioned neuropathologies and brain injuries, it is possible that increased levels of PBR would be required for increased transport of cholesterol (45) and protein import (8,46) needed for biogenesis of the inner mitochondrial membrane. In addition to understanding the role of PBR in neuropathology, the studies reported above demonstrated the consistent and specific increase in PBR during neurodegeneration and brain injury suggesting that PBR ligads may be suitable tools for brain imaging (47-50).
Within these researches, the 2 Units proposing this project have started in these past years an intense research activity aimed at synthesizing and characterizing a series of novel compounds active on the PBR. The results of these studies have led to the identification of a series of 2-phenyl-imidazo[1,2-a]pyridinacetammide derivatives that have shown high capacity to bind with high affinity and high selectivity to PBR, and to effectively stimulate the biosynthesis of steroids in brain as well as peripheral organs (51-54).
In this new research project, we propose to synthesize a new series of derivatives obtained with introducing polar and ionisable moieties at position 2 and 8 of the imidazopyridineacetamide lead structure (for chemical structures, see the project of Bari’s unit) and evaluate their PBR binding affinity and selectivity, their effects of on the function of mitochondrial ion channels coupled PBR, and their efficacy in stimulating the steroidogenesis,. The overall objective will be to identify and characterize the pharmacological activity of new high affinity and selective ligands for PBR which may be useful for the pharmacological modulation of the different functions regulated by PBR, particularly the steroidogenesis, for obtaining antitumor-drug conjugates, and for the preparation of radiolabeled compounds as markers of PBR in positron emission tomography (PET) for the diagnosis of different neurodegenerative diseases.

Elements and criteria proposed for the assessment of the results obtained
Chemical synthesis and characterization of novel compounds useful in biomedical research and in clinical practice, both as therapeutical agents that modulate the synthesis of neuroactive steroids and the site-directed delivery of antitumor drugs, and as markers for diagnostic purposes in different tumor and neurodegenerative pathologies.
Publications in qualified international journals. <<<