Richard D. Egleton

Nicotinic acetylcholine receptors in cancer: multiple roles in proliferation and inhibition of apoptosis.

Nicotinic acetylcholine receptors (nAChRs) constitute a heterogeneous family of ion channels that mediate fast synaptic transmission in neurons. They have also been found on non-neuronal cells such as bronchial epithelium and keratinocytes, underscoring the idea that they have functions well beyond neurotransmission. Components of cigarette smoke, including nicotine and NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], are agonists of nAChRs. Given the association of tobacco use with several diseases, the non-neuronal nAChR signaling pathway has considerable implications for cancer and cardiovascular disease. Recent studies have shown that alpha7 is the main nAChR subunit that mediates the proliferative effects of nicotine in cancer cells. As a result, alpha7 nAChR might be a valuable molecular target for therapy of cancers such as lung cancer and mesothelioma. Future studies involving the design of nAChR antagonists with improved selectivity might identify novel strategies for the treatment of tobacco-related cancers. Here we review the cellular roles of non-neuronal nAChRs, including regulation of cell proliferation, angiogenesis, apoptosis, migration, invasion and secretion.

Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability

Peptides have the potential to be potent pharmaceutical agents for the treatment of many central nervous system derived maladies. Unfortunately peptides are generally water-soluble compounds that will not enter the central nervous system, via passive diffusion, due to the existence of the blood-brain barrier. Peptides can also undergo metabolic deactivation by peptidases, thus further reducing their therapeutic benefits. In targeting peptides to the central nervous system consideration must be focused both on increasing bioavailability and enhancing brain uptake. To date multiple strategies have been examined with this focus. However, each strategy comes with its own complications and considerations. In this review we assess the strengths and weaknesses of many of the methods currently being examined to enhance peptide entry into the central nervous system.

Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution.

The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.

Bioavailability and Transport of Peptides and Peptide Drugs into the Brain

Rational drug design and the targeting of specific organs has become a reality in modern drug development, with the emergence of molecular biology and receptor chemistry as powerful tools for the pharmacologist. A greater understanding of peptide function as one of the major extracellular message systems has made neuropeptides an important target in neuropharmaceutical drug design. The major obstacle to targeting the brain with therapeutics is the presence of the blood-brain barrier (BBB), which controls the concentration and entry of solutes into the central nervous system. Peptides are generally polar in nature, do not easily cross the blood-brain barrier by diffusion, and except for a small number do not have specific transport systems. Peptides can also undergo metabolic deactivation by peptidases of the blood, brain and the endothelial cells that comprise the BBB. In this review, we discuss a number of the recent strategies which have been used to promote peptide stability and peptide entry into the brain. In addition, we approach the subject of targeting specific transport systems that can be found on the brain endothelial cells, and describe the limitations of the methodologies that are currently used to study brain entry of neuropharmaceuticals.

Development of neuropeptide drugs that cross the blood-brain barrier

SummaryIn recent years, there have been several important advancements in the development of neuropeptide therapeutics. Nevertheless, the targeting of peptide drugs to the CNS remains a formidable obstacle. Delivery of peptide drugs is limited by their poor bioavailability to the brain due to low metabolic stability, high clearance by the liver, and the presence of the blood brain barrier (BBB). Multiple strategies have been devised in an attempt to improve peptide drug delivery to the brain, with variable results. In this review, we discuss several of the strategies that have been used to improve both bioavailability and BBB transport, with an emphasis on antibody based vector delivery, useful for large peptides/small proteins, and glycosylation, useful for small peptides. Further development of these delivery methods may finally enable peptide drugs to be useful for the treatment of neurological disease states.

Improved bioavailability to the brain of glycosylated Met-enkephalin analogs

The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys(2,5),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the delta-opioid receptor, and mixed effect on binding to the mu-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C.

Fluorescence imaging of blood-brain barrier disruption:

Pathological alterations of the blood-brain barrier (BBB) can be topographically heterogeneous. The goal of this study was to develop a method to assess rapidly the magnitude and spatial distribution of permeability changes. Rats were perfused via the common carotid arteries with Ringers solution containing sodium fluorescein (NF) and Evans Blue albumin (EB). Global NF uptake was determined by fluorimetry and EB uptake was determined by absorbance spectroscopy. NF uptake was linear in control animals and at a rate comparable to sucrose, whereas uptake of EB was negligible. Infusion of 1.6 M mannitol immediately prior to perfusion significantly increased uptake of NF while EB uptake was unchanged. BBB disruption was confirmed by confocal microscopy of fresh-frozen sections. In control animals, NF and EB staining were limited to the edges of slices and to the circumventricular organs. In mannitol-treated animals, heavy NF staining was observed throughout the brain, and EB staining was localized around some microvessels. In animals given a approximately 500 microl air embolus prior to perfusion, a discrete area of NF and EB staining could be observed near the ventral midline, while the rest of the brain remained unaltered. We find that brain perfusion with NF/EB enables a rapid, reliable, and highly sensitive assessment of global BBB permeability and microscopic visualization of discrete BBB disruptions.

Protection against hypoxia-induced increase in blood-brain barrier permeability: Role of tight junction proteins and NFκB

Co-culture with glial cells and glia-conditioned media can induce blood-brain barrier properties in microvessel endothelial cells and protect against hypoxia-induced blood-brain barrier breakdown. We examined the effect of two types of glia-conditioned media on brain microvessel endothelial cell permeability and tight junction protein expression, and studied potential mechanisms of action. We found that C6-glioma-conditioned media, but not rat astrocyte-conditioned media, protected against an increase in permeability induced by exposure to 1% oxygen for 24 hours. This hypoxic stress caused an increase in the expression of tight junction proteins claudin-1 and actin, particularly in cells treated with C6-conditioned media. We found that C6-conditioned media has a significantly higher level of both basic fibroblast growth factor and vascular endothelial growth factor. Treatment with C6-conditioned media for 1 or 3 days protects against hypoxia-induced permeability increases, and this protective effect may be mediated by signal transduction pathways terminating at the transcription factor NFκB.

Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P-glycoprotein

P‐glycoprotein (Pgp, ABCB1) is a critical efflux transporter at the blood–brain barrier (BBB) where its luminal location and substrate promiscuity limit the brain distribution of numerous therapeutics. Moreover, Pgp is known to confer multi‐drug resistance in cancer chemotherapy and brain diseases, such as epilepsy, and is highly regulated by inflammatory mediators. The involvement of inflammatory processes in neuropathological states has led us to investigate the effects of peripheral inflammatory hyperalgesia on transport properties at the BBB. In the present study, we examined the effects of λ‐carrageenan‐induced inflammatory pain (CIP) on brain endothelium regulation of Pgp. Western blot analysis of enriched brain microvessel fractions showed increased Pgp expression 3 h post‐CIP. In situ brain perfusion studies paralleled these findings with decreased brain uptake of the Pgp substrate and opiate analgesic, [3H] morphine. Cyclosporin A‐mediated inhibition of Pgp enhanced the uptake of morphine in λ‐carrageenan and control animals. This indicates that the CIP induced decrease in morphine transport was the result of an increase in Pgp activity at the BBB. Furthermore, antinociception studies showed decreased morphine analgesia following CIP. The observation that CIP modulates Pgp at the BBB in vivo is critical to understanding BBB regulation during inflammatory disease states.

Angiogenic activity of nicotinic acetylcholine receptors : Implications in tobacco-related vascular diseases

Cigarette smoking bears a strong etiological association with many neovascularization-related diseases like cancer, cardiovascular disease and macular degeneration. Although cigarette smoke is a complex mixture of many compounds, nicotine is the major active and addictive component of tobacco. Recent studies have shown that nicotine can enhance angiogenesis and arteriogenesis in several experimental systems and animal models. The pro-angiogenic activity of nicotine is mediated by nicotinic acetylcholine receptors, which have been found to be expressed on several types of cells in the vasculature like endothelial cells, smooth muscle cells and immune cells. The present review summarizes the pro-angiogenic activity of nicotine in neoplastic and non-neoplastic disease. The present article focuses on the role of nAChRs, particularly alpha7-nAChR in mediating the pro-angiogenic effects of nicotine. The expression patterns of nAChRs on various components of the vasculature are discussed. The complex signaling pathways underlying the angiogenic effect of nAChRs are described. The review also takes a look at the therapeutic potential of nAChR agonists and antagonists in angiogenesis-related diseases. More basic research as well as patient-oriented clinical studies is needed to firmly establish the clinical potential of nAChR ligands in angiogenesis-based therapies. Also the side effects of targeting nAChRs remain to be established in patients. The development of selective nAChR agonists and antagonists with improved specificity may represent novel therapeutic regimens in the treatment of angiogenesis-related diseases.