| Biography: Nigel Greig was trained as a pharmacologist with a background in medicinal chemistry and physiology and gained his Ph.D. from the University of London; specifically, from the Pharmacology Department of the Royal College of Surgeons, England. Leaving the Cancer Chemotherapy Department of the Imperial Cancer Research Fund, London, he joined NIA in 1982. His initial studies focused on optimizing the delivery to and action of drugs within the brain. This resulted in the development of drug candidates for the treatment of brain tumors, and cancers of the breast, lymphatics and ovaries, as well as agents for the treatment of drug abuse and technology for the delivery of neuropeptides, antisense oligonucleotides and proteins to the brain. Leaving NIA in 1989, Dr. Greig was involved in the initiation of the successful California biotechnology company, Athena Neurosciences, now Elan Pharmaceuticals.The company was launched on technology from Dr. Greig's program. Returning to NIA as a tenured scientist in 1991, his research has evolved into his present interest, the design and development of drugs and diagnostics for the treatment of neurodegenerative diseases, with particular emphasis on Alzheimer's disease, and of type 2 diabetes. He heads the Drug Design and Development Section of the Laboratory of Neurosciences that extensively collaborates within NIA, academia and industry. This has resulted in the development of several agents from concept in the laboratory, through the required U.S. Government regulatory requirements to the bedside. Patents covering a variety of novel compounds of clinical interest have now been licensed from the NIA to industry and are in preclinical and clinical development, and new research within his program is providing both publications and patent applications to support potential drugs of the future. |
| Design of Drugs and Diagnostics: The goal of the Drug Design and Development Section is to develop novel agents against rate-limiting steps involved in the pathophysiology of diseases associated with aging with emphasis on nervous system diseases such as Alzheimer's disease (AD). |
| Alzheimer's Disease: |
| Acetylcholinesterase Inhibition: Although the neuropathological quantification of b-amyloid plaques and neurofibrillary tangles in the AD brain is the basis for confirming disease diagnosis after death, it is the neocortical synapses rather than the plaques and tangles that correlate best with psychometric indices of cognitive performance in AD. The loss of cholinergic synaptic markers in selected brain regions remains one of the earliest events leading to AD, with the cholinergic system being the most affected of the neurotransmitters and intimately involved in memory processing. Additionally, there are numerous mechanistic-based interactions linking the cholinergic system to Ab genesis, Tau phosphorylation, apoptotic cell death and inflammatory process that form a self-propagating cycle that drives AD pathogenesis. We have therefore focused our expertise on pivotal targets in each of these diverse but linked elements in order to develop mechanism-based strategies to not only slow or halt AD, but additionally to impact other neurodegenerative diseases. |
| Anticholinesterases: One of our efforts has focused on augmenting the cholinergic system, but maintaining the normal temporal pattern of neurotransmitter release by selectively inhibiting the enzyme acetylcholinesterase (AChE), acetylcholine's (ACh) degrading enzyme, in brain. Extensive studies involving synthetic chemistry, X-ray crystallography, molecular modeling, biochemistry and pharmacology resulted in our development of "selective cholinesterase inhibition technology" (SCIT). This has provided us the basis for the development of novel drugs to selectively and reversibly inhibit either AChE or its sister enzyme, butyrylcholinesterase (BChE), in the brain for an optimal time duration for the potential treatment of AD, age-associated memory impairment and other dementias. In addition, incorporation of charged moieties to restrict the brain entry of resulting compounds has provided drug candidates for potential treatment of myasthenia gravis as well as prophylactics for nerve gas poisoning (in current assessment by the U.S. and British Army). |
| The targeting of selective and site-directed drugs to specific enzymes rather than to receptors is a conceptually attractive method to optimize drug action. The reason for this is that formation of a reversible drug/enzyme complex allows selective enzyme inhibition over a protracted time duration (numerous hours), which is independent of the pharmacokinetic half-life of the drug (often minutes). Once the drug has formed a slowly reversible drug/enzyme complex to inhibit its function, the presence of free drug is no longer required for continued action. In contrast, drug/receptor stimulation requires the continued presence of drug, and its time-dependent maintenance at the target receptor for continued activity. It is difficult to achieve steady-state drug target levels and, indeed, when achieved, it generally results in a high body exposure to drug and potential toxicity. Our use of the former method, targeted enzyme inhibition, enhances specificity, lowers total body drug exposure and dramatically reduces toxicity. This is important in the elderly, which represents the fraction of the population afflicted with AD. The high variability and slowing of drug metabolism, commonly associated with age, often results in a gradual overdosing and toxicity in the elderly as one dose is often administered before a prior one is fully cleared. The dissociation between pharmacokinetics and pharmacodynamics minimizes this, as drug clearance (measured in minutes) can change dramatically without impacting on drug action (measured in hours). Incorporating such concepts into our drug design has resulted in several novel compounds with dramatic sustained cognitive action for once or twice daily dosing with wide therapeutic windows and minimal toxicity. For example, the novel experimental drug, phenserine (licensed to Axonyx, New York, NY), a long-acting and brain-directed, selective AChE inhibitor, is now in phase 3 clinical assessment in AD patients. Thus far, it appears to be well tolerated in elderly individuals, particularly when compared to currently available prescription anticholinesterases. Specifically with regard to phenserine, multiple phase I clinical trials have now been completed (to assess single and multiple dose tolerability in the elderly, as well as bioavailability). One phase 2 study, to characterize tolerability and actions on cognition in AD, has been successfully completed, and a further phase 2 clinical trial to characterize actions on disease progression, with an emphasis on Ab levels in CSF and plasma as well as cognition, is presently ongoing, as is a phase 3 clinical trial that is focused on cognition. |
| Other novel agents from SCIT are presently being developed as the first available reversible, nontoxic and brain-directed selective inhibitors of the enzyme BChE. (Collaborators: Debomoy Lahiri, Ph.D., University of Indiana, IN; Kumar Sambamurti, Ph.D., Medical University of South Carolina; Jack Rogers, Ph.D., Harvard, Boston, MA; Judith Flippen-Anderson, Ph.D., Naval Research Center, Washington D.C.; Tony Giordano, Louisiana State University, Shreveport, LA; Mohammad Kamal, Ph.D., University of Sydney, Australia; Axonyx Inc., New York, NY; Donald Ingram, Ph.D., Laboratory of Experimental Gerontology, NIA). |
| Butyrylcholinesterase Inhibition: Inhibition of AChE is a characteristic shared by all cholinesterase inhibitors currently approved for the treatment of AD. In the brain, AChE is primarily associated with neurons, where it hydrolyses acetylcholine (ACh) to terminate its biological action. Although overlooked for many years, a second cholinesterase, butyrylcholinesterase (BChE), is likewise capable of hydrolyzing ACh and may play an important role in the pathophysiology and symptomatology of AD. BChE, unlike AChE and most other enzymes in the AD brain, has been found elevated early in the disease process, particularly in brain regions associated with AD, where it co-localizes both with Ab plaques and neurofibrillary tangles. The association of BChE with the AD neurotoxic peptide, b-amyloid, has been shown to dramatically amplify the toxicity of the peptide. In addition, a mutant variant of BChE, the K form, when found together with the ApoE 4 allele, is associated with an increased susceptibility of sporadic AD. Hence, inappropriate BChE activity can increase the risk of AD and accelerate the disease process. |
| Regarding its enzyme kinetics, an important feature distinguishing BChE from AChE is its kinetics toward concentrations of ACh. BChE is not inhibited by excess substrate. This is reflected in its Km for ACh, which makes it less efficient in its substrate hydrolysis at low concentrations but highly efficient at high substrate concentrations, at which AChE becomes substrate inhibited. Consequently, we hypothesize that one role of BChE in brain, particularly when associated with glia, is that of a supportive hydrolyzing enzyme for ACh. Under conditions of high brain activity, local synaptic ACh levels can reach µM levels, which are inhibitory for AChE activity. The close spatial relationship of glial BChE would allow compensatory ACh hydrolysis to occur. In addition, some 15% of cholinergic synapses in human brain have BChE rather than AChE as the metabolizing enzyme. A further important feature that distinguishes these two cholinesterase subtypes is that AChE is lost early in AD, by up to 85% in specific brain regions in line with the loss in presynaptic ACh, whereas BChE levels are elevated. This results in a mismatch between substrate and enzyme. Indeed, the ratio of BChE/AChE has been found to dramatically change in cortical regions from 0.2 to as high as 11. Clearly, such an altered ratio in the AD brain could jeopardize the normally supportive role of BChE to hydrolyze only excessive ACh, terminating its action too quickly. Selective inhibition of BChE may therefore be of value to normalize the BChE/AChE ratio in AD brain and augment cholinergic neurotransmission. |
| To elucidate the role of BuChE in AD, the first, reversible, selective carbamate inhibitors of BChE were developed (cymserine: (-)-4'-isopropylphenyl-carbamoyleseroline and analogues) and their effects on cognition were assessed by administering them to male aged Fischer-344 rats whose performance was quantitatively evaluated in a 14-unit T-Maze (Stone maze). This cognitive task has proved highly robust and sensitive in evaluating age-dependent declines in memory and pharmacological interventions in rodents. The action of selective BChE inhibition on brain levels of ACh, as measured by in vivo microdialysis, has also been studied, together with actions on the levels of AD neuropathological markers, amyloid precursor protein APP and Ab peptide. (Collaborators: Debomoy Lahiri, Ph.D., University of Indiana, IN; Kumar Sambamurti, Ph.D., Medical University of South Carolina; Jack Rogers, Ph.D., Harvard, Boston, MA; Judith Flippen-Anderson, Ph.D., Naval Research Center, Washington D.C.; Tony Giordano, Louisiana State University, Shreveport, LA; Mohammad Kamal, Ph.D., University of Sydney, Australia; Donald Ingram, Ph.D., Laboratory of Experimental Gerontology, NIA). |
| b-amyloid Precursor Protein (b-APP) and Amyloid-b (Ab ) Peptide Inhibitors: Another of our focuses to develop therapeutics for treating AD relates to reducing the production and secretion of Ab. It is widely believed that Ab plays a central role in the progressive neurodegeneration observed in AD; diminishing the level of Ab has therefore emerged as a critical goal in AD therapy. Ab is generated from a larger protein, APP, by a group of enzymes collectively identified as secretases. Specifically, APP is proteolytically cleaved at specific amino acid by three secretases (a-, b- and g-), to different protein fragments, including toxic Ab and other C-terminal fragments that are implicated in the pathogenesis of AD. A major focus has hence been to develop agents to alter amyloidogenic processing to produce non-amyloidogenic by-products. The secretases as well as strategies to augment the clearance of Ab are thus legitimate, albeit unvalidated, targets for drug discovery. Our program, together with collaborators (Prof. Debomoy Lahiri, Ph.D., Indiana University School of Medicine, Indianapolis, IN; Prof. Kumar Sambamurti, Ph.D., Medical University of South Carolina, Charleston, SC; and Prof. Jack Rogers, Ph.D., Harvard University, Boston, MA), is jointly engaged in studying various classes of agents that can reduce APP expression, as this is the precursor to all the Ab toxic fragments. |
| In this regard, we have focused on the pharmacophore of (-)-phenserine: a tricyclic hexahydropyrrolo[2,3b]indole with a phenylcarbamate. In cell culture studies, (-)-phenserine lowered APP and Ab levels in human neuroblastoma cells via a mechanism unassociated with its anticholinesterase action. In rats, it was shown to improve cognitive performance, and lower APP production in both naive and cholinergic lesioned animals. Likewise, in transgenic mice over-expressing human APP and Ab, it was found to significantly lower both. Interestingly, phenserine's action to lower APP occurs through modulation of protein expression at the post-transcriptional level. In this regard, there are an increasing number of reports of post-transcriptional regulation of diverse gene products. For example, small molecules can significantly modulate post-transcriptional processes involved in the production of tumor necrosis factor-alpha (TNF-a). (-)-Phenserine's actions on APP are mediated through the 5' untranslated region (5' UTR) of APP mRNA; the very same element previously shown to be up regulated in the presence of interleukin-1 and other cytokines. Post-transcriptional regulation of proteins such as APP by small molecules is hence a feasible approach to discover and develop new therapeutic agents that lower Ab levels. Utilizing the pharmacophore of (-)-phenserine, we have developed a novel series of compounds to optimize action against APP and Ab and to minimize anticholinesterase activity. (Collaborators: Debomoy Lahiri, Ph.D., University of Indiana, IN; Kumar Sambamurti, Ph.D., Medical University of South Carolina; Jack Rogers, Ph.D., Harvard, Boston, MA; Judith Flippen-Anderson, Ph.D., Naval Research Center, Washington D.C.; Tony Giordano, Louisiana State University, Shreveport, LA; Donald Ingram, Ph.D., Laboratory of Experimental Gerontology, NIA). |
| Inflammation and TNF-a Inhibition: Inflammatory processes associated with the over-production of cytokines, particularly of TNF-a, accompany numerous neurodegenerative diseases, such as Alzheimer's disease and ALS, in addition to numerous systemic conditions that are common in the elderly, such as rheumatoid arthritis, as well as diseases such as erythema nodosum leprosum (ENL), septic shock, graft-versus-host and Crohn's disease. TNF-a has been validated as a drug target with the development of the inhibitors Enbrel and Remicade as prescription medications. Both, however, are large macromolecules that require direct injection and have limited to negligible brain access. The classical drug, thalidomide is being increasingly used in the clinical management of a wide spectrum of immunologically-mediated and infectious diseases, and cancers. Its clinical value in treating ENL derives from its TNF-a inhibitory activity. Structural modification of thalidomide was hence undertaken towards the discovery of novel isosteric potent analogues that would be of potential utility in the conditions described above. These were synthesized and evaluated for their TNF-a inhibitory activity against lipopolysacharide (LPS) stimulated peripheral blood mononuclear cells (PBMC) in cell culture. Additionally, PBMC viability was quantified to differentiate reductions in TNF-a secretion from cellular toxicity. Specific analogues potently inhibited TNF-a secretion, compared to thalidomide. The mechanism underpinning this likely is post-transcriptional as they decreased TNF-a mRNA stability via its 3'-UTR, as determined by luciferase activity in stably transfected cells with and without the entire 3'-UTR of human TNF-a. The activity of these novel compounds in classical models of (i) neurodegeneration as well as cancer (with specific focus on angiogenesis) is the focus of current studies. (Collaborators: Prof. Tony Giordano, Ph.D., Louisiana State University, Shreveport, LA; William Douglas Figg, Ph.D., NCI, NIH, Bethesda, MD., and Prof. Debomoy Lahiri, Ph.D., Indiana University School of Medicine, Indianapolis, IN). |
| Neurodegeneration: Collaborative studies with Mark Mattson, Ph.D., (Chief, Laboratory of Neurosciences, NIA, NIH, Baltimore, MD) are focused on modifying the course of apoptotic cell death. Apoptosis is a major form of cell death that involves a stereotyped sequence of biochemical and morphological events. Inhibition of rate limiting biochemical steps within this cascade of events can halt and rescue cells from a variety of physiological and pharmacological insults that induce cell death via apoptosis. Studies have focused on the design, synthesis and assessment of a novel series of potent compounds that inhibit the intracellular protein, p53. These compounds protect cells of neuronal origin from toxic concentrations of a variety of insults, including the AD Ab peptide, in tissue culture, and largely protect the brain from ischemic insults in in vivo rodent studies. Additional studies have demonstrated potency in a widely used model of Parkinson's disease. The focus of our studies is to test the clinical utility of p53 inhibition with emphasis on neurodegenerative diseases such as AD, Parkinson's disease and stroke. However, p53 inhibitors hold potential in protecting normal tissue from the toxicities associated with chemotherapeutic agents and radiation therapy in cancer treatment, and form a further focus of future research. (Collaborators: Mark Mattson, Ph.D. LNS, NIA; Debomoy Lahiri, Ph.D., University of Indiana; Robert Rosenthal, M.D., University of Maryland). |
| GLP-1 Agonists, Type 2 Diabetes and Neurodegeneration: Collaborative studies with Josephine Egan, M.D., (Diabetes Section, Laboratory of Clinical Investigation, NIA, Baltimore, MD) are being undertaken on type 2 diabetes, a disease prevalent in the elderly that is caused by a relative refractoriness of the insulin receptor to its ligand and a deficiency in its normal release. The focus of these studies has been to optimize the performance of pancreatic islet cells both in vitro and in rodent diabetic models with peptides that stimulate insulin release to develop novel therapeutics. Extensive studies have been undertaken on the peptide, exendin-4 (Ex-4), which bears a 52% homology to the endogenous insulinotropic peptide, glucagon-like peptide-1 (GLP-1). GLP-1 is released from the gastrointestinal tract during eating to stimulate pancreatic insulin release and thereby lowers blood glucose levels. Like other endogenous hormones, it is short acting. In contrast, Ex-4 has a duration of action of some 16 hours, is more potent than GLP-1 and maintains blood glucose levels chronically without toxicity. Our studies have focused on the structure/activity relation of the GLP-1 amino acid sequence in relation to binding affinity, induction of cAMP levels and insulin release, as well as to metabolic processes involved in its cleavage and inactivation. Novel peptides have been synthesized around to cores of GLP-1 and Ex-4 to optimize the former processes and minimize the latter one. Additional research has supported the transition of Ex-4 from the laboratory and into clinical trials as an experimental therapeutic for type 2 diabetes. Studies in cell culture and rodents indicate that Ex-4 is some 13-fold more potent due to its higher GLP-1 receptor affinity, and it is considerably longer acting than GLP-1. In clinical trials Ex-4 peptide appears, thus far, to be both safe and effective in controlling blood glucose levels in subjects afflicted with type 2 diabetes. Current studies in the laboratory are focused on understanding the mechanism of action of Ex-4 and analogues, further optimizing their action and developing minimized peptides to allow the future design of peptidomimetics. |
| Although predominantly located on pancreatic islet cells, numerous reports now document GLP-1 receptor expression in both the rodent and human brain (for review see: Perry T and Greig NH, J Alzheimers Dis 2003 and Trends Pharmacol Sci 2003). It still remains to be established whether or not GLP-1 is produced by neural cells, but GLP-1 present in the bloodstream can enter brain; utilizing a blood-brain barrier peptide transport system. Intestinally derived peptides, such as GLP-1, are classified not only as hormones, but also as growth factors - peptides capable of regulating diverse cellular processes, including mitosis, growth, and differentiation. Our recent studies indicate that GLP-1 can stimulate the formation of new b-cells in rodents (partly by enhancing b-cell proliferation and partly by enhancing the differentiation of duct progenitor cells to mature b-cells). This fueled our interest to assess a neurological role for GLP-1. Based on the described action of GLP-1 on islet cell differentiation, we hypothesized a neurotrophic role for GLP-1 within the nervous system. Our focus has been to evaluate the role(s) of GLP-1 and related analogues, in vitro and in vivo, to test this hypothesis with a view to developing the most promising ones as an alternative and potentially valuable novel therapeutic intervention for central and peripheral degenerative disorders, such as stroke and peripheral neuropathy associated with type 2 diabetes mellitus. |
| Using cell culture techniques, we have established the presence of the GLP-1 receptor (GLP-1R) on neural cell lines, such as PC12 cells as well as primary rat hippocampal cells by RT-PCR analysis of RNA and GLP-1R-induced increases in intracellular cAMP. Furthermore, GLP-1R stimulation induced differentiation in neural cells in a manner similar to nerve growth factor (NGF), which was reversed by co-incubation with a selective GLP-1R antagonist. The cellular signaling pathways that are activated by GLP-1 in neural cells is a focus of current studies. In addition, GLP-1R agonism provided complete protection against cell death induced by glutamate neurotoxicity in cultured hippocampal neurons, as has been shown by other neurotrophic factors (e.g., NGF and BDNF), suggesting that GLP-1-like peptides may play a significant role in protecting hippocampal neurons against excitotoxic damage and potentially against other types of brain injury. Protection, likewise, was afforded against Ab (particularly Ab1-42) as well as cellular oxidative stress and membrane lipid peroxidation induced by iron. |
| Studies have been undertaken to elucidate whether or not these actions in cell culture models translate to animals. Specifically, using a well established rodent model of neurodegeneration, we have shown complete amelioration of an ibotenic acid induced cholinergic brain lesion following infusion of GLP-1R agonist administration, as assessed by quantitation of the cholinergic cell marker, choline acetyltransferase. Actions on other well established rodent neurodegenerative models are also being assessed and suggest that neuroprotective effects in cell culture translate to animal studies. (Collaborators: Debomoy Lahiri, Ph.D., University of Indiana, IN; Kumar Sambamurti, Ph.D., Medical University of South Carolina; Mark Mattson, Ph.D., Laboratory of Neurosciences, NIA; Josephine Egan, M.D., Diabetes Section, Laboratory of Clinical Investigation, NIA). |
