Design and Development of Drugs and Pharmacologic Probes The goal of the Drug Design & Development Section is to develop novel agents against rate-limiting steps involved in the pathophysiology of diseases associated with aging, with particular interest in neurological diseases, such as Alzheimer's disease (AD) and stroke, as well as in systemic diseases, such as diabetes. Alzheimer's Disease: Although the neuropathological quantification of beta-amyloid (Ab) 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 correlates best to 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. Anticholinesterases: Our efforts have 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. Extensive studies involving chemistry, X-ray crystallography, biochemistry and pharmacology resulted in the ability to differentiate between the cholinesterase subtypes. This has provided us the ability to develop novel agents to selectively and reversibly inhibit either AChE or butyrylcholinesterase (BChE) in either the brain or periphery for an optimal duration for the potential treatment of a variety of diseases, such as AD, Myasthenia Gravis, and as chemical warfare prophylactics. Acetylcholinesterase: Two of numerous novel synthesized AChE inhibitors are in development for the treatment of AD; specifically, the pure non-competitive inhibitors, phenserine and tolserine. Both are phenylcarbamates of physostigmine that are 70- and 190-fold selective for AChE vs. BChE. Compared to current agents for AD treatment, they have a favorable toxicologic profile and robustly enhances cognition in animal models. Studies were undertaken to determine their time-dependent effects on cholinergic function, AChE activity, brain and plasma drug levels and brain extracellular ACh concentrations in rodents to support clinical studies. They possess along duration of action, coupled with a short pharmacokinetic half-life, reduces dosing frequency, decreases body drug exposure and minimizes the dependence of drug action on the individual variations of drug metabolism commonly found in the elderly In a collaboration with Axonyx Corp. (New York), clinical studies were undertaken to evaluate the safety, maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of phenserine tartrate, in healthy elderly subjects to support efficacy trials. Specifically, a Phase I, blinded, placebo controlled, dose escalation study, was undertaken in healthy elderly volunteers (n=32) that received single oral doses (5 to 20 mg). In conclusion, phenserine tartrate was safe and well tolerated as a single oral dose of either 5 or 10 mg. Further studies demonstrated safety following twice daily dosing for 5 consecutive days, and the agent is currently in phase II clinical efficacy trials in volunteers with mild to moderate AD. Butyrylcholinesterase: In the normal brain, approximately 80% of brain cholinesterase activity is AChE, 20% is BChE. AChE activity is concentrated mainly in neurons, while BChE is primarily associated with glial cells. Kinetic evidence indicates a role for BChE, in hydrolysing excess ACh. In advanced AD, however, AChE activity decreases to 15% of normal levels in specific brain regions, whereas BChE activity increases. The ratio of BChE to AChE is set in normal brain to achieve optimal brain activity and changes dramatically in cortical regions as AD progresses, thereby changing BChE?s role. Highly potent BChE inhibitors have been synthesized and are in preclinical assessment to evaluate their potential AD drug candidates. Molecular events associated with AD: The reduction in levels of the potentially toxic amyloid-b peptide (Ab) has emerged as an important therapeutic goal in AD. Key targets for this goal are factors that affect the expression and processing of the Ab precursor protein (bAPP). Our earlier research showed that our AChE inhibitor, phenserine, reduces bAPP levels in vivo. We therefore studied the mechanism of phenserine's actions to define the regulatory elements in bAPP processing. Phenserine treatment resulted in decreased secretion of soluble bAPP and Ab in cultured human neuroblastoma cells without toxicity. This activity was unrelated to its action as an anticholinesterase, but was post-transcriptional as it suppressed bAPP protein expression without altering bAPP mRNA levels. This was mediated via 5' untranslated region (5 UTR) of bAPP mRNA. We have synthesized novel agents to both characterize the target as well as to selectively and optimally regulate bAPP mRNA translation to reduce Ab synthesis. Stroke and Parkinson's disease: Drugs currently used in patients with stroke and Parkinson's disease (PD) provide temporary relief of symptoms, but do not prevent the cell death. Cell death in both results from a process called apoptosis, which may be triggered by mitochondrial impairment and oxidative stress. As up-regulation of p53 has been described as a common feature of several neurodegenerative disorders, and is a critical and rate-limiting step in the cascade of biochemical events that leads to apoptosis, it represents a potential target for interventive drug development. As a consequence, we recently designed and synthesized a novel series of tetrahydrobenzothiazole analogues that are based on the structure of the antihelminthic compound, pifithrin-a . Compounds are currently being assessed for neuroprotective action in tissue culture and in animal models to select agents of potential for evaluation as drug candidates. Diabetes: Type 2 diabetes is a prevalent disease in the elderly. It is caused by a relative deficiency of insulin and a decrease in insulin action at insulin-sensitive tissues. Present treatments are less than satisfactory. In collaborative studies with Josephine Egan, M.D. (Diabetes Section, LCS, NIA), we have extensive experience with GLP-1, a peptide that is actively secreted from the gut in response to food and is a potent secretagogue (i.e., insulinotropic), as a potential treatment for diabetes. When given continuously to diabetic subjects, pharmacological concentrations of GLP-1 can maintain blood glucose levels within their normal range. However, a major shortcoming of GLP-1 given exogenously as a potential therapeutic is its brief biological half-life, it is cleared within minutes in both rodents and humans. As a consequence, GLP-1 was used as a starting point to develop longer acting peptides Studies with exendin-4, an endogenous peptide from the Gila monster lizard that shares 53% homology with GLP-1, have shown that it, is (i) more potent and (ii) maintains higher plasma levels of insulin over, (iii), a longer time duration than does GLP-1, and (iv) have supported the development of exedin-4 into phase II efficacy studies for the treatment of type 2 diabetes. Novel, chimeric peptides that combine the best features of GLP-1 and exendin-4 have been design, synthesized and are under assessment in a variety of cell culture and animal models to assess their potential for clinical development.
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