Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients.

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.

Peripheral chemo-cytokine profiles in Alzheimer's and Parkinson's diseases.

The identification of peripheral biomarkers for neurodegenerative diseases is required to improve the accuracy of clinical diagnosis and monitor both disease progression and response to treatments. The data reviewed in this paper suggest that, in neurodegenerative disease, cytokines are links between peripheral immune system and nervous system dysfunction.

Glucagon-like peptide-1 can reverse the age-related decline in glucose tolerance in rats.

Wistar rats develop glucose intolerance and have a diminished insulin response to glucose with age. The aim of this study was to investigate if these changes were reversible with glucagon-like peptide-1 (GLP-1), a peptide that we have previously shown could increase insulin mRNA and total insulin content in insulinoma cells. We infused 1.5 pmol/ kg-1.min-1 GLP-1 subcutaneously using ALZET microosmotic pumps into 22-mo-old Wistar rats for 48 h. Rat infused with either GLP-1 or saline were then subjected to an intraperitoneal glucose (1 g/kg body weight) tolerance test, 2 h after removing the pump. 15 min after the intraperitoneal glucose, GLP-1-treated animals had lower plasma glucose levels (9.04+/-0.92 mmol/liter, P < 0.01) than saline-treated animals (11.61+/-0.23 mmol/liter). At 30 min the plasma glucose was still lower in the GLP-1-treated animals (8.61+/-0.39 mmol/liter, P < 0.05) than saline-treated animals (10.36+/-0.43 mmol/liter). This decrease in glucose levels was reflected in the higher insulin levels attained in the GLP-1-treated animals (936+/-163 pmol/liter vs. 395+/-51 pmol/liter, GLP-1 vs. saline, respectively, P < 0.01), detected 15 min after glucose injection. GLP-1 treatment also increased pancreatic insulin, GLUT2, and glucokinase mRNA in the old rats. The effects of GLP-1 were abolished by simultaneous infusion of exendin [9-39], a specific antagonist of GLP-1. GLP-1 is therefore able to reverse some of the known defects that arise in the beta cell of the pancreas of Wistar rats, not only by increasing insulin secretion but also by inducing significant changes at the molecular level.

Practical issues in stem cell therapy for Alzheimer's disease.

We have demonstrated that aged animals show significant improvements in cognitive function and neurogenesis after brain transplantation of human neural stem cells or of human adult mesenchymal stem cells that have been dedifferentiated by transfection of the embryonic stem cell gene. We have also demonstrated that peripheral administration of a pyrimidine derivative increased cognition, endogenous brain stem cell proliferation and neurogenesis. These results indicate a bright future for stem cell therapies in Alzheimer's disease (AD). Before this is realized, however, we need to consider the affect of AD pathology on stem cell biology to establish an effective stem cell therapy for this disease. Although amyloid-beta (Abeta) deposition is a hallmark of AD, an absence of a phenotype in the beta-amyloid precursor protein (APP) knockout mouse, might lead one to underestimate the potential physiological functions of APP and suggest that it is unessential or can be compensated for. We have found, however, that APP is needed for differentiation of neural stem cells (NSCs) in vitro, and that NSCs transplanted into a APP-knockout mouse did not migrate or differentiate -- indicating that APP plays an important role in differentiation or migration process of NSCs in the brain. Then again, treatment with high a concentration of APP or its over-expression increased glial differentiation of NSCs. Human NSCs transplanted into APP-transgenic mouse brain exhibited less neurogenesis and active gliosis around the plaque like formations. Treatment of such animals with the compound, (+)-phenserine, that is known to reduce APP protein levels, increased neurogenesis and suppressed gliosis. These results suggest APP levels can regulate NSC biology in the adult brain, that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases, and that a combination of stem cell therapy and regulation of APP levels could provide a treatment strategy for these disorders.

Metal specificity of an iron-responsive element in Alzheimer's APP mRNA 5'untranslated region, tolerance of SH-SY5Y and H4 neural cells to desferrioxamine, clioquinol, VK-28, and a piperazine chelator.

Iron closely regulates the expression of the Alzheimer's Amyloid Precursor Protein (APP) gene at the level of message translation by a pathway similar to iron control of the translation of the ferritin L- and H mRNAs by Iron-responsive Elements in their 5' untranslated regions (5'UTRs). Using transfection based assays in SH-SY5Y neuroblastoma cells we tested the relative efficiency by which iron, copper and zinc up-regulate IRE activity in the APP 5'UTR. Desferrioxamine (high affinity Fe3+ chelator), (ii) clioquinol (low affinity Fe/Cu/Zn chelator), (iii) piperazine-1 (oral Fe chelator), (iv) VK-28 (oral Fe chelator), were tested for their relative modulation of APP 5' UTR directed translation of a luciferase reporter gene. Iron chelation based therapeutic strategies for slowing the progression of Alzheimer's disease (and other neurological disorders that manifest iron imbalance) are discussed with regard to the relative neural toxic action of each chelator in SH-SY5Y cells and in H4 glioblastoma cells.

Increasing annual incidence of primary malignant brain tumors in the elderly.

Between 1973 and 1985, total age-adjusted cancer incidence in the United States (all races, men and women) rose by 10.7%, with an average annual percentage change of +0.9%. Analysis of reported age-specific incidence of primary malignant brain tumors over the same years demonstrates that incidence rates increased dramatically between 1973/1974 and 1985. In 1985, incidence rates for persons aged 75-79, 80-84, and 85 years of age and over were 187%, 394%, and 501%, respectively, of rates in 1973/1974. Similar increases were found in both men and women, analyzed separately and combined. Average annual percentage changes in primary brain tumor incidence were +7.0%, +20.4%, and +23.4% in these age ranges, respectively. Reported incidence in younger persons varied little over the same period of time. The most common histologic type of primary brain tumor in the elderly was of glial origin, predominantly the glioblastoma multiforme and astrocytoma. These tumors are highly malignant and invariably fatal. Two possible causes may explain the increased incidence in the elderly: the introduction and extensive use of x-ray computed tomography since 1973 and/or a true increase in incidence occurring independently of diagnostic advances.

Facilitated transport of melphalan at the rat blood-brain barrier by the large neutral amino acid carrier system.

Melphalan has been reported to be actively transported into tumor cells by two amino acid carrier systems. As amino acids are transported across cerebral capillaries by a facilitated mechanism, studies were undertaken to assess whether or not melphalan was transported similarly, and additionally to determine melphalan's plasma and brain pharmacokinetics. The brain uptake of [14C]melphalan was measured by an in situ brain perfusion technique in the anesthetized rat utilizing [14C]-melphalan. The cerebrovascular permeability-surface area product of [14C]melphalan was calculated at cold melphalan concentrations from O to 16.3 mumol/ml. The permeability-surface area product was concentration dependent and decreased from 10.8 +/- 0.6 (+/- SE) X 10(-4)S-1 at 0.02 mumol/ml melphalan to 5.4 +/- 0.3 X 10(-4)S-1 at 16.3 mumol/ml. The system became saturated at a concentration in excess of 0.1 mumol/ml. The Michaelis-Menten parameters Vmax and Km, determined by nonlinear regression analysis of the permeability-surface area product data, equaled 0.9 +/- 0.3 X 10(-4) mumol/s/g and 0.15 +/- 0.06 mumol/ml, respectively, for the saturable component of melphalan's brain uptake. The Kd of the nonsaturable component was 5.3 +/- 0.03 X 10(-4)S-1. Addition of the amino acid 1-phenylalanine to the brain perfusate inhibited the saturable component of melphalan's brain uptake. The analysis of the plasma and brain concentrations of melphalan by high-performance liquid chromatography, following i.v. melphalan administration, demonstrated that approximately 15% of the drug that was present in plasma entered the brain. These data suggest that the brain uptake of melphalan is facilitated, demonstrating concentration-dependent uptake, saturation, and inhibition, and that melphalan shares the large neutral amino acid carrier system at the blood-brain barrier.

Rapid high-affinity transport of a chemotherapeutic amino acid across the blood-brain barrier.

The therapeutic efficacy of many anticancer drugs against intracerebral tumors is limited by poor uptake into the central nervous system. One way to enhance brain delivery is to design agents that are transported into the brain by the saturable nutrient carriers of the blood-brain barrier. In this paper, we describe a nitrogen mustard amino acid, DL-2-amino-7-bis[(2-chloroethyl)amino/bd-1,2,3,4-tetrahydro-2-napthoi c acid, that is taken up into brain with high affinity by the large neutral amino acid carrier of the blood-brain barrier. Brain transport of DL-2-amino-7-bis[(2-chloroethyl)aminol-1,2,3,4-tetrahydro-2-naphth oic acid in the rat was found to be rapid (cerebrovascular permeability-surface area product approximately 2 x 10(-2) ml/s/g), saturable and inhibitable by large neutral amino acids. Maximal influx rate (Vmax) and half-saturation (Km) constants equaled 0.26 nmol/min/g and 0.19 microM, respectively, in the parietal cortex. Regional brain uptake of acid exceeded that of the clinical analogue, melphalan, by greater than 20-fold. The results demonstrate that drug modification to produce high-affinity ligands for the cerebrovascular nutrient carriers is a viable means to enhance drug delivery to brain for the treatment of brain tumors and other central nervous system disorders.

Organ distribution of liposomal formulations following intracarotid infusion in rats.

The blood-brain barrier (BBB) limits the ability of many hydrophilic solutes to pass from the circulatory system into the nervous system and significantly restricts brain drug delivery. This study examined brain uptake of liposomal formulations in rats. Radiolabeled liposomes less than 1 micron in diameter, prepared from phosphatidylcholine, cholesterol and containing p-aminophenyl-alpha-mannopyranoside or sulfatide, or covalently linked to cationized albumin were injected into the right carotid arteries of rats and organ distribution determined 10 min and 1 h after injection. In all formulations, liposomal retention in brain did not exceed one-tenth of 1% of the injected dose. It seems unlikely that liposomes can significantly bind to or cross the BBB; however, apparent brain uptake could result from liposomal entrapment in the small capillaries of the brain by liposomes larger than 1 micron in diameter.

In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger.

Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.

Forebrain neuronal specific ablation of p53 gene provides protection in a cortical ischemic stroke model.

Cerebral ischemic injury involves death of multiple cell types at the ischemic sites. As a key regulator of cell death, the p53 gene has been implicated in the regulation of cell loss in stroke. Less focal damage is found in stroke animals pre-treated with a p53 inhibitor or in traditional p53 knockout (ko) mice. However, whether the p53 gene plays a direct role in regulating neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 ko mice, we utilized a neuronal specific conditional ko mouse line (CamcreTRP53(loxP/loxP)) to achieve forebrain neuronal specific deletion of p53 and examined the role of the p53 gene in ischemia-induced cell death in neurons. Expression of p53 after stroke is examined using the immunohistochemical method and the outcome of stroke is examined by analysis of infarction size and behavioral deficits caused by stroke. Our data showed that p53 expression is upregulated in the ischemic region in neuronal cells in wildtype (wt) mice but not in CamcreTRP53(loxP/loxP) ko mice. Deletion of the p53 gene in forebrain neurons results in a decreased infarction area in ko mice. Locomotor behavior, measured in automated activity chambers, showed that CamcreTRP53(loxP/loxP) ko mice have less locomotor deficits compared to wt mice after middle cerebral artery occlusion (MCAo). We conclude that manipulation of p53 expression in neurons may lead to unique therapeutic development in stroke.

Post-trauma administration of the pifithrin-α oxygen analog improves histological and functional outcomes after experimental traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Programmed death of neuronal cells plays a crucial role in acute and chronic neurodegeneration following TBI. The tumor suppressor protein p53, a transcription factor, has been recognized as an important regulator of apoptotic neuronal death. The p53 inactivator pifithrin-α (PFT-α) has been shown to be neuroprotective against stroke. A previous cellular study indicated that PFT-α oxygen analog (PFT-α (O)) is more stable and active than PFT-α. We aimed to investigate whether inhibition of p53 using PFT-α or PFT-α (O) would be a potential neuroprotective strategy for TBI. To evaluate whether these drugs protect against excitotoxicity in vitro, primary rat cortical cultures were challenged with glutamate (50mM) in the presence or absence of various concentrations of the p53 inhibitors PFT-α or PFT-α (O). Cell viability was estimated by LDH assay. In vivo, adult Sprague Dawley rats were subjected to controlled cortical impact (CCI, with 4m/s velocity, 2mm deformation). Five hours after injury, PFT-α or PFT-α (O) (2mg/kg, i.v.) was administered to animals. Sensory and motor functions were evaluated by behavioral tests at 24h after TBI. The p53-positive neurons were identified by double staining with cell-specific markers. Levels of mRNA encoding for p53-regulated genes (BAX, PUMA, Bcl-2 and p21) were measured by reverse transcription followed by real time-PCR from TBI animals without or with PFT-α/PFT-α (O) treatment. We found that PFT-α(O) (10 µM) enhanced neuronal survival against glutamate-induced cytotoxicity in vitro more effectively than PFT-α (10 µM). In vivo PFT-α (O) treatment enhanced functional recovery and decreased contusion volume at 24h post-injury. Neuroprotection by PFT-α (O) treatment also reduced p53-positive neurons in the cortical contusion region. In addition, p53-regulated PUMA mRNA levels at 8h were significantly reduced by PFT-α (O) administration after TBI. PFT-α (O) treatment also decreased phospho-p53 positive neurons in the cortical contusion region. Our data suggest that PFT-α (O) provided a significant reduction of cortical cell death and protected neurons from glutamate-induced excitotoxicity in vitro, as well as improved neurological functional outcome and reduced brain injury in vivo via anti-apoptotic mechanisms. The inhibition of p53-induced apoptosis by PFT-α (O) provides a useful tool to evaluate reversible apoptotic mechanisms and may develop into a novel therapeutic strategy for TBI.

Exendin-4 decelerates food intake, weight gain, and fat deposition in Zucker rats.

Exendin-4 is a 39 amino acid peptide produced in the salivary gland of the Gila monster lizard. It has a 53% amino acid homology to the incretin hormone glucagon-like peptide-1 (GLP-1). Exendin-4 induces insulin release through activation of the GLP- 1 receptor but is a much more potent insulinotropic agent than GLP-1. Of critical importance for its potential use as a treatment for diabetes is its much longer biological effect in vivo. Previous studies involving once daily administration of exendin-4 over 13 weeks to db/db mice demonstrated that it lowers hemoglobin A1c (HbA1c), a marker of mean blood glucose levels. Food consumption in the treated animals dropped over the first 4 days and then increased to a level comparable with that of the untreated animals. In this study, we initially examined the effect of once daily injections (over 14 days) on the food consumption of Zucker fatty rats. We observed an immediate reduction in food intake which then leveled off(after 5 days) to match that of the untreated animals. Subsequently we injected the same animals twice daily (treatment period of 56 days in total) and observed a sustained reduction in food intake and weight-gain. This was matched by a reduction in the critical parameters of HbA1c, fasting blood glucose and plasma insulin. MRI imaging of the abdominal regions of the animals showed that initially only the amount of fat deposited in the sc region was reduced after 4 weeks exendin-4 treatment. At the 8-week time point there was a corresponding decrease in the amount of visceral fat deposition. The combination of appetite reduction, decreased fat deposition and an improvement in the parameters associated with glucose intolerance makes a case for the use of exendin-4 as a treatment for diabetes.

Insertion of an N-terminal 6-aminohexanoic acid after the 7 amino acid position of glucagon-like peptide-1 produces a long-acting hypoglycemic agent.

The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha(8), with the previously described GLP-1 8-glycine (GLP-1 Gly(8)) analog. GLP-1 Aha(8) (10 nM) was equipotent with GLP-1 (10 nM) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly(8), the binding affinity of GLP-1 Aha(8) for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC(50): GLP-1, 3.7 +/- 0.2 nM; GLP-1 Gly(8), 41 +/- 9 nM; GLP-1 Aha(8), 22 +/- 7 nM). GLP-1 Aha(8) was also shown to stimulate intracellular cAMP production 4-fold above basal at concentrations as low as 0.5 nM. However, it exhibited a higher ED(50) when compared to GLP-1 and GLP-1 Gly(8) (ED(50): GLP-1, 0.036 +/- 0.002 nM, GLP-1 Gly(8), 0.13 +/- 0.02 nM, GLP-1 Aha(8), 0.58 +/- 0.03 nM). A series of D-amino acid-substituted GLP-1 compounds were also examined to assess the importance of putative peptidase-sensitive cleavage sites present in the GLP-1 molecule. They had poor binding affinity for the GLP-1 receptor, and none of these compounds stimulated the production of intracellular cAMP in CHO/GLP-1R cells or insulin secretion in RIN 1046-38 cells. GLP-1 Aha(8) (24 nmol/kg) administered sc to fasted Zucker (fa/fa) rats (mean blood glucose, 195 +/- 32 mg/dl) lowered blood glucose levels to a nadir of 109 +/- 3 mg/dl, and it remained significantly lower for 8 h. Matrix-assisted linear desorption ionization-time of flight mass spectrometry of GLP-1 Aha(8) incubated with DPP IV (37 C, 2 h) did not exhibit an N-terminal degradation product. Taken together, these results show that insertion of Aha after the 7 position in GLP-1 produces an effective, long-acting GLP-1 analog, which may be useful in the treatment of type 2 diabetes mellitus.

Cholinesterase inhibitors, beta-amyloid precursor protein and amyloid beta-peptides in Alzheimer's disease.

The extracellular deposition of amyloid beta-peptide (Abeta) in the form of cerebrovascular amyloid and extracellular plaques is one of the major neuropathological manifestations of Alzheimer's disease (AD). Abeta is generated proteolytically from the large beta-amyloid precursor protein (APP). APP is cleaved by a group of proteases called "secretase" to generate soluble derivatives of APP (sAPP), which are secreted in human plasma, CSF and cultured cells. Neurochemically, there is a severe loss of cholinergic neurons and a decreased synthesis of acetylcholine in neocortex in AD. Current approved AD drugs, such as aricept and tacrine, are based on the use of cholinesterase inhibitors (ChEIs) and have been reported to improve memory deficits and cognitive decline in some patients with AD. To compare the effects of ChEIs on APP processing, we have tested a series of ChEIs such as tacrine, physostigmine, metrifonate, phenserine and cymserine in cultured human neuroblastoma cells. We analyzed levels of sAPP by immunochemical techniques with APP-specific antibodies and assayed levels of Abeta by a sensitive sandwich ELISA. Based on these results, ChEIs can be divided into three groups: the first group of ChEIs had no effect on sAPP secretion, the second decreased the sAPP secretion only, and third group affected the secretion of sAPP and Abeta. The difference in the action of metrifonate, physostigmine, phenserine and tacrine on APP processing is independent of their selectivity for the cholinesterase enzymes. This possibly is due to the different targets that are used by ChEIs. Studying the effects of ChEIs on different targets is useful to maximize the benefit of ChEIs for the treatment of AD subjects.

The experimental Alzheimer drug phenserine: preclinical pharmacokinetics and pharmacodynamics.

Phenserine, a phenylcarbamate of physostigmine, is a new potent and highly selective acetylcholinesterase (AChE) inhibitor, with a > 50-fold activity versus butyrylcholinesterase (BChE), in clinical trials for the treatment of Alzheimer's disease (AD). Compared to physostigmine and tacrine, it is less toxic and robustly enhances cognition in animal models. To determine the time-dependent effects of phenserine on cholinergic function, AChE activity, brain and plasma drug levels and brain extracellular acetylcholine (ACh) concentrations were measured in rats before and after phenserine administration. Additionally, its maximum tolerated dose, compared to physostigmine and tacrine, was determined. Following i.v. dosing, brain drug levels were 10-fold higher than those achieved in plasma, peaked within 5 min and rapidly declined with half-lives of 8.5 and 12.6 min, respectively. In contrast, a high (> 70%) and long-lasting inhibition of AChE was achieved (half-life > 8.25 h). A comparison between the time-dependent plasma AChE inhibition achieved after similar oral and i.v. doses provided an estimate of oral bioavailability of 100%. Striatal, in vivo microdialysis in conscious, freely-moving phenserine-treated rats demonstrated > 3-fold rise in brain ACh levels. Phenserine thus is rapidly absorbed and cleared from the body, but produces a long-lasting stimulation of brain cholinergic function at well tolerated doses and hence has superior properties as a drug candidate for AD. It selectively inhibits AChE, minimizing potential BChE side effects. Its long duration of action, coupled with its 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.

Clinical pharmacokinetics of physostigmine in patients with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetic and pharmacodynamic properties of physostigmine in subjects with Alzheimer's disease. METHODS: Plasma physostigmine concentration and butyrylcholinesterase inhibition were measured in blood samples collected during and after a single high-dose (1 to 1.5 mg for 45 to 60 minutes) and a sustained low-dose steady-state intravenous infusion in nine subjects with Alzheimer's disease. Escalating doses (0.5 to 25 mg/day) were administered during a 2-week period. A dose (2 to 12 mg/day) that optimized cognition in each subject was identified and then administered in a randomized, double-blind, placebo-controlled crossover design for 1 week. RESULTS: The elimination half-life of physostigmine was 16.4 +/- 3.2 (SE) minutes. Clearance and volume of distribution were 7.7 +/- 0.9 (SE) L/min and 2.4 +/- 0.6 (SE) L/kg, respectively. Butyrylcholinesterase inhibition half-life was 83.7 +/- 5.2 (SE) minutes. During sustained steady-state infusion, plasma physostigmine concentration (r = 0.95) and butyrylcholinesterase inhibition (r = 0.99) were linearly correlated with the dose. In five cognitive responders, the memory enhancement was significantly correlated (r = 0.86; p < 0.05) with butyrylcholinesterase inhibition. CONCLUSIONS: These results showed that, in cognitive responders, memory enhancement by physostigmine in Alzheimer's disease is correlated directly to the magnitude of plasma cholinesterase inhibition. Furthermore, during single-dose conditions, the dynamic half-life is five-fold longer than the kinetic half-life.

Clinical pharmacokinetics of arecoline in subjects with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease. METHODS: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS. RESULTS: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects. CONCLUSIONS: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Tight binding dopamine reuptake inhibitors as cocaine antagonists. A strategy for drug development.

The experiments reported in this study tested the hypothesis that tight binding dopamine (DA) reuptake inhibitors might act as cocaine antagonists. Binding studies demonstrated that the high affinity dopamine reuptake inhibitor, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine (GBR12909) produced a wash-resistant inhibition of the DA transporter in rat striatal membranes as labeled by [3H]cocaine or [3H]1-[2-(diphenyl- methoxy)ethyl]-4-(3-phenylpropyl)piperazine [( 3H]GBR12935), indicative of tight binding. In vivo microdialysis experiments showed that administration of 25 mg/kg GBR12909 to rats produced a modest, but not statistically significant, increase in the extracellular levels of striatal DA, while this same dose of GBR12909 inhibited the ability of cocaine to elevate extracellular DA levels by 64%. These data suggest that tight binding DA reuptake blockers may provide a fruitful approach for developing a cocaine antagonist.

Enhanced cerebrovascular permeability by Metrazol: significance for brain metastases.

Metrazol enhanced the penetration of two proteins (125I human serum albumin and horseradish peroxidase), and the anticancer agent, razoxane, into the central nervous system of anaesthetized rats. Penetration was increased throughout the whole brain. With the exception of the bladder, no peripheral tissue was affected. The increase in brain permeability was temporary and reversed within 4 hours; brain levels of drug and protein were increased by up to three times.