Raymond T. Bartus

On neurodegenerative diseases, models, and treatment strategies : Lessons learned and lessons forgotten a generation following the cholinergic hypothesis

Lifes Journey If life is indeed a journey, then poetry must be the map that reveals all its topographic possibilitiesellipsis while science is the compass that keeps us from getting lost. -R. T. Bartus, Simple Words for Complex Lives, (c) 1998 In the nearly 20 years since the cholinergic hypothesis was initially formulated, significant progress has been achieved. Initial palliative treatments for Alzheimers disease (AD) have proven beneficial and have gained FDA approval, the use of animal models for studying AD and other neurodegenerative diseases has achieved wider acceptance, and important insight into the potential causes and pathogenic variables associated with various neurodegenerative diseases continues to increase. This paper reviews the current status of the cholinergic hypothesis in the context of continuing efforts to improve upon existing treatments for AD and explores the role that animal models might continue to play. Using the benefit of hindsight, particular emphasis is placed on an analysis of the approaches, strategies, and assumptions regarding animal models that proved useful in developing the initial treatments and those that did not. Additionally, contemporary issues of AD are discussed within the context of the cholinergic hypothesis, with particular attention given to how they may impact the further refinement of animal models, and the development of even more effective treatments. Finally, arguments are presented that, despite the deserved enthusiasm and optimism for identifying means of halting the pathogenesis of AD, a clear need for more effective palliative treatments will continue, long after successful pathogenic treatments are available. This review, therefore, focuses on issues and experiences intended to: (a) facilitate further development and use of animal models for AD and other neurodegenerative diseases, and (b) accelerate the identification of newer, even more effective treatments.

Oncolytic virus therapy of multiple tumors in the brain requires suppression of innate and elicited antiviral responses.

The occurrence of multiple tumors in an organ heralds a rapidly fatal course. Although intravascular administration may deliver oncolytic viruses/vectors to each of these tumors, its efficiency is impeded by an antiviral activity present in complement-depleted plasma of rodents and humans. Here, this activity was shown to interact with complement in a calcium-dependent fashion, and antibody neutralization studies indicated preimmune IgM has a contributing role. Short-term exposure to cyclophosphamide (CPA) partially suppressed this activity in rodents and humans. At longer time points, cyclophosphamide also abrogated neutralizing antibody responses. Cyclophosphamide treatment of rats with large single or multiple intracerebral tumors substantially increased viral survival and propagation, leading to neoplastic regression.

Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration.

This research was performed to determine whether a selective inhibitor of the calcium-dependent protease, calpain, could reduce ischemia-associated brain damage when peripherally administered after a vascular occlusion. Methods A variation of the rat middle cerebral artery occlusion model was used. A range of doses of AK295 (a novel calpain inhibitor synthesized for this purpose) was continuously infused through the internal carotid artery, beginning 1.25 hours from the initiation of the occlusion. Rats were killed at 21 hours, and the infarct volume was quantified. Results Postocdusion (1.25-hour) infusion of the calpain inhibitor AK295 elicited a dose-dependent neuroprotective effect after focal ischemia. The highest dose tested (3 mg/kg per hour) afforded the maximum effect, illustrated by a 32% reduction in infarct volume 21 hours after the ischemia (vehicle, 81.7±4.7 mm3; AK295, 54.9±6.9 mm3; P<.007). Conclusions These data provide the first evidence that a peripherally administered calpain inhibitor can protect against ischemic brain damage. They offer further support for an important role of calpain proteolysis in the brain degeneration associated with cerebral ischemic events and suggest that selective calpain inhibitors provide a rational, novel, and viable means of treating such neurodegenerative problems.

Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7.

OBJECTIVEnIntracarotid infusion of the bradykinin analog, RMP-7, can increase permeability in brain tumor capillaries. This study sought to determine the following: 1) the unidirectional transport, Ki, of radiolabeled [14C]carboplatin into brain tumors with either intravenous or intracarotid RMP-7 infusions; 2) the duration and extent of increased permeability in tumor capillaries during continuous RMP-7 infusions; and 3) the effect on survival of carboplatin combined with RMP-7 treatment in rats with gliomas.nnnMETHODSnWistar rats with RG2 gliomas were used, and a unidirectional transfer constant, Ki, was determined using quantitative autoradiography. In the survival study, the rats were treated with intra-arterial carboplatin and RMP-7 at Days 5 and 7 after tumor implantation.nnnRESULTSnIntracarotid infusion of RMP-7 for 15 minutes increased the transport of [14C]carboplatin to tumors by 2.7-fold, as compared with saline infusion alone (P < 0.001). The transports of [14C]dextran and [14C]carboplatin into tumors were significantly higher with 15 minutes of intracarotid infusion of RMP-7 (0.1 microgram/kg/min), compared to those with 10-, 30-, or 60-minute infusions (P < 0.01). Rats treated at Days 5 and 7 after tumor implantation with carboplatin alone (10 mg/kg) exhibited a modest increase in survival at 31 days (37%, compared to < 10% of controls), while those given the combination of carboplatin with RMP-7 exhibited a significantly higher survival rate (74%).nnnCONCLUSIONnIntracarotid infusion of RMP-7 can selectively increase transport of carboplatin into brain tumors and results in higher survival in rats with gliomas. These findings support the use of intracarotid infusion of RMP-7 to enhance the delivery of carboplatin to patients with malignant brain tumors.

Calpain as a novel target for treating acute neurodegenerative disorders

Calpains are cytosolic, neutral proteases that normally exist in an inactive or quiescent state. They require higher than normal levels of calcium for activation which, once accomplished, lead to irreversible proteolysis of numerous cytoskeletal, membrane-associated and regulatory proteins. Because of these characteristics, calpain is gaining attention as a potentially important pathogenic variable in ischemic neuronal death. This manuscript explores this hypothesis by briefly reviewing current support for the role played by calpain in ischemic neurodegeneration, and then discussing a series of recently published studies which: 1. offer further evidence for the hypothesis, and 2. provide direct support for the idea that selective inhibition of calpain can greatly limit the neuronal damage that would normally occur following both global as well as focal brain ischemia. Thus, the data reviewed in this manuscript support the ideas that unregulated activation and proteolysis of intraneuronal calpain plays a significant role in the brain damage that occurs following an ischemic event and that delivering selective and membrane permeant calpain inhibitors to ischemic tissue may provide a powerfully effective therapeutic means of limiting neuronal damage.

Pathway across blood-brain barrier opened by the bradykinin agonist, RMP-7

The route taken by lanthanum (MW 139) across cerebral endothelium was delineated when the blood-brain barrier was opened by RMP-7, a novel bradykinin agonist. Balb C mice were infused through a jugular vein with LaCl3 with or without RMP-7 (5 micrograms/kg). Ten minutes later, the brains were fixed with aldehydes and processed for electron microscopy. The patency of the junctions between endothelial cells was estimated by counting the number of junctions penetrated by LaCl3. Tracer penetrated the junctions in about 25% of microvessels in vehicle infused, control mice and about 58% in the RMP-7 group, where more junctions per vessel were also penetrated. The LaCl3 then penetrated the basal lamina in about 20% of all microvessels in the RMP-7 group, versus 0.50% in the control group. From the basal lamina, the tracer entered perivascular spaces in about 13% of all microvessels in the RMP-7 group and about 0.07% in the controls. Very few endocytic pits or vesicles in the RMP-7 group were labeled, so LaCl3 did not cross endothelium by transcytosis. The increased number of tight junctions penetrated by tracer and its spread into periendothelial basal lamina and interstitial clefts indicated, therefore, a paracellular route of exudation in the RMP-7 treated animals.

Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain.

Changes in neocortex and striatum were characterized over time following focal ischemia to the brain. Rats were subjected to permanent middle cerebral artery occlusion (MCA-O) and sacrificed 1, 3, 6, 12, or 24 h later. The affected tissue was processed for tetrazolium chloride (TTC) and cresyl violet staining, as well as for Western blots to detect calpain-induced spectrin proteolysis. Significant changes in cell size and spectrin breakdown occurred within the first hour of occlusion, with further, dramatic changes in these two early markers continuing over time. Initial evidence of cell loss was noted at 1 h postocclusion in the striatum and at 3 h in the neocortex. However, even in the center of the most affected portion of the neocortex, the majority of cells appeared to be intact through 6 h. By this time, a significant TTC-defined infarct also emerged. These quantitative data indicate that calpain-induced proteolysis occurs very soon after the ischemic insult, is correlated with earliest changes in cell hypotrophy, and precedes or occurs in tandem with evidence of significant cell loss. They also demonstrate that, while some cell loss occurs earlier than previously believed, the majority of cells remains morphologically intact well beyond what is typically thought to be the window of opportunity for intervention. The results thus raise the question of how long after the ischemic event pharmaceutic intervention might be employed to salvage substantial numbers of neurons.

The Calpain Hypothesis of Neurodegeneration: Evidence for a Common Cytotoxic Pathway

Calpains general function and pathogenic role in the CNS are reviewed. Collectively, the literature indicates that calpain proteolysis plays a common and important role in a variety of acute neurodegenerative conditions, including focal ischemia (stroke), global ischemia, traumatic brain injury, and spinal cord injury. This evidence indicates that 1) calpain is activated in an abnormally sustained fashion during cellular events commonly associated with neurodegeneration (e.g., excessive interstitial glutamate and cytosolic calcium); 2) many of calpains preferred substrates are degraded as important components in these neurodegenerative conditions; 3) calpain activation occurs early in the pathogenic cascade of each, prior to onset of substantial cell death; and 4) calpain inhibitors can effectively reduce the severity of neuronal damage and loss of function normally associated with these acute neurodegenerative perturbations. Calpain proteolysis is also implicated in chronic neurodegenerative diseases, with the strength of current evidence varying among specific diseases. The evidence accumulated for a plausible role in Alzheimers disease (AD) is currently the strongest. For example, empirical links have been established between abnormal calpain proteolysis and 1) the cellular formation of classic Alzheimers pathology, such as β-amyloid plaques, neurofibrillary tangles, and Alz-50 immunoreactivity; 2) the brain regions with greatest concentrations of AD-related pathology; and 3) the degeneration of key brain pathways vulnerable in the early stages of the disease. Similar, though less extensive, evidence exists for a potential role of abnormal calpain proteolysis in Parkinsons disease. Finally, for several other chronic neurodegenerative conditions (e.g., Huntingtons disease and amyotrophic lateral sclerosis), early evidence is emerging that calpain may also play some pathogenic role. Thus, these data support the possibility that uncontrolled calpain proteolysis may contribute to and/or accelerate the loss of neurons associated with a wide range of neurodegenerative conditions and may, therefore, represent an important, final common cytotoxic pathway for many diverse forms of neurodegeneration. NEUROSCIENTIST 3:314–327, 1997

The development of the bradykinin agonist labradimil as a means to increase the permeability of the blood-brain barrier from concept to clinical evaluation

AbstractLabradimil (Cereport®; also formerly referred to as RMP-7) is a 9-amino-acid peptide designed for selectivity for the bradykinin B2 receptor and a longer plasma half-life than bradykinin. It has been developed to increase the permeability of the blood-brain barrier (BBB) and is the first compound with selective bradykinin B2 receptor agonist properties to progress from concept design through to tests of efficacy in patients.n In vitro studies demonstrate that labradimil has a longer half-life than bradykinin and selectively binds to bradykinin B2 receptors, initiating typical bradykinin-like second messenger systems, including increases in intracellular calcium and phosphatidylinositol turnover. Initial proof of principle studies using electron microscopy demonstrated that intravenous labradimil increases the permeability of the BBB by disengaging the tight junctions of the endothelial cells that comprise the BBB. Autoradiographic studies in rat models further demonstrated that labradimil increases the permeability of the BBB in gliomas. Intravenous or intra-arterial labradimil increases the uptake of many different radiolabelled tracers and chemotherapeutic agents into the tumour in a dose-related fashion. These effects are selective for the tumour and for the brain surrounding the tumour, and are particularly robust in tumour areas that are normally relatively impermeable. The increased chemotherapeutic concentrations are maintained for at least 90 minutes, well beyond the transient effects on the BBB.The increase in permeability with labradimil occurs rapidly but is transient, in that restoration of the BBB occurs very rapidly (2 to 5 minutes) following cessation of infusion. Even with continuous infusion of labradimil, spontaneous restoration of the barrier begins to occur within 10 to 20 minutes. Collectively, these data demonstrate that the B2 receptor system that modulates permeability of the BBB is highly sensitive and autoregulated and that careful attention to the timing of labradimil and the chemotherapeutic agent is important to achieve maximal effects.Survival studies in rodent models of both gliomas and metastatic tumours in the brain demonstrate that the enhanced uptake observed with the combination of labradimil and water-soluble chemotherapeutics enhances survival to a greater extent than achieved with chemotherapy alone. Finally, preliminary clinical trials in patients with gliomas provide confirmatory evidence that labradimil permeabilises the blood-brain tumour barrier and might, therefore, be used to increase delivery of agents such as carboplatin to tumours without the toxicity typically associated with dose escalation.

Biocompatibility of poly (DL-lactide-co-glycolide) microspheres implanted into the brain.

The delivery of therapeutic molecules to the brain has been limited in part due to the presence of the blood–brain barrier. One potential solution is the implantation of biodegradable polymers with sustained release of drugs. Poly (DL-lactide-co-glycolide) (PLG) is a bioerodible polymer with a long and successful history of use as a suture material. More recently, PLG has been investigated for localized and sustained delivery of molecules into both peripheral sites and the brain. Despite its well-defined safety profile for parenteral applications, little information exists concerning the safety of PLG when implanted into the brain. To further characterize the biocompatibility of PLG in the brain, we examined the gliotic response following implants of PLG into the brains of rats. As a control, each animal received an injection of the suspension medium into the contralateral hemisphere. Following implantation, PLG was well tolerated. GFAP-positive astrocytes were observed throughout the cerebral cortex and striatum on both the implanted and control sides, with the reaction being greatest within the heavily myelinated fiber tracts of the corpus callosum. Quantitative analyses revealed that this reaction occurred within 1 h postsurgery, reached its peak at 1 week following surgery, and then decreased markedly by 1 month postsurgery. A minimal gliotic reaction was still present 1 year postsurgery but was localized to the needle tract. No differences in GFAP reactivity were seen between the polymer-implanted and control sides at any time point. Histological analysis determined that the majority of the PLG disappeared between 1 and 4 weeks. A set of parallel studies in which PLG samples were retrieved from the brain at various time points corroborated these findings and determined that the majority of PLG degraded within 2 weeks following implantation. Together, these results demonstrate that PLG is well tolerated following implantation into the CNS and that the astrocytic response to PLG is largely a consequence of the mechanical trauma that occurs during surgery. The biocompatibility of PLG implanted into the CNS provides further support for its use in a wide range of new therapeutic applications for sustained and localized drug delivery to the brain.