A "bridging" (safety/tolerance) study of besipirdine hydrochloride in patients with Alzheimer's disease.

Besipirdine hydrochloride is a novel compound with cholinergic and adrenergic activity being investigated as a treatment for Alzheimer's disease (AD). The pharmacodynamics of some anti-dementia drugs are known to differ in patients with AD as compared with elderly normals. The present study was designed to determine the maximum tolerated dose (MTD) of multiple oral doses of besipirdine in AD patients. Twelve AD patients (NINCDS/ADRDA criteria; 7M, 5F, ages 58-75, mean age 65) were randomized to besipirdine (n = 9) or placebo (n = 3) in a double-blind, parallel-group, rising-dose design. Doses were 10, 20, 30, and 40 mg bid for 2 days each, followed by 50 and 60 mg bid for 5 days each. The most common adverse events were asymptomatic postural hypotension and asymptomatic bradycardia. Two patients on active drug developed severe adverse events: 1 after 3 days at 50 mg bid (nausea and vomiting); 1 after 3 days at 60 mg bid (angina). Due to the anginal episode, the study was terminated on Day 17. Plasma concentrations increased linearly with dose for besipirdine and its major metabolite. The two patients who developed severe adverse events had the highest plasma concentrations measured. Besipirdine 50 mg bid was considered the maximum tolerated dose (MTD).

The expression of phosphorylated and non-phosphorylated forms of MAP5 in the amphibian CNS.

MAP5 is a microtubule-associated protein that in rat and quail is more abundant in the developing than in the adult brain. Previous studies in our laboratory have shown that MAP5 can be resolved into two forms by SDS-PAGE, MAP5a and MAP5b (Mr 300,000-320,000 Da) with MAP5a representing a highly phosphorylated form of this protein. In the present study, the relationship between MAP5 expression and neuronal growth and plasticity was investigated by assessing the amount and distribution of MAP5a and MAP5b in both the developing Xenopus brain and in different regions of the adult brain where neurons of varying growth potential and plasticity are present. In the larval and metamorphic Xenopus brain, like the neonatal rat brain, MAP5 is present in the highly phosphorylated form, MAP5a, and in concentrated in neuronal processes. In the adult Xenopus brain, MAP5a remains high in the optic tectum but, like the situation in the adult rat brain, is undetectable in the telencephalon. Immunohistochemistry showed that MAP5 was concentrated in the outer layer of the tectum, where ingrowing and plastic retinal ganglion cell axons are found. The correlation between MAP5 expression and phosphorylation and growth potential suggests that this molecule plays an important role in the regulation and organization of the neuronal cytoskeleton during neurite outgrowth and plasticity.

The adult rat olfactory system expresses microtubule-associated proteins found in the developing brain.

We have compared the expression and localization of neuronal microtubule-associated proteins (MAPs) MAP5, MAP2, and tau in the adult rat olfactory system and cerebral cortex. Each of these MAPS is known to exist as distinct "early" and "late" forms in the developing and adult brain, respectively. Because axonal growth and dendritic reinnervation continue in the adult olfactory system, it can serve as a test of whether expression of early MAP forms is necessary for neuronal growth and plasticity. We found that for all three MAPs, the early forms continue to be expressed in the adult olfactory system, whereas the cerebral cortex switches to the late forms during neuronal maturation (between 10 and 20 days of age in the rat). For MAP2, the 6 kb mRNA and low-molecular weight MAP2c, both of which are typically found in embryonic tissue, persist in the adult olfactory bulb. For MAP5, an early highly phosphorylated form, MAP5a, is present throughout the brain and disappears during maturation simultaneously with a several-fold drop in the overall level of MAP5. However, in the olfactory bulb, MAP5 levels do not fall, and MAP5a persists in the adult. The early form of tau is also prominent in the adult olfactory bulb and, by immunohistochemistry, is mainly confined to a subset of olfactory axons, the vomeronasal nerve. Thus, in the adult olfactory bulb, both MAP protein synthesis and phosphorylation conform to a pattern associated with the developing brain. Immunohistochemistry also showed that MAP5 is concentrated in the olfactory nerve axons and the mitral cell dendrites of the olfactory bulb, i.e., exactly those elements that are involved in the olfactory nerve innervation that takes place in the adult. These results suggest that the expression of the early MAP forms is closely associated with neurite outgrowth and plasticity.

The sequential appearance of low- and high-molecular-weight forms of MAP2 in the developing cerebellum.

Mammalian microtubule-associated protein 2 (MAP2) exists in high-molecular-weight (Mr approximately 280,000) and low-molecular-weight (Mr approximately 70,000) forms, with the latter protein being more abundant in embryonic brain homogenates than in preparations from mature brain (Riederer and Matus, 1985). In the current study, we have shown that avian MAP2 also exists as both high- (Mr approximately 260,000) and low-molecular-weight (Mr approximately 65,000) forms whose relative abundance changes during brain maturation, indicating a conserved function for these proteins during vertebrate neuronal morphogenesis. Using indirect immunohistochemistry, we have determined the cellular distribution of the high- and low-molecular-weight forms of MAP2 in the developing avian cerebellum. In the embryonic cerebellum, low-molecular-weight MAP2 is found in the external granular layer and in epithelial cells. High-molecular-weight MAP2 is found only in neurons that have commenced dendrogenesis, i.e., Purkinje cells and neurons within the internal granular layer. Thus, low-molecular-weight MAP2 is not only more abundant in embryonic nervous tissue than in the adult, but it also appears in glia and in differentiating neurons before the high-molecular-weight form. We have also shown that in the mature cerebellum high-molecular-weight MAP2 cannot be detected with monoclonal antibodies or polyclonal antisera in Purkinje cell dendrites. Polyclonal antisera against the regulatory subunit of the cAMP-dependent protein kinase, which is associated with MAP2 in the Purkinje cell dendrites of the rat, also fail to stain Purkinje cell dendrites in the mature quail cerebellum. This suggests that high-molecular-weight MAP2 may be necessary for the establishment of dendrites but is not necessary for the maintenance of dendritic form.

Phylogenetic conservation of brain microtubule-associated proteins MAP2 and tau.

The major rat brain microtubule-associated proteins, MAP2 and tau, exhibit various properties that implicate them in the mechanisms underlying the growth of axons and dendrites during neuronal development. To determine if these properties represent fundamental morphogenetic mechanisms, we have examined the phylogenetic conservation of these proteins in Xenopus laevis, quail and rat with respect to their molecular form, cytological distribution and developmental expression. In all three species, the high-molecular weight form of MAP2 migrates as a pair of polypeptides (MAP2a and MAP2b); this doublet as well as the low-molecular weight form of MAP2 (MAP2c) and the tau proteins are markedly similar in size in the different classes of vertebrates. Immunohistochemical staining of the Xenopus and quail cerebellum showed that MAP2 is highly concentrated in dendrites whereas the tau proteins are predominantly confined to axons, exactly as they are in rat. The developmental regulation of these proteins in Xenopus and rat is also conserved. Between the larva and the adult (i.e. during metamorphosis) MAP2c undergoes a marked decrease while MAP2a undergoes a large increase. Thus, in both classes of vertebrates the timing of changes in MAP2 expression coincides with the maturation of neuronal morphology. Taken together, these conserved properties of MAP2 and tau in three phylogenetically divergent classes of vertebrates suggest that these proteins serve fundamental functions during neuronal morphogenesis.