Review article: tolevamer, a novel toxin-binding polymer: overview of preclinical pharmacology and physicochemical properties.

BACKGROUND: Tolevamer is a novel toxin-binding polymer that is currently being investigated in clinical trials for the treatment of patients who have Clostridium difficile-associated diarrhoea. AIMS: To summarize the results of in vitro and in vivo preclinical studies of tolevamer. In contrast to antibiotics, tolevamer binds C. difficile toxins to interrupt toxin-mediated intestinal inflammation and tissue damage, and does not demonstrate direct antimicrobial activity. METHODS: Pharmacokinetics/pharmacodynamics were studied in rats and dogs; efficacy was studied in a hamster model. RESULTS: Studies in rats and dogs indicate that tolevamer is essentially non-absorbed from the gastrointestinal tract and show that drug interactions with commonly used therapies are unlikely. Pharmacologic studies indicate that tolevamer reduces disease severity and recurrence rates in the hamster model of C. difficile-associated diarrhoea and blocks the enterotoxic effects of toxin A in rat ileum. The binding parameters calculated for the interaction of tolevamer with toxins A and B provide a reasonable physicochemical model that supports the potential clinical utility of tolevamer. CONCLUSIONS: These preclinical results are consistent with the effectiveness and safety profile of tolevamer observed in clinical studies in patients with C. difficile-associated diarrhoea.

Terbium binding to axonal membrane vesicles from lobster (Homarus americanus) peripheral nerve. A probe of calcium binding sites.

Tb3+, a fluorescent trivalent cation with physicochemical properties similar to Ca2+, binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster (Homarus americanus). Saturable binding is measured for at least two classes of binding site. Bound Tb3+ can be displaced by other cations in the order: Ca2+ greater than Mg2+ = Zn2+ greater than NH4+. The binding of Tb3+ to the lower affinity site (KD(app) = 6.0 microM) is inhibitable by Na+, Mg2+ and Ca2+, whereas the higher affinity site (KD(app) = 2.2 microM) is only sensitive to Ca2+. Using this spectral probe the role of Ca2+ in peripheral nerve membrane function can be investigated.

Local anesthetics noncompetitively inhibit terbium binding to the exterior surface of nerve membrane vesicles.

It has previously been shown that terbium binds to membrane vesicles prepared from the walking leg nerve of the lobster (Homarus americanus) with a high affinity Kd of 2.2 microM. Fluorescence of bound Tb3+ occurs via energy transfer from the aromatic residues of proteins (gamma ex = 280 nm; gamma em = 546 nm), and calcium inhibits Tb3+ binding competitively with a Ki of 1.8 mM. Displacement studies with EDTA demonstrate that more than 95% of the bound Tb3+ is at the vesicle exterior and is not being taken up by the vesicles. To investigate the putative role of Ca2+ in the interaction of local anesthetics with axonal membranes, lidocaine and the analogs GX-HCl and QX-314 were tested as inhibitors of Tb3+ binding. Inhibition by lidocaine is seen only at considerably higher doses (25 mM) than are required for conduction block of intact nerves (5 mM). Inhibition by lidocaine and the primary amine analog GX-HCl is entirely noncompetitive, whereas the quaternary ammonium derivative QX-314 appears to be a mixed competitive-noncompetitive inhibitor of Tb3+ binding. These data are not compatible with the hypothesis that there is a functionally essential cation binding site on the axonal membrane surface for which Ca2+ and local anesthetics compete, although local anesthetic action may be modified indirectly by altered calcium concentrations. Evidence is presented for a mechanism by which local anesthetics indirectly displace Tb3+ by altering the physical state of the axonal membrane.

Molecular forms of acetylcholinesterase in subcortical areas of normal and Alzheimer disease brain.

We have previously reported that the 10s molecular form (G4) of acetylcholinesterase (AChE) is selectively lost from several cortical areas of Alzheimer's disease (AD) brain. In the current follow-up study, we microdissected several areas of nondemented and AD brain, including the hippocampus, amygdala, and cingulate gyrus. Tissue homogenates were separated on sucrose density gradients and the resulting fractions were analyzed for AChE activity in order to define the ratios of the predominant AChE molecular forms (G4/G1). Both the hippocampus and amygdala exhibited distinct patterns of alterations in the G4/G1 ratio which correlate with the known distribution of histopathological changes in AD brain. In order to further define the major pool of AChE that is depleted in AD, we separated fractionated tissue homogenates into salt-soluble and detergent-soluble fractions. The G4/G1 ratios were only altered in the detergent-soluble fractions, indicating that the loss of the G4 AChE molecular form involves a selective depletion of the membrane pool. Available evidence would suggest that this form is the AChE molecular form physiologically relevant at the cholinergic synapse.

Cholinesterase activity in plasma, erythrocytes, and cerebrospinal fluid of patients with dementia of the Alzheimer type.

Cholinesterases, including pseudocholinesterase (BChE) of human plasma and acetylcholinesterase (AChE) of erythrocytes and cerebrospinal fluid (CSF), have been considered as possible markers in dementia of the Alzheimer type (DAT). Reported data, however, are widely varied, and no significant pattern emerges when total enzyme activity is assayed. In the present studies, we have reexamined the relationship of ChE activities in DAT and control patients. ChE activity was measured in plasma, erythrocytes, and CSF from DAT patients and compared with normal controls as well as with samples from patients with a diagnosis other than DAT. Early age onset (presenile) and late age onset (senile) DAT were also compared. No significant differences in total enzyme activity were found in any of the comparisons. Calculations of AChE/BChE ratios in CSF also provided no significant indication of any changes in ChE activities in DAT. It is suggested that measurements of total AChE or BChE activity in these biological materials do not provide a useful index of alterations in central cholinergic function in patients with DAT.

Cerebrospinal fluid levels of angiotensin-converting enzyme, acetylcholinesterase, and dopamine metabolites in dementia associated with Alzheimer's disease and Parkinson's disease: a correlative study.

Mean levels of the two hydrolases angiotensin-converting enzyme (ACE) and acetylcholinesterase (AChE), the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and total protein concentration were examined in cerebrospinal fluid (CSF) samples from a group of patients with dementia of the Alzheimer's type, a group of comparably demented patients with Parkinson's disease, and a neurologically healthy elderly control group. Both pathological groups exhibited a significant decrease in the mean levels of ACE activity and DOPAC per milliliter and were distinguishable from one another based on mean CSH HVA levels. Unlike the Parkinson's disease group, whose mean concentration of HVA was lower than, but not significantly different from that of the control group, the mean HVA concentration of the Alzheimer's disease group was significantly elevated. In contrast, comparisons of the mean CSF AChE activity (expressed per milliliter or per milligram of protein) and CSF total protein concentration did not reveal significant differences for any of the groups. Independent of CSF protein concentration, ACE activity per milliliter exhibited a positive correlation with AChE activity per milliliter within the control and Parkinson's disease groups, whereas a statistically significant correlation for these CSF hydrolases was not observed within the Alzheimer's disease group. Thus, the CSF profiles for patients with mild dementias associated with Alzheimer's or Parkinson's disease differed by at least two neurochemical criteria. Based on the levels of ACE activity, DOPAC, and HVA per milliliter of CSF, two discriminant functions were derived and resulted in the correct classification of 71% of all subjects (n = 38) into Alzheimer's disease, Parkinson's disease, and neurologically healthy control groups.

CSF cyclic nucleotides and somatostatin in Parkinson's disease.

Concentrations of cyclic adenosine 3',5' monophosphate (cAMP) were significantly lower in parkinsonian patients than in controls, but concentrations of guanosine 3',5' monophosphate (cGMP) were not altered. Both cAMP and cGMP levels were lower in patients with more severe symptoms on the left side of the body. Somatostatin-like immunoreactivity (SLI) was similar in parkinsonian patients and controls. Both cAMP and SLI were significantly related to acetylcholinesterase activity.

Synthesis and study of conformationally defined enantiomers of local anesthetics and conformationally defined enantiomers of fluorescent dyes designed to label electrically excitable membranes.

Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.

Pharmacological significance of acetylcholinesterase inhibition by tetrahydroaminoacridine.

Tetrahydroaminoacridine (THA; Tacrine) is a potent, non-competitive inhibitor of the neuronal enzyme acetylcholinesterase (AChE) and, consequently, a potent modulator of central cholinergic function. The compound reportedly improves the memory deficits of Alzheimer's dementia. Experiments were run with purified bovine caudate AChE to examine the kinetic properties of THA-AChE interaction within the scheme of multiple binding sites on the enzyme and a proposed "map" of the enzyme surface. The kinetic analyses were also designed to determine whether chemical modification of peripheral anionic sites on AChE may provide insight into mechanism for selective pharmacological alteration of cholinergic function in the brain. The studies demonstrated that THA is a reversible, non-competitive inhibitor with an I50 of 160 +/- 10 nM. THA bound primarily at a hydrophobic area outside of the catalytic sites, and binding of THA enhanced the effect of Ca2+ binding to a separate group of "accelerator" sites. Experiments with Al3+ demonstrated non-competitive inhibitor effects that were additive with THA inhibition and consistent with a model suggesting interaction of THA and Al3+ at the enzyme surface. In vitro enzyme inhibition studies also provide evidence for THA "protection" of the catalytic site against inhibition by the high-affinity phosphorylating agent, DFP (isoflurophate).

Noncompetitive inhibition by aluminum, scandium and yttrium of acetylcholinesterase from Electrophorus electricus.

Measurements of altered activity of soluble acetylcholinesterase from E. electricus electric organ by the inorganic cations aluminum, scandium and yttrium demonstrate that these ions are noncompetitive enzyme inhibitors. Al3+ inhibited enzyme activity at all substrate and inhibitor concentrations studied. Inhibition by Al3+ did not appear to be sensitive to the active site-specific, competitive ligand physostigmine or to calcium, a peripheral site-binding activator cation. Inhibition by another peripheral site-binding noncompetitive inhibitor, decamethonium, was not altered by Al3+. Al3+ appears thus to have interacted with a class of peripheral anionic sites on AChE distinct from the beta- or P1 peripheral anionic sites that bind Ca2+ and C-10 and may be a useful probe of a subclass of gamma- or P2-4 peripheral anionic sites. A possible mechanism for Al3+ neurotoxicity, via alterations of the enzymes of cholinergic neurotransmission, is also suggested.

Activation and inactivation of bovine caudate acetylcholinesterase by trivalent cations.

Kinetic analysis of the interaction of trivalent cations with mammalian brain acetylcholinesterase revealed at least three distinct concentration-dependent effects on enzyme activity. Acetylcholinesterase was purified from bovine caudate nucleus by affinity chromatography to a specific activity of 1.1 mmoles acetylthiocholine X hr-1 X (mg protein)-1. The cations studied included the chloride salts of lanthanum, terbium, yttrium and scandium in low and high ionic strength buffers (2 mM Pipes +/- 0.1 M NaCl). At low ionic strength, high affinity noncompetitive or allosteric activation was observed at very low cation concentrations (1-10 microM); at higher concentrations (50-200 microM) these cations were noncompetitive inhibitors; and at 200-500 microM they exerted a mixed competitive-noncompetitive inhibition. Activation by low cation concentrations was not evident in high ionic strength buffers, while enzyme inhibition by all the trivalent cations was similar at low and high ionic strength. Inhibition by all of the multivalent cations was fully reversed by a 10-fold excess of EDTA or by a 100-fold dilution of the inhibited enzyme. The water-soluble carboxyl group affinity reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, was shown to specifically block the activating effect of the multivalent cations supporting the suggestion that the beta- or "activator" peripheral anionic sites (P1) involve a carboxyl group outside the enzyme active site.

Toxicological evaluation of oligonucleotide therapeutics.

In this brief review, guidance is provided for the safety evaluation of phosphorothioate oligonucleotides. This novel class of compounds has proved to be challenging for the preclinical development scientist, as systemic exposure can evoke sometimes dramatic responses, in terms of general functional parameters, laboratory data (hematology, clinical chemistry) and histopathology of target organs. The polyanionic profile of these compounds is discussed vis-à-vis historical data for related structures. Dose-related responses in various laboratory test species are described. The liver, kidneys and immune system are major target organs, and an experimental perspective is provided for understanding each of these in determining a safe starting dose for clinical trials with novel compounds.

Ligand interactions of crustacean axonal membranes.

The relative abilities of a series of local anesthetics in which either or both the ester oxygens had been replaced with sulfur or selenium to block axonal or synaptic preparations are compared with their abilities to displace (3)H-nicotine from lobster axon plasma membrane fragments. The relative affinities of a series of cholinergic agonists and antagonists for synaptic and axonal membranes are discussed as is the salt sensitivity and reversibility of their interactions. The utility of a conjugate of ?-bungarotoxin and horse-radish peroxidase for the histochemical visualization of binding sites of intact axons or axonal vesicles is discussed. Labelling of a peptide isolated from axon plasma fragments with MBTA is compared with labelling of peptides isolated from synaptic membranes.

Organophosphate-induced alterations in muscarinic receptor binding and phosphoinositide hydrolysis in the human SK-N-SH cell line.

Low level, chronic exposure (0.1 nM, 24 hrs) to the organophosphate paraoxon significantly inhibits N-[3H]methylscopolamine ([3H]NMS) binding to muscarinic receptors in a noncompetitive manner in the SK-N-SH neuroblastoma cell line. The effect of paraoxon on muscarinic receptor-mediated phosphoinositide hydrolysis was also investigated. At 0.1 nM, paraoxon caused a time-dependent increase in the accumulation of inositol phosphates, while the classical muscarinic receptor agonist carbachol was virtually ineffective at low concentrations. In contrast, carbachol-induced increases in phosphoinositide hydrolysis at higher concentrations (1 mM) were markedly larger (five-fold) than the increases induced by PX. Approximately 50% of the maximal increase in the accumulation of inositol phosphates due to paraoxon treatment was antagonized by saturating concentrations of muscarinic receptor antagonists, while the response to carbachol was completely antagonized. In addition, chronic treatment (24 hrs) of SK-N-SH cells with pertussis toxin blocked 75% of the stimulatory effects of carbachol, but inhibited only 38% of the paraoxon-induced response. Neomycin, a phospholipase C inhibitor, completely blocked both the paraoxon and carbachol-induced stimulation of phosphoinositide hydrolysis. The results suggest that paraoxon can modulate signal transduction by indirect activation of muscarinic receptors as well as by acting at a distal site along the pathway.

Terbium binding to rat brain acetylcholinesterase. A fluorescence probe of anionic sites.

Studies are in progress to characterize the nature of ligand interactions at peripheral anionic sites on mammalian brain AChE, including the beta-anionic or "accelerator" anionic sites where enzyme activity is increased upon Ca2+ binding. Terbium was studied as a fluorescence probe of Ca2+ binding sites in partially purified AChE from whole rat brain. Scatchard analysis of Tb3+ binding in low ionic strength (2 mM) Pipes buffer revealed at least two populations of sites: high affinity sites with Kd(app) approximately 7.6 microM and low-affinity sites with a Kd(app) approximately 49.6 microM. Low-affinity binding was selectively inhibited by 50 mM NaCl; high-affinity binding was completely inhibited by 2 mM CaCl2; and all the bound Tb3+ could be displaced by 1 mM EDTA. The heterogeneity of Tb3+ binding sites is consistent with the multiple, concentration-dependent effects of Tb3+ on enzyme activity.