Diffusion, spread, and migration of botulinum toxin.

Botulinum toxin (BoNT) is an acetylcholine release inhibitor and a neuromuscular blocking agent used for the treatment of a variety of neurologic and medical conditions. The efficacy and safety of BoNT depends on accurate selection and identification of intended targets but also may be determined by other factors, including physical spread of the molecule from the injection site, passive diffusion, and migration to distal sites via axonal or hematogenous transport. The passive kinetic dispersion of the toxin away from the injection site in a gradient-dependent manner may also play a role in toxin spread. In addition to unique properties of the various BoNT products, volume and dilution may also influence local and systemic distribution of BoNT. Most of the local and remote complications of BoNT injections are thought to be due to unwanted spread or diffusion of the toxin's biologic activity into adjacent and distal muscles. Despite widespread therapeutic and cosmetic use of BoNT over more than three decades, there is a remarkable paucity of published data on the mechanisms of distribution and its effects on clinical outcomes. The primary aim of this article is to critically review the available experimental and clinical literature and place it in the practical context.

Botulinum toxin: description of injection techniques and examination of controversies surrounding toxin diffusion.

The benefits derived from botulinum toxin (BTX) injections may be negated by unintentional weakness of adjacent uninjected muscles. Such weakness may be the result of inaccurate targeting, or diffusion of BTX to surrounding muscles. Several techniques, using electromyographic, endoscopic or imaging guidance are purported to increase the accuracy of targeting. Diffusion of BTX is thought to be influenced by factors such as dose, concentration, injectate volume, number of injections, site and rate of injection, needle gauge, muscle size, muscular fascia, distance of needle tip from the neuromuscular junction, and protein content of the BTX formulation. This article describes techniques that aim to increase the accuracy of BTX injections and examines the controversies surrounding diffusion of BTX following injection.

Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25.

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.

Botulinum toxin as a therapeutic agent.

Botulinum toxin is a presynaptic neuromuscular blocking agent that, when injected intramuscularly in minute quantities, can produce selective muscle weakness. This property is employed therapeutically to provide symptomatic relief in conditions related to excessive muscle activities in strabismus, blepharospasm, hemifacial spasm, cervical dystonia, spasmodic dysphonia (adductor type), and jaw closing dystonia. It is investigational for a long list of medical conditions. It is a marketed drug in a number of countries in the world, but its use has only been approved by different regulatory agencies for use in a limited number of conditions. The long-term effects, appropriate dose for children, and in pregnancy, and maximum dose without causing toxicity remain unclear.

Clinical evidence of extraocular muscle fiber-type specificity of botulinum toxin.

OBJECTIVE: To compare the effects of botulinum toxin on static and dynamic aspects of eye movements, and thereby elucidate the mechanisms of its action on eye muscles. BACKGROUND: Laboratory evidence indicates that static alignment and saccades are subserved by different extraocular muscle fiber types, and botulinum toxin may cause specific dysfunction of the fibers controlling static alignment. Diplopia is a well-known side effect of periorbital botulinum toxin injections in humans, and may be a clinical correlate of the laboratory findings. METHODS: Search coil recording of eye movements was performed in one patient with systemic botulism, and in three patients with diplopia following periorbital injection of botulinum toxin A. RESULTS: In the patient with acute botulism, eye movement alignment, range, and saccadic velocity profiles were abnormal. In three patients with iatrogenic diplopia, static alignment was abnormal but movement range and saccadic velocities were within normal limits. Edrophonium improved the range of movements and saccadic velocities in the patient with systemic botulism but was ineffective in reversing ocular misalignment in the one iatrogenic patient to whom it was administered. CONCLUSIONS: Precise alignment is subserved by orbital singly innervated muscle fibers, and the effects of botulinum toxin are greatest on these fibers. This predilection is apparent when the toxin dose is very small, as must have been the case in our patients with iatrogenic diplopia. The lack of a response to edrophonium probably reflects structural damage to muscle fibers. In contrast, larger doses of toxin produce an acute dysfunction of all extraocular muscle fiber types, which is responsive to edrophonium and consequently reflects partial blockade at the neuromuscular junction.

Potencies of haloperidol metabolites as inhibitors of the human noradrenaline, dopamine and serotonin transporters in transfected COS-7 cells.

Extrapyramidal symptoms, such as tardive dyskinesia, often develop in patients on long-term treatment with haloperidol. It has been proposed that these symptoms could be caused by neurotoxic effects of haloperidol metabolites following uptake by monoamine transporters, in an analogous mechanism to the neurotoxic effect of MPP+ (1-methyl-4-phenylpyridinium) metabolised from MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In this study, the hypothesis was partially investigated by determining the potencies of haloperidol and reduced haloperidol and the corresponding pyridinium and tetrahydropyridine metabolites, compared with MPP+ and MPTP, as inhibitors of the noradrenaline transporter (NAT), dopamine transporter (DAT) and 5-HT transporter (SERT). Two days after COS-7 cells were transiently transfected with the cDNA for the human NAT, DAT or SERT (Lipofectamine method), the cells were incubated with 10 nM [3H]noradrenaline, dopamine or 5-HT, respectively, for 2 min at 37 C, in the absence or presence of various concentrations of the eight compounds or a specific uptake inhibitor (NAT: nisoxetine 1 microM; DAT: GBR 12909 1 microM; SERT: citalopram 10 microM). Specific amine uptake (fmol/ mg protein) was calculated as the difference in uptake in the absence and presence of the specific uptake inhibitor. Ki values were calculated for the eight compounds for inhibition of NAT, DAT and SERT. Haloperidol, its five metabolites and MPP+ and MPTP all inhibited NAT, DAT and SERT. For the pyridinium and tetrahydropyridine metabolites of haloperidol, there were not marked differences between their potencies as inhibitors between each other for NAT or DAT or between NAT and DAT, with all of the Ki values in the range of 5.8-16 microM. However, there were more marked differences for SERT, with all but one of the metabolites showing selectivity for inhibition of SERT relative to NAT and DAT. Haloperidol and reduced haloperidol had similar inhibitory potencies for all three transporters, and were clearly less potent than the other haloperidol metabolites only for inhibition of SERT. The lack of correlation between the inhibitory potencies of the haloperidol metabolites and their structural analogues, MPTP and MPP+, suggests that they are not likely to cause neurotoxicity by a mechanism analogous to that of the latter neurotoxin.

Botulinum toxin type B: an overview of its biochemistry and preclinical pharmacology.

Produced by Clostridium botulinum, botulinum toxins are high molecular weight protein complexes consisting of the neurotoxin and additional nontoxic proteins that function to protect the toxin molecule. The neurotoxin acts to inhibit the release of acetylcholine at the neuromuscular junction, causing muscle paralysis. Purified toxin complexes have found a niche in the treatment of clinical disorders involving muscle hyperactivity. The different serotypes are structurally and functionally similar; however, specific differences in neuronal acceptor binding sites, intracellular enzymatic sites, and species sensitivities suggest that each serotype is its own unique pharmacologic entity. Recently, botulinum toxin type B has been developed as a liquid formulation to avoid the lyophilization (vacuum-drying) and reconstitution processes associated with decreasing the potency and stability of current type A toxin preparations. Biochemical tests were conducted to evaluate the quality of toxin in this formulation. In 3 consecutive manufacturing lots, the botulinum toxin type B complex was found to be highly purified, intact, uniform, and consistent from lot to lot. Also, it showed long-term stability at refrigerator and room temperatures (2 to 25 degrees C). Electrophysiologic studies in cynomolgus monkeys showed that botulinum toxin type B is effective in paralyzing injected muscle groups, with minimal spread to relatively distant noninjected muscles.

The serotonin system: a potential target for anti-dyskinetic treatments and biomarker discovery.

L-DOPA-induced dyskinesia is a major problem in the treatment of Parkinson's disease. Today there are few anti-dyskinetic treatments available for the patients, and all of them have major limitations. Recent findings have revealed an important role of the serotonin system in L-DOPA-induced dyskinesia. In the parkinsonian brain, serotonin axon terminals can compensate for the dopamine loss by converting L-DOPA into dopamine and releasing it as a false neurotransmitter. However, the terminals represent an aberrant source of dopamine release, increasing the risk for dyskinesia. In line with this, a relatively high density of serotonin axon fibres in striatum has been reported in dyskinetic animals and patients. Furthermore, serotonin can influence dyskinesia by modulating glutamate or GABA signalling in the basal ganglia via receptors located on non-serotonergic neurons. Through either mechanism, modulation of certain serotonin receptors has been shown to reduce the severity of dyskinetic movements. The serotonin system represents an interesting target for developing anti-dyskinetic treatments. Future therapies may take advantage of the synergistic effect produced by the modulation of different serotonin receptors or pursue a region-specific modulation of certain receptors. Moreover, morphological or biochemical features of the serotonin system could be used to develop biomarkers for patient stratification in clinical trials of anti-dyskinetic compounds.

Pharmacological modulation of amphetamine-induced dyskinesia in transplanted hemi-parkinsonian rats.

Foetal cell transplantation in patients with Parkinson's disease can induce motor complications independent of L-DOPA administration, known as graft-induced dyskinesia. In the 6-OHDA lesioned rat model of Parkinson's disease, post-transplantation abnormal movements can develop in response to an amphetamine challenge, a behaviour which is used to model graft-induced dyskinesia. Although L-DOPA-induced dyskinesia has been well characterised pharmacologically, we lack knowledge on the modulation of post-transplantation amphetamine-induced dyskinesia which may shed light on the mechanisms underlying graft-induced dyskinesia. We assessed a series of drugs effective at reducing L-DOPA-induced dyskinesia against post-transplantation amphetamine-induced dyskinesia. Agents include: dopaminergic antagonists (D1: CP94253; D2: SCH-22390; D3: nafadotride), serotonergic agonists (5-HT(1A): 8-OH-DPAT; 5-HT(1B): CP94253), opioid antagonist (µ: naloxone), cannabinoid agonist (CB1: WIN55, 212-2), adrenergic antagonist (α1 and α2: yohimbine) and glutamatergic antagonists (NMDA: amantadine and MK-801; mGluR5: MTEP; AMPA: IEM1460). Abnormal involuntary movements in response to amphetamine were decreased by SCH-22390, raclopride, CP94253 and 8-OH-DPAT, yet were unaltered by naloxone, WIN55, 212-2, yohimbine, amantadine, MTEP and IEM1460. Unusually, MK-801 increased the appearance of amphetamine-induced dyskinesia. The results suggest that dopaminergic, serotoninergic and glutamatergic systems are likely to have a fundamental role in the development of graft-induced dyskinesias, which are mechanistically distinct from L-DOPA-induced behvaviours. Importantly, the expression of D1 and D2 receptors was unrelated to the severity of AIMs.

Absolute configuration of (+)-alpha-dihydrotetrabenazine, an active metabolite of tetrabenazine.

Chiral column liquid chromatography and enantiospecific enzymatic hydrolysis were utilized to separate the enantiomers of alpha- and beta-dihydrotetrabenazine and alpha-9-O-desmethyldihydrotetrabenazine, three benzo[a]quinolizines derived from the amine-depleting drug tetrabenazine. An X-ray crystal structure analysis of (-)-alpha-9-O-desmethyldihydrotetrabenazine gave an absolute structure of that compound as the 2S, 3S, 11bS isomer. Therefore, (-)-alpha-dihydrotetrabenazine also has the 2S, 3S, 11bS absolute configuration. (+)-alpha-Dihydrotetrabenazine, the single biologically active isomer from the metabolic reduction of tetrabenazine, thus has the absolute configuration of 2R, 3R, 11bR. For further in vitro and in vivo studies of the vesicular monoamine transporter, it is now possible to use the single enantiomer of radiolabeled alpha-dihydrotetrabenazine.

Effects of a quaternary pyridinium metabolite of haloperidol (HP+) on the viability and catecholamine levels of cultured PC12 cells.

Haloperidol has been found to be metabolized to a pyridinium ion (HP+; 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-pyridinium). HP+ is structurally similar to the toxic metabolite of the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), N-methyl-4-phenylpyridinium (MPP+). HP+ is toxic towards dopaminergic neurons and was proposed to be associated with some of the extrapyramidal side effects of haloperidol. We therefore investigated the neurotoxicity of HP+ towards cultured PC12 cells. At high concentrations, HP+ reduced the viability of PC12 cells as measured by trypan blue exclusion and the MTT method. However, HP+ decreased intracellular dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and dihydroxyphenylalanine (DOPA) levels at lower concentrations than those required to compromise cell viability. The immunoreactivity of tyrosine hydroxylase was not affected by the treatment with HP+. It was subsequently demonstrated that HP+ can release [3H]DA preloaded in rat striatum slices. Thus, it is proposed that HP+ decreases dopamine content in PC12 cells through actively releasing amines from the cells and (or) blocking the reuptake of the released amines.