Immunostaining of rat brain, spinal cord, sensory neurons and skeletal muscle for calcium channel alpha2-delta (alpha2-delta) type 1 protein.

Alpha2-delta (alpha2-delta) is a membrane-spanning auxiliary protein subunit of voltage-gated calcium channels found in muscle and brain. Of the four subtypes of alpha2-delta, only alpha2-delta types 1 and 2 (alpha2-delta-1 and alpha2-delta-2) bind the drugs gabapentin (Neurontin) and pregabalin (Lyrica). Although recent findings indicate that drug binding to alpha2-delta-1 is required for pharmacology of pregabalin and gabapentin, previous work has not addressed the location of alpha2-delta-1 protein within nervous tissues. A monoclonal antibody to alpha2-delta-1 revealed intense immunostaining in certain areas of rat brain, spinal cord, dorsal root ganglia, and skeletal muscle, with weaker staining in heart muscle, gut and liver. Little immunostaining was seen in spleen, kidney, thymus and lung. Staining was dense in some regions of the CNS including spinal dorsal horn, anterior olfactory nucleus, anterior amygdala, basolateral (ventral) amygdala and cortical amygdala, and the piriform, perirhinal, insular and entorhinal cortices. In hippocampus, staining was heterogeneous with greater density in areas of glutamate terminals (mossy fiber endings on CA3 pyramidal cells and perforant path endings on granule cells and CA1 stratum radiatum). Moderate staining occurred in the lateral posterior nucleus of the thalamus, superficial layers of neocortex, periaqueductal gray, substantia nigra, stria terminalis, nucleus accumbens shell and tegmental nucleus. We propose that areas of dense alpha2-delta-1 staining in brain and spinal cord are likely sites of action for the analgesic, anticonvulsant and anxiolytic-like actions of pregabalin and gabapentin in animal models.

MRSA pyomyositis complicating sickle cell anaemia.

A patient being treated for sickle cell crisis developed swollen, painful, indurated, discoloured thighs after several days in hospital. Imaging revealed the presence of multiple small abscesses in the muscle and methicillin resistant Staphylococcus aureus (MRSA) was cultured from aspirated fluid. Pyomyositis usually occurs in association with damaged muscle and impaired host defences. Staphylococcus is the most frequent organism involved. It is not a common complication of sickle cell disease, although it may be under diagnosed. Availability of advanced imaging techniques facilitates early diagnosis of pyomyositis.

Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons.

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.

Neuronal N-type calcium channel blockers: a series of 4-piperidinylaniline analogs with analgesic activity.

Several novel N-type voltage sensitive calcium channel blockers showed high affinity in the IMR32 assay and efficacy in the anti-writhing model. Herein, we describe the design, synthesis, SAR studies, biological data, physicochemical properties and pharmacokinetics of this 4-piperidinylaniline series.

The effects of gabapentin in the rat hippocampus are mimicked by two structural analogs, but not by nimodipine.

Gabapentin has been shown to reduce paired-pulse inhibition in the dentate gyrus of the urethane-anesthetized rat and has been shown to block calcium channels, but its not known how these possible mechanisms relate to its antiepileptic effect. Here, we tested two structural analogs of gabapentin for the ability to reduce seizure duration and to alter paired-pulse inhibition in the dentate gyrus in urethane anesthetized adult Sprague-Dawley rats. We compared with our results to those with diazepam, an anxiolytic and GABA(A) positive modulator and with nimodipine, a specific blocker of L-type Ca2+ channels. Both structural analogs of gabapentin caused a dose-dependent loss of paired-pulse inhibition and blocked the lengthening of the duration of the seizure discharge. Nimodipine also blocked the increase in duration of the seizure discharge, but increased paired-pulse inhibition. The effects of the GABA derivatives on paired-pulse inhibition and on seizure duration may have a common mechanism. Furthermore, our results indicate that gabapentin's postulated block of L-type calcium channels is not responsible for reducing paired-pulse inhibition. However, calcium channel block could still be the basis for the antiepileptic effect of gabapentin and its analogs.

The discovery of [1-(4-dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethylbutyl)-phen yl]-(3-methyl-but-2-enyl)-amine, an N-type Ca+2 channel blocker with oral activity for analgesia.

Our drug discovery efforts for N-type calcium channel blockers in the 4-piperidinylaniline series led to the discovery of an orally active analgesic agent 26.1-[4-Dimethylamino-benzyl)-piperidin-4-yl]-[4-(3,3-dimethyl-but yl)-phenyl]-(3-methyl-but-2-enyl)amine (26) showed high affinity to functionally block N-type calcium channels (IC50=0.7 microM in the IMR32 assay) and exhibited high efficacy in the anti-writhing analgesia test with mice (ED50=12 mg/kg by po and 4 mg/kg by iv). In this report, the rationale for the design, synthesis, biological evaluation, and pharmacokinetics of this series of blockers is described.

Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma. United Kingdom Children's Cancer Study Group.

Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping.

Structure-activity relationship at the leucine side chain in a series of N,N-dialkyldipeptidyl-amines as N-type calcium channel blockers.

Exploration of the SAR around the leucine side chain in a series of N,N-dialkyldipeptidylamines with potent functional activity at N-type VSCC is presented. A novel analog is disclosed which possesses improved aqueous solubility, in vivo activity in an audiogenic seizure model, and reversible blockade in electrophysiological assays.

Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin.

Spinal cord injury (SCI) often results in abnormal pain syndromes in patients. We present a recently developed SCI mammalian model of chronic central pain in which the spinal cord is contused at T8 using the NYU impactor device (10-g rod, 2.0-mm diameter, 12.5-mm drop height), an injury which is characterized behaviorally as moderate. Recovery of locomotor function was assessed with an open field test and scored using the open field test scale (BBB scale). Somatosensory tests of paw withdrawal responses accompanied by supraspinal responses to both mechanical punctate (von Frey hairs) and nonpunctate (4 mm diameter blunt probe) as well as thermal (radiant heat) peripheral stimuli were performed. Comparisons at the level of the individual animal between precontusion and postcontusion responses indicated significant increases in reactions to low threshold punctate mechanical stimuli, non-punctate stimuli and thermal stimuli (p < 0.05). To demonstrate the validity of this model as a central pain model, gabapentin, an agent used clinically for central pain, was given i.p. at 10 or 30 mg/kg. Gabapentin treatment significantly and reversibly changed the responses, consistent with the attenuation of the abnormal sensory behavior, and the attenuated responses lasted for the duration of the drug effect (up to 6 h). These results support the use of the spinal contusion model in the study of chronic central pain after SCI.

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists.

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.

Inherited protein C deficiency, protein S deficiency and hyperhomocysteinaemia in a patient with hereditary spherocytosis.

We report a family with hereditary spherocytosis in whom there is, in addition, a cluster of genetic predispositions to thrombosis. Although inherited prothrombotic abnormalities are prevalent in the general population, the likelihood of this combination of abnormalities being found in a single family is extremely low. The management of such high risk individuals is discussed.

Structure-activity relationship of N-methyl-N-aralkyl-peptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown efficacy in several animal models of stroke and pain. In the process of searching for small molecule N-type calcium channel blockers, we have identified a series of N-methyl-N-aralkyl-peptidylamines with potent functional activity at N-type VSCCs. The most active compound discovered in this series is PD 173212 (11, IC50 = 36 nM in the IMR-32 assays). SAR and pharmacological evaluation of this series are described.

N,N-dialkyl-dipeptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.

Oxygen/glucose deprivation in hippocampal slices: altered intraneuronal elemental composition predicts structural and functional damage.

Effects of oxygen/glucose deprivation (OGD) on subcellular elemental composition and water content were determined in nerve cell bodies from CA1 areas of rat hippocampal slices. Electron probe x-ray microanalysis was used to measure percentage water and concentrations of Na, P, K, Cl, Mg, and Ca in cytoplasm, nucleus, and mitochondria of cells exposed to normal and oxygen/glucose deficient medium. As an early (2 min) consequence of OGD, evoked synaptic potentials were lost, and K, Cl, P, and Mg concentrations decreased significantly in all morphological compartments. As exposure to in vitro OGD continued, a negative DC shift in interstitial voltage occurred ( approximately 5 min), whereas general elemental disruption worsened in cytoplasm and nucleus (5-42 min). Similar elemental changes were noted in mitochondria, except that Ca levels increased during the first 5 min of OGD and then decreased over the remaining experimental period (12-42 min). Compartmental water content decreased early (2 min), returned to control after 12 min of OGD, and then exceeded control levels at 42 min. After OGD (12 min), perfusion of hippocampal slices with control oxygenated solutions (reoxygenation) for 30 min did not restore synaptic function or improve disrupted elemental composition. Notably, reoxygenated CA1 cell compartments exhibited significantly elevated Ca levels relative to those associated with 42 min of OGD. When slices were incubated at 31 degreesC (hypothermia) during OGD/reoxygenation, neuronal dysfunction and elemental deregulation were minimal. Results show that in vitro OGD causes loss of transmembrane Na, K, and Ca gradients in CA1 neurons of hippocampal slices and that hypothermia can obtund this damaging process and preserve neuronal function.

A summary of mechanistic hypotheses of gabapentin pharmacology.

Although the cellular mechanisms of pharmacological actions of gabapentin (Neurontin) remain incompletely described, several hypotheses have been proposed. It is possible that different mechanisms account for anticonvulsant, antinociceptive, anxiolytic and neuroprotective activity in animal models. Gabapentin is an amino acid, with a mechanism that differs from those of other anticonvulsant drugs such as phenytoin, carbamazepine or valproate. Radiotracer studies with [14C]gabapentin suggest that gabapentin is rapidly accessible to brain cell cytosol. Several hypotheses of cellular mechanisms have been proposed to explain the pharmacology of gabapentin: 1. Gabapentin crosses several membrane barriers in the body via a specific amino acid transporter (system L) and competes with leucine, isoleucine, valine and phenylalanine for transport. 2. Gabapentin increases the concentration and probably the rate of synthesis of GABA in brain, which may enhance non-vesicular GABA release during seizures. 3. Gabapentin binds with high affinity to a novel binding site in brain tissues that is associated with an auxiliary subunit of voltage-sensitive Ca2+ channels. Recent electrophysiology results suggest that gabapentin may modulate certain types of Ca2+ current. 4. Gabapentin reduces the release of several monoamine neurotransmitters. 5. Electrophysiology suggests that gabapentin inhibits voltage-activated Na+ channels, but other results contradict these findings. 6. Gabapentin increases serotonin concentrations in human whole blood, which may be relevant to neurobehavioral actions. 7. Gabapentin prevents neuronal death in several models including those designed to mimic amyotrophic lateral sclerosis (ALS). This may occur by inhibition of glutamate synthesis by branched-chain amino acid aminotransferase (BCAA-t).

Pharmaceutical industry screening for new antiepileptic drugs.

It is clear that in coming years, many interesting new compounds will emerge from these and other strategies for drug discovery. In previous strategies of anticonvulsant discovery, whole-animal models of seizures were utilized from the initial stages; however, with the newer strategies, it will become necessary to select promising drug targets for clinical trials in epilepsy that have well-defined molecular targets of action but poorly defined actions in various animal seizure models. The new pharmaceutical discovery technologies of mass screening, cloned drug targets, and combinatorial chemistry will assist in the efficient discovery of novel drug candidates. Incidentally, these techniques also will minimize the use of large numbers of living laboratory animals for anticonvulsant drug screening.

Sodium channel modulators prevent oxygen and glucose deprivation injury and glutamate release in rat neocortical cultures.

Neocortical cultures were deprived of oxygen and glucose to model ischemic neuronal injury. We used a graded series of periods of oxygen and glucose deprivation, providing graded insults. Cell death was measured by release of lactate dehydrogenase (LDH). One hundred and twenty to 240 min of deprivation caused graded increases in glutamate overflow, LDH release and 45Ca influx. Curves of LDH release with respect to deprivation time were shifted to longer intervals by treatment with tetrodotoxin (TTX; 3, 30 or 300 nM), phenytoin (10, 30 or 100 microM), lidocaine (10, 30 or 100 microM) or the N-methyl-D-aspartate antagonist CPP [3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid, 3, 10, 30 or 100 microM]. Combined treatment with TTX and CPP caused pronounced rightward shifts of LDH deprivation curves. Our results indicate that Na+ channel blockade is neuroprotective in neocortex cultures. Our results also suggest that neuroprotection with Na+ channel blockers may be due to inhibition of glutamate release.