Costal cartilage graft for an unstable post-traumatic trapeziometacarpal intra-articular malunion in a pediatric patient: A case report.

Trapeziometacarpal dislocation is a rare traumatic lesion, especially in the pediatric population. Various treatments have been described for acute cases. We report the case of a 15-year-old boy who was admitted in the emergency department with chronic post-traumatic trapeziometacarpal dislocation due to an untreated first metacarpal base fracture (Bennett), with complete articular destruction. The patient was treated by open reduction, interposition of a costal cartilage graft, and intermetacarpal K-wire fixation. Follow-up at 2 years after the surgery showed excellent clinical and radiological results. This surgical technique had not been described previously in the pediatric population.

BAY36-7620: a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity.

L-Glutamate (Glu) activates at least eight different G protein-coupled receptors known as metabotropic glutamate (mGlu) receptors, which mostly act as regulators of synaptic transmission. These receptors consist of two domains: an extracellular domain in which agonists bind and a transmembrane heptahelix region involved in G protein activation. Although new mGlu receptor agonists and antagonists have been described, few are selective for a single mGlu subtype. Here, we have examined the effects of a novel compound, BAY36-7620 [(3aS,6aS)- 6a-Naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental[c]furan-1-on], on mGlu receptors (mGlu1-8), transiently expressed in human embryonic kidney 293 cells. BAY36-7620 is a potent (IC(50) = 0.16 microM) and selective antagonist at mGlu1 receptors and inhibits >60% of mGlu1a receptor constitutive activity (IC(50) = 0.38 microM). BAY36-7620 is therefore the first described mGlu1 receptor inverse agonist. To address the mechanism of action of BAY36-7620, Glu dose-response curves were performed in the presence of increasing concentrations of BAY36-7620. The results show that BAY36-7620 largely decreases the maximal effect of Glu. Moreover, BAY36-7620 did not displace the [(3)H]quisqualate binding from the Glu-binding pocket, further indicating that BAY36-7620 is a noncompetitive mGlu1 antagonist. Studies of chimeric receptors containing regions of mGlu1 and regions of DmGluA, mGlu2, or mGlu5, revealed that the transmembrane region of mGlu1 is necessary for activity of BAY36-7620. Transmembrane helices 4 to 7 are shown to play a critical role in the selectivity of BAY36-7620. This specific site of action of BAY36-7620 differs from that of competitive antagonists and indicates that the transmembrane region plays a pivotal role in the agonist-independent activity of this receptor. BAY36-7620 will be useful to further delineate the functional importance of the mGlu1 receptor, including its putative agonist-independent activity.

Cannabinoid CB(1) receptor upregulation in a rat model of chronic neuropathic pain.

Although cannabinoids are known to be more effective analgesics against chronic rather than acute pain, the mechanism underlying this phenomenon is still unclear. We report now that contralateral thalamic cannabinoid CB(1) receptors are upregulated after unilateral axotomy of the tibial branch of the sciatic nerve, a rat model of chronic neuropathic pain, and hypothesize that cannabinoid CB(1) receptor upregulation contributes to the increased analgesic efficacy of cannabinoids in chronic pain conditions.

Inhibition of evoked glutamate release by the neuroprotective 5-HT(1A) receptor agonist BAY x 3702 in vitro and in vivo.

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT(1A) receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC(50)=1 microM). This effect was blocked by the selective 5-HT(1A) receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT(1A) receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 microg/kg or 10 microg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

Effects of GR-89696 and the novel peripherally selective OP2 agonists, EMD-61569 and EMD-61747, against focal cerebral ischemia in the rat.

We examined whether increases in blood-brain barrier (BBB) permeability occurring after stroke can be exploited to apply protective substances selectively to ischemic tissue. To do this, the actions of the peripherally selective OP2 agonists, EMD-61569 and EMD-61747, have been compared with those of the centrally acting OP2 agonist, GR-89696, in the rat permanent focal ischemia model. EMD-61569, EMD-61747 and GR-89696 all bound with high affinity to OP2 receptors and were potent agonists in the rabbit vas deferens functional assay. These substances also potently inhibited electrically-induced overflow of dopamine from slices of rat nucleus accumbens. EMD-61747 and EMD-61569 penetrate poorly into the CNS under normal conditions and reverse haloperidol-induced L-DOPA accumulation in the nucleus accumbens of the rat only at high doses, in contrast to GR-89696. Permanent unilateral occlusion of the middle cerebral artery (MCAO) was associated with a disruption of the BBB and an increase in the concentration of EMD-61747 in the area of the infarct. GR-89696 at a dose of 0.1 mg/kg s.c. produced a reduction in infarct volume by 38% after MCAO, EMD-61569 and EMD-61747 had no influence on swelling and ischemic damage. We conclude that EMD-61747 and EMD-61569 are potent OP2 agonists, which usually have a limited ability to penetrate the BBB. The change in the properties of the BBB in ischemic tissue was not sufficient to elicit neuroprotection, since both EMD-61747 and EMD-61569 were inactive in the focal ischemia model. Conversely, GR-89696 had a robust protective action, and probably powerful OP2-typical side effects as a consequence of its unrestricted central activity.

Characterization of the aminomethylchroman derivative BAY x 3702 as a highly potent 5-hydroxytryptamine1A receptor agonist.

The aminomethylchroman derivative BAY x 3702 (R-(-)-2-[4-[(chroman-2-ylmethyl)-amino]-butyl]-1,1-dioxo-benzo[d] isothiazolone hydrochloride) is a new high affinity 5-hydroxytryptamine (5-HT)1A receptor ligand [calf hippocampus: Ki: 0.19 nM; reference compounds 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and ipsapirone: 0.98 and 2.56, respectively; rat cortex: 0.24 nM; rat hippocampus: 0.58 nM; human cortex and recombinant 5-HT1A receptors: 0.25 and 0.4 nM, respectively]. BAY x 3702 bound also with relatively high to moderate affinity to the following receptors: alpha-1 and alpha-2 adrenergic (Ki: 6 and 7 nM, respectively); 5-HT7- and 5-HT1D (7 and 36 nM); dopamine D2- and D4 (48 and 91 nM); sigma sites (176 nM) and 5-HT2C (310 nM); others: > 10 microM, as obtained in more than 50 different binding assays. In the forskolin-stimulated adenylate cyclase assay in rat hippocampal tissue, a model of postsynaptic 5-HT1A receptor function, BAY x 3702 was a potent 5-HT1A receptor full agonist (IC50: 1.9 nM; 8-OH-DPAT: 25.3 nM, full agonist; ipsapirone: partial agonist) and its effects could be completely blocked by the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohe xan e carboxamide trihydrochloride (WAY-100635). At those receptors where BAY x 3702 bound with lower affinity, the compound appeared to be either an agonist (5-HT1D receptors) or an antagonist (alpha-1, alpha-2 and D2 receptors). In a rat brain slice preparation containing the dorsal raphe nucleus (DRN), a model of somatodendritic 5-HT1A receptor function, BAY x 3702 inhibited potently (1 nM) neuronal firing. Also in vivo, BAY x 3702 (0.5 microgram/kg, i.v.) was found to suppress 5-HT neuronal firing in the DRN of anesthetized rats. In both electrophysiological assays BAY x 3702 was more potent than 8-OH-DPAT and ipsapirone; the potency difference being about 1 and 2 orders of magnitude, respectively. In rats trained to discriminate 8-OH-DPAT (0.1 mg/kg, i.p.) in a drug discrimination procedure, complete generalization was obtained with BAY x 3702 (ED50: 0.022 mg/kg, i.p. and 0.38 mg/kg, p.o.; 8-OH-DPAT: 0.028 mg/kg, i.p. and ipsapirone: 0.44 mg/kg, i.p.). In the rat hypothermia model BAY x 3702 induced a WAY-100635-reversible effect and the compound had a higher potency and intrinsic activity than 8-OH-DPAT and ipsapirone (ED50: 0.25 mg/kg, i.p. and 5.4 mg/kg, p.o., respectively; 8-OH-DPAT: 1.1 mg/kg, i.p. and ipsapirone: 6.2 mg/kg, i.p.). BAY x 3702 induced a stimulation of plasma ACTH levels in the rat; the effect being again more pronounced than that of ipsapirone (ED50: 7.5 and 25.3 mg/kg, p.o., respectively). It is concluded that BAY x 3702 is a relatively selective 5-HT1A receptor agonist with high potency and intrinsic activity.

A pharmacological profile of the novel, peripherally-selective kappa-opioid receptor agonist, EMD 61753.

1. The pharmacological properties of the novel diarylacetamide kappa-opioid receptor agonist, EMD 61753, have been compared with those of ICI 197067 (a centrally-acting kappa agonist) and ICI 204448 (a peripherally-selective kappa agonist). 2. EMD 61753 binds with high affinity (IC50 5.6 nM) and selectivity (kappa:mu:delta:sigma binding ratio 1:536:125: > 1,786) to kappa-opioid receptors and is a full and potent (IC50 54.5 nM) agonist in an in vitro assay for kappa-opioid receptors (rabbit vas deferens preparation). 3. Systemically-applied [14C]-EMD 61753 is found in high concentrations in the lungs, liver, adrenal glands and kidneys. Considerably less radioactivity is detected in the whole brain, and this radioactivity is concentrated in the region of the cerebral ventricles in the choroid plexuses. EMD 61753 penetrates only poorly into the CNS. 4. EMD 61753 was weakly effective in pharmacological tests of central activity. This compound reversed haloperidolol-induced DOPA accumulation in the nucleus accumbens of the rat only at a dose of 30 mg kg-1, s.c., (doses of 0.1, 1.0 and 10 mg kg-1, s.c., and 1.0, 10 and 100 mg kg-1, p.o., were inactive). Hexobarbitone-induced sleeping in mice was prolonged by EMD 61753 at threshold doses of 10 mg kg-1, s.c., and 100 mg kg-1, p.o., whereas the motor performance of rats in the rotarod test was impaired by EMD 61753 with an ID50 value of 453 mg kg-1, s.c. 5. EMD 61753 produced dose-dependent, naloxone-reversible antinociception in the mouse formalin test (1st phase ID50 1.9 mg kg-1, s.c., and 10.4 mg kg-1, p.o.; 2nd phase ID50 0.26 mg kg-1, s.c., and 3.5 mg kg-1, p.o.) and rodent abdominal constriction test (ID50 mouse 1.75 mg kg-1, s.c., and 8.4 mg kg-1, p.o.; ID50 rat 3.2 mg kg-1, s.c., and 250 mg kg-1, p.o.). EMD 61753 was inactive, or only weakly effective, in the rat pressure test under normalgesic conditions. After the induction of hyperalgesia with carrageenin, however, this compound elicited potent, dose-dependent (ID50 0.08 mg kg-1, s.c., and 6.9 mg kg-1, p.o., after remedial application, and 0.2 mg kg-1, s.c., and 3.1 mg kg-1, p.o., after prophylactic application) and naloxone-reversible antinociception. The antinociceptive action of systemically-applied (50 mg kg-1, p.o.) EMD 61753 in the hyperalgesic pressure test was completely inhibited by injection of the K-opioid antagonist norbinaltorphimine (100 Lg) into the inflamed tissue, a result which indicates that this opioid effect is mediated peripherally.6. Cutaneous plasma protein extravasation produced by antidromic electrical stimulation of the rat saphenous nerve was dose-dependently inhibited by systemically-applied EMD 61753 (ID13 values 3.7 mg kg-1, s.c., and 35.8 mg kg-1, p.o.), and this effect was completely antagonized by intraplantar application of norbinaltorphimine (50 microg). Extravasation elicited by the intraplantar application of substance P (10 microg) was not influenced by the administration of EMD 61753.7. EMD 61753 produced dose-dependent diuresis in non-hydrated rats at doses of and above 1.0 mg kg-1, s.c., and 10 mg kg-1, p.o., and in saline-loaded rats at doses of and above 10 mg kg-1, s.c.,and 30mgkg-1, p.o.8. The prostaglandin-mediated fall in mean arterial blood pressure elicited in anaesthetized rats by i.v.application of arachidonic acid was not inhibited by prior treatment with EMD 61753 (10mg kg-1,p.o.). Thus, a blockade of prostaglandin synthesis via inhibition of cyclo-oxygenase activity does not contribute to the in vivo effects of EMD 61753 and its metabolites.9 The present experiments therefore indicate that EMD 61753 is a potent, selective and orally-effective full ic-opioid receptor agonist which has a limited ability to penetrate the blood-brain barrier and elicit centrally-mediated sedation, putative aversion, diuresis, and antinociception. The inhibitory actions of systemically-applied EMD 61753 against hyperalgesic pressure nociception and neurogenic inflammation are mediated peripherally, probably by opioid receptors on the endings of sensory nerve fibres.

Central and peripheral actions of the novel kappa-opioid receptor agonist, EMD 60400.

1. The pharmacological characteristics of the kappa-opioid receptor agonist, EMD 60400, have been investigated, with particular reference to its central and peripheral sites of action and its ability to influence nociception. The kappa agonists ICI 197067 and ICI 204448 were tested for purposes of comparison. 2. EMD 60400 and ICI 197067 bind with high affinity (IC50 values of 2.8 and 1.5 nM, respectively) and high selectivity to kappa-opioid receptors. ICI 204448 has a lower binding affinity (IC50 13.0 nM) and selectivity for kappa-opioid receptors. 3. EMD 60400, ICI 197067, and ICI 204448 are full and potent agonists in the rabbit vas deferens in vitro assay for kappa-opioid receptors (IC50 values of 41.8, 15.7 and 15 nM, respectively). 4. Ex vivo binding experiments in mice revealed that EMD 60400 and ICI 197067 were well taken up after s.c. administration. Brain levels of EMD 60400 were lower than those of ICI 197067 at comparable doses, indicating that EMD 60400 does not penetrate into the CNS as well as ICI 197067. 5. Haloperidol-induced DOPA accumulation in the nucleus accumbens of the rat was dose-dependently reversed by s.c. application of EMD 60400 and ICI 197067 at doses of and above 3 and 0.3 mg kg-1, respectively. ICI 204448 had no effect on DOPA accumulation at 30 mg kg-1, s.c. 6. Prolongation of hexobarbitone-induced sleeping time in mice and motor impairment in the rat rotarod test were observed for EMD 60400 at doses above 3 and 2.5 mg kg-1, s.c., respectively, and for ICI 197067 at doses above 0.3 and 0.25 mg kg-1, s.c., respectively. ICI 204448 was inactive in these tests at doses of 30 and 100 mg kg-1, s.c., respectively.7. EMD 60400 applied s.c. produced dose-dependent naloxone-reversible antinociception in the mouse formalin test (1st and 2nd phase ID50 0.44 and 0.47 mg kg-1, respectively) and rodent writhing test (ID50 mouse 0.55 mg kg-1 and rat 0.3mg kg-1). Furthermore, EMD 60400 was considerably more potent in the rat pressure pain test after the induction of inflammation with carrageenin than under normalgesic conditions (ID50 values 0.1 Microg kg-1 and 4.0 mg kg-1, s.c., respectively). The action of EMD 60400 (50 microgkg-1, s.c.) in the hyperalgesic pressure pain test was completely antagonized by injection of the K-opioid antagonist, norbinaltorphimine (100 microg) into the inflamed tissue, thus demonstrating the peripheral opioid nature of this effect.8. EMD 60400 produced dose-dependent inhibition of neurogenic plasma extravasation elicited byantidromic electrical stimulation of the rat saphenous nerve (ID50 value 0.3 mg kg-1, i.v.). This inhibition was completely antagonized by intraplantar injection of norbinaltorphimine (50 microg).9. EMD 60400, ICI 197067, and ICI 204448 have diuretic effects in rats at doses of and above 0.1, 0.01,and 0.3 mg kg-1, s.c., respectively. An antidiuretic action was also observed with ICI 197067 at very low doses (3 and 6 microgkg-1, s.c.).10. Pharmacological and biochemical data therefore indicate that the three K-opioid receptor agonists tested here have different tendencies to elicit centrally-mediated sedation and putative aversion(ICI 197067 > EMD 60400 > ICI 204448) which correspond to their ability to cross the blood-brain barrier. EMD 60400 combines high affinity and selectivity for the K receptor with a degree of peripheral selectivity. The peripheral actions of systemically-applied EMD 60400 against hyperalgesic pressure pain and neurogenic inflammation are very probably mediated by opioid receptors on the endings of sensory nerve fibres.

A comparative biochemical, pharmacological and immunological study of Clostridium novyi alpha-toxin, C. difficile toxin B and C. sordellii lethal toxin.

The three clostridial cytotoxins, i.e. alpha-toxin of C. novyi (Tox alpha-nov), toxin B of C. difficile (ToxB-dif) and lethal toxin of C. sordellii (LT-sor) consist of single peptide chains of about 200,000 (Tox alpha-nov), 250,000 (LT-sor) and 275,000 (ToxB-dif) mol. wts. ToxB-dif and LT-sor but not Tox alpha-nov cross-reacted with rabbit polyclonal antibodies. Toxicity upon i.v. injection in mice was similar (LD50, 100 hr, 50-200 ng/kg) and was characterized by a slowly developing fluid loss into the interstitial space. When injected into the rat paw the toxins caused a delayed local edema lasting for days. In vitro the three toxins provoked a persistent retraction of endothelial cells cultured from pig pulmonary artery. ToxB-dif and Tox alpha-nov triggered the accumulation of F-actin in the perinuclear region at the expense of the tight peripheral bands whereas LT-sor led to a random loss of microfilament structure. The toxins inhibited uridine incorporation into endothelial or chicken embryonic cells whereas T 84 cells responded by an about 10-fold increase of uridine incorporation. Neither toxin ADP-ribosylated actin. The similarities between the three cytotoxins warrant their arrangement into a common group which perturbs the microfilament system.

Microinjection of alpha-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes.

Microinjection of purified alpha-toxin into Xenopus laevis oocytes induced meiotic maturation provided insulin was present in the medium. Induction of maturation as indicated by breakdown of the germinal vesicle depended on the amount of intracellular alpha-toxin (with a detection limit of 2 ng/oocyte) and on the concentration of insulin. The hormone concentrations used were inactive when given alone and so was alpha-toxin (1 micrograms/ml) applied from the outside. The results demonstrate an intracellular target for alpha-toxin whereas an extracellular target is apparently lacking in oocytes.

Chains and fragments of tetanus toxin, and their contribution to toxicity.

1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological and biochemical studies of cytotoxicity of Clostridium novyi type A alpha-toxin.

The actions of apparently homogeneous alpha-toxin from Clostridium novyi type A were studied in order to develop an in vitro system which closely mimics its in vivo effects and to search for the mode of poisoning. Time to death (by intravenous injection of mice) was inversely related to dose, with a detection limit of about 200 ng/kg of body weight at 100 h. Injections of 2.5 ng or more into the rat paw led to a slowly (maximum after about 30 h) developing, dose-dependent edema which was useful as a quantitative in vivo assay based on volumetry. Vascular leakage was due to gap formation between endothelial cells. Similarly, endothelial cells cultured from pig pulmonary artery lost their "cobblestone" arrangement after a dose-dependent lag period of some hours after poisoning. The morphological changes were accompanied by depression of uptake or incorporation of [3H]uridine. A quantitative in vitro assay was established on the inhibition of [3H]uridine incorporation. As in animals, the action of alpha-toxin started with a few nanograms per milliliter and proceeded slowly for at least 1 day but became resistant to antitoxin within 2 h of exposure. The toxin action is not limited to endothelial cells, since chicken embryonic cells, a mouse fibroblast line (L-929), and a rat phaeochromocytoma line (PC-12) behaved similarly. Alpha-toxin was found to differ from other bacterial toxins investigated whose modes of action are already known.

Tetanus toxin: biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis.

Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BE1) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT greater than BE greater than S) in hydrophobic interaction HPLC, and anionic behaviour (BC greater than BE greater than BT greater than S) in DEAE ion exchanger HPLC. The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD50, mouse s.c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i.e. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([3H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([3H]noradrenaline, with toxin BA, BC, BE and BT). Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)