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1.
Neuromuscul Disord ; 16(4): 237-48, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16542837

ABSTRACT

BN 82270 is a membrane-permeable prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor and anti-oxidant. Acute in vivo injection of dystrophic mdx mice (30 mg/kg, s.c.) fully counteracted calpain overactivity in diaphragm. A chronic 4-6 weeks administration significantly prevented in vivo the fore limb force drop occurring in mdx mice exercised on treadmill. Ex vivo electrophysiological recordings showed that BN 82270 treatment contrasted the decrease in chloride channel function (gCl) in diaphragm, an index of spontaneous degeneration, while it was less effective on both exercise-impaired gCl and calcium-dependent mechanical threshold of the hind limb extensor digitorum longus (EDL) muscle fibres. The BN 82270 treated mdx mice showed a marked reduction of plasma creatine kinase and of the pro-fibrotic cytokine TGF-beta1 in both hind limb muscles and diaphragm; however, the histopathological profile of gastrocnemious muscle was poorly ameliorated. In hind limb muscles of treated mice, the active form was detected by HPLC in the low therapeutic concentration range. In vitro exposure to 100 microM BN 82270 led to higher active form in diaphragm than in EDL muscle. This is the first demonstration that this class of chimeric compounds, dually targeting pathology-related events, exerts beneficial effects in muscular dystrophy. The drug/prodrug system may require posology adjustment to produce wider beneficial effects on all muscle types.


Subject(s)
Antioxidants/therapeutic use , Glycoproteins/therapeutic use , Muscular Dystrophy, Animal/drug therapy , Prodrugs/therapeutic use , Animals , Antioxidants/pharmacology , Biomechanical Phenomena , Body Weight , Calpain/physiology , Chloride Channels/drug effects , Creatine Kinase/blood , Diaphragm/drug effects , Glycoproteins/pharmacology , Hindlimb , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/drug effects , Muscular Dystrophy, Animal/physiopathology , Phenothiazines/therapeutic use , Physical Conditioning, Animal , Prodrugs/pharmacology , Rats , Rats, Sprague-Dawley , Transforming Growth Factor beta/blood , Transforming Growth Factor beta1
2.
Am J Pathol ; 166(2): 477-89, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15681831

ABSTRACT

Chronic inflammation is a secondary reaction of Duchenne muscular dystrophy and may contribute to disease progression. To examine whether immunosuppressant therapies could benefit dystrophic patients, we analyzed the effects of cyclosporine A (CsA) on a dystrophic mouse model. Mdx mice were treated with 10 mg/kg of CsA for 4 to 8 weeks throughout a period of exercise on treadmill, a protocol that worsens the dystrophic condition. The CsA treatment fully prevented the 60% drop of forelimb strength induced by exercise. A significant amelioration (P < 0.05) was observed in histological profile of CsA-treated gastrocnemius muscle with reductions of nonmuscle area (20%), centronucleated fibers (12%), and degenerating area (50%) compared to untreated exercised mdx mice. Consequently, the percentage of normal fibers increased from 26 to 35% in CsA-treated mice. Decreases in creatine kinase and markers of fibrosis were also observed. By electrophysiological recordings ex vivo, we found that CsA counteracted the decrease in chloride conductance (gCl), a functional index of degeneration in diaphragm and extensor digitorum longus muscle fibers. However, electrophysiology and fura-2 calcium imaging did not show any amelioration of calcium homeostasis in extensor digitorum longus muscle fibers. No significant effect was observed on utrophin levels in diaphragm muscle. Our data show that the CsA treatment significantly normalized many functional, histological, and biochemical endpoints by acting on events that are independent or downstream of calcium homeostasis. The beneficial effect of CsA may involve different targets, reinforcing the usefulness of immunosuppressant drugs in muscular dystrophy.


Subject(s)
Cyclosporine/pharmacology , Muscular Dystrophies/drug therapy , Animals , Body Weight/drug effects , Calcium/metabolism , Chlorides/metabolism , Coloring Agents/pharmacology , Creatine Kinase/blood , Electrophysiology , Fibrosis , Fura-2/pharmacology , Immunohistochemistry , Immunosuppressive Agents/pharmacology , Ions , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle, Skeletal/drug effects , Muscles/drug effects , Physical Conditioning, Animal , Spectrophotometry , Time Factors , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1
3.
Neuromuscul Disord ; 14(7): 405-16, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15210163

ABSTRACT

The antimyotonic activity of chiral derivatives of mexiletine and tocainide, selected as potent use-dependent blockers of skeletal muscle sodium channels, was evaluated in vivo acutely in myotonic ADR mice. The compounds had either aromatic (Me4 and Me6) or branched isopropyl groups (Me5 and To1) on the asymmetric centre, or had this latter one methylene apart from the amino group (Me2). Therapeutic doses of mexiletine (5-10 mg/kg) and tocainide (7-20 mg/kg) significantly reduced the long time of righting reflex (TRR), typical of ADR mice. Me4, Me5 and Me6 were 2-fold more potent than mexiletine. To1 fully normalised the TRR at 7 mg/kg. The electromyographic analysis confirmed a muscle-based activity for drug effectiveness on TRR. All the compounds reduced the myotonic hyperexcitability of intercostal muscle fibres when tested in vitro by current-clamp recordings, with a potency correlated with their action on sodium channels. On stimulus-evoked firing, the isopropyl analogues were 2-4-fold more potent than parent compounds, while the aromatic analogues were about 10-fold more potent than mexiletine. Patch-clamp recordings confirmed a normal-like pharmacological sensitivity of sodium channels of native ADR muscle fibres. Finally, the in vivo antimyotonic activity is due to the block of sodium channels and divergences with in vitro potency can be related to structure-based changes in drug pharmacokinetics.


Subject(s)
Anti-Arrhythmia Agents/therapeutic use , Mexiletine/therapeutic use , Myotonic Disorders/drug therapy , Tocainide/therapeutic use , Action Potentials/drug effects , Analysis of Variance , Animals , Anti-Arrhythmia Agents/blood , Disease Models, Animal , Dose-Response Relationship, Drug , Electric Conductivity , Electromyography/methods , Female , In Vitro Techniques , Inhibitory Concentration 50 , Male , Mexiletine/blood , Mice , Mice, Mutant Strains , Muscle Contraction/drug effects , Myotonic Disorders/blood , Patch-Clamp Techniques/methods , Sodium Channels/drug effects , Sodium Channels/physiology , Structure-Activity Relationship , Time Factors , Tocainide/analogs & derivatives , Tocainide/blood
4.
EMBO Rep ; 5(6): 584-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15167890

ABSTRACT

The highly homologous Cl(-) channels CLC-Ka and CLC-Kb are important for water and salt conservation in the kidney and for the production of endolymph in the inner ear. Mutations in CLC-Kb lead to Bartter's syndrome and mutations in the small CLC-K subunit barttin lead to Bartter's syndrome and deafness. Here we show that CLC-Ka is blocked by the recently identified blocker 2-(p-chlorophenoxy)-3-phenylpropionic acid of the rat channel CLC-K1 with an apparent K(D) approximately 80 microM. We also found that DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid), a generic Cl(-) channel blocker, inhibits CLC-Ka (K(D) approximately 90 microM). Surprisingly, the highly homologous channel CLC-Kb is fivefold to sixfold less sensitive to both compounds. Guided by the crystal structure of bacterial CLC proteins, we identify two amino acids, N68/D68 and G72/E72, in CLC-Ka and CLC-Kb, respectively, that are responsible for the differential drug sensitivity. Both residues expose their side chains in the extracellular pore mouth, delineating the probable drug binding site. These novel CLC-K channel blockers are promising lead compounds for the development of new diuretic drugs.


Subject(s)
2-Methyl-4-chlorophenoxyacetic Acid/analogs & derivatives , 2-Methyl-4-chlorophenoxyacetic Acid/pharmacology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Anion Transport Proteins/antagonists & inhibitors , Anion Transport Proteins/chemistry , Chloride Channels/antagonists & inhibitors , Chloride Channels/chemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , 2-Methyl-4-chlorophenoxyacetic Acid/chemistry , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/chemistry , Amino Acid Motifs/genetics , Anion Transport Proteins/genetics , Binding Sites/genetics , Chloride Channels/genetics , Dose-Response Relationship, Drug , Humans , Membrane Proteins/genetics , Patch-Clamp Techniques , Point Mutation , Protein Structure, Tertiary
5.
J Physiol ; 554(Pt 2): 321-34, 2004 Jan 15.
Article in English | MEDLINE | ID: mdl-14608015

ABSTRACT

Flecainide, a class IC antiarrhythmic, was shown to improve myotonia caused by sodium channel mutations in situations where the class IB antiarrhythmic drug mexiletine was less efficient. Yet little is known about molecular interactions between flecainide and human skeletal muscle sodium (hNa(v)1.4) channels. Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing wild-type (WT) and mutant hNa(v)1.4 channels (R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (HP) of -120 mV, flecainide use-dependently blocked WT and G1306E I(Na) equally but was more potent on R1448C channels. For WT, the extent of block depended on a holding voltage more negative than the activation threshold, being greater at -90 mV as compared to -120 and -180 mV. This behaviour was exacerbated by the R1448C mutation since block at -120 mV was greater than that at -180 mV. Thus flecainide can bind to inactivated sodium channels in the absence of channel opening. Nevertheless, all the channels showed the same closed-state affinity constant (K(R) approximately 480 microM) and the same inactivated-state affinity constant (K(I) approximately 18 microM). Simulations according to the modulated receptor hypothesis mimic the voltage-dependent block of WT and mutant channels by flecainide and mexiletine. All the results suggest similar blocking mechanisms for the two drugs. Yet, since flecainide exerts use-dependent block at lower frequency than mexiletine, it may exhibit greater benefit in all myotonic syndromes. Moreover, flecainide blocks hNa(v)1.4 channel mutants with a rightward shift of availability voltage dependence more specifically than mexiletine, owing to a lower K(R)/K(I) ratio. This study offers a pharmacogenetic strategy to better address treatment in individual myotonic patients.


Subject(s)
Flecainide/pharmacology , Muscle Proteins/antagonists & inhibitors , Mutation , Myotonia/drug therapy , Sodium Channel Blockers/pharmacology , Dose-Response Relationship, Drug , Flecainide/metabolism , Humans , Ion Channel Gating/drug effects , Muscle Proteins/metabolism , Myotonia/genetics , Myotonia/metabolism , NAV1.4 Voltage-Gated Sodium Channel , Sodium Channel Blockers/metabolism , Sodium Channels/metabolism
6.
Br J Pharmacol ; 139(7): 1255-64, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12890704

ABSTRACT

(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.


Subject(s)
2-Methyl-4-chlorophenoxyacetic Acid/analogs & derivatives , 2-Methyl-4-chlorophenoxyacetic Acid/chemistry , 2-Methyl-4-chlorophenoxyacetic Acid/pharmacology , Chloride Channels/biosynthesis , Muscle, Skeletal/drug effects , Quantitative Structure-Activity Relationship , Stereoisomerism , Animals , Binding Sites , Chloride Channels/drug effects , Chloride Channels/genetics , Humans , Male , Muscle, Skeletal/physiology , Oocytes/drug effects , Oocytes/metabolism , Rats , Rats, Wistar , Xenopus laevis
7.
Br J Pharmacol ; 139(3): 575-84, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12788817

ABSTRACT

(1) Growth hormone secretagogues (GHS) exhibit potent growth hormone (GH)-releasing activity through the activation of a pituitary receptor. Here, we consider the possibility that GHS can target a specific receptor in rat skeletal muscle and have a role in the control of muscle function. (2) By means of the intracellular microelectrode technique, we found that in vitro application of hexarelin and L-163,255 dose dependently reduced resting chloride (gCl) and potassium (gK) conductances in rat skeletal muscle. These effects were prevented by the GHS-receptor antagonist [D-Lys-3]-GHRP-6, and by either phospholipase C or protein kinase C (PKC) inhibitors. Ghrelin, a natural ligand of GHS receptors, also induced a reduction of muscle gCl and gK, which was antagonised by [D-Lys-3]-GHRP-6. (3) Both GHS shifted the mechanical threshold for the contraction of muscle fibres towards more negative voltages. Accordingly, by means of FURA-2 fluorescent measurements, we demonstrated that L-163,255 induced a resting [Ca(2+)](i) increase, which was reversible and not blocked by nifedipine or removal of external Ca(2+). (4) Ageing is a condition characterised by a deficit of GH secretion, which in turn modifies the electrical and contractile properties of skeletal muscle. In contrast to GH, chronic treatment of aged rats with hexarelin or L-163,255 failed to restore the electrical and contractile muscle properties. Moreover, the two GHS applied in vitro were able to antagonise the beneficial effect on gCl and gK obtained through chronic treatment of aged animals with GH. (5) Thus, skeletal muscle expresses a specific GHS receptor able to decrease gCl and gK through a PKC-mediated intracellular pathway. This peripheral action may account for the lack of restoration of skeletal muscle function in long-term GHS-treated aged animals.


Subject(s)
Aging/drug effects , Growth Hormone/pharmacology , Muscle Contraction/drug effects , Muscle, Skeletal/drug effects , Receptors, G-Protein-Coupled/physiology , Aging/physiology , Animals , Dose-Response Relationship, Drug , Electric Stimulation/methods , Ghrelin , Male , Muscle Contraction/physiology , Muscle, Skeletal/physiology , Oligopeptides/pharmacology , Peptide Hormones/pharmacology , Rats , Rats, Wistar , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, Ghrelin
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