WO2000053191A2 - Modulation de l'activation du precurseur du muscle squelettique - Google Patents

Modulation de l'activation du precurseur du muscle squelettique Download PDF

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WO2000053191A2
WO2000053191A2 PCT/CA2000/000255 CA0000255W WO0053191A2 WO 2000053191 A2 WO2000053191 A2 WO 2000053191A2 CA 0000255 W CA0000255 W CA 0000255W WO 0053191 A2 WO0053191 A2 WO 0053191A2
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muscle
cells
activation
nos
cell
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WO2000053191A3 (fr
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Judy E. Anderson
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The University Of Manitoba
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Priority to US09/936,609 priority Critical patent/US6967102B1/en
Priority to CA002371927A priority patent/CA2371927A1/fr
Priority to AU31395/00A priority patent/AU3139500A/en
Publication of WO2000053191A2 publication Critical patent/WO2000053191A2/fr
Publication of WO2000053191A3 publication Critical patent/WO2000053191A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/08—Oxides; Hydroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans

Definitions

  • the present invention relates generally to skeletal muscle proliferation. More specifically, the invention relates to nitric oxide as a modulator of skeletal muscle precursor cell activation, and to uses of nitric oxide to improve muscle formation and repair in normal and disease states.
  • Skeletal muscle arises after the induction of the mesoderm.
  • the dorsal mesodermal mesenchyme differentiates to form myotomes which, in turn, differentiate to form give rise to the myogenic precursor cells which ultimately form skeletal muscle.
  • the skeletal muscle precursors fuse side-to-side to form unbranched, multinucleated myofibers.
  • Some of the skeletal myogenic precursor cells do not differentiate and fuse into myocytes (also called myofibers) but, rather, attach to the outside of the plasmalemma of the myocytes.
  • satellite cells participate in muscle growth during maturation and typically thereafter will remain, throughout adulthood, as largely undifferentiated, quiescent skeletal muscle "satellite cells.”
  • myogenic precursor cells or muscle “stem cells”
  • muscle “stem cells” which proliferate and differentiate, again by fusion, into new and functional skeletal muscle.
  • the satellite cells of skeletal muscle provide a constant and renewable source of myogenic precursor cells which allows for skeletal muscle repair and regeneration throughout mammalian life.
  • the proliferation and differentiation of skeletal muscle satellite cells has been extensively studied in vitro.
  • TGF- ⁇ i is widely believed to inhibit satellite cell proliferation, as does contact with the myofiber plasmalemma, but not the basal lamina (Bischoff (1989) ; but see Hathaway et al. (1991) J. Cell Physiol.
  • satellite cells After muscle injury, satellite cells are activated and recruited to cycle as precursors for new muscle formation. Between injury and proliferation in vivo, satellite cells express immediate early genes after 3-6 hr., (Weiss, (1994) Acta Neuropathol . 87: 63-70; Kami, K. , Noguchi , K. , and Senba, E., (1995) Cell Tissue Res. 280: 11-19) and muscle regulatory genes after 6 hr. (Grounds, M.D., Garrett, K.L., Lai, M.C. Wright, W.E., and Bielharz, M.W. (1992) Cell Tissue Res.
  • hepatocyte growth factor also called scatter factor, HGF/SF
  • HGF/SF hepatocyte growth factor
  • satellite cells express c-met
  • HGF/SF While HGF/SF also plays a role in differentiation (Gal-Levi et al . (1998) Biochim. Biophys. Acta.
  • Satellite cells are activated without trauma and make DNA after exercise, training, stretch, cold, compression, hypertrophy, suspension and denervation (Bischoff (1986a) Dev. Biol. 115: 140-147; Bischoff (1986) Dev. Biol. Ill: 129-139; Bischoff (1990b) Development 109: 943-952; Darr and Schultz: (1987) J. Appl. Physiol. 63: 1816-1821; Darr and Schultz (1989) J. Appl. Physiol. 67: 1827-1834; Appell et al . (1988) Int. J.
  • Glucocorticoids e.g. deflazacort and prednisone
  • Glucocorticoids have been prescribed to Duchenne muscular dystrophy patients.
  • the glucocorticoids possess anti-inflammatory and immunomodulatory activities.
  • the major side effects of the glucocorticoids are hypertension, peptic ulcers, increased susceptibility to infections, osteoporosis, hyperglycemia, and vascular occlusion
  • muscle cells myoblasts
  • Another treatment aimed at muscle repair and formation involves the transfer of muscle cells (myoblasts) to the injured site.
  • muscle cells myoblasts
  • Autologous mouse skeletal muscle cells have been explanted from a healthy muscle, proliferated in vitro, and then implanted into a necrotized skeletal muscle site
  • skeletal muscle satellite cells to restore or regenerate injured skeletal muscle has led some researchers to test whether myogenic precursor cells could be used to replace lost or damaged myocardial muscle.
  • mouse fetal cardiomyocytes which are not terminally differentiated and retain the ability to divide, have been directly injected into the myocardium of a syngeneic adult mouse, and have been shown to form new and apparently functional myocardium (Soonpaa et al . (1994) Science 264: 98- 101) .
  • skeletal muscle satellite cells, explanted from adult canine skeletal muscle can be proliferated in vitro and implanted into a site of myocardial cryoinjury, where they appear to differentiate into
  • gene therapy has been used to provide, on an experimental basis, the active counterpart of the missing or mutated protein to the muscle precursor cells prior to injection of the precursor cells into the muscle (e.g. WO 91/12329) .
  • gene transfer in mammals has only limited success due to low level expression of the therapeutic protein in vivo (Partridge et al . (1991) Muscle Nerve 14: 197-212; Partridge et al . Nature Medicine (1998) 4: 1208-1209), difficulties with delivery of the gene to targetted myogenic cells (Feero WG et al . (1997) Gene Therapy 4: 664-674), and immune responses (Partridge TA. Myoblast transplantation.
  • Lanza RP et al . (eds) Yearbook of Cell and Tissue
  • the present invention is directed to methods, pharmaceutical compositions and kits, for modulating skeletal muscle precursor cell activation. Modulation is effected through the use of nitric oxide (NO) , donors of NO, inhibitors of NO activity (NO inhibitor) or regulators of NO production.
  • NO nitric oxide
  • the invention provides a use of NO, an NO donor, an NO inhibitor, or a regulator of NO production to modulate activation of muscle precursor cells. Local or systemic activation is further provided.
  • the invention provides a use of NO, an NO donor, or a regulator of NO production to increase activation of muscle precursor cells, thereby improving muscle regeneration and/or repair. Local or systemic activation is further provided.
  • the invention provides use of an inhibitor of NO activity or an inhibitor of NO production to decrease activation of skeletal muscle precursor cells, thereby limiting proliferation of skeletal muscle precursor cells. Local or systemic decrease is further provided.
  • the invention provides a method of amplifying muscle cells in culture, comprising placing NO, an NO donor, or a regulator of NO production into contact with muscle cells.
  • the invention provides a method for obtaining a muscle cell population in culture, comprising use of NO, an NO donor, or a regulator of NO production.
  • the invention provides a composition comprising muscle cells and a compound selected from the group consisting of NO, an NO donor and a regulator of NO production.
  • the invention provides use of NO, an NO donor, an NO inhibitor or a regulator of NO production to modulate the effects of steroid hormone on skeletal muscle.
  • NO has been used as "an agent to enhance the action of corticosteroids in the treatment of various diseases" (WO 98/41144) .
  • the use disclosed therein has been directed exclusively to treatment of anti- inflammatory, autoimmune or cardiovascular disease.
  • the invention provides a composition comprising any one of the group consisting of NO, an NO donor, an NO inhibitor and a regulator of NO production, and a diluent or carrier suitable for use in muscle, for modulating activation of muscle precursor cells.
  • the invention provides a composition
  • a composition comprising a compound selected from the group consisting of NO, an NO donor, an NO inhibitor and a regulator of NO production, and a component suitable for increasing concentration of the compound in muscle, for modulating activation of muscle precursor cells.
  • the invention provides a commercial package containing as an active ingredient NO, an NO donor, an NO inhibitor or a regulator of NO production, together with instructions for its use for modulating activation of muscle precursor cells.
  • the invention provides a method for validating a test wherein a change in activation state of muscle precursor cells is determined, comprising use of a DNA intercalator to determine that fibers associated with the precursor cells are intact.
  • the invention provides a method for validating a test wherein a fiber hypercontraction- dependent change in activation state of muscle precursor cells is determined, comprising use of a myotoxin and a DNA intercalator to determine fiber membrane damage.
  • the invention provides a method for identifying a compound which effects a change in activation state of muscle precursor cells, comprising: determining that fibers associated with the precursor cells are intact; determining the activation state of precursor cells in the absence of the compound; and determining the activation state of precursor cells treated with the compound; wherein the difference between the two activation states identifies the compound as a compound which effects a change in activation state of muscle precursor cells.
  • the invention provides a method for identifying a compound which effects a fiber hypercontraction-dependent change in activation state of muscle precursor cells, comprising: treating an intact fiber containing precursor cells with a myotoxin and a DNA intercalator to effect fiber hypercontraction; determining the activation state of precursor cells in the absence of the myotoxin, DNA intercalator and the compound; and determining the activation state of precursor cells treated with the compound in the absence of the myotoxin and DNA intercalator; wherein the difference between the two activation states identify the compound as a compound which effects a fiber hypercontraction-dependent change in activation state of muscle precursor cells.
  • the present invention offers a number of advantages.
  • the invention By allowing skeletal muscle precursor cells to be manipulated directly, the invention enables specific treatments to make more new muscle more quickly and avoid extensive use of immunosuppressive drugs for myoblast transfer.
  • the present invention also complements myoblast transfer protocols by reducing the need for an industrial tissue culture facility to amplify muscle precursors prior to transfer.
  • the present invention further extends the beneficial effects of glucocorticoid treatments by providing maximally available and activated precursor cells for the proliferative and fusion- promoting effects mediated by glucocorticoid drugs.
  • Treatments of normal muscle by physical therapy e.g. in aging persons, after a stroke or coma, post-surgery recovery, physical training in preparation for spaceflight and during weightlessness
  • promotion and acceleration of muscle growth by manipulating precursor cell activation could be of economic benefit.
  • Figure 1 Representative graphs from one experiment each on normal control (C57BL/6,A-H) and mdx mice (I-L) . Panels show the time course of changes in muscle weight to body weight
  • Figure 2 Time course of cell yield (cells/muscle) expressed as the ratio of RTA: LTA (mean + SEM) for normal mice (C57BL/6 and B6,129SF, 3 experiments, ⁇ ), normal mice treated with with L-NAME (C57BL/6, 3 experiments, A) and "NOS mutant" mice including mdx and B6, 129S-Nosl tm /P lh (NOS-I knockout) mice (3 experiments, ⁇ ) . Satellite cell activation (cell yield ratio of RTA:LTA) in normal mice begins at 0 min. and is significantly greater than in mice with NOS inhibition as a result of pharmacological treatment (by L-NAME) , a primary gene defect (NOS-I knockout mice) or secondary to dystrophin deficiency (mdx mice) .
  • Figure 3 Representative effects of crush in normal muscle at 0 min. (A, B) and 10 min. (C-E) after injury and after saline
  • A, B, E or L-NAME (C, D) pre-treatment .
  • A LTA section shows normal undamaged muscle.
  • B RTA section at 0 min. after crush injury.
  • C At low magnification, a dark band of hypercontraction in fiber segments (to the left) and extravasated blood cells between fibers are thin and retracted to the light of the hypercontracted region.
  • D Two delta lesions in a fiber after L-NAME and 10 min. after crush.
  • E Higher magnification view of muscle 10 min. after injury showing extravasated blood cells between hypercontracted and retracted fiber segments and segments with early sarcomere disruption.
  • FIG. 4 Satellite cell changes in vivo are delayed by NOS inhibition in normal mice treated wi th saline (A-H) or L-NAME (I-P) .
  • A-H wi th saline
  • I-P L-NAME
  • M-cadherin outlines a large satellite cell at 0 min. after injury.
  • B Large m-cadherin+ satellite cell on the external lamina 10 min. after injury.
  • C H&E-stained satellite cells (arrows) in low magnification RTA fibers at 0 min.
  • D At high magnification, hypertrophic satellite cells on fibers in RSOL (and RTA, not shown) at 10 min.
  • E&F Large satellite cell shows co-localized (yellow) staining for HGF/SF (Texas-red) and c-met (FITC) at 0 min. (E) and 10 min. (F) .
  • G Two resin sections (stained with toluidine blue) show large satellite cells (between arrowheads) at 0 min. in RTA.
  • H At 10. min. in RTA, satellite cells (arrows) with granulated cytoplasm and euchromatic nuclei are partially lifting off adjacent fibers.
  • a hypertrophic satellite cell (between arrowheads) is partly separated from an RTA fiber 10 min. after injury.
  • FIG. 5 L-NAME treatment over 6 days reduces normal muscle regeneration .
  • H&E normal muscle repair after saline pretreatment includes a small necrotic crushed region (right of panel A) , a region of adjacent mononuclear cells and myotubes (arrows) and surviving fiber segments (at the left) .
  • B New myotubes in the adjacent region contain many central nuclei and eosinophilic sarcoplasm after 6 days of regeneration.
  • C New myotubes (arrows) are also present among surviving fibers.
  • Figure 6 A single L-NAME injection before injury affects myogenic repair in normal muscle .
  • H&E high magnification
  • the RTA 6 days after injury shows a large necrotic region (to the right) , an adjacent area of mononuclear cells and small new myotubes (arrows) , and surviving fiber segments (to the left) .
  • B At high magnification a myotube (arrow) extends between mononuclear cells and a fiber segment.
  • C A very thin intensely eosinophilic myotube originates immediately beside a surviving fiber segment.
  • D At higher magnification, the same myotube has formed from the satellite cell position apparently inside the external lamina.
  • E An eosinophilic satellite cell (arrow) is elongated into a thin myotube.
  • F A column of apparently unfused centrally-nucleated cells with granular cytoplasm makes up a myotube.
  • G A BrdU-positive nucleus adjacent to a new myotube.
  • H&I Thin new myotube segments are positive for devMHC (Texas red florescence) whether they extend from a larger myotube (H) or are located among mononuclear cells near the crush (I) .
  • J A crimson satellite cell (arrow) on an EDL fiber.
  • K A large satellite cell (arrow) with crimson cytoplasm on a SOL fiber.
  • (L) M- cadherin is present between a satellite cell (arrow) and a small new myotube (arrowheads) .
  • (M) M-cadherin staining is intense on satellite cells located on the four intrafusal muscle fibers in a spindle complex.
  • Figure 7 A single treatment wi th L-NAME 30 min . before injury affects dystrophic muscle regeneration .
  • H&E At low magnification
  • B Many large new myotubes adjacent to the crush.
  • C Elongated mononuclear cells and myotubes are m-cadherin+.
  • D An elongated crimson cell is binucleate and located in the satellite position on a surviving fiber segment.
  • E A new myotube extends from a surviving segment and contains devMHC (Texas red fluorescence) .
  • devMHC Texas red fluorescence
  • HGF/SF+fibers (Texas red) in LTA.
  • I A large satellite cell
  • Figure 8 A model for the process of shear- induced, NO- mediated events that activate satelli te cells after skeletal muscle injury.
  • A In undamaged muscle with normal contraction and relaxation, thin quiescent satellite cells are demarcated by m-cadherin and contain few organelles. They are interposed between the overlying external lamina and the sarcolemma of a subjacent fiber, and are subject to pulsatile NO released from NOS-I ⁇ that is anchored to syntrophin. Normally, NO diffuses cylindrically out from the fiber to act on cells and enzymes in the interstitium or is neutralized by red cell hemoglobin in the vessels that wrap each fiber.
  • a released factor like HGF/SF enters the circulation and can transiently activate distant satellite cells on undamaged muscles, although normal pulsatile NO release will mostly attenuate that response. Capillaries dilate and blood cells extravasate into the interstitium.
  • Fiber segments fully retract and satellite cells become motile precursors as HGF/SF binds to c-met.
  • the external lamina remains as a scaffold for the satellite cells, now surrounded by less adhesive m- cadherin. The precursors may leave the fiber as the sequential expression of early immediate genes, muscle regulatory genes, proliferating cell nuclear antigen and later DNA synthesis begin prior to proliferation.
  • Figure 10 Identification of satellite cells on muscle fibers .
  • A) Fixed muscle fiber (phase contrast x520; bar 50 ⁇ m)
  • C) C-met immunostaining of the muscle fiber distinguishes between satellite cells and myonuclei by staining the cytoplasm of satellite cells, but not nuclei of satellite cells or myonuclei within the fiber or fiber sarcoplasm. Negative control fibers lacking primary antibody did not stain satellite cells.
  • Figure 11 Staining of fiber nuclei using ethidium bromide .
  • A) 2 fibers fixed using methanol immediately after plating and coverslipping (phase contrast x285; bar 50 ⁇ m) .
  • Membrane blebs appear on and close to the fiber due to hypotonic conditions during incubation in this experiment.
  • C) Unfixed live and hypercontracted fibers. (phase contrast ⁇ 260; bar 50 ⁇ m)
  • Figure 12 Addi tion of Marcaine + EtBr to fibers .
  • A) Unfixed live fiber (phase contrast x260; bar 50 ⁇ m)
  • Figure 13 Single fiber experiments to test the effects of L- Arginine at various concentrations on: A) proliferating satellite cells per fiber, and B) free satellite cells per fiber in culture after 48 hr .
  • CSR is serum replacement medium;
  • CME is crushed muscle extract.
  • Figure 14 Manipulation of NO augments deflazacort effects.
  • CNI central nucleaction index
  • LTA left tibialis anterior muscle
  • DIA diaphragm.
  • myogenic precursor cells refers to cells capable of myogenesis, or the process of proliferation and differentiation into new and functional muscle when present in a morphogenically permissive environment.
  • Myogenic precursor cells are variously referred to as “myoblasts,” “muscle stem cells” or “satellite cells”.
  • the present invention derives from, but is not limited to, the unexpected result that NO mediates satellite cell activation.
  • a model is presented in Figure 8 which broadens the field of NO signalling in muscle (reviewed by Grozdanovic and Baumgarten (1999) Histol. Histopathol. 14: 243-256) and hypothesizes that NO release mediates satellite cell activation by being responsive to shear.
  • normal cyclic loading of muscle produces pulsatile NO release (Tidball et al . (1998) Am. J. Physiol. 275: C260-C266) by rapid diffusion of NO down its concentration gradient, and maintains satellite cell quiescence.
  • the external lamina wrapping fibers may provide the potential for satellite cells to respond to shear between the sarcolemma and lamina. Satellite cells hug fibers across an even 15 nm cleft without obvious junctional complexes, and they associate closely with external lamina (Bischoff (1990) J. Cell Biol. Ill: 201-207; Schultz and McCormick (1994) Rev. Physiol. Biochem. Pharmacol. 123: 213-257).
  • Satellite cells have ideal topography to detect a rapid peak of NO release from underlying fibers after shear and also to be kept quiescent by normally continuous small pulses of NO from the fiber.
  • the speed of the NO-mediated signal for activation suggests that an initiating event such as mechanical shear forces acts on constitutive nitric oxide synthase (NOS-I) , since the response time is too short to induce expression or increase activity (McCall et al . (1991) Eur. J. Immunol.
  • a large release of NO is thus the primary signal that mediates or directly signals satellite cell activation.
  • Other secondary signals such as HGF/SG or other factors are then needed to maintain or complete activation.
  • Such secondary signals or the pathways that induce/initiate the secondary signals may become activated themselves, and circulate from the initiating site to initiate activation of satellite cells located outside the damaged muscle. Without the NO-mediated signal, however, normal fibers would repress activation and their satellite cells would return to quiescence. By contrast, satellite cells in damaged muscles, having received the secondary circulating signal or signals in addition to the primary signal, would complete the activation sequence.
  • satellite cell activation was defined structurally as cytoplasmic and organelle hypertrophy and dynamically as recruitment to cycle.
  • the close adherence of satellite cells to parent fibers must decrease during activation for satellite cells to move through the external lamina to form new fibers. Therefore the loss-of-adhesion feature was used as a simple index of activation.
  • the ability to isolate myogenic cells after brief standard digestion was a conservative estimate of available satellite cells and not an estimate of total myogenic cells. (Additional myogenic cells are found in the material collected on the Nitex filter during cell isolations) . NO is known to modulate leukocyte and platelet adhesion (Kubes et al . (1991) Proc . Natl. Acad. Sci.
  • one aspect of the present invention provides use of NO, an NO donor, an inhibitor of NO activity or a regulator of NO production to increase activation of skeletal muscle precursor cells, thereby improving muscle regeneration and/or repair. Localized, in situ or systemic activation is further provided.
  • the present invention further provides use of NO, an NO donor, an inhibitor of NO activity or a regulator of NO production to amplify populations of muscle cells in culture.
  • alteration of NO production is effected via changing NOS activity.
  • the present invention also has applications in the treatment of muscle dystrophic or degenerative disorders.
  • One such disorder is Duchenne muscular dystrophy (DMD) , an X-linked recessive disorder characterized by progressive and lethal muscle weakness.
  • DMD Duchenne muscular dystrophy
  • the deficiencies essentially weaken the fiber sarcolemma, increasing its susceptibility to contraction-induced fiber damage (Petrof et al. (1993) Proc. Natl. Acad. Sci.
  • cytoplasmic NOS-I in mdx muscle would act as a diffuse areal source of NO rather than the nearby linear source, subjacent and parallel to satellite cells found in normal muscle.
  • the normally steep NO gradient across the cleft between fiber and satellite cell would therefore be more shallow, diffuse more slowly, and the small NO transient would show attenuated responsiveness to shear forces.
  • Rapid repair by mdx muscle is consistent with the notion that mdx satellite cells are partly activated or on 'stand-by.' As well, it would follow that acute injury would not necessarily augment immediate activation for mdx and NOS-I knockout mice, as reported here in cell yield studies. By that reasoning, repair after imposed injury in the NOS-I X mdx double mutant should be less effective and/or delayed compared to mdx muscle repair. As well, dystrophy in that double mutant may be more severe than in mdx mice if it were assessed in mice younger than 12 months, before the index of repair (central nucleation) has reached its theoretical plateau.
  • cytoplasmic NOS-I in human fibers would serve as an even smaller non-linear NO source than in mdx muscle.
  • the resulting very shallow gradient or physiological NO transient across satellite cells could partly account for the severity of Duchenne dystrophy, almost as if the standby activation (like a "hair trigger") contributes to overly enthusiastic successive repair events and resulting in premature senescence (Decary et al . (1996) Human Gene Therapy 7: 1347-1350; Decary et al. (1997) Human Gene Therapy 8: 1429-
  • another aspect of the present invention provides use of an inhibitor of NO production to decrease activation of skeletal muscle precursor cells, thereby limiting proliferation of skeletal muscle precursor cells. Localized, in situ or systemic decrease is further provided.
  • inhibition of NO production is effected by changing NOS activity.
  • the present invention demonstrates that manipulating
  • NO-mediated activation can augment the beneficial effects of a steroid such as deflazacort (see Example 11) .
  • manipulation is effected by changing NOS activity.
  • another aspect of the present invention provides use of NO, an NO donor, an inhibitor of NO activity or a regulator of NO production to modulate the effects of a steroid hormone on skeletal muscle. Localized, in si tu or systemic modulation is further provided.
  • the steroid hormone is deflazacort.
  • the treatment is most effectively achieved by application in si tu of a compound for altering NO-mediated activation, or by delivering such a compound to specific tissue sites, since systemic treatment can affect to a different extent one muscle type or one phenotype of dystrophy, compared to another muscle phenotype.
  • the composition containing NO, an NO donor, an inhibitor of NO activity, a regulator of NO production or an inhibitor of NO production may include some component that is specific to the target tissue and organ, e.g. a muscle- targeting component.
  • the present invention further describes a technique for using isolated muscle fibers to monitor satellite cell activation and thereby identifying compounds that promote or decrease activation.
  • This technique allows tracking of individual satellite cells, as well as populations of cells, under closely monitored conditions.
  • the separation of satellite cells from fibroblasts and inflammatory cells is important since the latter cells are sources of cytokines and growth factors which also have a role in repair, and whose effects may interfere with effects of the compound to be identified.
  • Use of isolated muscle fibers to characterize activation involves (1) unambiguous distinction between precursor cells and myonuclei; and (2) determination of the time of fiber death under known conditions. Accordingly, one aspect of the present invention provides methods for monitoring the state of precursor cell activation and for determining whether a test compound effects activation.
  • the method of the invention is used to determine that the precursor cells are quiescent, i.e. in completely intact fibers without stimulus. In another embodiment, the method of the invention is used to determine that the precursor cells are in a state of activation which is independent of shear or hypercontraction. Accordingly, in another embodiment, the present method is used to determine whether a test compound affects hypercontraction-independent activation of the precursor cells. In another embodiment, the method of the invention is used to determine that the precursor cells are in a state of activation which is effected by shear-induced hypercontraction of the fiber. Accordingly, in another embodiment, the present method is used to determine whether a test compound affects activation which is effected by shear produced by hypercontraction of the fiber. Any compound of interest can be used as the test compound in this method.
  • the method of the invention for monitoring the state of precursor cell activation and for determining whether a test compound effects or affects activation is as follows: A. Fiber Isolation:
  • Fibers are isolated from muscles using a combination of fine dissection, enzyme digestion and physical disruption of a muscle cleaned of connective tissues, and are then plated on culture dishes coated in collagen (Vitrogen RT according to
  • the fibers may be isolated as detailed in Example 10. At least 16 dishes of fibers from a single experiment should be plated for culture.
  • satellite cell- containing fibers in controlled serum replacement medium non- stimulating basal medium
  • controlled serum replacement medium non- stimulating basal medium
  • the non-specific sites are blocked, typically in a 24 hr incubation, prior to immunostaining to identify muscle precursor satellite cells (according to published methods to identify c-met receptor in satellite cells or bcl-2 or CD-34
  • Satellite Cell Adhesion Molecule also called Neural Cell Adhesion Molecule, N-CAM
  • N-CAM Neural Cell Adhesion Molecule
  • the extent of proliferation activity by satellite cell nuclei can be examined using immunostaining procedures to localize Proliferating Cell Nuclear Antigen (PCNA) (Johnson and Allen, (1995) Exp. Cell Res.) or other molecules or epitopes which identify cell nuclei that are engaged in proliferation or DNA synthesis. Satellite cells can then be identified by microscopy using phase contrast optics and c-met or bcl-2, and the proliferation status assessed by the proportionate staining for PCNA in those cells.
  • PCNA Proliferating Cell Nuclear Antigen
  • This proportion serves as the basal level of activation, and is required for comparison between test situations (quality control) and serves as the negative control for the conditions in 3 and 4 below.
  • This basal level of activation must be low, however, in comparison to either "test A” or “test B” levels of activation (below) , in order for the test to be informative and meaningful .
  • C. Determination of the True Basal Activation Level In another set of culture dishes, satellite cell- containing fibers in controlled serum replacement medium containing a DNA intercalating substance (e.g. ethidium bromide or propidium iodide or other substances that intercalate into DNA of dead cells) are incubated typically for 30 min. This incubation is used to determine whether the sarcolemmal membranes of the fibers are intact (in which case the myonuclei inside such fibers will be non-fluorescent) or breached/porous (in which case the myonuclei, if ethidium bromide is used, will fluoresce red with the ethidium bromide having intercalated into the DNA) .
  • a DNA intercalating substance e.g. ethidium bromide or propidium iodide or other substances that intercalate into DNA of dead cells
  • nuclei within satellite cells will be non-fluorescent , since their membranes, which are not typically thought to be subject to damage, exclude the DNA intercalator.
  • Counter staining with antibodies specific to muscle precursor cells serves to confirm the identity of satellite cells, alone or in combination with each other and with immunostaining for m-cadherin, as explained above.
  • Proliferation status can be assessed by staining for PCNA or assaying other markers of proliferation as described above. Determination of fiber integrity and confirmation of proliferation status substantiate that the "basal level of activation" as determined above is the true basal level of activation.
  • Test A Determination of Fiber Hypercontraction-Independent Activation: Under certain conditions of fiber damage or stimuli, satellite precursor cells are activated without fiber hypercontraction (e.g. toxicity or stimulation by factors or proteins) . Such conditions or stimuli, including treatment with a test compound, are determined by comparing the "true basal level of activation" in fiber cultures in the absence of the test compound or test condition, with the level of activation observed after treatment with the test compound or test condition. The difference in the levels of activation determines the level of hypercontraction-independent activation (Test A) .
  • the compound or condition which produces activation of satellite precursor cells ultimately results in new DNA synthesis within the satellite cell nuclei.
  • incorporation of bromodeoxyuridme (BrdU) or other non-isotopic or isotopic nucleotide analogues into DNA of satellite cell nuclei can be used to monitor the level of proliferation.
  • the proportionate labelling of satellite cell nuclei is determined by immunostaining or exposure of emulsion after fiber fixation and appropriate staining/photo-identification as required.
  • Test A the difference between the proportionate labelling with the test compound or condition, and the proportionate labelling without the test compound or condition reflects the level of activation independent of fiber hypercontraction (shear) induced by the compound or condition.
  • satellite precursor cell activation is mediated by fiber hypercontraction or shear, either in addition to or distinct from activation mediated via hypercontraction-independent mechanisms (evidenced by "test A” above) .
  • the compound or condition which produces activation of satellite precursor cells ultimately results in new DNA synthesis within the satellite cell nuclei.
  • incorporation of bromodeoxyuridme (BrdU) or other non-isotopic or isotopic nucleotide analogues into DNA of satellite cell nuclei can be used to monitor the level of proliferation as described above in connection with Test A.
  • Teest B fibers in another set of culture dishes are incubated in a medium containing a myotoxin (e.g. Marcaine) plus a DNA intercalator (e.g.
  • the proportionate labelling of satellite cell nuclei is determined by immunostaining or exposure of emulsion after fiber fixation and appropriate staining/photo-identification as required.
  • Test B the difference between the proportionate labelling with the test compound, and the proportionate labelling without the test compound reflects effect of the test compound on the level of shear or hypercontraction-dependent activation.
  • the proportionate labelling without the test compound reflects the basal level of shear or hypercontraction-dependent activation which characterizes the innate responsiveness of the fibers. According to the above-described embodiment of the method, there are therefore four identifiable levels of activation which reflect the innate and the responsive activation states of skeletal muscle precursor cells.
  • the states denoted as “true basal” and “basal shear or hypercontraction- dependent” levels together characterize and determine the activation state of precursor cells in a muscle fiber, as an innate feature of fiber character. Quantifying these levels is important for quality control, i.e. establishing inter-test variability.
  • a particular compound may have effects on satellite cell activation via mechanisms and pathways dependent and/or independent of fiber shear or hypercontraction. Either (or both) mechanisms are targets of treatment and are likely affected by pathophysiologic mechanisms of diseases which directly or indirectly involve skeletal muscle.
  • Nitric oxide is a major freely diffusible endogenous mediator involved in diverse developmental and physiological processes (Annu. Rev. Biochem. (1994) 63: 175- 195) . In addition to controlling diverse cellular processes, NO also participates in certain pathophysiological conditions. In skeletal muscle NO has been shown to depress the muscle contractile function (Nature (1994) 372: 546-548). In the brain, nitric oxide plays important physiological role in neurotransmission and synaptic modulation. In primary cortical cultures, NO mediates glutamate neurotoxicity (Proc. Nat. Acad.
  • nitric oxide and compounds that release nitric oxide or otherwise directly or indirectly deliver or transfer nitric oxide to a site of its activity, such as on a cell membrane, in vivo.
  • nitric oxide encompasses uncharged nitric oxide (NO «) and charged nitric oxide species, particularly including nitrosonium ion (NO+) and nitroxyl ion (NO " ) .
  • the nitric oxide releasing, delivering, or transferring compounds include any and all such compounds which provide nitric oxide to its intended site of action in a form active for their intended purpose.
  • NO donor encompasses any of such nitric oxide releasing, delivering or transferring compounds .
  • NO donors include organic nitrates (e.g., glyceryl trinitrate (GTN) ) , organic nitrites (e.g., iso amyl nitrite), inorganic nitroso compounds (e.g., sodium nitroprusside (SNP) ) , sydnonimines (e.g., molsidomine (SIN-1) ) , furoxans and S-nitrosothiols (RSNO) (e.g., S- nitrosoglutathione, (GSNO) ) .
  • GTN glyceryl trinitrate
  • SNP sodium nitroprusside
  • RSNO S-nitrosothiols
  • GSNO S-nitrosoglutathione
  • S-nitrosothiols are compounds that include at least one -S-NO group.
  • Such compounds include S-nitroso-polypeptides (the term "polypeptide” is contemplated to include proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) , S-nitrosylated amino acids
  • S-nitroso hydrocarbons having one or more substituent groups in addition to the S-nitroso group, and heterocyclic compounds are described in U.S. Pat. No. 5,380,758, filed Sep. 14, 1992; Oae et al. (1983) Org. Prep. Proc. Int. 15(3): 165-198; Loscalzo et al. (1989) J. Pharmacol. Exp. Ther. 249(3): 726-729 and Kowaluk et al. (1990) J. Pharmacol. Exp. Ther. 256: 1256-1264.
  • Inhibitors of NO activity contemplated for use in the invention are compounds which chemically reacts with NO, binds to NO, or otherwise interacting with NO in such a way that the effective concentration of NO is reduced.
  • Such inhibitors of NO activity include, but are not limited to, NO scavengers such as membrane impermeable NO scavengers including MGD-FE (N-methosyl- D-glucamine dithiocarbamate/ferrous sulfate mixture) , carboxy
  • PTIO (2- (4-carboxyphenyl) 4 , 4 , 5 , 5-tetra methylimidazoline- 1-oxyl 3-oxide)
  • calcium chelator BAPTA/AJV1 S-nitroso- N-acetylpenicillamine (SNAP)
  • SNAP S-nitroso- N-acetylpenicillamine
  • SIN-1 3-morpholini sydnonimine
  • DEVI-1 diethyldithiocarbamate
  • melatonin and its precursors superoxide dismutase, glutathione peroxidase, glutathione reductase, dimethyl sufoxide .
  • regulators of NO production may be used in the practice of the present invention.
  • a regulator of NO production is the NOS I ⁇ enzyme.
  • NOS nitric oxide synthase
  • eNOS endothelial
  • nNOS neuronal NOS
  • iNOS inducible NOS
  • nNOS and eNOS enzymes are discretely expressed in specific tissues and rapidly transduce signaling events in a calcium-dependent manner.
  • eNOS activity accounts for endothelium-dependent blood vessel relaxation, while nNOS occurs discretely in a variety of cell types, including neurons, epithelial cells, mesangial cells, and skeletal muscle cells.
  • Inducible iNOS is a calcium- independent form of NOS expressed at highest levels in immunologically activated cells. NO is produced constitutively at high levels in skeletal muscle by an isoform of neuronal nitric oxide synthase, NOS-I ⁇ .
  • NOS-I ⁇ is linked via ⁇ l-syntrophin to the dystroglycan complex especially in fast-twitch fibers.
  • NOS-I ⁇ is expressed at low levels and displaced to the cytoplasm (Brenman JE, Chao DS, Xia H, Aldape K, and Bredt DS . (1995) Cell 82: 743-752; Grozdanovic Z, and Baumgarten HG. (1999) Histol . Histopathol . 14: 243-256).
  • Regulators which increase NO production include, but are not limited to, the NOS enzyme or substances which result in an increase in NOS activity (e.g. sodium nitroprusside or
  • the scope of the present invention encompasses the use of gene therapy, pharmacologic and immunologic means to achieve systemic or local delivery of a product to target, produce and/or substantially result in increased local satellite cell activation (e.g. for muscle atrophy, muscle growth) via, e.g. over-expression of the enhancer region of the gene encoding
  • NOS-I ⁇ over-expression of a regulatory intronic region of the gene encoding NOS-I ⁇ ; over-expression of the promoter region of the gene encoding
  • NOS-I ⁇ anti-sense oligonucleotides or transcriptional regulatory sequences which bind DNA and modulate expression of the NOS-I ⁇ gene ; increasing production of NOS-I ⁇ by satellite cells; manipulation of the 5' untranslated region upstream of the gene for NOS-I ⁇ ;
  • S-nitrosomyoglobin (as an NO-donor) ; increased binding or expression of proteins that bind NOS-I ⁇ ;
  • Regulators which decrease NO production include inhibitors of the NOS enzyme.
  • Suitable nitric oxide synthase inhibitors which may be employed include, but are not limited to, arginine-based analogues such as N G" mono-methyl-L-arginine
  • L-NAME N-amino-L-arginine, and N-methyl-L-arginine
  • flavoprotein binders such as diphenylene iodonium and related iodonium derivatives, ornithine and ornithine derivatives such as N-iminoethyl-L-ornithine
  • redox dyes such as methylene blue
  • calmodulin binders such as trifluoropiperazine and calcinarin
  • heme binders and depleters of biopterin such as methotrexate .
  • the scope of the present invention encompasses the use of gene therapy, pharmacologic and immunologic means to achieve decreased local satellite cell activation (e.g. in muscle that is excessively activated as in genetic disease like DMD, and the diaphragm of mdx mice) via, e.g. anti-sense oligonucleotides and transcriptional regulatory sequences which bind polynucleotides encoding NOS-I ⁇ and inhibiting its production; decreasing production of NOS-I ⁇ by satellite cells; antibodies which bind and inhibit NOS activity.
  • gene therapy e.g. in muscle that is excessively activated as in genetic disease like DMD, and the diaphragm of mdx mice
  • anti-sense oligonucleotides and transcriptional regulatory sequences which bind polynucleotides encoding NOS-I ⁇ and inhibiting its production
  • decreasing production of NOS-I ⁇ by satellite cells antibodies which bind and inhibit NOS activity.
  • Increased systemic satellite cell activation (e.g. for widespread growth of muscle as in agriculture, chronic wasting diseases and athletic interests may be achieved via, e.g. analogues or homologues of NOS-I ⁇ , transfections of replication-defective adenoviral or retroviral vectors with a sequence that would substitute for NOS-I ⁇ activity and bind to the cytoskeleton inside skeletal muscle fibers without affecting the vasculature or neuronal NOS or inducible NOS expression or activity; activation of NOS-I ⁇ protein or its analogues or homologues that could substitute for NOS-I ⁇ activity and bind to the cytoskeleton inside fibers without affecting vasculature or neuronal NOS, or inducible NOS expression or activity; use of analogues or homologues of NOS which are active inside muscle fibers; increasing NOS-I ⁇ production by satellite cells; regulatory sequences or compounds (endogenous or exogenous) that increase HGF binding with c-met in satellite cells,
  • Decreased systemic satellite cell activation may be desired in certain circumstances. For example, a temporary halt in activation may be desired where the activation is a negative consequence of drug treatment inducing wasting by loss of the satellite cell or stem cell population in a skeletal muscle. A permanent halt may be desired in muscle-derived tumours, genetically altered muscle myogenic cells used to treat diabetes, short stature (due to loss of GH) , pancreatic insufficiency, osteoporosis, liver disease, genetic and metabolic neurotrophic abnormalities.
  • Such decreased activation may be effected via: blocking NOS-I ⁇ activity; blocking transcription of the NOS gene or inhibiting the activators of NOS-I ⁇ protein activity; absorbing compounds which give rise to NO from the region between the muscle fiber sarcolemma and the satellite cells (e.g. proteins in the m-cadherin cleft or analogues/homologues of m-cadherin that could be resistant to NO-induced loss of satellite cell adhesion) ; blocking binding of HGF with c-met in the satellite cell
  • the NO, NO donor, inhibitor of NO activity or regulator of NO production of the present invention may be provided to precursor muscle cells by any suitable means, preferably directly (e.g., in vitro by addition to culture medium, or locally by injection or topical administration at a treatment site) or systemically (e.g., parenterally or orally).
  • the NO, NO donor, inhibitor of NO activity or regulator of NO production comprises part of a physiologically acceptable solution so that in addition to delivery of the desired agent to the target cells, the solution does not otherwise adversely affect the cells' or subject's electrolyte and/or volume balance.
  • the NO, NO donor, inhibitor of NO activity or regulator of NO production of the present invention may be administered by any route which is compatible with the particular NO, NO donor, inhibitor of NO activity or regulator employed.
  • the agent is to be provided parenterally, such as by intravenous, subcutaneous, intramuscular intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or by aerosol administration
  • the agent preferably comprises part of an aqueous solution.
  • administration may be by periodic injections of a bolus of the NO, NO donor, inhibitor of NO activity or regulator of NO production, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir which is external (e.g., an i.v. bag) or internal (e.g., a bioerodable implant, or implanted NO-producing cells either singly or in colonies) .
  • a reservoir which is external (e.g., an i.v. bag) or internal (e.g., a bioerodable implant, or implanted NO-producing cells either singly or in colonies) .
  • a given NO donor or regulator of NO production or other agent may be adapted to different situations by association with a suitable molecule.
  • association or genetic fusion of NOS to another protein may improve NOS binding to the sarcolemma and/or cycloskeleton to effect increased NOS activity inside fibers in close proximity to the sarcolemma
  • NO donors or regulators may also be made more soluble or dispersible in physiological solutions than the corresponding original form.
  • Formulations for local or topical administration to a tissue or skin surface may be prepared by dispersing the NO, NO donor, inhibitor of NO activity or regulator of NO production with an acceptable carrier such as a lotion, cream, ointment or soap.
  • the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface.
  • tissue adhesive such as hydroxypropylcellulose or fibrinogen/thrombin solutions
  • tissue-coating solutions such as pectin-containing formulations may be used.
  • compositions comprising NO or NO donors can be administered by intranasal, oral, enteral, topical, vaginal, sublingual, rectal, intramuscular, intravenous, or subcutaneous means.
  • the compounds of this invention can be employed in combination with conventional excipients; i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxilliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxilliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories.
  • Ampules are convenient unit dosages.
  • particularly suitable are tablets, dragees or capsules having talc and/or a carbohydrate carrier binder or the like, the carrier preferably being lactose and/or corn starch and/or potato starch.
  • a syrup, elixir or the like can be used wherein a sweetened vehicle is employed.
  • Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc, or slow-release polymers or other compounds formulated with or without inherent complementary or tissue-specific physical intervention capabilities.
  • terapéuticaally effective amount of a pharmaceutical composition is an amount which is sufficient to achieve the desired pharmacological effect.
  • the dosage required to provide an effective amount of the composition will vary, depending upon the age, health, physical condition, sex, weight and extent of disease, of the recipient. Additionally, the dosage may be determined by the frequency of treatment and the nature and scope of the desired effect.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of modulating agent following administration) .
  • sustained release formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a modulating agent dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are bio-compatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of modulating agent release.
  • the amount of modulating agent contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • nitrated or nitrosylated polymers may be used as a source of NO (WO98/05689) .
  • the polymer is used to coat a device for implanting, or is used as a bolus for injecting, at a skeletal muscle site so that local delivery of NO is achieved.
  • the NO, NO donor, inhibitor of NO activity or regulator of NO production of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art.
  • the amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art .
  • the compound of the present invention can be administered in various ways.
  • the compound or as pharmaceutically acceptable salt can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants, vehicles and vectors.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneal, ophthalmic, intraocular and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.
  • the animal being treated is a warm-blooded animal or cold-blooded animal (e.g. fish) and, in particular, but not exclusively, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
  • the length of the treatment generally may be proportional to the length of the disease or process, and may further depend on the animal species, drug effectiveness and degree of effect required or recommended.
  • the doses may be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, gylcerol, propylene glycol, liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils, often but not always without any inherent effect on NO generation or action.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, availability e.g.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Retention of bioavailability in a tissue may be influenced by co-injection or co-administration with a stabilizing agent that would localize the invention as treatment to the fiber sarcoplasm or to the extracellular matrix as desired. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practising the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, vectors, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres .
  • suitable delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres .
  • delivery systems useful in the present invention include those described in: U.S. Patent Nos .
  • a pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.
  • the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level.
  • the patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.
  • the quantity to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably will be from lO ⁇ g/kg to mg/kg per day.
  • the compounds provided herein also may be associated with molecules capable of targeting the NO, NO donor, inhibitor of NO activity or regulator of NO production to the desired tissue.
  • an antibody, antibody fragment, or other binding protein that interacts specifically with a surface molecule on cells of the desired tissue may be used.
  • Molecules that identify muscle cells include molecular markers for muscle precursor cells (e.g. Bcl-2, disclosed in WO 98/44142; c-met receptor) or muscle fiber extracelllular matrix and external sarcolemma HGF, M-cadherin, HGF-activating enzyme or collagen IV.
  • An antibody may be generated against such a marker for targeting NO, NO donor, inhibitor of NO activity or regulator of NO production to the desired treatment site.
  • Targeting molecules may be covalently or non-covalently associated with the NO, NO donor, inhibitor of NO activity or regulator or NO production.
  • a targeting agent may be associated with NO, NO donor, inhibitor of NO activity or regulator of NO production to facilitate targeting to one or more specific tissues.
  • a “targeting agent,” may be any substance (such as a compound or cell) that, when associated with regulator of NO production enhances the transport of regulator of NO production to a target tissue, thereby increasing the local concentration of the modulating agent.
  • Targeting agents include antibodies or fragments thereof, receptors, ligands and other molecules that bind to cells of, or in the vicinity of, the target tissue.
  • Known targeting agents include serum hormones, antibodies against cell surface antigens, lectins, adhesion molecules, tumor cell surface binding ligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes and those drugs and proteins that bind to a desired target site.
  • An antibody targeting agent may be an intact (whole) molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments are F(ab') 2. -Fab 1 , Fab and F [v] fragments, which may be produced by conventional methods or by genetic or protein engineering.
  • Linkage is generally covalent and may be achieved by, for example, direct condensation or other reactions, or by way of bi- or multi-functional linkers.
  • it may also be possible to target a polynucleotide encoding a regulator of NO production to a target tissue, thereby increasing the local concentration of the regulator.
  • Such targeting may be achieved using well known techniques, including retroviral and adenoviral infection.
  • Antibodies may either monoclonal, polyclonal or recombinant. Conveniently, the antibodies may be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane,
  • Antibody fragments may also be prepared from the antibodies and include Fab, F(ab') 2 . and Fv by methods known to those skilled in the art .
  • a host such as a rabbit or goat
  • the immunogen or immunogen fragment generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera.
  • the polyclonal antibody can be absorbed such that it is monospecific . That is, the sera can be absorbed against related immunogens to that no cross-reactive antibodies remain in the sera rendering it monospecific .
  • the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • an appropriate donor with the immunogen
  • the immunogen generally a mouse
  • splenic antibody producing cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody.
  • the cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use.
  • RNAs from antibody producing B- lymphocytes of animals, or hybridomas are reverse-transcribed to obtain complimentary DNAs (cDNAs) .
  • Antibody cDNA which can be full or partial length, is amplified and cloned into a phage or a plasmid.
  • the cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker.
  • the antibody, or antibody fragment is expressed using a suitable expression system to obtain recombinant antibody.
  • Antibody cDNA can also be obtained by screening pertinent expression libraries.
  • the antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art. (For a general discussion of conjugation of fluorescent or enzymatic moieties, see Johnstone & Thorpe, Immunochemistry in Practice,
  • the detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ -galactosidase, peroxidase, urease, fluorescein, green or other-coloured fluorescent protein, rhodamine, tritium, 1 C, thallium, gadolinium and iodination.
  • fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, ⁇ -galactosidase, peroxidase, urease, fluorescein, green or other-coloured fluorescent protein, rhodamine, tritium, 1 C, thallium, gadolinium and iodination.
  • Targeted delivery of the NO, NO donor, inhibitor of NO activity or regulator of NO production of this invention may be achieved in conjunction with liposomes, which are artificial lipid vesicles.
  • liposomes which are artificial lipid vesicles.
  • the first consideration in the preparation of targeted liposomes is the design of the (non-protein-coupled) liposome itself.
  • the properties of liposomes are determined by two parameters: the method of liposome preparation and its lipid composition.
  • the former parameter determines liposome size and number of lamellae, whereas the latter determines a number of properties such as surface charge, fluidity, and in vivo stability.
  • MLVs multilamellar vesicles
  • SUVs small unilamellar vesicles
  • the small size of SUVs has been shown to confer decreased rates of clearance in vivo (Liu, D. and Huang, L. (1992) J. Lipsome Res. 2: 57-66); however, their correspondingly small inner volume limits the practical use of SUVs for the delivery of encapsulated aqueous materials.
  • LUVs Large unilamellar vesicles
  • LUVs Such properties of LUVs may simply be referred to as a high 'encapsulated volume/lipid' ratio; a quantity which is smaller in value for MLVs and SUVs, and which provides a simple index indicating the ability of liposomes to encapsulate soluble materials.
  • Solutes dissolved in the aqueous buffers used in the above methods become encapsulated upon liposome formation, which is often followed by gel filtration chromatography or dialysis to remove the unencapsulated ' free ' solute.
  • Vesicle 'sizing' by pressure-filtration is also often performed to optimize the cellular uptake of liposomes, which has been shown to occur primarily by clathrin-mediated endocytosis (Straubinger et al. (1983) Cell 32: 1069-1079), and therefore liposome size must not exceed that of a cell-surface coated pit for efficient uptake to occur.
  • liposomes with a diameter in the range of 50- 100 nm have been reported to exhibit efficient cellular uptake in vivo, which decreased as liposome diameter increased above 200nm (Allen et al . (1991) Biochim. Biophys. Acta 1061: 56-64). Large liposomes (> 200nm dia.) are also cleared more rapidly in vivo, where a size-dependent tissue distribution has also been observed since liposomes with a diameter of greater than 300nm preferentially accumulate in the spleen due to the filtration capability of this organ (Liu et al . , (1991) Biochim. Biophys Acta 1066: 159-165).
  • liposomes of ca. lOOnm diameter are often chosen for most applications since they combine sufficient levels of contents encapsulation with optimal cellular uptake and decreased rates of clearance in vivo .
  • Various other pharmacologically important properties of liposomes are determined by their specific lipid composition, which is selected with particular attention to the optimization of liposomal delivery to cells or tissues in vivo or in vi tro . Liposome surface charge has also been shown to have effects on liposome-cell interactions both in vi tro (Batzri, S. and Korn, E.D. (1975) J. Cell Biol. 66: 621-634;
  • 'Targeting' of liposomes refers to the attachment of a ligand (e.g., protein) to the liposome surface, which, due to its ability to recognize a specific cell-surface determinant (s) , 'targets' the liposome to a specific tissue or cell type (reviewed in Papahadjopoulos, D. (1993) in Liposome Technology (Gregoriadis G. , ed.), Vol. 3, pp.1-14, CRC Press, Boca Raton, Fla.).
  • Various types of liposome-attached targeting ligands have been exploited, ranging from small ligands such as folate to larger, protein ligands such as transferrin.
  • Ligands such as those described are typically limited to a single ligand-receptor system, and methods for efficient coupling of one such ligand may not be readily transferable to other, even related ligands. It is for this reason that antibodies (and their fragments) have been chosen as the liposome-attached 'targeting' ligand in many reports (reviewed in Papahadjopoulos (1993) in Liposome Technology (Gregoriadis G., ed.), Vol. 3, pp.1-14, CRC Press, Boca Raton, Fla.), since a number of monoclonal and polyclonal antibodies exist against a variety of cell-surface proteins.
  • the coupling of antibodies to liposomes may be accomplished via the chemical and biosynthetic approaches noted above.
  • the most common chemical method of antibody-liposome coupling involves the use of amine-reactive agents, which by modifying protein lysine residues, typically in a non-specific manner, can perturb antigen binding and/or create neoepitopes .
  • Another concern in the use of intact antibodies is the presence of the Fc portion of the antibody, which contains regions involved in classical complement activation and Fc receptor- mediated clearance, both of which may enhance the rate of clearance of the liposome-antibody complexes in vivo .
  • Fab 1 fragments are ideal for chemical modification using thiol- specific reagents, since they contain a circumscribed coupling site consisting of antibody 'hinge ' -derived cysteine residue (s), which is distant from the antigen-binding regions and therefore do not perturb the latter.
  • gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired or which has the effect of increasing production of a second active product of interest.
  • the genetic material of interest can encode a hormone, receptor, gene sequence, virus (and other vectors) , enzyme, polypeptide or peptide which affects the level of NO.
  • ex vivo and (2) in vivo gene therapy are removed from a host, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the host subject, rather the genetic material to be transferred is introduced into the cells (to the nucleus, cytoplasm or other organelles with DNA (e.g. mitochondria)), of the recipient organism in situ, that is within the recipient.
  • the host gene if the host gene is defective, the gene is repaired in situ (Culver, (1998) Antisense DNA & RNA Based Therapeutics. Coronado, CA. Abstract) . These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle may include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene may be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle may, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible or promoter to allow selective transcription or a conditional promoter (e.g. under control of a tetracycline-responsive element and subject to on/off control by the use of tetracycline) .
  • Enhancers that may be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non- translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below. Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art .
  • nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells.
  • Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated or immune response-mediated events.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject .
  • Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of an antibiotic or other formulations. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.
  • Lateral infection is inherent in the life cyle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighbouring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical- type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells. As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if DMD is to be treated then a vector specific for muscle cells could often but not exclusively be used.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector will depend upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site or sites. However, local aministration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection.
  • An alternate mode of administration can be by direct inoculation locally at the site of treatment or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells.
  • local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area.
  • transfection vehicles are known by one skilled within the art .
  • NO-related treatments of the present invention are applicable in conjunction with steroid hormone treatment.
  • glucocorticoid effects by deflazacort and prednisone
  • Suggested mechanisms include motivation, increased muscle mass, immunosuppression, myoblast proliferation (Angelini et al . , (1994) Muscle Nerve. 17: 386- 391) , or greater satellite cell recruitment and fatigue resistance (Khan M.A. (1993) J. Neurol . Sci. 120: 8-14).
  • Other anti-inflammatory drugs do not improve muscle strength.
  • Glucocorticoids can affect the expression of laminin and matrix proteins in non-muscle tissues (Lannes-Vieira et al . , (1993) Int. Immunol.
  • the muscle repair processes are affected by an increase or decrease in thyroid hormone, (Anderson JE et al . Muscle Nerve (1994) 17: 64-73; Mclntosh LM et al . (1994) Muscle Nerve 17: 444-453.) likely in part through thyroid response elements on the muscle regulatory genes, myoD and myo-genin.
  • anabolic steroids Krahn MJ et al. (1994) J Neurol. Sci. 125: 138-146) and cyclosporine therapy (Sharma KR et al. Neurology (1993) 43: 527-532) can positively affect muscle repair.
  • immunosuppression or lower rates or forces of contraction might decrease dystrophic damage in DMD.
  • PR Prednisone
  • Prednisone also enhances myogenesis (myotube formation) of mdx muscle cultures (Metzinger L et al. (1993) Neurosci Lett. 155: 171- 174) and increases strength and endurance in mdx mice (Hudicki MS et al. Res Commun Chem Pathol Pharmacol (1993) 79: 45-60).
  • the side effects of prednisone treatment include (Khan MA. (1993) J Neurol. Sci. 120: 8-14) Cushingoid appearance, irritability, and decreased bone density, and limit its therapeutic usefulness, especially in young people.
  • Deflazacort an oxazoline derivative of prednisone, has similar treatment effects to date in increasing muscle strength but fewer side effects (Angelini C et al (1994) Muscle Nerve 17: 386-391; Khan MA. (1993) J Neurol. Sci. 120: 8-14) than prednisone, although it is certainly not without important side effects that may cause discontinuation of treatment.
  • deflazacort has shown dramatic benefits to dystrophic skeletal muscle in mice or DMD patients, a result matched by no other drug treatment.
  • Deflazacort treatment on mdx mice produced a significant, 1.5-2 fold increase in precursor cell proliferation and formation of new muscle fibers.
  • Deflazacort promotes muscle repair by actions: (a) on the cycling of proliferative myoblasts, (b) on the differentiation and attachment of newly formed muscle fibers, and (c) on secondary involvement of myofibers adjacent to sites of primary fiber injury. The three actions combine to improve muscle function and prevent fibrotic tissue overgrowth of skeletal muscles (particularly diaphragm) in the mdx dystrophic mouse model of Duchenne muscular dystrophy.
  • prednisone or methyl-prednisolone there are a large number of potential derivatives or prednisone or methyl-prednisolone, and other steroids that may have similar effects; (e.g. anabolic steroids are known to increase muscle mass in conjunction with a positive nitrogen balance (protein) and ongoing muscle activity (training) ) .
  • anabolic steroids are known to increase muscle mass in conjunction with a positive nitrogen balance (protein) and ongoing muscle activity (training) ) .
  • NO activity or a regulator of NO production is used to augment the beneficial effects of glucocorticoids and could thereby be used to decrease the effective dose of deflazacort or other emerging glucocorticoids.
  • the regulator of NO production is used to augment the beneficial effects of glucocorticoids and could thereby be used to decrease the effective dose of deflazacort or other emerging glucocorticoids.
  • NO NO, NO donor, inhibitor of NO activity or a regulator of NO production is used with deflazacort.
  • the NO-related treatments of the present invention are applicable to any condition where regeneration or growth of muscle is desired.
  • the invention may be used as part of pre- or post-surgical procedures, to promote, encourage or allow optimal or efficient repair of muscle damage by muscle regeneration rather than formation of scar tissue and fibrosis.
  • the invention may be used as part of rehabilitation procedures by stimulating muscle formation, and thereby increasing muscle function after muscle disuse or wasting, e.g. after bedrest or confinement, stroke or coma induced incapacitation, arthritis, casting, peripheral nerve section and regrafting) and in anticipation of a requirement to prevent permanent atrophy using rehabilitation strategies in anticipation of secondary or curative surgical or medical/pharmacologic treatment.
  • the NO-related treatments of the present invention are useful particularly in the treatment of diseases and conditions in which muscle disease-specific processes frustrate muscle regeneration (e.g. genetic mutations of pathways involving the genes for muscle regulatory proteins (MyoD, myf5, myogenin, MRF4) , growth factors (e.g. basic fibroblast growth factor, hepatocyte growth factor/scatter factor, insulin-like growth factors, insulin, and their relevant receptors) , and in conditions characterized by muscle fiber instability or rigidity or loss of or excessive adhesion between the satellite cell and the fiber (e.g. originating from genetic mutation or toxic exposure) .
  • muscle regulatory proteins MyoD, myf5, myogenin, MRF4
  • growth factors e.g. basic fibroblast growth factor, hepatocyte growth factor/scatter factor, insulin-like growth factors, insulin, and their relevant receptors
  • muscle fiber instability or rigidity or loss of or excessive adhesion between the satellite cell and the fiber e.g. originating from genetic mutation or toxic exposure
  • important linkages may be weakened a) between the fiber cytoskeleton, sarcolemma and extracellular matrix/external lamina, b) between the sarcolemma, M-cadherins and other adhesion proteins and satellite cells, c) between fusing muscle precursors, d) between extracellular matrix and growth factors, and e) between enzymes/proteins and extracellular matrix or sarcolemma.
  • NO-related treatments of the present invention are useful for regenerating damaged muscle tissue, in particular in dystrophic muscles such as Duchenne, Becker, Emery-Dreifuss, Landouzy-Dejerine, Scapulohumeral of Seitz, Limb-girdle (Erb) , von Graefe-Fuchs, Oculopharyngeal, Myotonic (Steinert) and Congenital dystrophies or any condition where atrophy and/or fiber loss are prevalent and contributory to decreased functional capacity.
  • dystrophic muscles such as Duchenne, Becker, Emery-Dreifuss, Landouzy-Dejerine, Scapulohumeral of Seitz, Limb-girdle (Erb) , von Graefe-Fuchs, Oculopharyngeal, Myotonic (Steinert) and Congenital dystrophies or any condition where atrophy and/or fiber loss are prevalent and contributory to decreased functional capacity.
  • the NO-related treatment of the present invention can be used to increase muscle mass in normal muscle, e.g. during aging and athletic activity in humans or animal species (e.g. horse, dog).
  • the manipulation of muscle precursor cell activation may be directed differentially to different muscles which are distinctly susceptible to various conditions such as injury, disease, functional demands, muscle-specific endurance training and individual use .
  • EXAMPLE 1 Preliminary procedures for manipulating NO level Male normal mice (C57BL/6 and B6, 129SF (Jackson
  • mice C57BL/10 ScSn, Central Animal Care Services, University of Manitoba
  • NOS-I knockout mice C57BL/10 ScSn, Central Animal Care Services, University of Manitoba
  • mice were injected with saline or saline containing one of three drug treatments as follows: the NOS inhibitor N ⁇ - nitro-L-arginine methyl ester (L-NAME, 7.5, 10, or 15 mg/kg), the NO donor L-arginine (L-Arg, 225 mg/kg) or combined L-NAME (7.5mg/kg) plus L-Arg. Fifteen min. later, animals were anesthetised (ketamine :xylazine ip) .
  • the crush injury was delivered to the right TA muscle (RTA) using a hemostat clamp closed for 3 sec (Mclntosh et al . 1994. Muscle Nerve 17:444- 453) . Skin was held closed or sutured for longer recovery (see below) .
  • the time course study from 0-30 min. after injury was completed in one day for each treatment group, treatments were coded, and each set of experiments were carried out by the same individual (s) .
  • the time course of treatment effects was determined at two intervals; during the early response 0, 5, 10 and 30 min after injury, and over the longer term after 6 days recovery. Short term experiments were repeated at least twice.
  • the longer term animals were maintained either on plain drinking water or water containing fresh L-NAME at 12.5 mg/lOOml (30 mg/kg/day) , based on an intake of 6-7 ml/day/mouse (Mclntosh et al . , 1994. 17:444-453).
  • Tissues were rapidly harvested within 1-2 min. after cervical dislocation under anesthesia. Whole muscles were carefully dissected from animals in order: RTA, left TA (LTA), left extensor digitorium longus (LEDL) , left (LSOL) and right soleus (RSOL) , and weighed (TAs and RSOL) . Muscles were used to determine cell yield or embedded for cryosectioning (7 ⁇ m thick) to examine morphology.
  • Satellite cells from RTA, LTA (representative fast-twitch muscles) and RSOL (a representative slow-twitch muscle) were isolated by standard procedures (Allen et al . ,
  • a 100 ⁇ l aliquot of cell suspension was diluted in 10 ml isotone for Coulter counting.
  • the number of cells isolated per muscle was calculated and plotted over time.
  • cells were counted using a hemocytometer, to ensure that they were nucleated cells and not isolated myonuclei or red blood cells.
  • the LSOL and LEDL were embedded for cryosectioning to monitor effects of treatment or remote injury on tissue histology, as visualized by fresh hematoxylin and eosin staining (H&E) and immunostaining for c-met and m-cadherin (see below) .
  • Sections and cultures were viewed on an Olympus microscope equipped with epifluorescence and phase contrast optics. Observations were based on systematic viewing of 2-4 longitudinal sections per muscle (separated by >100 ⁇ m) . In the case of muscle regenerating from crush injury, observations (without knowledge of treatment group) were made in pre-set fields of muscle from the central crush region, the adjacent regenerating region and the surviving region as reported (Mclntosh et al . (1994) Muscle Nerve 17: 444-453) .
  • the yield from RSOL (an uninjured slow- twitch muscle ipsilateral to RTA, and included for comparison with fast-twitch TA) was lower than from LTA on a per muscle basis (although 2-7-fold higher expressed as cells/mg) , and did not change over the 30 min. time course.
  • the data compiled from three repeat experiments on normal mice treated with saline are presented as the ratio of cell yield in RTA/LTA (mean ⁇ SEM) in Figure 2 and demonstrate the consistent large immediate rise in cell yield at 0 min.
  • L-NAME treatment substantially changed the time course of cell yield, preventing the initial injury- induced rise in RTA yield ( Figure IF) and delaying the increased cell yield until 10 min. after injury.
  • the yields from LTA and RSOL were lower at 0 min. than in the saline- treated mice (15 and 50%, respectively) .
  • 30% fewer cells were isolated at 0 min. from RTA than LTA.
  • yields from RTA and LTA were higher (3.5 and 2 -fold, respectively) than at 0 min.
  • cell yield from both RTA and LTA had dropped once again.
  • EXAMPLE 3 Effects of NOS inhibition in mdx dystrophic muscle vs. NOS-I knockout in muscle
  • satellite cells are intimately contoured to fibers and often stay attached to the external lamina as the sarcolemma buckles after injury (Schultz and McCormick, 1994 Rev. Physiol. Biochem. Pharmacol. 123, 213-257), they are ideally positioned to be "first responders" to a shear-induced release of NO from the subjacent NOS-I ⁇ .
  • first responders As activation would increase the harvest of myogenic cells from a single muscle by reducing their adhesion to fibers and lamina, and would also affect subsequent muscle repair, the release of myogenic cells from single crush-injured muscles was used as an index of the collective process in muscle.
  • the mdx mouse displays X-linked dystrophin-deficient myopathy.
  • fiber injury in the limb muscles is followed by dramatic repair of muscle structure (e.g. Anderson, J.E. et al. (1987) Anat. Rec. 219: 243-257) and largely successful recovery of limb muscle function (e.g Anderson, J.E. et al. (1988) J. Muscle Res. Cell Motil. 9: 499-515) compared to DMD.
  • the diaphragm muscle of mdx mice shows severe damage, fibrosis and poor repair very similar to DMD (Stedman, H.H. et al.
  • myoblasts are dividing, and new muscle fibers (called myotubes) are present. If a drug did improve repair, more new muscle fibers (marked by developmental myosin and containing central nuclei) should form, possibly by increased recruitment and proliferation of myoblasts cells.
  • mdx mouse is similar to humans with Duchenne muscular dystrophy (DMD) in that muscles in both lack dystrophin, (Hoffman EP, Brown RH Jr, Kunkel LM. (1987) Cell 51: 919-928) which is crucial to muscle integrity.
  • DMD Duchenne muscular dystrophy
  • mdx limb muscles respond to muscular dystrophy with an active myoproliterative response (Anderson JE, Ovalle WK, Bressler BH.
  • Some small caliber myofibers such as extraocular fibers, are spared from DMD and mdx dystrophy, (Karpati G, Carpenter S, Prescott S. (1988). Muscle Nerve. 11:795-803) possibly due to substitution by utrophin. (Matsumura K, Ervasti
  • 360:588-591 does not account for the successful recovery of limb muscles from dystrophy, in contrast to the progression of
  • the myogenic proportions of cells isolated from mdx muscles were very high in LTA and RTA (95% and 96% respectively, 295 cells counted) and likely included both satellite cells from fibers and myoblasts from the interstitium of dystrophic muscles.
  • Muscle weight as a proportion of body weight had a different profile in mdx than in normal mice ( Figure II) .
  • RTA weight increased later (after 5 min) and was maintained over 30 min. in saline-treated mdx mice, while L- NAME abolished the increase in RTA weight for 30 min. ( Figure 1J) .
  • mdx RTA muscles were subjectively less hemorrhagic after L-NAME than after saline treatment .
  • the basal level of LTA yield was about 30% more in mdx than normal mice.
  • RTA yield did not show an immediate rise at 0 time. Instead, counts for LTA and RTA were similar. Over 10 min., the RTA yield doubled and then levelled off somewhat . The cell yield from LTA did not change over time, while RSOL yields dropped by half from 0-10 min.
  • NOS-I knockout mice showed a time course of cell yield from LTA, RTA and RSOL that was very similar to that in mdx mice, summarized in Figure 2 (3 experiments, pooled data from mdx and NOS-I knockout mice) .
  • the immediate increase in cell yield in RTA of normal mice was absent in RTA muscle of both mdx and NOS-I knockout mice.
  • EXAMPLE 4 Effects of NOS inhibition on early muscle and satellite cell responses to injury
  • M-cadherin was interposed between fibers and all satellite cells observed in undamaged muscle, and typically surrounded large satellite cells on fibers in saline- treated RTA at 0 and 10 min. (Figure 4A) . At 10 min. , large m- cadherin+cells were very often observed on the empty external lamina sheaths present after fiber retraction ( Figure 4B) . Interestingly satellite cells were easily visible on nearly every fiber by H&E staining at 0 min. at the RTA fiber periphery ( Figure 4C) and were often prominent in the RSOL, LEDL, LTA and RTA at 10 min.
  • adjacent regions contained many mononuclear cells and capillaries between the long myotubes .
  • Surviving tissue at the ends of RTA contained fibers interspersed or continuous with new myotubes. Many mononuclear cells (over half of 20-30 satellite cells clearly identified per section) stained for both c-met and HGF/SF, while myotubes did not stain for either protein.
  • Satellite cells in mdx LTA were very large and c-met+ (Figure 7H) as were satellite cells in NOS-I-knockout LTAs although their extensive cytoplasm was not as granulated or as distinct from fiber sarcoplasm by H&E staining as in normal undamaged muscles after L-NAME treatment ( Figure 71, compare with Figures 6J, 6K) .
  • HGF/SF As an activator of satellite cells, the nature of activation was elusive as it was studied with later markers, such as regulatory gene expression or DNA synthesis.
  • EXAMPLE 7 Satellite cell morphology from mice 0 and 10 minutes after crush injury and after pretreatment with saline or L-NAME Measurements of cell area (size increases in activated cells) , nucleus-to-cytoplasm ratio (a measure that decreases with activation) , and cytoplasmic density (a measure that decreases in activation with the increase in cell size, despite the hypertrophy of cytoplasmic organelles) were made from electron micrographs without knowledge of their source .
  • Cytoplasmic density was determined as [the integrated cell density minus the integrated nuclear density] divided by [cell area minus nuclear area] .
  • a computer was used to scan micrograph negatives, and digital images were analyzed using the program NTH Image for morphometry. Table 1:
  • * indicates a significant difference from the same group at 0 minutes post-crush injury.
  • 0 indicates a significant difference from saline-treated group at same time post-crush injury.
  • EXAMPLE 8 Role of NO in satellite cell activation Results from the preceding Examples show that satellite cell activation occurs immediately upon muscle injury, is mediated by NO release, is briefly transmitted to distant muscles and is prevented under pharmacologic and genetic conditions that reduce the activity or expression of NOS-I. Time-course studies of myogenic cell yield and morphology showed two aspects of activation, namely altered adhesion and morphological changes. Prior to identifying HGF/SF as an activator of satellite cells, the nature of activation was elusive as it was studied with later markers, such as regulatory gene expression or DNA synthesis. The present demonstration in satellite cells of a rapid shift by HGF/SF to its "mitogenic and motogenic" receptor (Rong, S., Segal, S., Anver, M.
  • the external lamina wrapping fibers may provide the potential for satellite cells to respond to shear between the sarcolemma and lamina. Satellite cells hug fibers across an even 15 nm cleft without junctional complexes, and they associate closely with external lamina (Bischoff, R. (1990a) .
  • Satellite cells have the ideal topography to detect a rapid peak of NO release from underlying fibers after shear and also to be kept quiescent by normally continuous small pulses of NO from the fiber.
  • the speed of the NO-mediated signal for activation suggests that an initiating event such as mechanical shear forces acts on constitutive NOS-I, since the response time is too short to induce expression or increase activity (McCall, T.B., et al . , (1991). Eur. J. Immunol. 21, 2523-2527; Rubinstein, I., et al., (1998). J. Clin. Invest. 101, 1325- 1333) . Effects of L-NAME on edema (and hemorrhage) were congruent with NO effects on vascular tone (Busse, R. , and
  • HGF/SF may become activated themselves, and circulate from RTA to initiate activation of satellite cells located outside the damaged muscle. Without the NO-mediated signal, however, normal fibers would repress activation and satellite cells would return to quiescence. By contrast, satellite cells in RTA, having received the secondary circulating signal in addition to the primary signal would complete the activation sequence.
  • Combined treatment with an NO donor and a NOS inhibitor partly reversed effects of NOS inhibition on RTA yield and prevented the temporary increase in LTA yield.
  • signals involved in fully activating precursor cells likely include both NO-mediated and NO-independent mechanisms.
  • Shear produced by layers that shift laterally against each other would be strong during segmental retraction within the external lamina.
  • intramuscular injection of muscle fibers and their adherent satellite cells is a form of shear which could maximize shear-induced satellite cell activation and supply crushed muscle extract containing (HGF) directly to the site of fiber implantation.
  • HGF crushed muscle extract containing
  • satellite cell activation was defined structurally as cytoplasmic and organelle hypertrophy and dynamically as recruitment to cycle.
  • the close adherence of satellite cells to parent fibers must decrease during activation for satellite cells to move through the external lamina to form new fibers. Therefore the loss-of-adhesion feature was used as a simple index of activation.
  • the ability to isolate myogenic cells after brief standard digestion was a conservative estimate of available satellite cells and not an estimate of total myogenic cells. (Additional myogenic cells are found in the material collected on the Nitex filter during cell isolations.) NO is known to modulate leukocyte and platelet adhesion (Kubes et al 1991 Proc. Natl. Acad. Sci. USA.
  • NO has a broad impact on glucose uptake, insulin resistance, exercise, blood flow and contractility (Balon and Nadler, 1994. J. Appl. Physiol. 77:2519-2521; 1997 J. Appl. Physiol. 82:359-363; Shen et al . , 1995. Med. Sci. Sports Exerc . 27:1125-1134; Joyner and Dietz . 1997. J. Appl. Physiol. 83:1785-1796; Kapur et al . , 1997. Diabetes 46:1691-1700; Chen et al . , 1998. Am. J. Physiol. 274 (Regulatory Integrative Comp. Physiol. 43), R822-R829; Young and Leighton, 1998.
  • results from studies in isolated fiber culture model using DNA synthesis as the marker of completed activation indicate that manipulation of NO is a viable treatment option.
  • in situ hybridization experiments show that satellite cells themselves express NOS-I ⁇ . This suggests that satellite cells may direct (in an autocrine fashion) their own activation by shear or other stimuli, in addition to receiving paracrine signals from fibers.
  • the present results address for the first time the initial steps of satellite cell activation.
  • a single exposure to NOS inhibition had subtle effects on myotube formation that echo NO-stimulated myoblast fusion in vitro. Longer NOS inhibition reduced the effectiveness of repair and restricted its distribution, in agreement with the idea that shear-induced responses become attenuated longitudinally away from the injury.
  • a model proposes the hypothesis that NO mediates rapid satellite cell activation, including hypertrophy and altered adhesion inside the fiber-lamina complex, and that distant muscle precursors may be transiently activated by circulating factors released from injured muscle.
  • the rapidity of activation and the notion that immediate satellite cell responses to muscle injury may be contributed by the physical character of the external lamina and mechanical shear.
  • the signalling mechanism underlying NOS-I activity in response to shear can also be determined, and may involve Akt/PKB-dependent phosphorylation of NOS-I as recently reported for NOS-III
  • NOS-I ⁇ is subsarcolemmal and in mdx muscle is reduced and displaced to the cytosol due to the absence of dystrophin.
  • Mdx muscle pathology was recently reported to be independent of nNOS (NOS- I) perturbation (Chao et al., 1998. J. Neurochem. 71:784-789; Crosbie et al . , 1998. Hum. Mol . Genet. 7:823-829).
  • Cytoplasmic NOS-I in mdx muscle would act as a diffuse areal source of NO rather than the nearby linear source, typically subjacent and parallel to satellite cells found in normal muscle fibers.
  • the normally steep NO gradient across the cleft between fiber and satellite cell would therefore become more shallow, diffuse more slowly, and the small NO transient would be manifest as an attenuated responsiveness to shear forces.
  • pulsatile NO acts to maintain quiescence, a smaller gradient in dystrophy from the pulsatile NO of cytoplasmic origin, could release mdx satellite cells from what is normally full quiescence, and account for the greater proliferative activity and larger satellite cells in mdx muscle and primary cultures (Mclntosh and Anderson, 1995. Biochem. Cell Biol.
  • dystrophy in that double mutant may be more severe than in mdx mice if it were assessed in younger mice ( ⁇ 12 months) , before the index of repair (central nucleation) has reached its theoretical plateau.
  • cytoplasmic NOS-I in human fibers would serve as an even smaller non-linear NO source than in mdx muscle.
  • the resulting very shallow gradient or physiological NO transient across satellite cells could partly account for the severity of Duchenne dystrophy, almost as if the standby activation (like a "hair trigger") contributes to overly enthusiastic successive repair events and early senescence (Decary et al . , 1996. Human Gene Therapy 7:1347- 1350; Decary et al . , 1997 Human Gene Therapy 8:1429-1438). It is now clear that satellite cell activation needs to be considered separately from dystrophy.
  • NOS-I knockout mice also had a modest focal myopathy (segmental muscle fiber damage and inflammation) in TA and diaphragm. That myopathy was not present in the control strain (B6, 129SF) , and may relate to the absence of NOS-I expression in the nervous system or a constitutive heightening of satellite cell activation.
  • the mdx and NOS-I knockout experiments suggest that increased satellite cell activation from reduced or absent NOS expression may benefit myogenesis in the short term (and through a few cycles) by facilitating standing activation and precursor recruitment to cycle. However, in the longer term, that standby activation appears to be detrimental. Accordingly, dystrophy may be reduced by either increasing the local (not systemic) pulsatile
  • EXAMPLE 10 Isolated fiber studies of satellite cell activation on regenerating muscle
  • Single intact fibers can be used to examine the dynamic time course of gene expression by satellite cells on fibers in culture (e.g. Bischoff, (1986a) Dev. Biol. 115:140- 147; Yablonka-Reuveni, Z, and Rivera, (1994) Dev. Biol. 164:588-603; Yablonka-Reuveni, Z, Seger R, and Rivera
  • Fiber preparations are used to model aspects of development (Cornelison DD, and Wold BJ. (1997). Dev. Biol. 19:270-283) and recruitment-repair sequences, through study of gene transcripts or labelled products that mark recruitment to cycle (PCNA) , activity
  • HGF/SF hepatocyte growth factor/scatter factor
  • This technique allows for the tracking of individual satellite cells, as well as populations of cells, under closely monitored conditions away from fibroblasts and inflammatory cells - each sources of cytokines and growth factors also affecting repair.
  • To properly use the isolated fiber to characterize satellite cell activation we must 1) positively distinguish satellite cells from myonuclei and 2) determine the exact time of fiber death under known conditions. Once these are determined, experiments can be devised to monitor the action of satellite cells immediately after their activation.
  • Bundles were gently triturated to separate single fibers using a wide mouth glass pipette with a polished end.
  • the fibers were plated onto vitrogen-coated 35 mm dishes and grown in 1.5 ml of DMEM + 10% Horse Serum (HS) + 1% antimyotics + 0.1% gentamycin.
  • HS Horse Serum
  • Ethidium bromide is a fluorescent chemical which intercalates into the DNA of dead cells but is not taken up into live fibers. Media was mostly decanted from freshly plated fibers and the fibers were located using a microscope.
  • Ethidium bromide stained the nuclei of both fixed fibers and hypercontracted fibers, but did not stain the nuclei of live fibers.
  • the fixed fibers with immediate staining of myonuclei act as the positive control, as do fibers that hypercontracted during a rough preparation, while live fibers immediately after isolation do not have EtBr-fluorescent myonuclei (even after 30 minutes incubation)
  • Satellite cells were identified on intact fibers and were distinguished from myonuclei using c-met antibody.
  • EXAMPLE 11 Satellite cell activation and NOS-I ⁇ activity Muscle repair can be manipulated by changes in NO synthase (NOS-I ⁇ ) activity and/or expression that affect satellite cell activation and ultimately proliferation.
  • NOS-I ⁇ NO synthase
  • the time course and properties of activation in normal, dystrophic, NOS-I (-/-) and double mutant mdx X NOS-I (-/-) muscle are studied.
  • single isolated fibers are used to see the effect of activation and satellite cell manipulation after NOS activity or NO concentration is manipulated.
  • In vivo studies of activation Activation in vivo (cell release and hypertrophy) is monitored after 2 different stimuli to the tibialis anterior muscle.
  • a traumatic injury is applied, and compared to activation that occurs after more physiologic injury from repeated electrical stimulation to fatigue (mimicking severe exercise) .
  • Another group of mdx mice half pretreated with daily deflazacort for 3 weeks) receives either a traumatic or physiological stimulus.
  • Activation of satellite cells from dystrophic muscle with and without deflazacort therapy are used to test whether the drug therapy modulates NOS activity/expression in concert with any effects on activation.
  • mice Normal C57BL/6 mice, 6-8 weeks of age are divided into three equal groups to receive electrical stimulus, traumatic stimulus or no injury/activating stimulus.
  • the time course of cell yield (activation) has tissues collected at 0,5,10, or 30min. after stimulus.
  • mice receive one of 4 treatments (by intra peritoneal (ip) injection) 30min before stimulus: saline, the NOS inhibitor L-NAME (N ⁇ -Nitro-L-arginine methyl ester, 7.5 mg/kg), the NO donor L-Arginine (225 mg/kg), or L-NAME plus L-Arginine.
  • L-NAME N ⁇ -Nitro-L-arginine methyl ester, 7.5 mg/kg
  • L-Arginine 225 mg/kg
  • L-NAME plus L-Arginine L-NAME plus L-Arginine.
  • mice are treated (ip) and are anesthetised and the right tibialis anterior (TA) muscle is prepared for surgery or electrical stimulus 30 min later. These treatments are coded and surgery is routine.
  • the crush injury enables precise studies of a response like activation or repair since it is rapid and synchronized to all TA fibers .
  • the nerve is exposed and positioned over a silver electrode. Pulses are delivered (120 Hz for 5 min) to produce fused tetanic contractions to fatigue the fast muscle, as reported (Anderson, J.E. et al (1988). J. Muscle Res. Cell Motil. 9:499-515) .
  • cell yield time course Cells are isolated from muscles in time course experiments, each run in one day (4 mice). Mice are killed immediately (0 min.) or at 5,10,30 min after injury or electrical (fatigue) stimulus, revealing time-dependent changes in cell yield and satellite cell hypertrophy (assays of activation) .
  • the very rapid rise in the cell yield ratio of RTA: LTA at 0 min. (typically within 1 min) is dramatic.
  • Cell yield after brief digestion (after (Allen et al . (1998). Methods Cell Bio. 52:155-162) are determined by Coulter counting of cells per muscle, since treatments differentially affect muscle weight (edema) after injury. Logistically, including preparation, surgery and analyses, only 2-3 experiments per week of this type are possible.
  • the RTA LTA ratio of cell yield ( ⁇ SEM) is calculated to find the effects of stimuli or treatment on activation.
  • b) morphologic changes results of satellite cell activation are assessed using tissues collected from stimulated and unstimulated Tas . Muscle sections are used to identify satellite cells, study NOS activity and expression and examine immediate early gene expression as follows . i) satellite cells are identified and examined for hypertrophy and position using immunostaining for two c-met receptor, m-cadherin, neural cell adhesion molecule (N-CAM or leul9) and CD34, as markers of muscle satellite precursor cells (Irintchev A, Zeschnigk M. Starzinski-Powitz A, and Wernig A. (1994). Dev.
  • NOS expression is determined using in situ hybridisation (and Northern blotting) with a riboprobe specific for NOS-I ⁇ (the muscle isoform of NOS-I) , made with custom primers (Kobzik L, Reid MB, Bredt DS, and Stamler JS . (1994). Nature. 372:546-548; Meltzer JC, Sanders V, Grimm PC, Stern E, Rivier C, Lee S, Rennie SL, Gietz RD, Hole AK, Watson PH,
  • c-fos an immediate early gene is determined by in situ hybridisation and Northern blotting. c-fos expression increases within 15 min. of liver injury or neurons (Meltzer JC, Sanders V, Grimm PC, Stern E, Rivier C, Lee S, Rennie SL, Gietz RD, Hole AK, Watson PH, Greenberg AH, and Nance DM. (1998). Brain Res. Protocols 2:339-351). Satellite cell c-fos expression after stimulus may corroborate activation.
  • NO levels will be increased by L-Arginine (50-1000 nM) .
  • Initial results show maximal activation at 500 nM.
  • Longer in vivo manipulation (1 wk) of NOS or [NO] with L-NAME (12.5 mg/mL) or L-arginine (325 mg/mL) in water are examined for effects of treatment on full activation of satellite cells in vitro.
  • Experiments include double fluorescence staining for pairs of myoD, c-fos, c-jun, myogenin, c-met, NOS, PCNA, HGF, and BrdU, and DAPI (to mark nuclei) to study the expression of muscle regulatory and early immediate genes in activated and quiescent satellite cells under different conditions.
  • CNI central nucleation index
  • the CNI is a measurement well known in the art (see Karpati and Carpenter. 1988. Muscle Nerve. 11:795-803 and Anderson et al . 1996. Muscle Nerve. 19:1576-1585).
  • CNI is the proportion of all fibers in a section or a muscle which show a centrally-located nucleus (often given as a percentage) . Since muscular dystrophy causes ongoing damage of muscle fibers, the successful repair of those fibers is marked by centrally-nucleated fibers which accumulate as a proportion of total fibers. Unsuccessful repair can be viewed as a loss of fibers in a section of a whole muscle; this too can produce a change in the ratio of centrally nucleated to total number of fibers (expressed as a percentage) .
  • a segment viewed in cross section has a central nucleus inside it (which is always a myonucleus, postmitotic) or a peripheral nucleus (which can be a postmitotic myonucleus, as in an uninjured (intact) fiber, or a satellite cell nucleus which can be seen at the periphery of the fiber by light microscopy and is usually considered indistinguishable from the normal myonuclei at the fiber periphery) .
  • a central nucleus inside it which is always a myonucleus, postmitotic
  • a peripheral nucleus which can be a postmitotic myonucleus, as in an uninjured (intact) fiber, or a satellite cell nucleus which can be seen at the periphery of the fiber by light microscopy and is usually considered indistinguishable from the normal myonuclei at the fiber periphery
  • Figure 14 shows that in the mdx mouse, the CNI in placebo-treated animals is about 0.6 (i.e. 60% of fibers) show a centrally located nucleus in a cross section of the muscle. This is similar for the tibialis anterior muscle (LTA) and diaphragm at the age shown in the graph (which is 8 weeks of age) and is reliably used to monitor the progressive effect of dystrophic fiber injury on a muscle over time as the disease progresses. CNI will increase with age in the mdx mice (until the plateau discussed above) . Mice are treated from 4-8 weeks of age with placebo, Deflazacort, D+L-NAME or D+L-Arginine.
  • LTA tibialis anterior muscle
  • L-NAME treatment was then added to deflazacort to see if part of the effect of deflazacort was mediated by NO.
  • the animals were given L-NAME in drinking water, at the same time as they got daily deflazacort injections, both over the 4 week treatment time. In these animals, the LTA CNI was higher than with deflazacort alone, which means the muscles with the less severe dystrophy needed the activation to have the full beneficial effect of deflazacort to reduce CNI.
  • Arginine caused no change in LTA CNI from deflazacort alone; (though CNI in deflazacort treatment alone is still significantly lower than placebo treatment) .
  • the NO donor did raise the CNI of DIA from the level seen with D+L- NAME (i.e. it negated the benefit of NOS inhibition in the diaphragm) .
  • deflazacort did significantly reduce the CNI in both the LTA and diaphragm (DIA) .
  • the effect was counteracted by L-NAME in LTA, indicating that the deleterious systemic effects of L-NAME (e.g. on the vasculature) prevailed over the local effects on satellite cell activation.
  • L-NAME augmented the beneficial effects of deflazacort in diaphragm, presumably because the persistent unregulated activation of satellite cells in mdx dystrophic muscle ("standby" mode) is reduced there.

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Abstract

L'invention concerne des méthodes, des compositions pharmaceutiques et des trousses permettant de moduler l'activation du précurseur du muscle squelettique. Pour effectuer cette modulation, on utilise du monoxyde d'azote (NO), des donneurs de NO, des inhibiteurs d'activité du NO (inhibiteurs de NO) ou des régulateurs de production de NO, soit au niveau local, soit au niveau systémique. L'invention concerne également l'utilisation de NO, d'un donneur de NO, d'un inhibiteur de NO ou d'un régulateur de production de NO afin de moduler les effets des stéroïdes hormonaux sur le muscle squelettique. L'invention concerne également une méthode permettant d'identifier un composé qui provoque un changement de l'état d'activation des précurseurs du muscle. L'invention apporte un certain nombre d'avantages évidents. En permettant de manipuler directement les précurseurs du muscle squelettique, l'invention permet d'élaborer des traitements spécifiques afin de régénérer et de réparer le muscle.
PCT/CA2000/000255 1999-03-11 2000-03-10 Modulation de l'activation du precurseur du muscle squelettique WO2000053191A2 (fr)

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US6872751B2 (en) * 1999-06-11 2005-03-29 Centre National De La Recherche Scientifique - Cnrs Composition and method for augmenting or restoring the production of fetal protein in patient in need thereof
US7378438B2 (en) 2002-04-19 2008-05-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Beta-agonist compounds comprising nitric oxide donor groups and reactive oxygen species scavenger groups and their use in the treatment of respiratory disorders
WO2007088123A2 (fr) 2006-02-03 2007-08-09 Nicox S.A. Utilisation de derives nitro-oxydes de medicaments pour le traitement de dystrophies musculaires
WO2007088123A3 (fr) * 2006-02-03 2007-09-20 Nicox Sa Utilisation de derives nitro-oxydes de medicaments pour le traitement de dystrophies musculaires
WO2007088050A3 (fr) * 2006-02-03 2007-10-11 San Raffaele Centro Fond Procédé de traitement de la dystrophie musculaire
US8247460B2 (en) 2006-02-03 2012-08-21 Giulio Cossu Method of treatment for muscular dystrophy
AU2007211508B2 (en) * 2006-02-03 2012-08-23 Nicox Science Ireland Use of nitrooxyderivative of drug for the treatment of muscular dystrophies
US8575222B2 (en) 2006-02-03 2013-11-05 Nicox S.A. Use of nitrooxyderivatives of drug for the treatment of muscular dystrophies
CN101378739B (zh) * 2006-02-03 2014-06-04 尼科克斯公司 药物的硝基氧基衍生物用于治疗肌肉营养不良症的应用
EP2786749A1 (fr) 2006-02-03 2014-10-08 Nicox S.A. Utilisation de dérivés nitro-oxydes de médicaments pour le traitement de dystrophies musculaires
WO2010108843A1 (fr) 2009-03-27 2010-09-30 Nicox S.A. Utilisation d'un dérivé nitro-oxy du paracétamol pour le traitement de dystrophies musculaires

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