EP1185258A1 - Utilisations therapeutiques d'inhibiteurs d'oxyde nitrique - Google Patents

Utilisations therapeutiques d'inhibiteurs d'oxyde nitrique

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Publication number
EP1185258A1
EP1185258A1 EP00937605A EP00937605A EP1185258A1 EP 1185258 A1 EP1185258 A1 EP 1185258A1 EP 00937605 A EP00937605 A EP 00937605A EP 00937605 A EP00937605 A EP 00937605A EP 1185258 A1 EP1185258 A1 EP 1185258A1
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European Patent Office
Prior art keywords
cells
tissue
hematopoietic
differentiation
inhibitor
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EP00937605A
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German (de)
English (en)
Inventor
Grigori Enikolopov
Natalia I. Peunova
Boris A. Kuzin
Hollis Cline
Tatyana Michurina
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Cold Spring Harbor Laboratory
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Cold Spring Harbor Laboratory
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Priority claimed from US09/315,929 external-priority patent/US6372796B1/en
Application filed by Cold Spring Harbor Laboratory filed Critical Cold Spring Harbor Laboratory
Publication of EP1185258A1 publication Critical patent/EP1185258A1/fr
Withdrawn legal-status Critical Current

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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
    • A61K31/21—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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • 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
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • 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
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • Organ development requires a tightly controlled program of cell proliferation followed by growth arrest and differentiation and, often, programmed cell death.
  • the balance between the number of cell divisions and the extent of subsequent programmed cell death determines the final size of an organ (reviewed by Bryant and Simpson, Quart. Rev. ofBiol, 59:387-415 (1984); Raft, Nature, 555:397-400 (1992)).
  • the present invention is based on the discovery that nitric oxide (NO) is an important growth regulator in an intact developing organism.
  • the present invention relates to a method of increasing in a mammal a population of hematopoietic cells (e.g., hematopoietic stem cells), including precursors to myeloid, lymphoid and erythroid cells, which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, wherein the hematopoietic tissue is contacted with multiple doses of at least one inhibitor of NO, such as multiple doses of one or more inhibitors of nitric oxide synthase (NOS), thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
  • NOS nitric oxide synthase
  • the present invention relates to a method of increasing in a mammal a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, comprising contacting the hematopoietic tissue with two inhibitors of nitric oxide synthase, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
  • the method can be carried out in vivo or ex vivo.
  • the method can be used to prevent differentiation of erythroid cells and/or myeloid cells in the mammal.
  • the method can further comprise contacting the hematopoietic tissue with at least one agent (e.g., a hematopoietic growth factor) which induces differentiation of a selected hematopoietic stem cell population.
  • at least one agent e.g., a
  • the present invention also relates to a method for treating a mammal to increase a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue of the mammal.
  • the hematopoietic tissue of the mammal is contacted with multiple doses of at least one inhibitor of NOS, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
  • the present invention relates to a method for treating a mammal to increase a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue of the mammal, comprising contacting the hematopoietic tissue of the mammal with two inhibitors of nitric oxide synthase, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
  • the method can further comprise contacting the hematopoietic tissue with at least one agent which induces differentiation of a selected hematopoietic stem cell population.
  • hematopoietic tissue which is to be transplanted is obtained, wherein the hematopoietic tissue to be transplanted can be obtained from the mammal being treated (autologous transplantation) or from another mammal (heterologous transplantation).
  • the hematopoietic tissue to be transplanted is contacted with multiple doses of at least one inhibitor of NOS.
  • the hematopoietic tissue which is to be transplanted is transplanted into the mammal being treated, thereby providing the mammal with hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation.
  • two NOS inhibitors are used.
  • the method can further comprise treating the mammal with an inhibitor(s) of NOS before or after transplanting the hematopoietic tissue.
  • the method can further comprise treating the mammal with an enhancer (one or more) of NOS before or after transplanting the hematopoietic tissue.
  • the present invention also relates to a method of increasing a population of progenitor blood cells (e.g., red blood cells, white blood cells) which are capable of undergoing normal hematopoiesis, differentiation and maturation comprising contacting progenitor cells (stem cells) of blood with multiple doses of at least one inhibitor of NO (e.g., an inhibitor of NOS).
  • progenitor blood is contacted with two inhibitors of NOS.
  • the present invention also relates to a method of increasing a population of dividing cells in a tissue of a mammal comprising contacting the cells with multiple doses of at least one inhibitor of nitric oxide.
  • the present invention also relates to a method of increasing a population of cells in S phase in a tissue of a mammal, comprising contacting the tissue with multiple doses of at least one inhibitor of NO, such as an inhibitor of NOS. In one embodiment, the method results in an increase in the size of an organ in which the tissue is occurs. Furthermore, as described herein the cells in S phase can be used in gene therapy.
  • the present invention also relates to a method of decreasing a population of cells in S phase in a tissue of a mammal and inducing differentiation of the cells, comprising contacting the tissue with an enhancer(s) of NO, such as an enhancer of NOS. In one embodiment, the method results in a decrease in the size of an organ with which the tissue is associated.
  • the present invention also relates to a method of coordinating developmental decisions of a cell type in a mammal, comprising introducing NO into the cell type or a precursor of the cell type, thereby inhibiting proliferation of the cell type or a precursor of the cell type and inducing differentiation of the cell type or a precursor of the cell type.
  • a method of inducing differentiation in a mammalian cell population comprising contacting the cell population with NO or a NO enhancer is also encompassed by the present invention.
  • the invention also pertains to a method of regenerating tissue in an adult mammal comprising contacting a selected tissue (e.g., blood, skin, bone and digestive epithelium), or precursor cells of the selected tissue, with multiple doses of at least one inhibitor of NO, thereby inhibiting differentiation and inducing proliferation of cells of the tissue, then contacting the selected tissue with a compound (e.g., nitric oxide, a growth factor or a combination of both) which inhibits proliferation and induced differentiation.
  • a selected tissue e.g., blood, skin, bone and digestive epithelium
  • a compound e.g., nitric oxide, a growth factor or a combination of both
  • the method involves repopulating an organ or tissue (e.g., muscle or nerve fiber) comprised of normally nondividing cells by contacting a selected organ or tissue, or precursor cells of the selected organ or tissue, with multiple doses of at least one inhibitor of NO, thereby inhibiting differentiation and inducing proliferation of cells of the organ or tissue, then contacting the selected organ or tissue with a compound which inhibits proliferation and induced differentiation.
  • an organ or tissue e.g., muscle or nerve fiber
  • the invention also encompasses a method of producing a subpopulation of hematopoietic cells.
  • hematopoietic tissue is contacted with multiple doses of at least one inhibitor of NOS, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation; and at least one agent (e.g., a hematopoietic growth factor) selected to induce specific differentiation of the hematopoietic stem cell population, thereby producing a subpopulation of hematopoietic cells.
  • the hematopoietic tissue is contacted with two inhibitors of NOS.
  • Identification of NO as an important growth regulator in an organism provides for various therapeutic applications in humans and other mammals.
  • NO nitric oxide
  • the present invention relates to a method of increasing in a mammal a population of hematopoietic cells (e.g., hematopoietic stem cells), including precursors to myeloid, lymphoid and erythroid cells, which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue, by contacting the hematopoietic tissue with multiple doses of at least one inhibitor (one or more) of NO, such as an inhibitor of NOS.
  • hematopoietic tissue is tissue involved in hematopoiesis.
  • the present invention includes a method for treating a mammal to increase a population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation in hematopoietic tissue of the mammal, in which the hematopoietic tissue of the mammal is contacted with multiple doses of at least one inhibitor of NOS.
  • the invention also pertains to a method of producing a subpopulation of hematopoietic cells by contacting hematopoietic tissue with multiple doses of at least one inhibitor of NOS, thereby producing hematopoietic tissue having an increased population of hematopoietic stem cells which are capable of undergoing normal hematopoiesis, differentiation and maturation; and at least one agent selected to induce specific differentiation of the hematopoietic stem cell population, thereby producing a subpopulation of hematopoietic cells.
  • two inhibitors of NO such as two inhibitors of NOS, are used in the methods.
  • a combination of L- NAME and ETU can be contacted with the hematopoietic tissue to increase a population of hematopoietic stem cells in a mammal, to treat a mammal to increase a population of hematopoietic stem cells in hematopoietic tissue in a mammal or to produce a subpopulation of hematopoietic cells.
  • the present invention also relates to a method of increasing a population of progenitor blood cells comprising contacting progenitor cells of blood with multiple doses of at least one inhibitor (one or more) of NO (e.g., inhibitor of NOS).
  • the present invention relates to a method of increasing a population of progenitor blood cells comprising contacting progenitor cells of blood with two inhibitors of NOS.
  • the sources of progenitor cells of blood include, for example, bone marrow, peripheral blood, umbilical cord vein blood, fetal liver, and long term hematopoietic cell culture.
  • red blood cells and white blood cells e.g., granulocytes (neutrophils, basophils, eosinophils), monocytes, lymphocytes
  • white blood cells e.g., granulocytes (neutrophils, basophils, eosinophils), monocytes, lymphocytes
  • the present invention also relates to a method of increasing a population of dividing cells in a tissue of a mammal comprising contacting the cells with multiple doses of at least one inhibitor of NO.
  • the present invention can also be used to increase a population of cells (targeted cells) in S phase in a tissue of a mammal relative to a similar tissue in an untreated mammal, by contacting the tissue with multiple doses of at least one inhibitor of NO, such as an inhibitor of NOS.
  • the method results in an increase in the size of an organ with which the tissue is associated.
  • the present invention can also be used to decrease a population of cells in S phase in a tissue of a mammal and inducing differentiation of the cells, comprising contacting the tissue with at least one enhancer of NO, such as an enhancer of NOS.
  • the method results in a decrease in the size of an organ with which the tissue is associated.
  • the cells in S phase can be used in gene therapy.
  • the present invention also relates to a method of coordinating developmental decisions of a cell type in a mammal, comprising introducing NO into the cell type or a precursor of the cell type, thereby inhibiting proliferation of the cell type or a precursor of the cell type and inducing differentiation of the cell type or a precursor of the cell type.
  • a method of inducing differentiation in a mammalian cell population comprising contacting the cell population with NO or a NO enhancer is also encompassed by the present invention.
  • the invention also pertains to a method of regenerating tissue in an adult mammal.
  • the method comprises contacting a selected tissue with multiple doses of at least one inhibitor of NO, thereby inhibiting differentiation and inducing proliferation of cells of the tissue, then contacting the selected tissue with a compound which inhibits proliferation and induces differentiation of the proliferated cells to cells characteristic of the tissue.
  • the method involves repopulating an organ or tissue (e.g., muscle or nerve fiber) having normally nondividing cells comprising contacting a selected organ or tissue with multiple doses of at least one inhibitor(s) of NO, thereby inhibiting differentiation and inducing proliferation of cells of the organ or tissue, then contacting the selected organ or tissue with a compound which inhibits proliferation and induces differentiation of the proliferated cells to cells characteristic of the organ or tissue.
  • an organ or tissue e.g., muscle or nerve fiber
  • a compound which inhibits proliferation and induces differentiation include NO, an enhancer of NO and a growth factor.
  • One or more these compounds can be used to inhibit proliferation and induce differentiation.
  • Tissue which can be regenerated using the methods described herein include blood, skin, bone and digestive epithelium, nerve fiber, muscle, cartilage, fat or adipose tissue, bone marrow stroma and tendons.
  • the methods described herein can further comprise the step of contacting the hematopoietic tissue target cells (e.g., bone marrow) with at least one agent which induces differentiation of a selected hematopoietic stem cell population to a particular cell type (e.g., erythrocytes, macrophages, lymphocytes, neutrophils and platelets).
  • a particular cell type e.g., erythrocytes, macrophages, lymphocytes, neutrophils and platelets.
  • a mammal is treated to increase a population of hematopoietic stem cells in the hematopoietic tissue of the mammal by contacting the hematopoietic tissue of the mammal with multiple doses of at least one inhibitor of NOS
  • the increased population of hematopoietic tissue can be contacted with an agent, such as a hematopoietic growth factor, which will cause or promote differentiation of the cells of a particular cell type.
  • Agents which can be used in the methods of the present invention to induce differentiation of the increased or expanded number of cells produced by contacting cells with a NOS inhibitor include, for example, erythropoietin, G-CSF, GM-CSF and interleukins such as IL-1, IL-2, IL-3 and IL-6.
  • the methods described herein can further comprise the step of contacting the hematopoietic tissue with at least one agent which further induces or maintains proliferation of the selected hematopoietic stem cell population to a particular cell type (e.g., erythrocytes, macrophages, lymphocytes, neutrophils and platelets).
  • Inhibitors of NO for use in the present invention include, for example, NO scavengers such as 2-phenyl-4,4,5,5-tetraethylimidazoline-l-oxyl-3-oxide (PTIO), 2- (4-carboxyphenyl)-4,4,5,5-tetraethylimidazoline-l-oxyl-3-oxide (Carboxy-PTIO) and N-methyl-D-glucamine dithiocarbamate (MGD); and NOS inhibitors such as N-nitro-L-arginine methyl-ester (L-NAME), N-monomethyl-L-arginine (L-NMMA), 2-ethyl-2-thiopseudourea (ETU,), 2-methylisothiourea (SMT), 7-nitroindazole, aminoguanidine hemisulfate and diphenyleneiodonium (DPI).
  • NO scavengers such as 2-phenyl-4,4,5,5-tetra
  • multiple doses of at least one inhibitor of NO can be used.
  • the inhibitors can be the same or different.
  • two inhibitors of NO such as two inhibitors of NOS (e.g., L-NAME and ETU), are used in the methods of the present invention.
  • the NO inhibitor(s) can be administered in a single dose or in multiple doses.
  • multiple doses refers to at least two doses of at least one inhibitor of NO.
  • the multiple doses can be administered in a day or over a period of days (e.g., a period of about 2 days to a period of about 15 days or months).
  • the NO inhibitor(s) can be administered over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.
  • a mixture of two NO inhibitors e.g., L-NAME and ETU
  • L-NAME and ETU are administered to the mammal or contacted with the cells twice a day for 9 days.
  • one or more enhancers of NO can be used.
  • Enhancers of NO include, for example, NOS enhancers, and NO donors such as sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (SNOG, GSNO), diethylamine NONOate (DEA/NO), DETA/NO (NOC-18), 3-mo ⁇ holinosydnonimine (SIN-1) and spermine NONOate (Sper/NO).
  • SNP sodium nitroprusside
  • SNAP S-nitroso-N-acetylpenicillamine
  • SNOG S-nitrosoglutathione
  • DEA/NO diethylamine NONOate
  • DETA/NO NOC-18
  • SIN-1 3-mo ⁇ holinosydnonimine
  • Sper/NO spermine NONOate
  • NO is a diffusible multifunctional second messenger that has been implicated in numerous physiological functions in mammals, ranging from dilation of blood vessels to immune response and potentiation of synaptic transmission (Bredt and Snyder, Annu. Rev. Biochem., 65:175-195 (1994) ; Nathan and Xie, Cell, 75:915-918 (1994); Garthwaite and Boulton, Annu. Rev. Physiol, 57:683-706(1995)).
  • NO is produced from arginine by NOS in almost all cell types.
  • a group of three chromosomal genes, giving rise to numerous isoforms of NOS, have been cloned from mammalian cells (Knowles and Moncada, Biochem.
  • results demonstrate the activity of NO as an antiproliferative agent during Drosophila development, controlling the balance between cell proliferation and cell differentiation.
  • results shown here demonstrate that NO acts as a crucial regulator of hematopoiesis after bone marrow (BM) transplantation. NO regulates the maturation of both the erythroid and myeloid lineages.
  • Imaginal discs specialized groups of undifferentiated epithelial cells that are recruited during embryogenesis, are formed in the first larval instar as integuments of the larval epidermis. Disc cells divide rapidly throughout the larval development and cease proliferating at the end of the third instar period. In leg, wing, and haltere discs, progression through the cell cycle stops in G2 phase 3-4 hours before puparium formation. It resumes 15-18 hours later (12-14 hours after pupariation) and then stops again in a defined spatial pattern after 12-14 hours (10-14 hours of pupal development) (Fain and Stevens, Dev.
  • Transformation of imaginal precursors in adult structures during fly metamorphosis involves transition from cell proliferation to cell differentiation. Cessation of cell division is a necessary, although not sufficient, condition for cell differentiation to proceed. A temporary cytostasis occurs at the end of the larval period, and permanent arrest of cell division occurs during pupal development. NO, a diffusible messenger molecule, is capable of efficiently blocking cell division. Induction of NOS initiates a switch to growth arrest prior to differentiation of cultured neuronal cells (Peunova and Enikolopov, Nature, 575:68-73 (1995)). Thus, NOS can act as a permissive factor, making the further development of the fully differentiated phenotype possible. Results described herein show that NOS acts as an antiproliferative agent during normal Drosophila development, indicating that NO is an important growth regulator in the intact developing organism.
  • NADPH-diaphorase staining becomes particularly intense, and it gradually decreases during prepupal and pupal development.
  • other structures with intense NADPH-diaphorase staining include imaginal rings, histoblasts and the brain. These structures undergo radical changes during metamorphosis before giving rise to adult organs.
  • imaginal discs are released from the G2 block and reenter S phase 12-15 hours after pupariation, at the time when diaphorase staining is diminished to low levels in adult flies.
  • the final number of cells in an organ or a segment is determined both by cell multiplication and cell death, which the forming structures of the fly undergo as a normal step in development (especially at the late stages of pupal development).
  • Results described herein indicate that the changes in the size of the leg segments after manipulation of NOS activity correlated directly with the changes in DNA synthesis and the number of dividing cells.
  • no significant changes in apoptosis were detected in the larval and prepupal leg discs after inhibition or ectopic expression of NOS, compared with the control discs, when cell death was monitored by acridine-orange staining or by the TUNEL assay. This suggests that it is cell multiplication, rather than changes in programmed cell death that leads to the changes in the size of the appendage.
  • apoptotic death may conceal excessive cell proliferation in other developing organs.
  • the effect of the absence of programmed cell death on potential excessive cell proliferation was also assessed.
  • Transgenic flies were used in which programmed cell death in the developing eye was suppressed by recombinant p35, an inhibitor of apoptosis, to reveal excessive proliferation after NOS inhibition. Under these circumstances, several cell types and structures are overrepresented, the most noticeable change being an overall increase of the size of the eye due to the increased number of ommatidia.
  • NO may be more important for the induction of growth arrest and subsequent differentiation of already committed cells than for the developmental commitment and establishment of the cell identity in the embryo or larvae.
  • NOS expression can be induced to high levels in a large number of tissues and cell types by appropriate stimulation (Bredt and Snyder, Annu. Rev. Biochem., 65:175-195 (1994); Forstermann et al, Adv. Pharmacol, 54:171186 (1995)).
  • the pattern of NOS distribution in a developing organism differs strongly from the distribution in the adult organism.
  • transient elevation of NOS expression in a given tissue often coincides with the cessation of division of committed precursor cells.
  • the developing mammalian brain provides an especially apt demonstration of this (Bredt and Snyder, Neuron, 75:301-313 (1994); Blottner et al, Histochem. J., 27:785-811 (1995)).
  • NOS activity in the developing cerebral cortical plate and hippocampus at days 15-19 of prenatal development correlates with the timecourse of cessation of precursor cells proliferation, tight growth arrest, and cell differentiation; notably, NOS activity goes down after the proliferation of committed neuronal precursors is completed. NOS levels are also transiently increased in developing lungs, bones, blood vessels, and nervous system (Blottner et al, Histochem. J., 27:785-811 (1995); Collin-Osdoby et al, J. Cell Biochem., 57:399-408 (1995); Cramer et al, J. Comp. Neurol, 555:306-316 (1995); S aul, Adv.
  • results described herein support the position that production of NO is required during embryonic development and during tissue regeneration in the adult organism for the proper control of cell proliferation.
  • the antiproliferative properties of NO are particularly important in situations in which terminal differentiation of committed cells is temporally separated from cell proliferation and is strictly dependent on cessation of cell division. Given the multiplicity of the NOS isoforms and their overlapping tissue distribution, it is conceivable that any group of cells in the embryo and fetus can be exposed to NO action.
  • recent data showing that NO can be transferred within the organism by hemoglobin raise the possibility that a developing mammalian embryo can be also supplied with NO exogenously by the mother.
  • NO is a readily diffusible molecule, and it may therefore exert its antiproliferative properties not only in the cell that produces it but in the neighboring cells as well (Gaily et al, Proc. Natl. Acad. Sci. USA, 57:3547-3551 (1990)).
  • This property is important when one considers mechanisms for the coordinated development of a group of neighboring cells committed to form a particular structure. These cells have to generate an intrinsic signal that tells them to stop dividing in a coordinated fashion after they have reached a certain number. This cooperation and coordination is achieved in many instances by tightly controlled paracrine regulation, which involves signaling between adjacent cells via gap junctions or secreted proteins.
  • Results described herein show that yet another way of coordinating developmental decisions in groups of cells is by diffusible antiproliferative second messenger molecules, which can spread without a need for surface receptors or specialized systems for secretion and exert their influence within a Umited domain.
  • An efficient source of readily diffusible molecules may induce synchronized changes in the adjacent cells within a limited volume of a tissue.
  • several adjacent cells producing easily diffusible antiproliferative messenger molecules may share the total pool of these molecules produced by the neighbors as well as by themselves. If a particular threshold level of a signal is needed to initiate a signaling chain that eventually leads to growth arrest, then the cells in this group could stop dividing when a certain number of cells and, therefore, a certain local concentration of messenger molecules, is reached.
  • NADPH-diaphorase cells The morphology of the NADPH-diaphorase cells suggests that they are largely of the granulocyte-macrophage lineage at different stages of differentiation. This is in accordance with numerous data showing that NOS is present in the cells of the myeloid lineage, and can be induced to high levels by appropriate stimulation.
  • a mouse model of syngeneic BM transfer was used to evaluate the role of NO in hematopoiesis.
  • Mice were irradiated to inhibit hematopoiesis in the recipient animal, BM was transplanted from syngeneic animals, and the animals were treated with specific NOS inhibitors. This procedure permits the proliferation, differentiation and survival of only the transplanted cells.
  • NOS inhibitors the colonies in the spleen were monitored to test the differentiation of erythroid cells, and the formation of colonies on the membranes placed in the peritoneal cavity of the recipients were monitored to test the differentiation of cells of the granulocyte-macrophage lineage.
  • NO-based approach can be focused on renewable and regenerating tissues, such as blood, bone, skin, and digestive epithelium.
  • a similar strategy can be used to repopulate organs with normally nondividing cells such as muscle and nerve cells.
  • the work described herein can also be used to enhance gene therapy methods.
  • NOS can be used to drive a population of cells into the S phase wherein the cells are replicating.
  • replicating cells are more responsive to gene therapy methods (e.g., introduction of genes via live vectors) than non-replicating cells.
  • the present invention provides for a method of converting cells into a state which renders the cells more receptive to gene therapy methods, wherein the cells are contacted with a NO inhibitor (e.g., NOS inhibitor).
  • a NO inhibitor e.g., NOS inhibitor
  • the present invention provides for a method of converting cells into a state which renders the cells resistant to gene therapy methods. That is, the present invention provides for a method of converting cells into a state which renders the cells more resistant to gene therapy methods, wherein the cells are contacted with NO and/or a NO enhancer (e.g., NOS enhancer).
  • NO and/or a NO enhancer e.g., NOS enhancer
  • NO regulates maturation of both erythroid and myeloid cell lineages By interfering with NO production in the recipient animal after BMT, the number of undifferentiated stem and blast cells which are then capable of further differentiation along the erythroid or myeloid lineages can be dramatically increased. The blast cells enrichment reaches 80-fold for the myeloid lineage, and 20-fold for the erythroid lineage.
  • the data described herein demonstrates that manipulations of NOS activity and NO levels during hematopoiesis can be used for therapeutic purposes to influence self renewal and differentiation of hematopoietic stem cells, and to replace damaged or defective cells.
  • Areas of application include enhancement of blood cell and myeloid cell formation following high dose chemotherapy in cancer treatment; improved engraftment following bone marrow or stem cell transplantations, and gene therapy; stem cell therapy by amplifying the undifferentiated cells of erythroid and myeloid lineages and applying appropriate factors to induce te ⁇ ninal differentiation; and regulation of formation of various blood cell components for treating hematological and autoimmune disorders.
  • the data also shows that changing the levels of NO production interferes with osteoblast and chondrocyte differentiation.
  • manipulation of NO production can regulate growth and differentiation of osteoblasts, chondrocytes, or mesenchymal stem cells. This can be used for amplification and further differentiation of cells in the injured tissue, or for cell implants (in combination with biocompatible carriers, if necessary).
  • an NO-based approach can be used for regeneration therapy of the damaged tissue, post injury repair, age related diseases such as osteoporosis and osteoarthritis, and for reconstituting marrow stroma following high dose cancer chemotherapy.
  • the data shows that changing the levels of NO production interferes with keratinocyte differentiation.
  • the results described herein demonstrate that regulation of NO production can be used when increased proliferation and subsequent differentiation of skin tissue is required (e.g., during burns and wound healing).
  • NO can be used to control disorders caused by hyperproliferation of keratinocytes during psoriasis.
  • Yet another potential application is to use NO-based preparations as exfoliant agents in cosmetic therapy.
  • NO has been shown to act as a regulator of cell differentiation in neuronal cells. It has been demonstrated that NO regulates brain development in animals and contributes to controlling the size of the brain in intact animals.
  • these studies of the role of NO in neurons suggest that NO may be used to control proliferation and subsequent differentiation of nerve cells in replacement therapy after neurodegenerative disorders caused by aging (e.g., Alzheimer's or Parkinson's), stroke, or trauma.
  • NO is actively produced in smooth muscle cells of the blood vessels and is subject to complex physiological regulation. These cells are highly susceptible to suppression of DNA synthesis by NO.
  • the very strong antiproliferative activity of NO can be used for inhibition of smooth muscle cells proliferation and neointima formation for treatment of restenosis following angioplasty.
  • NO-based therapy has application for treatment of ailments characterized by destruction of specific sets of cells. This includes hepatocyte regeneration after toxic injury of the liver, treatment of reproductive system disorders, and administration of differentiated pancreatic tissue for treatment of type 1 diabetes.
  • the methods of the present invention can be carried out in vivo or ex vivo.
  • Administration of the NO inhibitor, NO enhancer and/or agent which induces differentiation can performed using various delivery systems known in the art.
  • the routes of administration include intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural and intranasal routes. Any other convenient route of administration can be used such as, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings.
  • the NO inhibitor, NO enhancer and/or agent which induces differentiation can be admimstered with other components or biologically active agents, such as adjuvants, pharmaceutically acceptable surfactants, excipients, carriers diluents and vehicles.
  • Administration can be systemic or local, e.g., direct injection at the site containing the cells to be targeted.
  • the NO inhibitor, NO enhancer and/or agent which induces differentiation are protein or peptides
  • they can be administered by in vivo expression of genes or polynucleotides encoding such into a mammalian subject.
  • expression systems such as live vectors, are available commercially or can be reproduced according to recombinant DNA techniques for use in the present invention.
  • the amount of NO inhibitor, NO enhancer and or agent which for use in the present invention which will be effective in the treatment of the particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgement of the practitioner and each patient's circumstances.
  • the amount of NO inhibitor(s) for use in the methods of the present invention can be from about 1 mg/kg body weight to about 1000 mg/kg body weight, from about 5 mg/kg body weight to about 500 mg/kg body weight, and from about 25 mg/kg body weight to about 100 mg/kg body weight.
  • about 25 mg/kg body weight to about 1000 mg/kg body weight L-NAME and/or about 1 mg/kg body weight to about 100 mg/kg body weight can be used in the methods of the present invention.
  • about 300 mg/kg body weight L-NAME is used in combination with about 30 mg kg body weight ETU in the methods of the present invention.
  • Nitric Oxide Regulates Cell Proliferation During Drosophila
  • Transgenic GMR-P35 flies (3.5 and 2.1 alleles, Hay et al, Dev., 120:2121-2129 (1994)) were a generous gift from B. Hay and G.M. Rubin.
  • Transgenic flies carrying mouse macrophage NOS (NOS2) gene under heat-shock promoter (hs-mNOS20(2) and hs-mNOS 15(2) alleles) were generated by P-element-mediated germline transformation.
  • NOS2 mouse macrophage NOS
  • hs-mNOS20(2) and hs-mNOS 15(2) alleles were generated by P-element-mediated germline transformation.
  • a 4100 base pair Notl fragment from the plasmid CL-BS-mac-NOS containing the entire mouse macrophage NOS gene (Lowenstein et al, Proc. Natl. Acad. Sci.
  • NADPH-diaphorase staining was performed as described by Dawson et al, Proc. Natl. Acad. Sci. USA, 55:7797-7801 (1991) and Hope et al, Proc. Natl Acad. Sci. USA, 55:2811-2814 (1991), with minor modifications.
  • Fixation-insensitive NADPH-diaphorase staining reflects activity of various NOS isoforms in mammals and Drosophila. Imaginal discs were mounted in 80% glycerol and photographed in a Zeiss Axiophot microscope under Nomarski optics.
  • L-NAME L-nitroarginine methyl ester
  • D-NAME D-nifroarginine methyl ester
  • ETU 2-ethyl-2-thio ⁇ seudourea
  • Timing of the injection of NOS inhibitors that gave the highest efficiency was determined in trial experiments and was found to be most efficient when performed 5-12 hours before pupanation. This treatment did not affect the onset of pupanation and hatching.
  • NOS is expressed in imaginal discs during larval development.
  • cells of imaginal discs undergo temporary cell cycle arrest. Cytostasis is released 12-14 hours after pupariation and is established once again (permanently) in the late pupae and the pharate adult.
  • the ability of NO to reversibly halt cell division and establish temporary growth arrest makes it a plausible candidate for mediating cytostasis in imaginal discs.
  • imaginal discs of the third instar and early pupae were examined for NOS presence.
  • Drosophila NOS (dNOS) gene which is preferentially expressed in the adult head, has recently been cloned and characterized (Regulski and Tully, Proc. Natl.
  • NOS-related mRNA species are present in the embryo, larvae and adult flies. These mRNAs may be produced by the cloned dNOS gene or by other potential Drosophila NOS genes, making the detection of the relevant RNA species difficult. Therefore, to visualize the expression of NOS in Drosophila during larval development, histochemical staining for the NADPH-diaphorase (reduced nicotinamide adenine dinucleotide phosphate-diaphorase) activity of NOS was used, which reflects the distribution of the total enzyme activity in a tissue (Dawson et al, Proc. Natl.
  • NADPH-diaphorase staining was observed in all imaginal discs, imaginal rings, histoblasts and the brain of the larvae, beginning in the third instar. Staining became more intense as development proceeded, and in late third instar larvae and early pupae, a highly specific and reproducible pattern of very intense staining was evident.
  • NADPH-diaphorase staining was initially seen at the very beginning of the third instar. Staining was confined to the center of the disc, corresponding to the presumptive distal tip of the leg.
  • leg imaginal discs As with the leg imaginal discs, other imaginal discs, imaginal rings and histoblasts exhibited increasingly intense NADPH-diaphorase staining as larval development proceeded. Wing, eye, haltere and genital discs in the third instar had distinct and reproducible patterns of intense staining, which gradually decreased in a specific spatial pattern during early pupal development.
  • Synthesis of DNA is affected by manipulations of NOS activity.
  • NO acts as an antiproliferative agent during Drosophila development at stages when the cells of imaginal discs enter temporary cytostasis
  • its action might directly affect DNA synthesis in the discs. Inhibition of NOS would then be expected to relieve the block and increase the number of cells in S-phase; conversely, high levels of NO would lead to a decrease in the number of dividing cells.
  • DNA synthesis in larval and prepupal discs was monitored while the levels of NOS activity were manipulated.
  • specific NOS inhibitors were injected into developing larvae.
  • NOS2 is a calcium-independent form of NOS that is capable of efficient constitutive NO production.
  • Imaginal discs were labelled with 5-bromo-deoxyuridine (BrdU), and the extent and distribution of labeling of S- phase nuclei in leg imaginal discs from larvae after inhibition of NOS, from NOS2 transformants after heat shock induction, and from control untreated larvae were compared.
  • NOS activity was inhibited by injecting specific NOS inhibitors in the developing larvae at the end of the third instar, several hours before metamo ⁇ hosis. Most of the larvae completed metamo ⁇ hosis successfully, giving rise to adult flies within the normal time frame. The resulting adults differed from normal flies in many respects, the most dramatic being enlargements of the appendages and other structures of the fly body.
  • Ectopic expression of a mouse NOS transgene results in reduced size of leg segments.
  • the ability of NO to inhibit DNA synthesis and cell proliferation suggests that overexpression of NOS in developing larvae may lead to diminished cell proliferation in the imaginal discs and to a reduction in the size of organs of the adult fly.
  • Transformed flies that express the mouse NOS2 transgene under the control of the heat-shock promoter were tested.
  • Transgenic larvae were heat-shocked within one hour after pupariation to induce ectopic expression of NOS before the final cell divisions take place. This resulted in a reduction in the size of the limbs of the fly.
  • the distal segments of the legs were affected most frequently and to the greatest degree.
  • p35 is a strong inhibitor of apoptosis, which acts by inhibiting the interleukin IB-converting enzyme-like proteases and is able to prevent apoptosis in multiple contexts.
  • GMR-P35 flies express p35 under the transcriptional control of multimerized glass-binding site from the Drosophila Rhl promoter. Glass promoter directs expression of the transgene in all cells in and posterior to the mo ⁇ hogenetic. furrow in the eye disc (Ellis et al, Dev., 779:855-865 (1993)).
  • Wild type ommatidia contain, in addition to eight photoreceptor cells, a set of four cone cells and two primary pigment cells, surrounded by an array of six secondary pigment cells, three tertiary pigment cells, and three bristles (Wolff and Ready, Pattern formation in the Drosophila retina, in The Development of Drosophila melanogaster, M. Bate and A. Martinez- Arias, eds. (Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY), 1277-1326 (1993)).
  • the number of photoreceptor and accessory cells is normally constant, and variations in this arrangement in the eyes of the normal flies are very rare.
  • the number of secondary and tertiary pigment cells was increased from 12 to 25 (25+ ⁇ 4) cells per sample area (defined as described in Hay et al, Cell, 55: 1253-1262 (1995)) as a result of suppressed programmed cell death. Inhibition of NOS in these flies resulted in a further increase in the number of secondary and tertiary pigment cells to more than 35 (36+/-8) per sample area.
  • the number of cone cells was increased from four in normal and untreated GMR-P35 ommatidia to five and six in many ommatidia of GMR-P35 flies after NOS inhibition. Clusters of ommatidia were also found which contained one, two, or three cone cells, which may correspond to improperly formed supernumerary ommatidia that did not attain the proper set of cells.
  • Nitric Oxide Regulates Hematopoiesis in Animals Erythroid Differentiation
  • BM cells were flushed from the femurs of syngeneic donors and injected intravenously (10 5 BM cells per mice) in the recipients. The animals received twice a day injections of 100 mg/kg of L-NAME and D-NAME and 10 mg/kg of ETU for 7-10 days.
  • mice were analyzed 9-10 days after transplantation.
  • the differentiation status of the colonies in the spleen was evaluated by mo ⁇ hological criteria and by immunohistochemical tests for the presence of receptors to various cytokines, which are present only at specific stages of the erythroid cells' maturation.
  • the analysis of the colonies in the spleen of control animals and animals treated with inactive enantiomere D-NAME (Table 1) showed that in agreement with numerous-data, most of the colonies in the spleen (>60%) contained erythroid colonies, smaller fractions contained undifferentiated blasts cells (14%) or both erythroid and blast cells (13%), and small fractions of colonies contained megakaryocytes (7.5%) and granulocytes (4%).
  • cellulose acetate membranes were implanted in the peritoneal cavity of mice. After 7 days, when a layer of fibroblasts had covered the membranes, the mice were irradiated as described above. BM cells from syngeneic donors were injected (10 5 BM cells per mice) in the peritoneal cavity of the recipients. Animals received injections of NOS inhibitors as described above. Membranes with growing colonies were isolated and analyzed 7-8 days later.
  • the differentiation status of the colonies in the spleen was evaluated by mo ⁇ hological criteria, myeloperoxidase reaction, and by immunohistochemical tests for the presence of receptors to various cytokines, which are present only at specific stages of the myeloid cells' maturation.
  • the analysis of the colonies on the membranes in control animals and animals treated with inactive enantiomere D-NAME showed that in agreement with numerous data, most of the colonies (92%) contained granulocytic colonies. A much smaller fraction contained undifferentiated blasts cells (6%), and a very small fraction of colonies contained erythroid cells (1.3%).
  • the results of the analysis demonstrate that the blast cells in the spleen colonies (representing erythroid differentiation) have accumulated mostly at the stage of differentiation where they have already acquired the receptor for IL-3, but not for erythropoietin, GM-CSF or G-CSF, whereas the colonies with mo ⁇ hological signs of erythroid differentiation had accumulated EpoR.
  • the blast cells in the colonies on the membranes have accumulated mostly at the stage of differentiation where they have already acquired the receptor for IL-3, but not for erythropoietin, GM-CSF or G-CSF, whereas the myeloperoxidase-positive colonies with mo ⁇ hological signs of myeloid differentiation had accumulated GM-CSF-R and G-CSF-R.
  • the animals were treated as described above and the BM cells from the femurs of syngeneic donors were injected intravenously (10 5 BM cells per mice) in the recipients.
  • the animals received injections of NOS inhibitors
  • mice were analyzed 7-10 days after transplantation.
  • the BM cells were tested for the presence of various growth factor receptors which serve as markers of the differentiation stage and indicate the presence of stem cells and multipotent precursor cells.
  • the BM preparations were tested for cells expressing receptors to HSF (ligand of c-kit), GM-CSF, G-CSF and IL-3.
  • HSF ligand of c-kit
  • GM-CSF GM-CSF
  • G-CSF IL-3
  • the critical question is whether undifferentiated stem cells which accumulate in the bone marrow as a result of treatment with NOS inhibitors have the capacity to revert to normal state and resume normal hematopoiesis process once the action of NOS inhibitors is suspended. The failure to do so might indicate that the cells become stranded in their undifferentiated status, similar to various pathological conditions.
  • the treatment of mice with NOS inhibitors was halted 7-9 days after the BM transfer and checked the BM cells for the presence of hematopoiesis markers 1-7 days after termination of injections.
  • NO nitric oxide
  • NOS NO synthase
  • the Xenopus NOS gene was cloned and the distribution of NOS-positive neurons in the developing brain was studied. It was found that inhibition of NOS dramatically increases the number of cells in the developing brain, and increases the overall size of the brain. The results suggest that NOS is directly involved in the control of cell proliferation and neuronal differentiation in the developing vertebrate brain.
  • the NOS cDNA from Xenopus was cloned. Analysis of its primary structure suggests that the cloned gene represents the homologue of the Ca 2 + -dependent neuronal NOS isoform of mammals. Analysis of the gene reveals a remarkable degree of evolutionary conservation with long stretches of amino acid sequences identical to those of humans, mice, rats, and Drosophila.
  • the cloned gene produces enzymatically active protein when transfected in cultured cells.
  • the primary structure of the gene made it possible to obtain a specific antibody, and the immunofluorescence analysis indicates that the diaphorase staining of the developing Xenopus correctly represents the distribution of the XnNOS enzyme. This notion is supported by in situ hybridization analysis of XnNOS transcripts in the tadpole brain. The cloned gene is now being used to isolate other putative NOS genes from Xenopus.
  • NOS is expressed in a consistent spatio-temporal pattern in the developing Xenopus brain
  • the Xenopus brain undergoes histogenesis starting at stage 39-40; prior to that, the neural tube consists of rapidly dividing undifferentiated neuroepithelial cells.
  • new cells arise in the narrow zone of the germinal layer in a defined pattern, which can be revealed by labeling with BrdU.
  • the distribution of NADPH-diaphorase staining (which is indicative of NOS expression) in Xenopus brain from stage 40 through stage 50 was analyzed. Zones of staining first appeared at stage 43, the time of migration of young neurons off the neural tube and their differentiation.
  • Staining appeared outside of the germinal layer and became more intense as development of tadpoles proceeded. The most intense staining was observed in single large differentiated neurons in the tectum and spinal cord, and in the marginal zone of the tectum composed of processes of differentiated neurons. The gradient of diaphorase staining was latero-medial and reciprocal to the pattern of proliferation, suggesting that zones of active proliferation in the germinal layer remained free of NOS activity through these stages.
  • Neuronal differentiation in the brain is affected by inhibition of NOS
  • NOS NOS inhibitors
  • antibodies to specific neuronal markers which have a specific and highly reproducible pattern of expression during Xenopus development were used. It was found that the distribution of neurons positive for Islet- 1, N-tubulin, and N CAM was changed after inhibition of NOS. In particular, the neurons were displaced into the marginal zone, neurons in the intermediate layer were more heterogeneous and with shorter branches than in control brains, and the distinct layered structure of the tectum was altered. In addition, the number of Islet- 1 positive motor neurons was increased after inhibition ofNOS.
  • Inhibition of NOS leads to ectopic proliferation of neuronal precursors.
  • the Xenopus brain has a fine cytoarchitecture. Groups of neighboring cells share the place and time of birth and become involved in common local circuits. The position of young and mature neurons in the brain is strictly dependent on the place of their birth, migration, and final differentiation, and compose a characteristic pattern. In the brains of animals treated with inhibitors of NOS, it was found, in addition to extra layers of young dividing neuronal precursors, numerous ectopic sites of neuronal proliferation. Large clusters of cells were observed in atypical location, occupying the marginal zone, various areas of the tectum, the telencephalon and the hindbrain.
  • Inhibition of NOS activity in the brains of developing tadpoles resulted in increased number of cells in the S-phase, accompanied by a modest increase in programmed cell death at late stages. Together, this increased the total number of cells in the brain and consequently increased the overall size of the brain. The most affected areas are the optic tectum and the area immediately adjacent to the ventricle where the impregnated piece of plastic was inserted. In cases when the source of the NOS inhibitor was shifted in the ventricle towards the telencephalon or hindbrain regions in the developing brain, an increase in size of the anterior the posterior parts of the brain, respectively was observed.
  • mice Female mice were used at 8-12 week of age, and were of the following strains: C57B1/6, B6 CBAFl/J, CBAB6F1/J, DBA (purchased from Jackson Laboratories or Taconic Farms). All mice were bred and maintained at the Animal Care facility of CSHL on standard food diet and acidified water ad libidium.
  • Recipient mice were exposed to 8.2 - 9.5 Gy total body gamma irradiation using Marc I irradiator from Cesium- 137 source (Atomic Energy of Canada, Ottawa) at a dose rate of 1.06 Gy/min 3-20 hours before bone marrow transplantation.
  • the dose of irradiation is enough to suppress hematopoiesis in recipient mice. NO action on hematopoiesis was studied by BM transfer after total body irradiation.
  • the donor mice were sacrificed by CO2 asphyxiation or cervical dislocation and the femures and tibiae were isolated.
  • the bone marrow cells were extracted from the femures and tibiae were isolated.
  • the bone marrow cells were extracted from the femures and tibiae by repeatedly flushing the bones with Dulbecco modified Eagle medium (DMEM) (GibcoBRL).
  • DMEM Dulbecco modified Eagle medium
  • Single cell suspensions were prepared by drawing the bone marrow through a 21 -gauge needle followed by a 26-gauge needle and through 70 mkm nylon cell strainer. Cells were counted using a hematocytometer. 3-5xl0 4 nuclear bone marrow cells were injected into tail vein or lxl 0 6 cells were injected intraperitoneally. Spleens or testis were cut into pieces, then drawn through a 21- gauge needle and a 70 mkm nylon cell strainer to obtain a single cell suspension.
  • DMEM Dulbecco modified Eagle medium
  • N-omega-Nitro L-arginine (L-NAME) (Sigma)
  • N-omega-Nitro D-arginin N-omega-Nitro D-arginin
  • mice were killed by cervical dislocation and bone marrow cells from both femurs and tibiae were flushed out using a 2mL syringe with 21 -gauge needle followed by 26-gauge needle. Spleens and testis were cut into pieces, then drawn through a 21 -gauge needle and a 70 mkm nylon cell strainer to obtain a single cell suspension. Bone marrow, spleen, or testis cells were counted using a hematocytometer.
  • hemapoietic cells (bone marrow or spleen cells) were resuspended in PBS (phosphate buffered saline) containing 3% fetal bovine serum.
  • Erythrocytes were lysed with ammonium chloride-potassium bicarbonate buffer (154 mM ammonium dichloride, 10 mM potassium bicarbonate, 0.082 mM EDTA) 5 minutes at room temperature. After washing, cell suspensions were filtered through a 70 mkm pore size nylon cell strainer and were counted using a hemacytometer.
  • Antibodies The antibodies used in immunofluorescence staining included E13-161.7
  • Anti-SCA-1 [Ly-6A E] conjugated with phycoerithrin (PE) (PharMingen), 2B8 (anti-c-kit), conjugated with FITC (PharmMingen), V-18 (anti-IL-3R alfa) (Santa Cruz Biotechnology, Inc.), M-20 (anti-EpoR) (Santa Cruz Biotechnology, Inc.), M- 20 (anti-G-CSFR) (Santa Cruz Biotechnology, Inc.), anti-rabbit IgG-fluorescein conjugated (FITC).
  • Anti-nNOS mAb, anti-macNOS mAb, anti-eNOS mAb and polyclonal anti-nNOS antibodies were purchased from Transduction Laboratories. Polyclonal anti-nNOS antibodies were also purchased from Zymed.
  • mice were injected intraperitoneally with 50 ug/ml 5-Bromo-deoxyuridine (BrdU) (Beckton-Dickinson) once a day for 5 days. Bone marrow cell smears were prepared and fixed with 4% formaldehyde. BrdU- labeled S phase nuclei were visualized after denaturing DNA in 2M HCI, 0.5% Triton for 2 hours, and incubation with fluorescein-conjugated antibodies to 5-BrdU (Becton Dickinson) as suggested by the manufacturer. Samples were analyzed on a Zeiss Axiphot fluorescent microscope. For nuclei visualization, smears were stained with DAPI, a fluorescent DNA stain (Molecular Probes), at 1 uM. TUNEL
  • NADPH-diaphorase staining was performed essentially as described (Dawson, T.M., et al, Proc. Natl. Acad. Sci. USA, 88:1191 (1991) and Hope, B.T., et al, Proc. Natl Acad. Sci. USA, 55:2811 (1991)) with minor modifications.
  • Cells were fixed in 3.7% paraformaldehyde for 1 hour, washed in PBS, and incubated for 60 min at 37°C in the staimng solution containing 1 mM NADPH, 0.025% Nifroblue tetrazolium salt and 0.3% Triton.
  • Peripheral blood was analyzed using standard methods. Leukocytes were counted in the hematocytometer and in methanol-fixed and Giemza stained smears of peripheral blood.
  • This protocol has dramatically increased the proportion of early progenitor cells in the bone marrow. At first the increase is minimal or actually reversed compared to the control animals. However, several days after cessation of treatment, the proportion of progenitor cells (c-kit-positive) became much higher than in control animals which received the saline solution.
  • the content of c-kit-positive cells in the bone marrow was increased from 5.1% to 23.9%o.
  • the content of IL-receptor-positive cells was increased from 4.3% to 25%.
  • the content of Sea-positive cells in the bone marrow was increased from 1.7% to 5.1%.
  • the content of Sea- and c-kit-positive cells (Sca + c-kit + cells) in the bone marrow was increased from 0.4% to 1.48%.
  • the highest increase for c-kit in the bone marrow was at day 1 after cessation of treatment with NOS inhibitors which was ongoing for 9 days.
  • the highest increase for IL3-R in the bone marrow was at days 2-3 after cessation of treatment with NOS inhibitors which was going for 9 days.
  • the highest increase for Sea in the bone marrow was at days 1-2 after cessation of treatment with NOS inhibitors, also ongoing for 9 days.
  • NOS is especially effective for enrichment of bone marrow and spleen in early hematopoietic progenitors.
  • the proportion of BrdU-labeled cells was significantly higher (up to 10 fold) compared to the control bone marrow. This indicates that treatment with NOS inhibitors has a direct effect on DNA synthesis in the hematopoietic cells in bone marrow.
  • treating animals with NOS inhibitors after bone marrow transfer dramatically increased the number of cells which express stem and progenitor cells' markers. This indicates that as a result of treatment with NOS inhibitors, bone marrow becomes enriched in stem and early progenitor cells. However, it was possible that this procedure only affected the levels of expression of the markers, or it increased the proportion of immediate progenitor cells but not of multipotent hematopoietic stem cells.
  • the proportion of colony forming units in the bone marrow and spleen of experimental animals was tested by transferring the bone marrow or spleen cells to secondary recipients.
  • mice were irradiated at a dose 8.2-9, 0 Gy and injected intravenously 3-5xl0 4 or intraperitoneally lxlO 6 with bone marrow cells from syngeneic donors.
  • Mice in the control group were injected with saline solution. After 9 days, injections were suspended. Experimental and control mice were sacrificed 1 or 3 days after termination of NOS inhibitor injections.
  • CFUs multipotent stem cell
  • pre-CFUs content 3xl0 4 bone marrow cells or lxlO 6 spleen cells from experimental or control mice were transferred into secondary irradiated recipient mice intravenously.
  • aliquots of bone marrow or spleen cells from the experimental animals were tested by FACS for the presence of c-kit or Sca-1 molecules or both of them on the cell surface. After 8 and 12 days the secondary recipients were sacrificed and the hematopoietic colonies in their spleen were counted.
  • the number of day- 12 spleen colonies was 3.5 fold greater in mice which received bone marrow cells from experimental animals (primary recipients, treated with mixture of two NOS-inbibitors for 9 days and left untreated for 1 day) (3.5 ⁇ 0.22) than from the control primary recipients which received saline solution (1.0 ⁇ 0.15).
  • the number of day-8 spleen colonies was 2.9 fold less in secondary recipients which received bone marrow cells from experimental mice (1.50 ⁇ 0.23), than in the secondary recipients which received bone marrow cells from control mice (4.38 ⁇ 0.76). This indicates that under these experimental conditions the number of more primitive CFUs-12 in bone marrow of primary recipients is increased, whereas the number of more committed CFUs-8 is decreased.
  • the increase after 12 days corresponded to the increase in the number of c-kit- positive cells in bone marrow from the primary recipients as determined by FACS analysis.
  • the following parameters in the optic tectum were analyzed: the total number of cells (as measured by the number of DAPI-stained nuclei), the number of S phase proliferating cells (as measured by BrdU inco ⁇ oration), the number of apoptotic cells (as measured by TUNEL assay), the relative numbers of BrdU- and TUNEL-positive cells per 10 3 total cells, the size of the optic tectum, and the cell density in the tectum (Table 5).
  • the data show that exogenously supplied NO drastically decreases the number of proliferating cells at day 1 to only 5% of the control value (Table 5).
  • NOS activity in the brain was inhibited by using Elvax matrix impregnated with a NOS inhibitor, either L-NAME or ETU, or with saline as a control. Pieces of impregnated matrix were inserted into the brain ventricle of stage 45 tadpoles.
  • the number of apoptotic cells was assayed with the TUNEL assay to test the effect of inhibition of NOS on programmed cell death in the developing tadpole brain. While the number of BrdU- positive cells and the total number of DAPI-stained nuclei significantly increased after a 3 day exposure to L-NAME, there was no corresponding change in the absolute or even the relative number or distribution of TUNEL-positive apoptotic cells (Table 6). This demonstrates that excessive proliferation of neuronal precursors due to inhibition of NOS was not accompanied by an immediate and significant change in cell death, indicating that programmed cell death did not significantly contribute to the increase in the total cell number which was the most important consequence of NOS inhibition.
  • the distribution of tectal neurons expressing the pan-neuronal markers ⁇ -II tubulin and N-CAM was determined.
  • ⁇ -U tubulin and N-CAM immunoreactivity was concentrated in the tectal neuropil, however both antibodies also label differentiated tectal cell bodies.
  • the staining pattern suggest that su ⁇ lus cells in L-NAME-treated brains differentiate and express neuronal antigens.
  • the antibody staining revealed a gross distortion of the overall organization of the optic tectum. The cell body region of the treated brains was thicker, whereas the greatly disorganized neuropil was more loosely and chaotically packed with neurites, and the border between the neuropil and the cell body region was markedly irregular.
  • Albino Xenopus laevis tadpoles were obtained by human chorionic gonadofrophin induced matings and raised under standard conditions.
  • animals were anesthetized in 0.02% 3- aminobenzoic acid (MS-222; Sigma) and a tiny piece (100 ⁇ m 2 x 30 ⁇ m) of slow release Elvax plastic polymer (Dupont) was inserted into the tectal ventricle through an incision made in the overlying skin with a 30 gauge needle.
  • Elvax was prepared with stock concentrations of the NOS inhibitors, 2-ethyl-2-thiopseudourea (ETU, Sigma) or L-nitro-arginine methyl ester (L-NAME, Sigma) or the NO donor S- nitroso-acetylpenicillamnin (SNAP, Sigma) prepared as a 1 : 10 ratio of chemical to polymer matrix.
  • Low molecular weight molecules, including L-NAME are released at a constant rate over a period up to 30 days as the lyophilized Elvax matrix slowly hydrates.
  • NO donor SNAP decreases volume, cell density, and total cell number in the tadpole brain.
  • NOS inhibitors increase volume, cell density, and total cell number in the tadpole brain.
  • Brains were implanted with Elvax impregnated with NOS inhibitors L-NAME and ETU or saline solution as a control. Each group contained at least 5 animals. Changes in cell counts and brain volume were determined from 3 sequential 20 ⁇ m sections through the midbrain (see methods for details). Differences of means of cell number, brain volume, and cell density were analyzed by ANOVA.
  • NOS neuropeptides
  • Various isoforms of NOS are expressed in the developing nervous system of mammals (Bredt, D. and Snyder, S.H., Neuron, 13: 301-313 (1994)).
  • NOS is present in cells which bear the characteristics of the neural stem cells (e.g., can form neurospheres, a hallmark of multipotential neural stem cell) (Wang et al, Cell Tissue Res. 296, 489-497 (1999)).
  • the distribution of NOS in the developing and adult brain is consistent with the antiproliferative role of NO during mammalian neurogenesis.
  • NOS activity in the rat brain was inhibited.
  • the solution of NOS inhibitors was introduced in the lateral ventricles of the adult rat brain using osmotic micropumps. 50 mM solution of L-NAME, D-NAME, or saline as a control was injected over the time course of 7 days. 4 days after treatment rats were injected with 60 ⁇ g/g solution of 8-bromo-deoxyuridine (BrdU) 7 times at 12 hour intervals. Animals were sacrificed and brain sections were analyzed for the number of BrdU- positive cells using anti-BrdU antibodies (Beckton-Dickinson).
  • NOS inhibitors Treatment with NOS inhibitors increased the number of BrdU-positive cells in the subventricular zone, rostral migratory stream, and hippocampus by 52.8%, 39.3%, and 12%, respectively. An increase in the number of BrdU-positive cells was also observed in the cortex, co ⁇ us callosum and other areas of the brain. Together, these data indicate that NO acts as a negative regulator of cell proliferation in the adult rat brain and that it is possible to increase the extent of neurogenesis in the adult brain by blocking the production of NO.
  • EXAMPLE 7 Mouse Hematopoiesis. NOS expression in the bone marrow. Various isoforms of NO synthase are produced in most of the types of tissues in humans and rodents. To examine the presence of NOS protein or rnRNA isoforms in the mouse bone marrow, the preparations of the isolated bone marrow were analyzed by Western blots and RT- PCR. NOS protein isoforms were not detected when probing the total bone marrow lysates with antibodies to neuronal, endotheUal, or inducible forms of NOS, probably due to their low content in the preparations of the total bone marrow. However, mRNA of each NOS form were detected when using RT-PCR.
  • RT-PCR signals were reliable and specific for each of the NOS genes tested, since they were reproducibly obtained with several combinations of primers for each gene, produced by DNA fragments of the expected nucleotide sequence, and generated DNA fragments of the expected size after digestion with restriction endonucleases. These results indicate that all three genes coding for NOS isoforms are expressed in the bone marrow.
  • HUCB human umbilical cord blood

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Abstract

L'oxyde nitrique (NO) constitue un important régulateur de croissance dans un organisme en développement intact. Cette invention se rapporte à un procédé permettant d'accroître chez un mammifère la population de cellules souches hématopoïétiques qui sont capables de subir une hématopoïèse, une différenciation et une maturation normales du tissu hématopoïétique, lequel est mis en contact avec des doses multiples d'au moins un inhibiteur de NO, tel que des inhibiteurs d'oxyde nitrique synthase (NOS). Cette invention se rapporte également à un procédé qui sert à augmenter la population de cellules en phase S dans un tissu d'un mammifère et consistant à cet effet à mettre en contact le tissu avec des doses multiples d'au moins un inhibiteur de NO, tel qu'un inhibiteur de NOS. Cette invention concerne en outre un procédé servant à régénérer des tissus chez un mammifère adulte et consistant à mettre en contact un tissu sélectionné (par exemple sang, peau, os et épithélium digestif) ou des cellules précurseurs, avec des doses multiples d'au moins un inhibiteur de NO, qui inhibe la différenciation et induit la prolifération de cellules de ce tissu.
EP00937605A 1999-05-20 2000-05-18 Utilisations therapeutiques d'inhibiteurs d'oxyde nitrique Withdrawn EP1185258A1 (fr)

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US09/315,929 US6372796B1 (en) 1996-11-13 1999-05-20 Therapeutic uses for nitric oxide inhibitors
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PCT/US2000/013685 WO2000071112A1 (fr) 1997-11-13 2000-05-18 Utilisations therapeutiques d'inhibiteurs d'oxyde nitrique

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