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WO2004071429A2 - Traitement d'une inflammation pulmonaire - Google Patents

Traitement d'une inflammation pulmonaire Download PDF

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Publication number
WO2004071429A2
WO2004071429A2 PCT/US2004/003470 US2004003470W WO2004071429A2 WO 2004071429 A2 WO2004071429 A2 WO 2004071429A2 US 2004003470 W US2004003470 W US 2004003470W WO 2004071429 A2 WO2004071429 A2 WO 2004071429A2
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ppt
gene
subject
antagonizing
accordance
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WO2004071429A3 (fr
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J. Julio Perez Fontan
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University of Washington
Washington University in St Louis WUSTL
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University of Washington
Washington University in St Louis WUSTL
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to lung inflammation treatments, and more particularly to methods of treating and preventing acute, ventilation-induced inflammation in the lung.
  • acute pulmonary injury refers to an injury to the lung that involves a rapid inflammatory response.
  • Conditions such as adult (acute) respiratory distress syndrome (ARDS) and aspiration pneumonia, as well as immune-complex mediated injuries such as acute pulmonary rejection are acute pulmonary injuries.
  • ARDS adult respiratory distress syndrome
  • immune-complex mediated injuries such as acute pulmonary rejection are acute pulmonary injuries.
  • acute pulmonary injuries are associated with extensive inflammation, small blood vessel injury, infiltration of immune cells, cytokine production and increased vascular permeability.
  • protein migrates from blood into air spaces.
  • Acute pulmonary injuries often have a high rate of mortality. For example, ARDS has a fatality rate of approximately 40 percent despite recent advances in clinical treatment.
  • VILI ventilation induced lung injury
  • Newborn infants and others with a variety of respiratory disordeis are typically treated with positive pressure mechanical ventilation and oxygen therapy. Such treatment is therapeutic, yet sometimes initiates a potent, acute inflammatory response leading to ventilation-induced acute lung injury, subsequent Bronchopulmonary Dysplasia (BPD) and ultimately chronic lung disease.
  • BPD Bronchopulmonary Dysplasia
  • the biochemical and molecular bases underlying VILI are not clear. However, proposed mechanisms have invoked mechanical trauma as the primary underlying cause.
  • the mechanical trauma has been proposed to be the result of overdistention of aerated lung units (volutrauma), or the result of sheer stresses on the alveolar walls due to the existence of atelectatic (airless or non-aerated) lung units.
  • Atelectatic lung units may result in mechanical stress due to repeated opening and closing of unstable alveoli, or may produce mechanical stress at the margins between atelectatic and aerated lung units.
  • tidal volumes the volume of air moved during breathing
  • the relatively high tidal volumes of about 10-15 ml/kg that are typically used in standard mechanical ventilation practice.
  • the standard, higher tidal volumes compensate for the increased intrapulmonary dead space and pulmonary shunt in ventilated patients.
  • the higher tidal volumes can also cause overdistention of the remaining aerated lung airspaces.
  • Clinical signs of the resulting pulmonary trauma include acute inflammation, increased pulmonary vascular permeability, shunt (non-aerated lung units forming routes through which pulmonary blood bypasses other, aerated lung units), and hypoxemia (decreased blood oxygen content).
  • Atelectrauma is related to the diffuse microatelectasis that frequently exists in ventilated subjects. Atelectasis generally refers to an airless state of all or part of a lung. The airlessness may be due to block or shunt, infection, destruction, , or a combination of any of these.
  • the diffuse and small areas of atelectatic lung include alveoli that are subjected to cyclic opening and closing with each breath. It is believed that excessive stress at the margins between aerated and atelectatic or flooded lung units, as the aerated lung units expand during inspiraion, produces mechanical injury that initiates an acute inflammatory cascade.
  • PEEP positive end-expiratory pressure
  • Fi02 inspired oxygen
  • stretch-mediated inflammation has been proposed as a mechanism of injury during positive pressure mechanical ventilation.
  • Recent recommendations on the use of lower tidal volumes in patients with acute lung injury are based on a recognition of stretch-mediated inflammation as an underlying mechanism of injury during ventilation.
  • Neurogenic inflammation the process by which injured or irritated sensory nerves promote or perpetuate inflammation, was initially identified over thirty years ago and has since been widely studied. Neurogenic inflammation is now known to involve the activity of molecules known as tachykinins or neurokinins.
  • Neurokinins are stored primarily in nonmyelinated afferent C-fibers and are released antidromically in response to noxious stimuli, such as capsaicin.
  • the family of neurokinin pepides includes substance P, neurokininA, neurokinin B, neuropeptide y, neuropeptide K, and neuropeptide Y.
  • the mRNAs that encode substance P, neurokinin A, neuropeptide y and neuropeptde K are derived from the same gene, the preprotachykinin A (PPT-A) gene. See, e.g,. Krause etal. (1987).
  • the preprotachykinin B (PPT-B) gene encodes neurokinin B. Kotani et al., (1986).
  • Alternative RNA splicing of the PPT-A primary gene transcript produces three different mRNAs, ⁇ -PPT, ⁇ -PPT and ⁇ -PPT mRNA.
  • Substance P precursor sequences are encoded by all three of these, while Neurokinin A precursor sequences are encoded in the ⁇ -PPT and ⁇ -PPT mRNAs.
  • Neuropeptide K and Neuropeptide Y are the N-terminally extended derivatives of Neurokinin A and are thought to be the post-translational products of ⁇ -PPT and ⁇ -PPT mRNAs.
  • multiple neurokinin peptide molecules with related biological activities are derived from the single PPT-A gene.
  • the effects of the various neurokinins are mediated through three different known G-protein-coupled receptors that demonstrate differential relative affinities for the agonists substance P, neurokinin A and neurokinin B.
  • the Neurokinin 1 receptor (NK1) has the highest affinity for substance P
  • the Neurokinin 2 receptor (NK2) binds preferentially to neurokinin A
  • the Neurokinin 3 receptor (NK3) binds primarily to neurokinin B.
  • the present invention is directed toward a method for the treatment or prevention of ventilation-induced pulmonary inflammation in a subject in need of such treatment or prevention, wherein the method includes antagonizing PPT-A gene activity in the subject.
  • the present invention is directed toward a method for preventing the development of ventilation-induced pulmonary inflammation in a subject in need of such prevention, wherein the method includes antagonizing the activity of the PPT-A gene in the subject after discontinuation of mechanical ventilation of the subject.
  • the present invention is directed toward a method for controlling the proliferation of ventilation-induced pulmonary inflammation in a subject in need of such control, wherein the method includes antagonizing the activity of the PPT-A gene in the subject during ventilation and after discontinuation of mechanical ventilation of the subject.
  • the present invention is directed toward a composition for treating or preventing ventilation-induced pulmonary inflammation, the composition including a first compound that comprises a high-affinity antagonist of NK1 , and a second compound that comprises a high affinity antagonist of NK2.
  • the present invention is directed toward a method of making a therapeutic composition for the treatment or prevention of ventilation-induced pulmonary inflammation, the method including selecting a plurality of compounds according to the ability of each compound to antagonize the activity of one or more PPT-A gene-encoded peptides.
  • Figure 1A shows photomicrographs (20x) of hematoxylin-stained sections of broncho-alveolar lavage fluid obtained from wild type (WT)and PPT-A gene deficient mice (PPT-A -/-) 24 hours after a 4 hour period of mechanical ventilation;
  • Figure 1 B is a photomicrograph (40x) of broncho-alveolar lavage fluid obtained from wild type and PPT-A gene deficient mice 24 hours after a 4 hour period of mechanical ventilation
  • Figure 1C is a graphic representation of neutrophil, macrophage and erythrocyte cell counts in broncho-alveolar lavage fluid samples obtained from wild type and PPT-A gene deficient mice 24 hours after mechanical ventilation at high tidal volumes
  • Figure 1 D is a graphic representation of ratios of Evans blue concentrations in broncho-alveolar lavage fluid and serum from wild type and PPT-A gene deficient mice 24 hours after mechanical ventilation at high tidal volumes;
  • Figure 1E is a graphic representation of TNF-alpha and MIP 1-alpha levels in broncho-alveolar lavage fluidfrom wild type and PPT-A gene deficient mice both at the end of a period of 4 hours of mechanical ventilation and 24 hours after the end of mechanical ventilation, at high tidal volumes;
  • FIG. 2A shows photographs of broncho-alveolar lavage fluid from wild type (WT), PPT-A gene deficient (PPT -/-) and capsaicin-selective denervated mice after intravenous injection of chicken ovalbumin and intratracheal instillation of rabbit polyclonal antibody against ovalbumin;
  • Figure 2B is a casein zymogram comparing protease activity in broncho-alveolar lavage fluid obtained from wild type mice, PPT-A gene deficient mice, and capsaicin denervated mice;
  • Figure 2C is a graphic representation of erythrocyte and neutrophil counts, Evans blue lavage/serum concentration ratios, and TNF-alpha levels in broncho-alveolar lavage fluid taken from control wild type, PPT-A gene deficient mice, and capsaicin denervated mice after immune complex formation;
  • Figure 3A is a schematic diagram of experimental groups including wild type mice with bone marrow reconstituted with cells from wild type litter mates, mice with homozygous deletion of the PPT-A gene with bone marrow reconstituted with cells from wild type litter mates, and wild type mice with bone marrow reconstituted with cells from litter mates with homozygous deletion of the PPT-A gene; and
  • Figure 3B is graphic representation of cell counts and Evans blue ratios for broncho-alveolar lavage fluid and serum from wild type and PPT-A gene deficient after immune complex formation.
  • PPT-A gene Targeted deletion of the preprotachykinin A gene (PPT-A gene) protects lungs against acute pulmonary injury, and particularly ventilation-induced pulmonary inflammation. Accordingly, in one embodiment the present invention is directed towards a method of treatment and prevention of ventilation-induced pulmonary inflammation in a subject in need of such treatment or prevention, by antagonizing PPT-A gene activity in the subject.
  • the examples herein establish that PPT-A gene- encoded peptides are required for acute ventilation-induced inflammatory pulmonary injury to occur, and also for acute immune-complex-mediated injury to occur.
  • the activity of neurokinin B did not reveal itsef to be a necessary or even noticeable component in generating acute ventilation-induced pulmonary injury.
  • alleles are an alternative form of the gene encoding PPT-A. Alleles may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments thereof, and to naturally occurring or synthetic molecules. Oligopeptides of PPT-A are preferably about 5 to about 15 amino acids in length and retain the biological activity or the immunological activity of PPT-A. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence, and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which, when bound to PPT-A or to a receptor of a PPT-A gene-encoded neurokinin, decreases the amount or the duration of the effect of the biological activity of PPT-A, including expression of PPT-A gene-encoded neurokinins, through and including binding of PPT-A gene-encoded neurokinin to receptor proteins.
  • Antagonists may include antibodies, proteins, nucleic acids, carbohydrates, or any other molecules which decrease the effect of PPT-A.
  • the term "antibody” refers to intact molecules as well as fragments thereof, such as Fab F(ab') 2 , and Fv, which are capable of binding the epitopic determinant.
  • Antibodies that bhd PPT-A polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal can be derived from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled pep ⁇ de is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • high affinity refers to a characteristic of an antagonist to bind selectively or preferentially to one neurokinin receptor type in comparison to other neurokinin receptor types.
  • a high affinity NK1 blocker refers to a compound that binds selectively or preferentially to NK1 relative to NK2 and NK3
  • a high affinity NK2 blocker refers to a compound that binds selectively or preferentially to NK2 relative to NK1 and NK3, as is determined according to standard affinity binding analysis.
  • neurokinin 1 and "NK1" as used interchangeably herein refer to the G-protein coupled receptor protein that binds substance P preferentially over NKA and NKB.
  • neurokinin 2 and “NK2” as used interchangeably herein refer to the G-protein coupled receptor protein that binds NKA preferentially over substance P and NKB.
  • neurokinin 3 and “NK3” as used interchangeably herein refer to the G-protein coupled receptor protein that binds NKB preferentially over substance P and NKA.
  • PPT-A refers to the amino acid sequences of substantially purified preprotachykinin-A obtained from any species, particularly mammalian, including bovine, murine, rat and human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • preprotachykinin-A gene and "PPT-A gene” as used interchangeably herein refer to any one of multiple homologous nucleic acid sequences that have been isolated and des ⁇ ibed in various mammalian species including human, rat, mouse, and bovine sequences and identified as encoding at least substance P and neurokinin A.
  • Human PPT-A gene is described in A.J. Harmar et al., FEBS Lett. 208:67-72 (1986).
  • Rat PPT-A gene is described in M.S. Carter and J. E. Krause, J. Neurosci. 10(7):2203-U (1990).
  • Mouse PPT-A gene is described in K. Kako et al, Blomed. Res. 14, 253-59 (1993).
  • Bovine PPT-A gene is described in Nawa et al. Nature 312:729-34 (1984).
  • PPT-A gene activity refers to any one of, any combination of, or all of the multiple biochemical and molecular events constituting the cascade that exists from PPT-A gene transcription, translation and expression ultimately through and including binding of PPT-A gene-encoded peptides to receptor molecules.
  • Antagonism of PPT-A gene activity thus defined can occur at any one or more of multiple possible points. For example, interfering with PPT-A gene expression at the level of any PPT-A gene-expressing cell is one possible approach.
  • Another approach is to target a specific PPT-A gene-expressing cell population, such as migratory hematopoietic cells, whose presence and PPT-A gene expression are required, as demonstrated below, for acute inflammatory pulmonary injury to occur.
  • Another approach is to block the activity of PPT-A gene-encoded peptides, for example by blocking a receptor or receptors of the PPT-A gene- encoded products substance P, neurokinin A, neuropeptide K, and neuropeptide Y, using ligands that act as antagonists of NK1 and NK2.
  • PPT-A gene-encoded peptide refers to any one of substance P, neurokinin A, neuropeptide K, and neuropeptide Y.
  • respiration-induced refers to a characteristic of an acute pulmonary inflammation that is clinically observed to arise during or after treatment of a mammalian subject using positive pressure mechanical respiration to supplement or provide respiration to the subject.
  • a method for treating or preventing ventilation-induced pulmonary inflammation involves antagonizing PPT-A activity in the subject.
  • Antagonizing PPT-A gene activity will involve at least one of multiple possible actions.
  • antagonizing PPT-A gene activity is accomplished by antagonizing the activity of at least one PPT-A gene-encoded peptide, such as antagonizing the activity of at least one of, or any combination of two or more of substance P, neurokinin A, neuropeptide Y, neuropeptide K, and neuropeptide Y.
  • antagonizing PPT-A gene activity in the subject includes administering to the subject a pharmaceutical composition including at least one PPT-A gene-encoded neurokinin antagonist.
  • the PPT-A gene-encoded neurokinin antagonist is, for example, an antagonist of at least one receptor of PPT-A gene-encoded peptides, such as, for example, a composition including an NK1 receptor blocker, a composition including an NK2 receptor blocker, or a composition including a combination of an NK1 receptor blocker and an NK2 receptor blocker.
  • a suitable pharmaceutical composition including an NK1 receptor blocker, a NK2 receptor blocker, or a combination of an NK1 receptor blocker and an NK2 receptor blocker is formulated as known in the art with pharmaceutically acceptable carriers in a formulation that permits administration of the composition by inhalation, and the composition is administered to the subject by inhalation.
  • a vector expressing an antisense sequence to the polynucleotide PPT-A gene may be administered to a subject.
  • any of the antagonists, or antisense sequences may be administered in combination w ' rth other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may demonstrate synergism to effect treatment or prevention of acute ventilation-induced pulmonary inflammation. Using compounds or agents that act synergistically may provide therapeutic efficacy at a lower dosages of each agent, thereby thus reducing the risk of adverse side effects.
  • Antagonists or inhibitors of the PPT-A gene may be produced using methods which are general ⁇ known in the art.
  • purified PPT-A gene may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind the PPT-A gene.
  • Antibodies to the PPT-A gene may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the PPT-A gene or any fragment or oligopeptide thereof which has immunogenic properties.
  • adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to the PPT-A gene have an amino acid sequence consisting of at least five amino acids and more preferably at least 10 amino acids, they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of PPT-A gene amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule.
  • Monoclonal antibodies to the PPT-A gene may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. Nature 256:495-497 (1975) ; Kozbor, D. et al, J. Immunol. Methods 8131-42 (1985); Cote, R. J. et al, Proc. Natl. Acad. Sci. 80:2026-2030 (1983); Cole, S. P. et al, Mol. CellBiol. 62:109-120 (1984) ).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al, Proc. Natl. Acad. Sci. 86:3833-3837 (1989); Winter, G. et al. Nature 349:293-299 (1991) ).
  • Antibody fragments which contain specific binding sites for the PPT-A gene may also be generated.
  • such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. Science 254:1275-1281 (1989)).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • the polynucleotides encoding the PPT-A gene, or any fragment or complement thereof may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding the PPT-A gene may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding the PPT-A gene.
  • complementary molecules or fragments may be used to antagonize PPT-A gene activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotidesor larger fragments can be designed from various locations along the coding or control regions of sequences encoding the PPT-A gene.
  • Expression vectors derived from retro viruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence which is complementary to the polynucleotides of the gene encoding PPT-A. These techniques are described both for example in Sambrook et al, supra.
  • Genes encoding PPT-A can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes PPT-A. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions of the gene encoding PPT-A (signal sequence, promoters, enhancers, and introns).
  • Oligonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site, are preferred.
  • An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee etal, Nucl. Acids, Res, 3:173 (1979); Cooneyet al. Science 241 :456 (1988); and Dervan et al.
  • RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the imRNA molecule into PPT-A gene- encoded products (Okano, J. Neurochem. 56:560 (1991)).
  • the antisense constructs can be delivered to cells be procedures known in the art such that the antisense RNA or DNA may be expressed in vivo.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of PPT-A mRNA by preventing the transcript from binding to ribosomes. Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding PPT-A.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding PPT-A. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient Delivery by transfection, by liposome injections or polycationic amino polymers (Goldman, C. K. et al. Nature Biotechnology 15:462-66 (1997)) may be achieved using methods which are well known in the art. Any of the therapeutic methods described above may be applied to any subject in need of such therapy for the treatment or prevention of ventilation- induced pulmonary inflammation, including, for example, mammals such as dogs, cats, cows, horses, rabbits, is monkeys, and most especially, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of the PPT-A gene, antibodies to the PPT-A gene, mimetics, agonists, antagonists, or inhibitors of the PPT-A gene, as well as antibodies to PPT-A gene-encoded peptides, and mimetics, agonists, antagonists, or inhibitors of PPT-A gene-encoded peptides.
  • compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • agent such as stabilizing compound
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, inhalation, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton, Pa.).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optional ⁇ grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubiizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids etc. Salts tend to be more soluble in aqueous or ofrier protonic solvents than are the corresponding free base forms.
  • Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example of antibodies to PPT-A gene-encoded peptides, or of antagonists or inhibitors of the PPT-A gene or PPT-A gene-encoded peptides, which ameliorates or prevents the ventilation-induced pulmonary inflammation.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
  • ED 50 the dose therapeutically effective in 50% of the population
  • LD 50 the dose lethal to 50% of the population
  • the dose ratio of or to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 /ED 5 o.
  • compositions that exhibit large therapeutic indices are especially suitable.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Compounds suitable for use as antagonists of PPT-A gene-encoded peptides in pharmaceutical compositions include, for example, various compounds described in: U.S. Pat. No. 6,365,602;
  • the invention is directed toward a method for preventing the development of ventilation-induced pulmonary inflammation in a subject in need of such treatment or prevention, by antagonizing the activity of the PPT-A gene in the subject after cessation or discontinuation of mechanical ventilation of the subject.
  • the invention embraces a method for controlling the proliferation of ventilation-induced pulmonary inflammation in a subject in need of such control, by antagonizing the activity of the PPT-A gene in the subject during ventilation and after discontinuation of mechanical ventilation of the subject.
  • the invention is directed toward a method of making a therapeutic composition for the treatment or prevention of ventilation- induced pulmonary inflammation, by selecting a plurality of compounds according to the ability of each compound to antagonize the activity of one or more PPT-A gene- encoded peptides. More preferably, compounds are selected for the composition so that the composition includes at least one component that antagonizes NK1 and at least one component that antagonizes NK2, so that the receptors most involved in mediating the effects of at least substance P and neurokinin A are antagonized.
  • compounds as described above that act to selectively or preferentially antagonize the NK1 receptor, and compounds that act to selectively or preferentially antagonize the NK2 receptor as described above are combined in a composition that is administered to the subject.
  • multiple compounds that each demonstrate mixed but varying selectivity for NK1 and NK2 as' described above are combined in a composition to administer to the subject.
  • the selected compounds are then combined in amounts that together comprise a composition effective for the treatment or prevention of ventilation- induced pulmonary injury.
  • compounds that act to selectively or preferentially antagonize the vanilloid receptor-1 are used alone, or in combination with NK1 receptor blockers and/or NK2 receptor blockers, to block the progression of neurokinin-dependent inflammation that arises from PPT-A activity.
  • the compounds can be formulated in pharmaceutical compositions suitable for administration by the various known routes of pharmaceutical administration including oral, buccal, inhalation, rectal, topical, and injection including intravenous, intraperitoneal, and intradermal.
  • PPT-A gene deficient mice are described in Y.Q. Cao, et al. Nature 192, 390 (1998), which is herein incorporated by reference together with the primary references contained therein.
  • PPT-A gene deletion prevents synthesis of PPT-A gene-encoded peptides including Substance P, neurokinin A, neuropeptide K and neuropeptide Y. See, e.g., Cao et al. .
  • neurokinin B which is encoded by the PPT-A gene, is not affected by PPT-A gene deletion and remains available as a ligand at least for the NK3 receptor. See, e.g., U.S. Pat. No. 6,008,194.
  • mice The lungs of wild type and PPT-A gene deficient mice were ventiated mechanically for a period of four hours using a tidal volume of either 6 ml/kg (low tidal volume, peak airway pressure 9-10 cm H 2 0) or 20 ml/kg kg (high tidal volume, peak airway pressure 16-17 cm H 2 0). All ventilation experiments were performed in inbred C57BL/6 mice which are obtainable from, for example, Charles River Laboratories, Wilmington, Massachusetts, U.S.A.
  • Figure 1 A shows photomicrographs (20x) of hematoxylin-stained sections of broncho-alveolar lavage fluid obtained from wild type and PPT-A gene deficient mice 24 hours after a 4-hour period of mechanical ventilation at 20 ml/kg tidal volume. The sections reveal edema and infiltration of the pulmonary interstitium by inflammatory cells in wild type mice, but not in PPT-A gene deficient mice.
  • Figure 1 B is a higher power (40x) photomicrograph of broncho- alveolar lavage fluid obtained from each group of mice 24 hours after the end of the 4-hour period of mechanical ventilation at 20 ml/kg.
  • FIG. 1 C is a graphic representation of neutrophil, macrophage and erythrocyte cell counts in broncho-alveolar lavage fluid samples obtained from the two groups of mice 24 hours after mechanical ventilation at high tidal volumes. Increased numbers of neutrophils (hatched bars), macrophages (open bars), and erythrocytes (closed bars) were evident in wild type but not in PPT-A gene deficient mice.
  • Figure 1 D is a graphic representation of ratios of Evans blue concentrations in broncho-alveolar lavage fluid and serum from the two groups of mice 24 hours after mechanical ventilation at high tidal volumes. Samples from wild type mice demonstrated an increased alveolar-capillary permeability to proteins, which was not evident in samples from PPT-A gene deficient mice.
  • Figure 1E is a graphic representation of TNF-alpha (closed bars) and MIP 1-alpha levels (open bars; measured with a radioligand assayfrom R&D Systems, Minneapolis, Minnesota) in broncho-alveolar lavage fluid from wild type and PPT-A gene deficient mice, both at the end of a period of 4 hours of mechanical ventilation, and 24 hours after the end of mechanical ventilation, at high tidal volume.
  • TNF-alpha levels were higher in wild type than in PPT-A gene deficient mice both at the end of the 4-hour ventilation period, and 24 hours later.
  • MIP 1-alpha levels were higher in wild type mice than in PPT-A gene deficient mice only 24 hours after the end of the 4-hour ventilation period.
  • Capsaicin injection induces immediate release of neurokinins from sensory nerve fibers expressing the vanilloid receptor-1 (M. J. Caterina et al. Science 288, 306 (2000)), which causes permanent sensory denervation of the fibers.
  • Wildtype (WT), PPT-A gene deficient (PPT -/-), and capsaicin-selective denervated mice received simultaneous intravenous injection of chicken ovalbumin and intratracheal instillation of rabbit polyclonal antibody against ovalbumin.
  • Figure 2A shows photographs of broncho-alveolar lavage flu ' d from wild type (WT), PPT-A gene deficient and capsaicin-selective denervated mice after intravenous injection of chicken ovalbumin (20 mg/kg in 0.5 ml PBS) and intratracheal instillation of rabbit polyclonal antibody against ovalbumin (10 mg/kg in 0.1 ml PBS).
  • Figure 2A shows alveolar hemorrhage in wild type mice, but not in PPT-A gene deficient mice.
  • Figure 2B is a casein zymogram comparing protease activity in broncho-alveolar lavage fluid obtained from wild type mice, PPT-A gene deficient mice, and capsaicin denervated mice.
  • the zymogram demonstrates increased protease activity in the broncho-alveolar lavage fl ⁇ d obtained in from wild type mice, as compared to capsaicin denervated mice and PPT-A gene deficient mice.
  • the most prominent proteolytic band (white arrows) has the electrophoretic mobility profile of macrophage metalloelastase. Black arrowheads indicate molecular weight markers.
  • Figure 2C is a graphic representation of erythrocyte and neutrophil counts, Evans blue lavage/serum concentration ratios, and TNF-alpha levels (measured with a radio-ligand assay from R&D Systems, Minneapolis, MN) in broncho-alveolar lavage fluid taken from control wild type, PPT-A gene deficient mice, and capsaicin denervated mice after immune complex formation.
  • Control wild type mice (results indicated by "c" on top of bars) were injected with ovalbumin intravenously and normal saline intratracheally.
  • FIG. 3A is a diagram of the experimental scheme, showing the three experimental groups.
  • a first group included wild type mice with bone marrow reconstituted with cells from wild type litter mates.
  • a second group included mice with homozygous deletion of the PPT-A gene (shown by crossing of affected cells), but with bone marrow reconstituted with cells from wild type litter mates so that the animals were restored with hematopoietic cells capable of producing PPT-A gene-encoded peptides.
  • a third group included wild type mice with bone marrow reconstituted with cells from litter mates with homozygous deletion of the PPT-A gene, so that the animals in the third group lost hematopoietic cells capable of producing PPT-A gene-encoded peptides.
  • a period of at least 145 days was allowed for bone marrow engraftment and the turnover of any hematopoietic cells that may have survived the conditioning irradiation.
  • FIG. 3B is graphic representation of cell counts and Evans blue ratios for broncho-alveolar lavage fluid and serum from wild type and PPT-A gene deficient after bone marrow reconstitution and immune complex formation. Wild type mice with reconstituted wild type bone marrow developed intense inflammation after immune-complex formation, similar to that observed in non-transplanted wild type mice (not shown). In contrast, wild type hematopoietic bone marrow did not reestablish the wild type inflammatory response in PPT-A gene deficient mice.
  • PPT- A gene deficient mice retained protection against immune complex-mediated injury even after their bone marrow was reconstituted with wild type bone marrow, showing that PPT-A gene expression by hematopoietic cells is not sufficient on its own to produce injury.
  • a surprising and unexpected result was that wild type mice with bone marrow reconstituted with PPT-A gene deficient cells gained protection against injury, indicating that PPT-A gene expression in hematopoietic cells is necessary for immune complex-mediated inflammation in the lungs.
  • EXAMPLE 4 Examples 1-3 supra show that the progression of immune complex and stretch mediated inflammation in murine lungs depends on an unexpected synergy between PPT-A-expressing neurons and hemopoietic cells. These results indicate that PP7 " -/A-encoded neurokinins released by sensory fibers activate neurokinin receptors in local hemopoietic cells (macrophages), which then amplify the inflammatory response through a chain of autocrine and paracrine neurokinin- mediated interactions.
  • Wild type mice reconstituted with PPT-A +/- bone marrow had substantial reductions in hemorrhage (63% fewer red cells in the alveolar lavage), inflammation (77% fewer neutrophils), and permeability injury [24% lower Evans Blue (EB) lavage/serum ratio] as compared to wild type mice reconstituted with PPT>A +/+ bone marrow.
  • Mice with a targeted disruption of the TrpV-1 gene were protected against both immune complex- (68% fewer red cells, 80% fewer neutrophils, and 45% lower EB permeability) and stretch-mediated inflammation (86% fewer red cells, 93% fewer neutrophils, and 75% lower EB permeability).

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Abstract

L'invention a trait à une méthode permettant de traiter ou de prévenir une inflammation pulmonaire induite par la ventilation, chez un sujet ayant besoin d'un tel traitement. Ledit procédé consiste à antagoniser l'activité de PPT-A.
PCT/US2004/003470 2003-02-06 2004-02-06 Traitement d'une inflammation pulmonaire Ceased WO2004071429A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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WO2021245512A1 (fr) * 2020-06-02 2021-12-09 Nerre Therapeutics Limited Antagonistes du récepteur de la neurokinine (nk)-1 destinés à être utilisés dans le traitement d'états de fibrose pulmonaire favorisés par une lésion mécanique des poumons

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Title
CHAVOLLA - CALDERON M ET AL: 'Bone marrow transplantation reveals an essential synergy between neuronal and hemopoietic cell neurokinin production in pulmonary inflammation' THE JOURNAL OF CLINICAL INVESTIGATION vol. 111, no. 7, April 2003, pages 973 - 980, XP002903525 *
COLTEN H ET AL: 'Pulmonary Inflammation' THE NEW ENGLAND JOURNAL OF MEDICINE vol. 336, 10 April 1997, pages 1094 - 1096, XP002982439 *
FONTAN J ET AL: 'Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat' AMERICAN JOURNAL OF PHYSIOLOGY LUNG, CELLULAR AND MOLECULAR B. vol. 278, 2000, pages L344 - L355, XP002903524 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021245512A1 (fr) * 2020-06-02 2021-12-09 Nerre Therapeutics Limited Antagonistes du récepteur de la neurokinine (nk)-1 destinés à être utilisés dans le traitement d'états de fibrose pulmonaire favorisés par une lésion mécanique des poumons
CN115697332A (zh) * 2020-06-02 2023-02-03 尼尔医疗有限公司 用于治疗由肺的机械损伤促进的肺纤维化状况的神经激肽(nk)-1受体拮抗剂
JP2023531863A (ja) * 2020-06-02 2023-07-26 ネッレ セラピューティクス リミテッド 肺の機械的損傷によって促進される肺線維症状態の治療で使用するためのニューロキニン（ｎｋ）－１受容体拮抗薬
US11872222B2 (en) 2020-06-02 2024-01-16 Nerre Therapeutics Limited Uses

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