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WO1999064015A1 - Compositions pharmaceutiques contenant de l'alpha-tocopherylphosphocholine - Google Patents

Compositions pharmaceutiques contenant de l'alpha-tocopherylphosphocholine Download PDF

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Publication number
WO1999064015A1
WO1999064015A1 PCT/US1998/011818 US9811818W WO9964015A1 WO 1999064015 A1 WO1999064015 A1 WO 1999064015A1 US 9811818 W US9811818 W US 9811818W WO 9964015 A1 WO9964015 A1 WO 9964015A1
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WO
WIPO (PCT)
Prior art keywords
tocopherylphosphocholine
composition
effective
acceptable salt
pharmaceutically
Prior art date
Application number
PCT/US1998/011818
Other languages
English (en)
Inventor
Andrew C. Peterson
Parvin I. Yazdi
Thaddeus P. Pruss
Original Assignee
Clarion Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Clarion Pharmaceuticals, Inc. filed Critical Clarion Pharmaceuticals, Inc.
Priority to AU78261/98A priority Critical patent/AU7826198A/en
Priority to PCT/US1998/011818 priority patent/WO1999064015A1/fr
Priority to CA002334313A priority patent/CA2334313A1/fr
Priority to EP98926417A priority patent/EP1085881A1/fr
Publication of WO1999064015A1 publication Critical patent/WO1999064015A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin

Definitions

  • the present invention relates to novel compositions comprising ⁇ - tocopherylphosphocholine and salts thereof and novel methods of using these compounds.
  • the compounds have been discovered to form liposomes and to possess antiviral, anti-fungal, anti-inflammatory, PAF-antagonist, and UV radiation-blocking activities.
  • the invention specifically includes the use of ⁇ -tocopheryl-phosphocholine in medicaments to treat inflammation of the respiratory system, as a pulmonary surfactant, and as a mucolytic agent to thin or lower the viscosity of tenacious mucus.
  • ⁇ -tocopherol with phosphocholine
  • ⁇ -tocopherylphosphocholine also known as ⁇ -tocopherolphosphocholine
  • pharmaceutically acceptable salts thereof have been described in the literature. See, e.g. , Japanese Public Patent Disclosure No. 1-211578, published August 24, 1989. It has now been found that ⁇ -tocopherylphosphocholine, also referred to herein as CPR-2001 , and its pharmaceutically acceptable salts (collectively the "subject compounds”) are useful chemopreventative and adjuvant agents in several aspects. Further, it has been found that the subject compounds form liposomes.
  • respiratory disease(s) encompasses diseases involving the parenchyma (alveoli), such as adult respiratory distress syndrome (ARDS), respiratory distress syndrome (RDS), emphysema, pneumonia and pulmonary fibrosis, and diseases involvling the conducting airways, such as asthma, bronchitis, chronic obstructive pulmonary disease and cystic fibrosis, among others.
  • ARDS adult respiratory distress syndrome
  • RDS respiratory distress syndrome
  • emphysema emphysema
  • pneumonia and pulmonary fibrosis emphysema
  • diseases involvling the conducting airways such as asthma, bronchitis, chronic obstructive pulmonary disease and cystic fibrosis, among others.
  • the lung is an organ comprising 6% of the mammalian body volume.
  • the primary purpose of the lung is to facilitate gas interchange and as a consequence has a large surface area which is constantly in contact with a hostile environment.
  • Lung surfactant is a material ordinarily secreted onto the surface of lung alveoli to facilitate gas exchange.
  • the natural surfactant coats the surface of the alveoli and provides the proper mechanical stability by reducing the surface tension at the air-alveolar interface
  • RDS is the leading cause of death and disability among premature infants. In addition, about 150,000 cases of ARDS are reported annually with 60-80% mortality.
  • a number of natural surfactants (human and bovine) and synthetic surfactants have been administered to the lungs of human subjects, for example, by inhalation of an aerosol formulation.
  • a complex array of compounds comprise natural mallian pulmonary surfactant; the approximate composition is 90 wt. % lipids, 8 wt. % proteins and 2 wt. % carbohydrates.
  • Adult bovine lung surfactant extract which is obtained from adult bovine lung lavage, has been shown to contain 84% phosphatidyl cholines ⁇ F.R. Rana et al. , Biochemistry (1993) 32: 27-31 ⁇ . Dipalmitoyl phosphatidylcholine
  • DPPC DPPC is the major component (about 80%) of natural human lung surfactant.
  • disorders of the upper airways involve altered secretion of lung surfactant and/or alteration in the composition of the mucus Asthma, chronic bronchitis and cystic fibrosis, in particular, are associated with hypersecretion of lung surfactant Alterations in mucous composition usually accompany this hypersecretion and lead to increases in the viscosity of the mucus As a consequence, patients suffering from these airways disorders experience a progressive difficulty in clearing mucus from their lungs The patient experiences airway obstruction, oftentimes severe, and will essentially drown in mucus secretions if left untreated There are several reported drugs which have been shown to have a beneficial effect on airway clearance of mucus, for example, through their effect on cilia or by altering the physical properties of mucous secretions One class of such compounds are based on iodide compounds Treating the lungs with a saturated solution of potassium iodide or lodinated glycerol, is thought to stimulate the secretion of "thinner" secretions that are easier
  • Inflammation is a pervasive symptom which accompanies respiratory diseases and occurs in response to local trauma, bacterial infection, viral infection, allergic and immunogenic reactions, and the like Inflammatory cells in the airways release mediators which can trigger or block the secretion of components of lung surfactant Infection of the airways in cystic fibrosis, for instance, is associated with abnormal rheological and transport properties of airway secretions which may be responsible for the severity of the disease ⁇ E. Puchelle et al , Eur I Clin Invest (1985) 15 389- 394 ⁇
  • Pulmonary fibrosis is a pathological condition caused by viral pneumonias, drug reactions, chemical vapors and dusts
  • the early stages of pulmonary fibrosis are marked by the appearance of fluid and cellular debris in the alveoli This exudate promotes an inflammatory reaction characterized by interstitial edema and invasion by leukocytes, macrophages, neutrophils, fibroblasts and eosinophils. These inflammatory cells also produce reactive oxygen species. Destruction of the alveoli occurs progressively as the reactive oxygen species and inflammation continues.
  • the present invention relates to the surprising and unexpected discoveries that the subject compounds are effective to treat, control or prevent viral and fungal infections and inflammation and to antagonize the activities of PAF (platelet-activating factor).
  • the subject compounds are also effective in blocking ultraviolet (UV) radiation, especially UV-B radiation.
  • the invention also relates to the surprising and unexpected discovery that the subject compounds form liposomes.
  • the invention provides a method of treating a susceptible viral or fungal infection or inflammation or antagonizing PAF-activities in a mammal, which method comprises administering to the mammal an amount of ⁇ - tocopherylphosphocholine or a pharmaceutically acceptable salt thereof that is effective to treat the infection or inflammation or antagonize the PAF-activities.
  • compositions which comprise an antiviral-effective, antifungal-effective, anti-inflammatory-effective, PAF-activity- antagonizing-effective or UV radiation-blocking amount of ⁇ - tocopherylphosphocholine or a pharmaceutically acceptable salt thereof and a pharmaceutically effective carrier.
  • the invention provides a liposome which comprises ⁇ - tocopherylphosphocholine or a salt thereof as a structural component of the bilayer of the liposome.
  • the liposomes of the invention can be employed as vehicles to deliver ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof in a therapeutic use thereof or as vehicles to deliver other therapeutically active substances carried by the liposomes.
  • the invention is also directed to methods and compositions to treat a respiratory disease in a mammal afflicted with the same.
  • the method comprises administering to the mammal a therapeutically-effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically-acceptable salt thereof, the amount being effective to make respiration easier.
  • the methods and pharmaceutical compositions are drawn to treating, without limitation, various respiratory diseases including asthma, bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, pneumonia, pulmonary fibrosis, respiratory distress syndrome (RDS), adult respiratory distress syndrome (ARDS), and combinations thereof.
  • the invention is further directed to methods and pharmaceutical compositions to improve the clearance of mucus from the lungs in a mammal afflicted with a pulmonary disorder involving thickened or accumulated pulmonary mucous secretions.
  • the method comprises delivering to the lung airways of the mammal an effective mucus-clearing or mucolytic amount of ⁇ -tocopheryl-phosphocholine or a pharmaceutically-acceptable salt thereof.
  • mucolytic are used synonymously herein and refer to the ability of the subject compounds to decrease the viscosity of tenacious mucus and to increase the mobility of mucus across the surface of underlying pulmonary tissues.
  • the ⁇ -tocopheryl-phosphocholine is delivered to the lung airways of the subject being treated in the form of an aerosolized liquid, an aerosolized powder, or a micronized dry powder.
  • Humans are among the mammals that can be treated by the methods of the invention.
  • the present invention provides methods and pharmaceutical compositions for treating viral and fungal infections, inflammation, and respiratory diseases in a mammal suffering therefrom by administering to the mammal an antiviral-effective, anti-fungal-effective, or anti-inflammation-effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a method for antagonizing PAF-activities (and treating the related pathological conditions -such as inflammation or bronchial asthma) in a mammal which comprises administering to the mammal a PAF-activity- antagonizing-effective amount of ⁇ -tocopherylphosphocholine or pharmaceutically acceptable salt thereof.
  • the invention provides pharmaceutical compositions comprising ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof, i.e. , "active ingredient", together with a pharmaceutically acceptable carrier therefor and, optionally, other therapeutically active ingredients.
  • the pharmaceutical compositions of the invention comprise an amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof that is effective to treat a viral or fungal infection or inflammation in a mammal suffering therefrom, to which the composition is administered, or effective to antagonize PAF-activities (and treat the related pathological conditions) in a mammal, to which the composition is administered, or to block UV radiation (when applied topically).
  • the carrier In a pharmaceutical composition of the invention, the carrier must be pharmaceutically acceptable in the sense of being compatible with other ingredients in the pa ⁇ icular composition and not deleterious to the recipient thereof.
  • the compositions include those suitable for oral, topical (including compositions suitable for ophthmalogic and nasal administration), rectal or parenteral (including subcutaneous, intramuscular, intradermal and intravenous) administration. Humans are among the mammals that can be treated by the methods and with the compositions of the invention.
  • the invention also provides methods and pharmaceutical compositions to alleviate respiratory distress due to tenacious pulmonary mucous secretions in a patient suffering from such distress.
  • the method is accomplished by the intermittent inhalation of an aerosol or dry powder formulation of ⁇ -tocopheryl-phosphocholine to deliver an effective mucolytic amount of ⁇ - tocopheryl-phosphocholine to the lung airways.
  • the mucolytic activity of the subject compounds are demonstrated in a standard animal model m the Examples provided below
  • the in vivo mucolytic assay is similar to that described by Chand et al Agents and Actions, (1993) 38 165-170, incorporated herein by reference
  • the invention also provides a method of administering a pulmonary surfactant to a patient in need thereof
  • the method is accomplished by the intermittent inhalation of an aerosol or by directly pouring a liquid composition of ⁇ -tocopheryl-phosphochohne into the lungs of a patient in need of pulmonary surfactant, thereby delivering an effective surface-active amount of ⁇ -tocopheryl-phosphochohne
  • the effectiveness of the subject compounds as surfactants is demonstrated m the Examples using a standard animal model of RDS, a disease which features a deficiency of pulmonary surfactant
  • the invention also provides a method ot alleviating respiratory distress due to pulmonary inflammation in a patient suffering from pulmonary inflammation
  • the method is accomplished, most preferably, bv the intermittent inhalation of an aerosol or dry powder formulation of ⁇ -tocopheryl-phosphochohne to deliver an effective anti-inflammatory amount of ⁇ -tocopheryl-phosphocholine to the patient
  • the anti-inflammatory activity ol the subject compounds is demonstrated in the Examples using a standard in vitro assay which measures the inhibition of superoxide anion release from phorbol 12 my ⁇ state- 13 acetate(PMA)-st ⁇ mulated macrophages
  • Still another aspect of the present invention is liposomes which comprise ⁇ - tocopherylphosphochohne or a salt thereof as a structural component of the bilayer
  • preferred salts are pharmaceutically acceptable salts
  • a compound in a hposome is a ' structural component ' of the bilayer of the hposome if the compound, when the only lipophilic or amphophilic substance suspended in an aqueous medium, forms liposomes when the suspension is subjected to hposome- formmg conditions
  • the invention comprehends liposomes of which the structural components of the bilayer consist essentially (for hposome integrity) of ⁇ - tocopheryl-phosphochohne or a salt thereof or comprise ⁇ -tocopherylphosphochohne or a salt thereof together with other compounds which are also structural components
  • the liposomes of the invention may comprise, in the bilayer or the non-bilayer (e g , aqueous) compa ⁇ ment, compounds that are not "structural components" as defined herein
  • ⁇ -tocopherylphosphocholine is represented by the general Formula (I):
  • ⁇ -tocopherylphosphocholine shown in Figure (I) has three asymmetric carbon atoms in its structure and. consequently, has 8 stereoisomers (4 pairs of enantiomers).
  • the compound as used in accordance with the present invention may be one or a mixture of two or more of the biologically-active stereoisomers. Each of the stereoisomers is biologically active. See, for example. Kiyose et al. (1995) Lipids 30(11): 1015-1018.
  • the invention encompasses use of all of the stereoisomers of the compound of Formula (I), as well as the pharmaceutically acceptable salts of the compounds (including the stereoisomers) of Formula (I). Unless specifically qualified otherwise, reference herein to "compound of Formula (I)" or to " ⁇ -tocopherylphosphocholine” means any combination of the stereoisomers.
  • a substantially pure stereoisomer may be obtained, substantially free of the other 7 stereoisomers, by the application of art-known resolution methodology such as, for example, chromatography using chiral columns or using in the preparation stereoisomerically appropriate precursors.
  • ⁇ -tocopherol as it occurs in nature and can readily be obtained from natural sources, and which is commonly referred to as - ⁇ -tocopherol, is the substantially pure 2R,4'R,8'R - stereoisomer (the one with the R configuration at all of the asymmetric centers, employing the standard Cahn-Ingold-Prelog system for designating configurations).
  • ⁇ -tocopherylphosphocholine can be carried out by art- recognized procedures, beginning with ⁇ -tocopherol. See, for example, Japanese Public Patent Disclosure No. 1-211578, published August 24, 1989.
  • the compound of Formula (I), as a substantially equimolar mixture of the 8 stereoisomers, can be prepared from commercially available, synthetic ⁇ -tocopherol, so-called "JJ- ⁇ -tocopherol, " as a starting material.
  • This d, /- ⁇ -tocopherol is a substantially equimolar mixture of the 8 stereoisomers of ⁇ -tocopherol.
  • the salts of ⁇ -tocopherylphosphocholine are pharmaceutically acceptable salts and include acid addition salts, for example, those made with hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, carbonic, acetic, citric or lactic acids, as well as salts made with bases, such as, for example, sodium hydroxide, potassium hydroxide or calcium hydroxide.
  • acid addition salts for example, those made with hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, carbonic, acetic, citric or lactic acids
  • bases such as, for example, sodium hydroxide, potassium hydroxide or calcium hydroxide.
  • the salts of the invention are made by conventional means well known to those of ordinary skill in the art.
  • Formula (I) and salts thereof may be used alone or in combination with other compatible medicaments.
  • A. Antiviral ⁇ -tocopherylphosphocholine and its pharmaceutically acceptable salts possess antiviral activity and can be used in the control or prevention of viral infections, e.g. , of herpes simplex and polio viral infections.
  • the in vitro activity of the compound CPR-2001 in inhibiting Herpes simplex virus type-1 and Poliovirus type III is demonstrated in the assays illustrated in Examples 1A and IB.
  • the first assay is for inhibition of cytopathic effects (CPE) caused by viral- infection of the cells and the second is a standard viral yield reduction assay.
  • This yield reduction assay is a modification of the general method described by Erlich et al. , Ann. NY Acad. Sci. 130, 5-16 (1965).
  • the two assays demonstrate the marked antiviral activity of compound CPR-2001. No apparent cytotoxicity at antivirally effective levels has been observed.
  • CPR-2001 and its pharmaceutically acceptable salts are effective particularly for the treatment of an infection by Herpes viruses (particularly Herpes simplex, including its two immunological types, HSV-1 and HSV-2), and Poliovirus (all immunological variants), although infections caused by other viruses, such as, for example, Varicella-zoster virus.
  • Herpes viruses particularly Herpes simplex, including its two immunological types, HSV-1 and HSV-2
  • Poliovirus all immunological variants
  • viruses such as, for example, Varicella-zoster virus.
  • CMV Cytomegalovirus
  • EBV Epstein-Barr virus
  • Picornaviruses Picornaviruses
  • Rhinovirus Human papilloma viruses and Hepatitis viruses
  • This invention thus provides a method of treating a host mammal having a susceptible viral infection comprising administering to said mammal an antiviral effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof.
  • the invention also provides pharmaceutical compositions comprising an antiviral-effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • Anti-Fungal ⁇ -tocopherylphosphocholine and its pharmaceutically acceptable salts possess antifungal activity and can be used in the control or prevention of fungal infections.
  • infections caused by other fungi such as, for example, Aspergillus, Rhizopus, Dermatophytes and Histoplasma spp. , among others, may also be effectively treated.
  • the subject invention thus provides a method of treating a host mammal having a susceptible fungal infection which comprises the administration to said mammal of an antifungal effective amount of ⁇ -tocopherylphosphochohne or a pharmaceutically acceptable salt thereof
  • the invention also provides pharmaceutical compositions comprising an antifungal-effective amount of ⁇ - tocopherylphosphochohne or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier
  • Inflammation is a complex process, involving a variety of cell types including macrophages
  • macrophages See, for example, S L Kunkel, ' Inflammatory Cytokines' , pp 1-15, in Manual of Vascular Mediators, P A Ward, Editor, produced by the publishers of Hospital Practice References relative to macrophages are numerous, including, for example, J Immunology 119, 950-954 (1977) and Cell 15, 261-267 (1978)
  • Macrophages are activated by injection and by a wide variety of no n- infectious irritants and proinflammatory agents Upon activation, macrophages participate in a variety of reactions They may phagocuize bacteria and kill them by either oxygen-dependent or oxygen-independent pathways They are activated to increase oxygen consumption and production of reactive oxygen species (for example, superoxide) In addition, they release a va ⁇ erv of inflammatory cytokines including several interleukins and tumor necrosis factor (TNF-0) Inhibition of any of these pathways can lead to reduced inflammation
  • the RAW 264 7 cell line (ATCC TIB 71) is a mu ⁇ ne monocyte/macrophage line that shows many of the differentiative functions of a macrophage
  • the cells are capable of phagocytosis and undergo an oxidative burst in response to appropriate signals Agents that inhibit the activation of these cells in vitro are therefore inhibitors of critical steps in inflammatory processes
  • Activation of macrophages and other phagocytic cell types initiates a cascade of actions that include increased oxygen consumption (respiratory burst) and production of oxygen radicals These events can be measured in a variety of ways, including chemiluminescence based on the addition of luminol (see M A Trush et al ,
  • CPR-2001 exhibits marked dose related anti-inflammatory activity
  • CPR-2001 and its pharmaceutically acceptable salts may thus be utilized for ameliorating inflammatory conditions such as for example, rheumatoid arthritis, osteoarthritis, acute bursitis, tendinitis and allied inflammatory disorders
  • Platelet-activating factor (PAF, l-O-C 16 . 18 alkyl-2(R)acetyl-sn-glyceryl-3- phosphocholine), its effect on platelets and its biological effects have been the subject of many reported studies since its discovery in the early 1970's.
  • PAF literature reviews include those of Dean A. Handley, Pharmacological Methods in the
  • PAF has been shown to be a mediator of inflammation and has been found in lung fluids of asthma patients. It is a chemoattractant and encourages the migration of neutrophils and eosinophils to sites of inflammation and to the airways of asthmatic patients. Moreover, PAF has been shown to be a powerful bronchoconstrictor of the airways of asthmatic patients. In addition PAF has been found in the psoriatic lesions of psoriasis patients. Accordingly, antagonists of PAF have potential utility in treating inflammatory states including rheumatoid arthritis, asthma and psoriasis, and immediate and delayed type hypersensitivity reactions.
  • Example 4 demonstrates the inhibition of the constrictor activity of PAF on the test animal airway.
  • the subject compounds are thus useful in ameliorating PAF-related disease states, including PAF-induced bronchial asthma.
  • the subject invention thus provides a method of inhibiting PAF activity in a host mammal having a susceptible PAF-induced pathophysiological condition which comprises the administration to said mammal of a PAF-antagonist effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof.
  • the invention also provides pharmaceutical compositions comprising a PAF-antagonist- effective amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • UV Radiation Blocker When an electron in a molecule is promoted to a higher energy level, the absorption of UV or visible radiation occurs. As a consequence, the electronic structure of a molecule governs both the efficiency and electromagnetic frequency of the absorption.
  • conjugated double bonds are the key feature of molecules which are effective UV-absorbers (i.e. , blockers). Molecules which lack conjugated double bonds rarely absorb in the UV-A region (320 to 400 nm) or UV-B region
  • UV-A and UV-B regions Compounds which efficiently absorb radiation in either or both of the UV-A and UV-B regions are useful in sunscreen and other dermatological products, including make-up, because they block the damaging effects sun exposure has on the skin.
  • CPR 2001 shows a marked absorption in the UV-B region, the absorption stretching from about 270 nm to about 310 nm and having a maximum absorption at about 295 nm.
  • the invention includes a pharmaceutical composition for blocking UV radiation, the composition comprising one or more of the subject compounds in combination with a pharmaceutically-suitable carrier, preferably a carrier suitable for topical application to the skin, lips, or hair.
  • a pharmaceutically-suitable carrier preferably a carrier suitable for topical application to the skin, lips, or hair.
  • Aerosolized delivery of drugs to the lung airways has been employed in clinical practice for many years
  • a variety of medicinal agents have been utilized in aerosol therapy including, for example, mucolytics such as N-acetylcysteine and synthetic surfactant formulations contaimng DPPC and DSPC for treating patients with RDS and ARDS
  • mucolytics such as N-acetylcysteine
  • synthetic surfactant formulations contaimng DPPC and DSPC for treating patients with RDS and ARDS
  • the advantage of an aerosol delivery system is its wide-spread drug delivery to all lung regions intermittently over extended periods of time
  • ⁇ -tocopheryl-phosphocholine be utilized as a dispersion m an aerosolized liquid formulations, ready for use It is also much preferred that the formulation be suitable for use with art-recognized inhalation devices, such as a nebulizer or a metered-dose inhaler, without anv further manipulation by the user
  • the subject compounds may also be prepared as a sterile lyophihzed powder of appropriate average diameter for direct inhalation or, alternatively, the powder is first reconstituted with an aqueous carrier such as water or, preferably, a saline solution of about 0 4 to about 0 9 percent sodium chloride as a dispersion and delivered via an appropriate nebulizer or inhalant system
  • the recommended average diameter of the particulate dispersed powder along any axis is about 1 to 10 microns ( ⁇ M) and, preferably, about 5 to 10 ⁇ M
  • ⁇ -tocopheryl-phosphocholine is included in an amount from about 0 1 to
  • ⁇ -tocopheryl-phosphochohne can be utilized in formulations which contain other ingredients suitable for inhalation therapy, for example, phosphohpids, such as phosphatidylchohnes, phosphatidylethanolamines, phosphatidylglycerols and the like
  • formulations which contain the subject compounds and tyloxapol, glycerol and/or fatty alcohols, such as hexadecanol, vitamin E, all-trans-retinol, cholesterol and the like are intended to be utilized when it is found to be desirable to do so
  • formulations which comprise ⁇ -tocopheryl-phosphochohne and pulmonary surfactant proteins such as pulmonary surfactant protein A (SP-A), pulmonary surfactant protein B (SP-B), pulmonary surfactant protein C (SP-C), pulmonary surfactant protein D (SP-D), and the like are intended to be utilized when it is found to be desirable to do so
  • formulations which comprise the subject for example, phosphohpids,
  • the compounds of Formula I are efficacious in the treatment of respiratory diseases in which mucus viscosity or accumulated mucus and/or pulmonary inflammation is a major or contributing symptom
  • respiratory diseases include, without limitation, bronchitis, cystic fibrosis and asthma (see American Thoracic Society Symposia Excerpts, 1994 International Conference)
  • the subject compounds are also efficacious in the treatment of chronic obstructive pulmonary disease, a disease also associated with increased pulmonary mucous secretion (see Ferguson and Chermiack (1993) Management of COPD, New Eng J Med , pp 1017-1021)
  • the subject compound are efficacious m treating respiratory diseases characterized by a deficiency of pulmonary surfactant
  • diseases include without limitation, ARDS, RDS, emphysema, and pulmonary fibrosis
  • the invention also entails pharmaceutical compositions comprising ⁇ -tocopherylphosphochohne or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier therefor and, optionally, other therapeutically active substances in addition to the ⁇ -tocopherylphosphochohne or salt thereof
  • the pharmaceutical compositions of the invention comprise an amount of ⁇ -tocopherylphosphocholine or a pharmaceutically acceptable salt thereof that is effective to treat a viral or fungal infection or inflammation in a mammal suffering therefrom, to which the composition is administered, or effective to antagonize PAF- activities (and treat the related pathological conditions) in a mammal, to which the composition is administered.
  • the carrier In a pharmaceutical composition of the invention, the carrier must be pharmaceutically acceptable in the sense of being compatible with other ingredients in the particular composition and not deleterious to the recipient thereof.
  • the compositions include those suitable for oral, topical, rectal or parenteral (including subcutaneous, intramuscular, intradermal and intravenous) administration.
  • the pharmaceutical compositions comprise the active ingredient ( ⁇ -tocopherylphosphocholine or pharmaceutically acceptable salt thereof) presented in unit dosage form.
  • the term "unit dosage” or "unit dose” is denoted to mean a predetermined amount of the active ingredient sufficient to be effective for treating each of the indicated activities.
  • Preferred unit dosage formulations are those containing a daily dose, daily sub-dose, or an appropriate fraction thereof, of the administered active ingredient.
  • compositions may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid or solid carrier and then, if necessary, shaping the product into the desired unit dosage form.
  • compositions of the present invention suitable for oral administration may be presented as discrete unit dosages, e.g. , as capsules, cachets, tablets, boluses, lozenges and the like, each containing a predetermined amount of the active ingredient: as a powder or granules; or in liquid form, e.g. , as a collyrium, suspension, solution, syrup, elixir, emulsion, dispersion and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g. , a powder or granules, optionally mixed with accessory ingredients or excipients, e.g. , binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
  • Compositions suitable for parenteral administration conveniently comprise a sterile injectable preparation of the active ingredient in, for example, a solution which is preferably isotonic with the blood of the recipient.
  • Useful formulations also comprise concentrated solutions or solids containing the active ingredient which upon dilution with an appropriate solvent give a solution suitable for parenteral administration.
  • the parenteral compositions include aqueous and no n- aqueous formulations which may contain conventional adjuvants such as buffers, bacteriostats, sugars, thickening agents and the like.
  • the compositions may be presented in unit dose or multi-dose containers, for example, sealed ampules and vials.
  • Compositions suitable for topical or local application comprise the active ingredient formulated into pharmaceutically-acceptable topical vehicles by conventional methodologies.
  • compositions suitable for inhalation administration include drops, collyriums, aerosol sprays, lotions, gels, ointments, plasters, shampoos, transferosomes, liposomes and the like.
  • Compositions suitable for inhalation administration include a micronized powder or liquid formulation having a particle size in the range of from about 5 microns or less to about 500 microns, for rapid inhalation through the nasal or oral passage from a conventional inhalation squeeze or spray container.
  • Suitable liquid nasal compositions include conventional nasal sprays, nasal drops and the like, of aqueous solutions of the active ingredient and optional adjuvants.
  • the active ingredient may be provided in the form of a liposome.
  • compositions of this invention may further include one or more optional accessory ingredients(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, colorants, binders, surfactants, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
  • accessory ingredients e.g., diluents, buffers, flavoring agents, colorants, binders, surfactants, thickeners, lubricants, suspending agents, preservatives (including antioxidants) and the like.
  • the amount of active ingredient required to be effective for each of the indicated activities will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner.
  • the factors to be considered include the species and sex of the mammal, the condition being treated, the route of administration, the nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered.
  • the pharmaceutical compositions of this invention contain from about 0.5 to about 500 mg and, preferably, from about 5 to about 350 mg of the active ingredient, preferably in a unit dosage form, for each of the indicated activities.
  • a suitable effective dose is in the range of about 0.1 to about 200 mg/kg body weight per day, preferably in the range of about 1 to about 100 mg/kg per day, calculated as the non-salt form of ⁇ -tocopherylphosphocholine.
  • the total daily dose may be given as a single dose, multiple doses, e.g. , two to six times per day, or by intravenous infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary.
  • a dose range would be about 7.5 to about 1500 mg per day, and a typical dose would be about 800 mg per day. If discrete multiple doses are indicated, treatment might typically be 200 mg of a compound of Formula (I) given 4 times per day.
  • the subject compounds are preferably utilized at concentrations of from about 0.1 % to about 5.0% by weight.
  • LIPOSOMES As also indicated above, still another aspect of the present invention is ⁇ - tocopherylphosphocholine or a pharmaceutically acceptable salt thereof in the novel physical form of a liposome (i.e., as a structural component of the bilayer of the liposome).
  • Liposomes which are microscopic, man-made spheres of fatty material, were first formed from phospholipids. Although it has been known for a long time that other lipids, such as cholesterol, dicetyl phosphate, and stearylamine can be incorporated into liposome membranes (i.e. , liposome bilayers) that contain phospholipids, only phospholipids were known to be "structural components" of the membranes, i.e. , to form liposomes when suspended in aqueous media without any other lipophilic or amphophilic substance. Subsequently, it was found that certain ammonium salts could also form liposomes.
  • dialkyl dimethyl ammonium salts will form liposomes if the alkyl chains are 12 carbons or longer.
  • a series of novel structures based on the chemical structure of phospholipids, that also form liposomes.
  • much information has been gained on the type of chemical structure necessary for liposome formation (i.e. , to be a structural component of a liposome bilayer).
  • the liposome-forming molecule must be one with two alkyl chains and a polar headgroup, although there are some exceptions to this rule.
  • fatty acids which have only a single alkyl chain, will form liposome structures under conditions where they dimerize through hydrogen-bonding.
  • ⁇ -tocopherylphosphocholine does not suggest that it, or a salt thereof, would be capable of forming a liposome when suspended alone (i.e. , without another lipophilic or amphophilic compound) in an aqueous medium subjected to liposome-forming conditions, ⁇ -tocopherylphosphocholine has only a single alkyl-like moiety, namely the tocopheryl group, connected to a phosphocholine group.
  • Molecules of this type usually form micelles.
  • the ability, first disclosed in this application, of ⁇ - tocopherylphosphocholine to form a liposome might be explained retrospectively by the bulky nature of the tocopheryl group, which in molecular terms might be functionally equivalent to a dialkyl structure containing only straight hydrocarbon chains. Even this consideration, however, would not lead to a prediction that ⁇ - tocopherylphosphocholine or a salt thereof would form a liposome. It might form a number of more complex micellar structures, or might not even suspend at all appropriately for liposome formation in an aqueous medium. Therefore, the capacity of ⁇ -tocopherylphosphocholine and salts thereof to form a liposome by themselves
  • Example 6 hereafter details the preparation of the subject liposomes of the invention.
  • This novel liposome form of ⁇ -tocopherylphosphocholine or a salt (preferably pharmaceutically acceptable salt) thereof has beneficial pharmaceutical applications.
  • it advantageously may be incorporated into topical pharmaceutical compositions of the subject compounds such as ointments, salves, creams and creamy liquids, dispersions and the like.
  • the subject liposomes are also suitable for transdermal application of other therapeutic agents by enhancing the absorption of such agents through the skin of humans and animals. As with other known liposomes, such agents would be encapsulated within the internal aqueous compartment of the liposome structure.
  • liposomes may also be prepared from mixtures of the subject compounds with other amphophilic therapeutic agents, such as, for example, phospholipids, cholesterol and certain drugs such as Amphotericin B and Doxorubicin.
  • the preparation strategy would require the mixing of the subject compound with the other component in an organic solvent. This initial mixing would then be followed, for example, by evaporation of the solvent to give a mixed film of the components. This film would then be swollen in aqueous medium as described in Example 6.
  • Other techniques known in the art could be employed to make liposomes with ⁇ -tocopherylphosphocholine or salts thereof.
  • Liposome formation with ⁇ -tocopherylphosphocholine and salts thereof would also respond to known size-control methods in addition to sonification. Principal among these is the method of extrusion through polycarbonate membranes, or on an industrial scale, filtration with ceramic filters.
  • the liposomes can also be used to encapsulate medicinal compounds displaying inflammatory side-effects, thereby mitigating the unwanted inflammatory activity. Encapsulating such compounds within liposomes comprising the subject compounds as structural components of the liposomes diminishes inflammatory side- effects.
  • the subject invention thus provides ⁇ -tocopherylphosphocholine or a salt thereof in the form of a liposome and provides liposomes which comprise this compound or a salt thereof as a structural component of the bilayer (membrane).
  • the invention also provides pharmaceutical compositions comprising said liposome in an effective amount for each of the indicated activities and a pharmaceutically effective carrier, particularly for topical application, as well as such pharmaceutical compositions wherein the ⁇ -tocopherylphosphocholine liposome contains another drug encapsulated within the liposome 's structure.
  • Example 1 A- Antiviral: Antiviral Assay: Herpes Simplex Virus Type 1
  • the cultures are incubated at 37 °C with 5% CO 2 until a monolayer of cells is formed on the bottom of each well (usually overnight). 3.
  • the media is removed from the wells by aspiration.
  • the cells are refed with media containing the desired concentration of test compound CPR-2001 (100 ⁇ M final concentration), media only (cell control and virus control samples), or solvent (9: 1, DMSO:ethanol) at a final concentration of 0.33 % .
  • Compound CPR-2001 is dissolved at a stock concentration of 30 mM and diluted 300- fold.
  • the cell count wells receive media only.
  • the cultures are incubated for 24 hr at 37°C with 5 % CO 2 .
  • each well is scored for the presence of toxicity (cells exposed to compound but not infected) as indicated by rounding and/or detachment of the cells from the plate. Scoring is as follows: 1 + , ⁇ 25% of cells rounded; 2 + , 25 to 50% of cells rounded; 3 + , 75% of cells rounded; 4 + , 100% of cells rounded. 6.
  • the media is removed from the cell count wells by aspiration.
  • the monolayer is rinsed 2x with 100 ⁇ l of trypsin:EDTA solution and is incubated at 37°C until the cells can be suspended (usually 2-5 minutes). The cells are counted in a hemocytometer to determine the number of cells per well.
  • the remaining wells, except the cell control samples, are infected with 2 plaque forming units per cell of the standard HSV-1 KOS strain of virus using the following procedure.
  • the media is aspirated from the wells.
  • Media (50 ⁇ l) with 2% serum containing the desired amount of virus is added to each well.
  • the cultures are then incubated at 37°C with 5 % CO 2 for 60 minutes.
  • the plate is gently shaken at 15 to 30 minute intervals to insure adequate distribution of the virus and the cells are exposed to the virus.
  • the excess viral solution is removed by aspiration and the cells are refed (200 ⁇ l/well) with media containing 2% serum (cell control and virus control samples), media containing 2% serum and compound CPR- 2001, or media containing the vehicle (solvent control) at the same concentration in the test compound wells.
  • the plate is incubated at 37°C with 5% C0 2 for 48 hr.
  • each well is scored for the presence of CPE as indicated by rounding and/or detachment of the cells from the plate. Scoring is as follows: 1 + , ⁇ 25 % of cells rounded; 2 + , 25 to 50% of cells rounded; 3 + , 50 to 75% of cells rounded; 4 + , 100% of cells rounded.
  • the yield of infectious virus in each well is measured by standard plaque assay on Vero cells (ATCC #CCL81). Virus is released from the cells by freezing and thawing (-80°C and 37°C) three times. Serial 10-fold dilutions of each sample are made in media containing 2% serum. Vero cell monolayers of Buffalo Green cells in 6 well cell culture plates (lxlO 6 cells/well) are infected with 100 ⁇ l of each dilution (separate well per dilution) . Following a 1 hr adsorption period with gentle rocking at 37°C, the virus inoculum is removed, the monolayer is overlaid with 2 ml of
  • the titer plates are incubated at 37 °C with 5% CO, until viral plaques become visible (3-4 days).
  • the cells are fixed by adding 2 ml of phosphate buffered saline (PBS) containing 10% (v:v) formalin.
  • PBS phosphate buffered saline
  • the methylcellulose, media and formalin are removed by aspiration. Two mis of PBS
  • Table 1A shows the results of the CPE inhibition assay. Exposure of the cells to compound CPR-2001 markedly inhibits the development of viral cytopathic effect and is not toxic. The DMSO vehicle used for dissolving the compound is not toxic and does not prevent the development of viral CPE, indicating the vehicle has no antiviral activity. The cell control and virus controls also give the expected results. Table 1A also shows the data from the yield reduction assay. As expected, the DMSO (vehicle control) has no effect on viral yield (lxlO 6 pfu/ml) compared to the virus control (lxlO b pfu/ml). In contrast, treatment of the cells with compound CPR- 2001 reduces the yield to 5.5 x 10 4 . This represents an 18-fold reduction in infectious virus compared to the virus and vehicle control samples and represents a substantial antiviral effect.
  • Poliovirus Type III The antiviral effect against Poliovirus Type III is demonstrated in similar assays by following the foregoing steps 1 through 10 of Example 1A except for step
  • the cells are then infected.
  • the Poliovirus Type III vaccine strain is obtained from Dr. G. Sedmak, City of Milwaukee, Public Health Department Virology Laboratory.
  • the cells are infected as follows.
  • the medium is aspirated and replaced with 50 ⁇ l of medium (2% serum) containing 100 TCID50 doses of Poliovirus Type III.
  • the cultures are then incubated at 37°C with 5 % CO 2 for 60 minutes.
  • the plate is shaken gently at 15 minute intervals to insure adequate distribution of the virus and the cells are exposed to the virus.
  • the media containing virus is removed by aspiration and replaced with 200 ⁇ l of media containing compound CPR-2001 (100 ⁇ M), or media only (virus control and vehicle control wells).
  • the cell control wells received media only (no virus).
  • Table IB shows the results of the CPE inhibition assay. Exposure of the cells to compound CPR-2001 inhibits the development of viral CPE compared to the virus control and vehicle control samples. The cell control and virus control samples give the expected results. Table IB also shows the data from the yield reduction assay.
  • Compound CPR-2001 reduces the yield of infectious virus by 10.7-fold compared to the virus control, which represents a substantial antiviral effect.
  • Cells (either C. albicans or C. neoformans) are placed in wells of a 96- well plate within a total volume of 90 ⁇ l. Cells are grown in RPMI-1640 plus 10% FCS.
  • Fungizone (2.5 ⁇ g/ml and 5.0 ⁇ g/ml); and CPR-2001 (1 ⁇ g/ml, 10 ⁇ g/ml and 100 ⁇ g/ml). 3 Incubate for 48 hours at 37 °C
  • Chemiluminescence (CL) Assay 1 Cell line RAW 264 7 (ATCC TIB 71),
  • Retinoic acid is used topically to treat acne, especially inflammatory acne. An increase in the inflammation occurs before the acne improves. When encapsulated within CPR 2001 , the inflammatory action of the retinoic acid is significantly reduced.
  • PAM-212 cells were exposed to either retinoic acid or retinoic acid encapsulated within CPR 2001 liposomes. Encapsulating retinoic acid within CPR 2001 liposomes had no effect on the activity of the retinoic acid to inhibit cell proliferation of PAM-
  • 5-fluorouracil In dermatology, 5-fluorouracil (5-FU) is used topically to treat actinic keratinosis, a premalignant skin condition caused by excessive exposure to the sun. Like retinoic acid, when 5-FU is applied to the skin it also causes a primary inflammatory response prior to reversing the actinic keratinosis. When applied topically to the ear of the mouse, 5-FU increases the inflammatory response due to
  • alveolar macrophages Activation of alveolar macrophages is a critical component of pulmonary inflammation In their activated state, alveolar macrophages secrete a variety of inflammatory mediators and reactive oxygen species In the present in vitro model of inflammation, mouse macrophages are activated by phorbol- 12-my ⁇ state-13-acetate
  • the intensity of macrophage activation in the presence or absence of the subject compounds is determined by measuring the respiratory burst (release of reactive oxygen species) of the macrophages.
  • the RAW 264.7 cell line (American Type Culture Collection, Rockville, Maryland, USA, Accession No. TIB 71) is a murine monocyte/macrophage line, the cells of which show many of the differentiative functions of a macrophage. Like macrophages, the cells are capable of phagocytosis and undergo an oxidative burst
  • Cell line RAW 264.7 (ATCC TIB-71 , attachment dependent); Culture medium: Dulbecco's Modified Eagle's Medium (DMEM, Sigma Chemical Co. Cat. No. D-7777) with 10% Fetal Bovine Serum (FBS);
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • Cell line is passaged when approximately 80% confluent; with trypsin (1 mg/mL) and ethylenediamine tetraacetic acid (EDTA) (1 mM in Ca-Mg free Hank's balanced salt solution); at 1:4 to 1:5 split;
  • trypsin (1 mg/mL)
  • EDTA ethylenediamine tetraacetic acid
  • test compound dissolved in phosphate buffered saline (PBS), or in dimethyl sulfoxide (DMSO) for final concentration levels ranging from 1 to 30 mM;
  • PBS phosphate buffered saline
  • DMSO dimethyl sulfoxide
  • CPR 2001 has utility in respiratory diseases in which inflammation is a major or contributing factor, such as in bronchial asthma, pulmonary fibrosis, ARDS, emphasema, pneumonia, and cystic fibrosis.
  • respiratory diseases in which inflammation is a major or contributing factor, such as in bronchial asthma, pulmonary fibrosis, ARDS, emphasema, pneumonia, and cystic fibrosis.
  • administration of CPR 2001 inhibits inflammation in an accepted in vitro model in a dose-dependent fashion.
  • the procedure, which delivers the test compound to mice as an aerosol, is a modified assay for the evaluation of mucolytic activity in mice as described by Chand et al , previously cited. 1.
  • the test animals are Hsd: lCR(CD-l) mice that weigh approximately
  • test compound is diluted in normal saline to a concentration of 25 mg/mL and administered using a human De Vilbiss atomizer Model No. 15 at approximately 100 mg/kg.
  • the control group receives saline.
  • the nozzle is placed in the mouth of the mouse and 2 spray doses are given.
  • the amount of phenol red secreted into the trachea is quantitated photometrically at 546 nm.
  • Calibration of the dose delivered is done by measuring the volume of five sprays per tube in five separate tubes. The average amount is 60.4 ⁇ L/spray with a range of 58-63 ⁇ L.
  • a common in vivo model for the evaluation of surfactants is lung compliance in premature fetal rabbits This method is described in Revak et al , Am Rev Respir Dir (1986) 134 1258-1265 In this assay, the effectiveness of the subject compounds as lung surfactants is determined by evaluating dynamic lung compliance in premature fetal rabbits which are known to have difficulty initiating breathing due to low levels of natural lung surfactant
  • test animals are fetal rabbits delivered prematurely at 27 days of gestation
  • test solution is prepared by suspending the Formula I compound (120 g) in 6 mL of 10 mM HEPES buffer with sodium chloride (150 mM) at a pH of 7 4
  • test solution 3 0 2 mL is administered to the fetal rabbit before breathing commenced by endotracheal instillation and then monitored for breathing
  • control group receives saline
  • the fetal rabbit is ventilated and monitored for breathing
  • the following is a protocol for the evaluation of anti-histamine activity in anesthetized guinea pigs.
  • Guinea pigs of 650-1000 g are used to facilitate catheterization of the jugular vein and carotid artery.
  • the guinea pigs are anesthetized with 35-
  • pentobarbital sodium 45 mg/kg pentobarbital sodium.
  • anesthetic additions are made during the course of the intervention.
  • the cutdown is a ventral medial incision over the cervical area so that the trachea, jugular vein and carotid artery can be cannulated.
  • the animals are immediately attached to a volume regulated Harvard ® rodent respirator,
  • Model 683 via a tracheostomy and the respirator is set at 60 respirations per minute and a volume of 8 ml/kg to maintain a normal arterial P-CO 2 of approximately 40 mmHg.
  • Pancuronium a muscle relaxant, is then given IV at a dose of 0.2 mg/kg to prevent spontaneous breathing.
  • a tube is connected to the respirator pump and the endotracheal catheter is attached to a pressure transducing strain gauge and then to a 2-channel Gilson ® physiological recorder.
  • One channel of the recorder inscribes the pressure tracing from the airway; the second channel inscribes the pressure tracing from a similar strain gauge attached directly to a catheter inserted into the carotid artery.
  • TPR Total pulmonary resistance
  • Example 8 - Anti-PAF Activity The following is a protocol for the evaluation of anti-PAF activity in anesthetized guinea pigs. 1. Guinea pigs of 650-1000 g are used to facilitate catheterization of the jugular vein and carotid artery. The guinea pigs are anesthetized with 35- 45 mg/kg pentobarbital sodium. When or if the recordings are unstable, anesthetic additions are made during the course of the intervention. The cutdown is a ventral medial incision over the cervical area so that the trachea, jugular vein and carotid artery can be cannulated.
  • the animals are immediately attached to a volume regulated Harvard ® rodent respirator, Model 683, via a tracheostomy and the respirator is set at 60 respirations per minute and a volume of 8 ml/kg to maintain a normal arterial P-CO 2 of approximately 40 mmHg.
  • Pancuronium a muscle relaxant, is then given IV at a dose of 0.2 mg/kg to prevent spontaneous breathing.
  • a tube is connected to the respirator pump and the endotracheal catheter is attached to a pressure transducing strain gauge and then to a 2-channel Gilson ® physiological recorder.
  • TPR Total pulmonary resistance
  • the animal After the anesthetic and muscle relaxant are given, the animal is allowed to stabilize. The airway is gently suctioned with a syringe, and the lungs are briefly inflated by closing the expiratory port on the ventilator until the pressure is approximately three times resting pressure. When the pressure returns to a steady state, this TPR is considered control pressure.
  • compositions containing CPR- 2001 or a pharmaceutically acceptable salt thereof as the active ingredient, are each prepared according to art recognized methodologies in pharmacy.
  • the active ingredient is dissolved in the water and the pH is adjusted to the proper value by the buffering agent.
  • the solution is filtered and sterilized by autoclaving before being sealed under sterile conditions in ampoules.
  • the components are thoroughly mixed and then packed into a two-part gelatin capsule.
  • Active ingredient (10-50 microns) 5.0 mg
  • the active ingredient premicronized to a fine particle size between 1-10 ⁇ m in diameter, is blended with normal tabletting grade lactose in a high energy mixer.
  • the powder blend is filled into appropriately sized hard gelatin capsules on a suitable encapsulating machine.
  • the contents of the cartridges are administered using a powder inhaler.
  • the active ingredient ⁇ -tocopheryl-phosphocholine
  • a sufficient quantity of sterile isotonic saline to yield a solution which is 5 % by weight Formula I compound.
  • the solution is administered in the form or an aerosolized metered-dose via a suitable nebulizing device.
  • the active ingredient is dissolved in some of the water with stirring.
  • the sorbitol solution, flavoring agent and the rest of the water are added.
  • the syrup is clarified by filtration through suitable cellulosic filter pads.
  • Plasticized Hydrocarbon Gel 100 " ⁇ " " ⁇ “” ⁇ M ⁇ m TM ⁇ ⁇ ⁇ ” ⁇ " ⁇ — - ⁇ —
  • the active ingredient finely sieved, is uniformly blended with the plasticized hydrocarbon gel, commercially available as Plastibasee, a polyethylene and mineral oil gel base.
  • CPR-2001 Five micromoles of CPR-2001 are dissolved in about 1 ml of chloroform. The solution is transferred to a 16 x 120 mm screw-capped tube. The tube is placed, uncapped, inside a 1 x 8 inch boiling tube with B24/40 standard glass fitting. Inside the boiling tube, in the space between the boiling tube and the smaller screwcapped tube, there is a small amount of distilled water to aid thermal contact between the inner and outer tube.
  • the boiling tube is placed on a rotary evaporator under reduced pressure to evaporate the chloroform and leave the lipid as a thin film in the lower portion of the screw-capped tube.
  • the boiling tube is partly immersed in a water bath at 37 °C during the evaporation to aid the process.
  • the screw-capped tube is removed from the boiling tube, and one ml of aqueous solution is added to it.
  • the tube is capped and immersed in a water bath at 75 °C to aid swelling of the lipid. Swelling of the film is observed to occur when the film becomes cloudy, and is, with agitation, dislodged from the walls of the tube to give a cloudy suspension in the aqueous medium.
  • This suspension is found to consist of liposomes by the application of two techniques. First, laser-light scattering, or QELS, reveal the presence of particles of 658 nm in diameter in one determination, 1468 nm in another. Second, electron microscopy shows that these particles have the characteristic concentric lamellar structure of liposomes.
  • the bath is switched on and the exact position of the tube, and the level of water in the bath, are adjusted to achieve maximum agitation of the tubels contents.
  • the bath is left to sonicate the sample for about 10 minutes.
  • the solution becomes less turbid and viscous, and QELS reveal a particle size of the liposomes of 102 nm. Electron microscopy of the liposomes show that the particles are still multilamellar, but smaller.
  • Liposomes of the hydrogen iodide acid addition salt of ⁇ - tocopherylphosphocholine are obtained by following the foregoing procedures.

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Abstract

On a découvert que l'α-tocophérylphosphocholine et ses sels possédaient des pouvoirs antiviraux, antifongiques, anti-inflammatoires, antagonistes du PAF, et bloquants du rayonnement ultraviolet. Ces composés sont efficaces pour traiter une grande variété d'affections, notamment des maladies des voies aériennes et des voies pulmonaires. Ce composé et ses sels se sont également avérés pouvoir former des liposomes. L'invention décrit ainsi des méthodes pour traiter des infections virales et fongiques, des troubles respiratoires, des troubles inflammatoires et des états pathophysiologiques causés par l'activité de PAF chez un mammifère en lui administrant de l'α-tocophérylphosphocholine ou un sel pharmaceutiquement acceptable de celle-ci. L'invention concerne également des compositions pharmaceutiques comprenant de l'α-tocophérylphosphocholine ou un sel pharmaceutiquement acceptable de celle-ci et un excipient pharmaceutiquement acceptable. D'autre part, l'invention concerne des liposomes renfermant de l'α-tocophérylphosphocholine ou un sel de celle-ci comme constituant structural de la bicouche liposomique.
PCT/US1998/011818 1998-06-08 1998-06-08 Compositions pharmaceutiques contenant de l'alpha-tocopherylphosphocholine WO1999064015A1 (fr)

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PCT/US1998/011818 WO1999064015A1 (fr) 1998-06-08 1998-06-08 Compositions pharmaceutiques contenant de l'alpha-tocopherylphosphocholine
CA002334313A CA2334313A1 (fr) 1998-06-08 1998-06-08 Compositions pharmaceutiques contenant de l'alpha-tocopherylphosphocholine
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001247585A (ja) * 2000-03-03 2001-09-11 Nof Corp トコフェロール誘導体、その中間体、その製造方法及び用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01211578A (ja) * 1988-02-19 1989-08-24 Fuji Kagaku Kogyo Kk ビタミンｅ誘導体及びその製造法
US5114957A (en) * 1990-05-08 1992-05-19 Biodor U.S. Holding Tocopherol-based antiviral agents and method of using same
WO1997045098A2 (fr) * 1996-05-31 1997-12-04 Giorgio Panin Esters de la vitamine e pour application locale

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01211578A (ja) * 1988-02-19 1989-08-24 Fuji Kagaku Kogyo Kk ビタミンｅ誘導体及びその製造法
US5114957A (en) * 1990-05-08 1992-05-19 Biodor U.S. Holding Tocopherol-based antiviral agents and method of using same
WO1997045098A2 (fr) * 1996-05-31 1997-12-04 Giorgio Panin Esters de la vitamine e pour application locale

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 8940, Derwent World Patents Index; Class B03, AN 89-288873, XP002082121 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001247585A (ja) * 2000-03-03 2001-09-11 Nof Corp トコフェロール誘導体、その中間体、その製造方法及び用途

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