U.S. patent application number 11/220968 was filed with the patent office on 2006-08-24 for use of lipid conjugates in the treatment of infection.
Invention is credited to Saul Yedgar.
Application Number | 20060189571 11/220968 |
Document ID | / |
Family ID | 46322606 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189571 |
Kind Code |
A1 |
Yedgar; Saul |
August 24, 2006 |
Use of lipid conjugates in the treatment of infection
Abstract
This invention provides compounds and methods of use thereof in
suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell, including, inter alia, infection with
intracellular pathogens. Also provided are compounds and methods of
use thereof in suppressing, inhibiting, preventing, or treating an
infection in a subject.
Inventors: |
Yedgar; Saul; (Jerusalem,
IL) |
Correspondence
Address: |
PEARL COHEN ZEDEK, LLP
1500 BROADWAY 12TH FLOOR
NEW YORK
NY
10036
US
|
Family ID: |
46322606 |
Appl. No.: |
11/220968 |
Filed: |
September 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11220965 |
Sep 8, 2005 |
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11220968 |
Sep 8, 2005 |
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10952496 |
Sep 29, 2004 |
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11220965 |
Sep 8, 2005 |
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10627981 |
Jul 28, 2003 |
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10952496 |
Sep 29, 2004 |
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09756765 |
Jan 10, 2001 |
7034006 |
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10627981 |
Jul 28, 2003 |
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60174905 |
Jan 10, 2000 |
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60174907 |
Jan 10, 2000 |
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Current U.S.
Class: |
514/54 ;
514/56 |
Current CPC
Class: |
A61K 47/544 20170801;
A61K 31/727 20130101; A61K 31/739 20130101; A61K 47/61 20170801;
A61K 31/727 20130101; A61K 2300/00 20130101; A61K 31/739 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/054 ;
514/056 |
International
Class: |
A61K 31/739 20060101
A61K031/739; A61K 31/727 20060101 A61K031/727 |
Claims
1. A method of suppressing, inhibiting, preventing, or treating a
human immunodeficiency virus (HIV) infection in a subject
comprising the step of administering to said subject an effective
amount of a lipid or phospholipid moiety bound to a physiologically
acceptable monomer, dimer, oligomer, or polymer, and/or a
pharmaceutically acceptable salt or a pharmaceutical product
thereof.
2. The method according to claim 1, wherein said phospholipid
moiety is phosphatidylethanolamine and said physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan.
3. The method according to claim 1, wherein said phospholipid
moiety is dipalmitoyl phosphatidylethanolamine and said
physiologically acceptable monomer, dimer, oligomer, or polymer is
heparin.
4. The method according to claim 1, wherein said phospholipid
moiety is dipalmitoyl phosphatidylethanolamine and said
physiologically acceptable monomer, dimer, oligomer, or polymer is
chondroitin sulfate.
5. The method according to claim 1, wherein said phospholipid
moiety is dipalmitoyl phosphatidylethanolamine and said
physiologically acceptable monomer, dimer, oligomer, or polymer is
hyaluronic acid.
6. The method according to claim 1, wherein said phospholipid
moiety is dimyristoyl phosphatidylethanolamine and said
physiologically acceptable monomer, dimer, oligomer, or polymer is
hyaluronic acid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/952,496 filed Sep. 29, 2004, and a
continuation-in-part of U.S. application Ser. No. 10/627,981, filed
Jul. 28, 2003, which are continuation-in-part applications of U.S.
application Ser. No. 09/756,765, filed Jan. 10, 2001, which claims
priority from U.S. Provisional Application Ser. No. 60/174,905,
filed Jan. 10, 2000, and U.S. Provisional Application Ser. No.
60/174,907 filed Jan. 10, 2000, which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] This invention provides compounds and methods of use thereof
in suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell, including, inter alia, infection with
intracellular pathogens. Also provided are compounds and methods of
use thereof in suppressing, inhibiting, preventing, or treating an
infection in a subject.
BACKGROUND OF THE INVENTION
[0003] Lipid-conjugates are thought to inhibit the enzyme
phospholipase A2 (PLA2, EC 3.1.1.4). Phospholipase A2 catalyzes the
breakdown of phospholipids at the sn-2 position to produce a fatty
acid and a lysophospholipid. The activity of this enzyme has been
correlated with various cell functions, particularly with the
production of lipid mediators such as eicosanoid production
(prostaglandins, thromboxanes and leukotrienes), platelet
activating factor and lysophospholipids. Lipid-conjugates may offer
a wider scope of protection of cells and organisms from injurious
agents and pathogenic processes, including the prevention and
treatment of microbial infections.
[0004] Microbial infections (e.g., infections by viral or bacterial
species) account for significant morbidity and mortality throughout
the world. Although significant resources have been dedicated to
identifying compounds having antimicrobial properties, microbial
infections continue to present a significant human health risk.
[0005] There are relatively few effective pharmaceutical
compositions intended or adapted for antiviral, antifungal, or
antiparasitic therapy. A major obstacle in the development of
antiviral agents is the difficulty in distinguishing viral
replicative mechanisms from host replicative processes. An
additional limitation of existing antiviral drugs is that they have
a narrow antiviral spectrum and are often ineffective against the
latent virus.
[0006] There are a much larger number of existing antibacterial
agents, which has led to a significant decrease in morbidity and
mortality from infectious diseases in this century. This important
public health contribution has been largely due to the widespread
use of antibiotics that target specific nutrient, cell wall, DNA,
RNA and protein biosynthetic pathways that are particular to
pathogenic bacteria. However, in recent years the capacity to
manage infectious diseases has been threatened by the emergence of
bacterial strains that are no longer susceptible to currently
available antimicrobial agents. The widespread use of available
antibacterial agents has led to the development of increasing
numbers of antibiotic resistant bacteria.
[0007] In fact, the usefulness of most existing antimicrobial
treatments are limited by the development of multidrug resistance
and the emergence of long-term toxicities. Other challenges include
creating a drug that is broadly applicable in combating many
different types of microbial infections, which is especially
important in the treatment of immunocompromised individuals.
SUMMARY OF THE INVENTION
[0008] In one embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell, comprising the step of contacting the cell with a
compound comprising a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0009] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating an infection in a
subject, comprising the step of administering an effective amount
of a lipid or phospholipid moiety bound to a physiologically
acceptable monomer, dimer, oligomer, or polymer, and/or a
pharmaceutically acceptable salt or a pharmaceutical product
thereof to an infected subject.
BRIEF DESCRIPTION OF FIGURES
[0010] FIG. 1.1: Effect of Lipid-conjugates on HIV infectivity.
[0011] FIG. 2.1: Effect of Lipid-conjugates on injection of HeLa
cells by Chlamydia.
[0012] FIG. 2.2: Effect of Lipid-conjugates on Chlamydia-induced
apoptosis of HeLa cells.
[0013] FIG. 3.1: Inhibition of endothelin-1 (ET)-induced
contraction of rat tracheal rings by Lipid-conjugates. A:
Contraction of rat trachea by Endothelin-1. B: Effect of a
Lipid-conjugate on ET-induced contraction of rat trachea.
[0014] FIG. 3.2: Effect of Lipid-conjugates on ET-1 induced
contraction of rat trachea.
[0015] FIG. 3.3: Effect of Lipid-conjugates on Acetylcholine
(AcCh)-induced contraction of isolated rat trachea rings.
[0016] FIG. 3.4: Effect of a Lipid-conjugate, administered
subcutaneously, on early asthmatic reaction (EAR) induced by
ovalbumin (OVA) inhalation.
[0017] FIG. 3.5: Effect of a Lipid-conjugate on sPLA.sub.2
expression in lung of rats with OVA-induced asthma.
[0018] FIG. 3.6: Effect of a Lipid-conjugate on cysteinyl
leukotriens (LTC.sub.4, LTD.sub.4 and LTE.sub.4) level in the
broncho-alveolar lavage (BAL) of OVA-induced asthmatic rats.
[0019] FIG. 3.7: Effect of Lipid-conjugate inhalation on early and
late asthmatic reaction (EAR and LAR, respectively) in
OVA-sensitized asthmatic rats.
[0020] FIG. 3.8: Effect of Lipid-conjugate inhalation on cysteinyl
leukotriens (LTC4, LTD4 and LTE4) level in the BAL, of
OVA-sensitized asthmatic rats.
[0021] FIG. 3.9: Effect of Lipid-conjugate inhalation on NO
production by macrophages collected from the BAL of OVA-sensitized
asthmatic rats.
[0022] FIG. 3.10: Effect of Lipid-conjugate inhalation on
structural change in airways (airway remodeling) of OVA-sensitized
asthmatic rats.
[0023] FIG. 3.11: Effect of a Lipid-conjugate on the remodeling of
asthmatic rat airway; histological morphometry.
[0024] FIG. 3.12: Effect of Lipid-conjugate inhalation on
TNF.alpha. production by macrophages collected from the BAL of
OVA-sensitized asthmatic rats.
[0025] FIG. 3.13: Amelioration of OVA-induced broncho-constriction
by Lipid-conjugate inhalation before challenge.
[0026] FIG. 3.14: Amelioration of OVA-induced broncho-constriction
by Lipid-conjugate inhalation after challenge.
[0027] FIG. 4.1 I-II: Effect of Lipid-conjugates on LPS-induced
production of TNF.alpha. in human whole blood.
[0028] FIG. 4.2: Effect of a Lipid-conjugate on rat survival in
LPS-induced endotoxinemia.
[0029] FIG. 4.3: Effect of a Lipid-conjugate on serum levels of
TNF-.alpha. and IL-6 in septic rats.
[0030] FIG. 4.4: Effect of a Lipid-conjugate on TNF-.alpha.
production after i.p. administration of LPS and simultaneous i.v.
administration of a Lipid-conjugate.
[0031] FIG. 4.5: Effect of a Lipid-conjugate on serum cytokine
levels in rats injected with LPS or LPS+LTA.
[0032] FIG. 4.6: Effect of a Lipid-conjugate on mRNA expression of
IL-1, TNF-.alpha. and IL-6 genes in lung and kidney of rats with
LPS-induced sepsis.
[0033] FIG. 4.7: Effect of a Lipid-conjugate on mRNA expression of
sPLA.sub.2-IIA and iNOS genes in kidney and lung of rats with
LPS-induced sepsis.
[0034] FIG. 4.8: Effect of a Lipid-conjugate on ICAM-1 expression
in lung and kidney of rats with LPS-induced sepsis.
[0035] FIG. 6.1: A Lipid-conjugate protects BGM cells from membrane
lysis induced by combined action of hydrogen peroxide produced by
glucose oxidase (GO) and exogenous phospholipase A.sub.2
(PLA.sub.2).
[0036] FIG. 6.2: A Lipid-conjugate protects BGM cells from
glycosaminoglycan degradation by hydrogen peroxide produced by
glucose oxidase (GO).
[0037] FIG. 6.3: A Lipid-conjugate protects LDL from copper-induced
oxidation.
[0038] FIG. 7.1: Lipid-conjugates inhibit the secretion of
PGE.sub.2 from glial cells stimulated by LPS.
[0039] FIG. 7.2: Lipid-conjugates inhibit the secretion of
PGE.sub.2 from glial cells stimulated by pardaxin (PX).
[0040] FIG. 7.3: Lipid-conjugates inhibit the production of nitric
oxide (NO) by LPS-stimulated rat glial cells.
[0041] FIG. 7.4: Lipid-conjugates inhibit the production of nitric
oxide (NO) by PX-stimulated PC12 cells.
[0042] FIG. 7.5: Lipid-conjugates inhibit the secretion of
LPS-stimulated sPLA.sub.2 (expressed as fatty acid release) from
glial cells.
[0043] FIG. 7.6: Lipid-conjugates inhibit PX-induced activation of
PLA.sub.2 (expressed as fatty acid release) in PC12 cells.
[0044] FIG. 7.7: Effect of a Lipid-conjugate on LPS-induced oleic
acid (OA) release.
[0045] FIG. 7.8: Lipid-conjugates inhibit PX-induced dopamine
release by PC12 cells.
[0046] FIG. 7.9: Lipid-conjugates inhibit PX-induced production of
5-HETE by PC12 cells.
[0047] FIG. 7.10: Effect of Lipid-conjugates on T-cell permeation
through a monolayer of endothelial cells.
DETAILED DESCRIPTION OF THE INVENTION
Methods of Treating Disease Based on Phospholipid Conjugates
[0048] In one embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell, comprising the step of contacting the cell with a
compound comprising a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0049] In one embodiment, the compounds for use in the present
invention (for e.g., a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer)
are referred to herein as "Lipid-conjugates".
[0050] In one embodiment, "suppressing, inhibiting, preventing, or
treating" refers to delaying the onset of symptoms, reducing the
severity of symptoms, reducing the severity of an acute episode,
reducing the number of symptoms, reducing the incidence of
disease-related symptoms, reducing the latency of symptoms,
ameliorating symptoms, reducing secondary symptoms, reducing
secondary infections, prolonging patient survival, preventing
relapse to a disease, decreasing the number or frequency of relapse
episodes, increasing latency between symptomatic episodes,
increasing time to sustained progression, expediting remission,
inducing remission, augmenting remission, speeding recovery, or
increasing efficacy of or decreasing resistance to alternative
therapeutics.
[0051] In one embodiment, symptoms are primary, while in another
embodiment, symptoms are secondary. In one embodiment, "primary"
refers to a symptom that is a direct result of infection with a
pathogen, while in one embodiment, "secondary" refers to a symptom
that is derived from or consequent to a primary cause. In another
embodiment, "symptoms" may be any manifestation of a disease or
pathological condition, comprising inflammation, swelling, fever,
pain, bleeding, itching, runny nose, coughing, headache, migraine,
difficulty breathing, weakness, fatigue, drowsiness, weight loss,
nausea, vomiting, constipation, diarrhea, numbness, dizziness,
blurry vision, muscle twitches, convulsions, etc., or a combination
thereof.
[0052] In one embodiment, "treating" refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or lessen the targeted pathologic condition or
disorder as described hereinabove. Thus, in one embodiment,
treating may include suppressing, inhibiting, preventing, treating,
or a combination thereof.
[0053] In one embodiment, a pathogenic effect is apoptosis,
necrosis, membrane blebbing/protrusion, cell death, permeabilized
cell membrane, cell enlargement, dilated organelles, ribosome
dissociation from endoplasmic reticulum, nuclear disintegration,
chromatin condensation, pyknotic or fragmented nuclei, leakage of
cellular contents, tissue inflammation, expression of
apoptosis-specific proteins, cell shrinkage, formation of apoptotic
bodies, expression of pathogen antigens, granularity, ragged edges,
filmy appearance, cell rounding or a combination thereof. In
another embodiment, a pathogenic effect is caused by infection with
any of the pathogens described hereinbelow. In one embodiment, a
pathogenic effect is a cytopathic effect.
[0054] Thus, in one embodiment of the present invention, the
compounds for use in the present invention are directed towards the
resolution of symptoms of a disease or disorder that result from a
pathogenic infection as described hereinabove. In another
embodiment, the compounds affect the pathogenesis underlying the
pathogenic effect described hereinabove.
[0055] In one embodiment, a pathogenic effect on a cell could be a
cell of any tissue, in one embodiment, a vertebrate cell, in
another embodiment, a mammalian cell, and in another embodiment, a
human cell. In one embodiment, a pathogen may infect a plurality of
cell types, tissues or organs. In another embodiment, pathogens
have preference for infecting specific cell types, tissues, or
organs. It is to be understood that agents of the present invention
may be efficacious in treating any cell type in which the pathogen
may exert an effect. In one embodiment, a compound for use in the
present invention may localize to or act on a specific cell type.
In one embodiment, a compound for use in the present invention may
be cytoprotective. In one embodiment a compound for use in the
present invention may be inserted or partially inserted into a cell
membrane. In another embodiment a compound for use in the present
invention may be effective in treating a plurality of cell
types.
[0056] In another embodiment, the cell exhibiting a pathogenic
effect described hereinabove is present in a subject with a
pathogenic infection.
[0057] In one embodiment, the invention provides a method of
treating a subject suffering from a pathogenic effect, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a pathogenic
effect.
[0058] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating an infection in a
subject comprising the step of administering to said subject an
effective amount of a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0059] In another embodiment, the invention provides a method of
treating a subject suffering from a pathogenic infection,
comprising the step of administering to a subject any one of the
compounds for use in the present invention, or any combination
thereof, in an amount effective to treat the subject suffering from
a pathogenic infection.
[0060] In one embodiment, the pathogenic effect is due to an
infection of the cell described hereinabove by a pathogen. In one
embodiment, the pathogen is a virus and in another embodiment, the
pathogen is a bacterium. In one embodiment, the pathogenic effect
is the result of a viral infection and in another embodiment, the
pathogenic effect is the result of a bacterial infection. In
another embodiment, the pathogenic effect is the result of an
infection with influenza, HIV, poxvirus, chlamydia, or a
combination thereof, as is described hereinbelow.
[0061] In another embodiment, the pathogenic effect is due to a
cytopathic effect of a pathogen in a cell. In another embodiment,
the pathogenic effect in the cell is due to a cell-to-cell spread
of a pathogen. In another embodiment, the pathogenic effect is the
result of obstructive respiratory disease, cytokine overproduction,
sepsis, hemolysis, oxidative injury, central nervous system insult,
conjunctivitis, or a combination thereof, as is described
hereinbelow. In another embodiment, the pathogenic effect is the
result of cancer. In another embodiment, the pathogenic effect is
due to toxic products produced by the pathogen. In one embodiment,
the toxic product may be worm eggs.
[0062] In one embodiment, the invention provides a method of
treating a subject suffering from a viral infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a viral infection.
[0063] In one embodiment, the invention provides a method of
treating a subject suffering from a bacterial infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a bacterial
infection.
[0064] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
viral infection.
[0065] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
bacterial infection.
[0066] In another embodiment, the viral pathogenic effect,
infection, or combination thereof is mediated by any one or more of
the following pathogens: hepatitis B virus, hepatitis C virus,
human immunodeficiency virus, human herpesviruses, herpes simplex
virus-1, herpes simplex virus-2, human cytomegalovirus,
Epstein-Barr virus, Varicella-Zoster virus, human herpesvirus-6,
human herpesvirus-7, human influenza, measles virus, hantaan virus,
pneumonia virus, rhinovirs, poliovirus, human respiratory syncytial
virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps
virus, malaria (Plasmodium falciparum), Bordetelia pertussis,
Diptheria, Rickettsia prowazekii, Borrelia bergdorferi, Ebola
virus. In one embodiment, the viral pathogenic effect, infection or
combination thereof is mediated by Pichinde virus, while in another
embodiment, it is mediated by Punta Toro virus.
[0067] In one embodiment, the pathogenic effect, infection or
combination thereof is mediated by one or more of the following
pathogens: Helminths, Bacillus anthracis (anthrax), Clostridium
botulinum, Yersinia pestis, Variola major (smallpox) and other pox
viruses, Francisella tularensis (tularemia), Arenaviruses,
Lymphocytic choriomeningitis, Junin virus, Machupo virus, Guanarito
virus, Lassa Fever, Bunyaviruses, Hantaviruses, Rift Valley Fever,
Flaviruses, Dengue, Filoviruses, Ebola, Marburg, hemorrhagic fever
viruses, Tickborne hemorrhagic fever viruses, Crimean-Congo
Hemorrhagic fever virus, Tickborne encephalitis viruses, Yellow
fever, Tuberculosis, Multi-drug resistant tuberculosis, Influenza,
Rickettsias, Rabies virus, Severe acute respiratory
syndrome-associated coronavirus (SARS), Burkholderia pseudomallei,
Coxiella burnetii (Q fever), Brucella species (brucellosis),
Burkholderia mallei (glanders), Ricin toxin (from Ricinus
communis), Epsilon toxin of Clostridium perfringens, Staphylococcus
enterotoxin B, Typhus fever (Rickettsia prowazekii), Diarrheagenic
E. coli, Pathogenic Vibrios, Shigella species, Salmonella, Listeria
monocytogenes, Campylobacter jejuni, Yersinia enterocolitica),
Caliciviruses, Hepatitis A, Cryptosporidium parvum, Cyclospora
cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma.
Microsporidia, West Nile Virus, LaCrosse, California encephalitis,
Western Equine Encephalitis, Eastern Equine Encephalitis,
Venezuelan Equine Encephalitis, Japanese Encephalitis Virus, and
Kyasanur Forest Virus.
[0068] In another embodiment, the pathogenic effect, infection, or
combination thereof is mediated by one or more of the following
microorganisms: Actinobacillus pleuropneumoniae, Aeropyrum pernix,
Agrobacterium tumeficians, Anopheles gambiae, Aquifex aeolicus,
Arabidopsis thaliana, Archeglobus fulgidis, Bacillus anthracis,
bacillus cereus, Bacillus halodurans, Bacillus subtilis,
Bacteroides thetaiotaomicron, Bdellovibrio bacteriovorus,
Bifidobacterium longum, Bordetella bronchiseptica, Bordetella
pertussis, Borrelia burgdorferi, Bradyrhizobium japonicum, Brucella
melitensis, Brucella suis, Bruchnera aphidicola, Brugia malayi,
Caenorhabditis elegans, Canipylobacter jejuni, Candidatus
blochmanniafloridanus, Caulobacter crescentus, Chlorobium tepidum,
Chromobacterium violaceum, Clostridium acetobutylicum, Clostridium
perfringens, Clostridium tetani, Corynebacterium diphtheriae,
Corynebacterium efficiens, Corynebacterium glutamicum, Coxiella
burnetii, Danio rerio, Dechloromonas aromatica, Deinococcus
radiodurans, Drosophila melanogaster, Eimeria tenella, Eimeria
acervulina, Entamoeba histolytica, Enterococcus faecalis,
Escherichia coli, Fusobacterium nucleatum, Geobacter
su6rurreducens, Gloeobacter violaceus, Haemophilis ducreyi,
Haemophilis influenzae, Halobacterium, Helicobacter hepaticus,
Helicobacter pylori, Lactobacillus johnsonii, Lactobacillus
plantarum, Lactococcus lactis, Leptospira interrogans serovar lai,
Listeria innocua, Listeria monocytogenes, Mesorhizobium loti,
Methanobacter thermoautotrophicus, Methanocaldocossus jannaschii,
Methanococcoides burtonii, Methanopyrus kandleri, Methanosarcina
acetivorans, Methanosareina mazei Goel, Mycobacterium avium,
Mycobacterium bovis, Mycobacterium leprae, Mycobacterium
tuberculosis, Mycoplasma gallisepticum strain R, Mycoplasma
genitalium, Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma
pulmonis, Nanoarchaeum equitans, Neisseria meningitidis,
Nitrosomonas europaea, Nostoc, Oceanobacillus iheyensis, Onion
yellows phytoplasm, Oryzias latipes, Oryza sativa, Pasteurella
multocida, Photorhabdus luminescens, Pirellula, Plasmodium
falciparum, Plasmodium vivax, Plasmodium yoelii, Porphyromonas
gingivalis, Prochlorococcus marinus, Pseudomonas aeruginosa,
Pseudomonas putida, Pseudomonas syringae, Pyrobaculum aerophilum,
Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii,
Ralstonia solanacearum, Rhodopseudomonas palustris, Rickettsia
conorii, Rickettsia prowazekii, Rickettsia rickettsii,
Saccharomyces cerevisiae, Salmonella enterica, Salmonella
typhimurium, Sarcocystis cruzi, Schistosoma mansoni,
Schizosaccharomyces pombe, Shewanella oneidensis, Shigella
flexneri, Sinorhizobium meliloti, Staphylococcus aureus,
Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus
agalactiae, Streptococcus mutans, Streptococcus pneumoniae,
Streptococcus pyogenes, Streptomyces avermitilis, Streptomyces
coelicolor, Suffiblobus tokodaii, Synechocystis sp., Takifugu
rubripes, Tetraodon fluviatilis, Theileria parva,
Thermoanaerobacter tengcongensis, Thernzoplasma acidophilum,
Thermoplasma voleanium, Thermosynechococcus elongatus, Aermotoga
maritima, Toxoplasma gondii, Treponema denticola, Treponema
pallidum, Tropheryma whipplei, Tryponosoma brucei, Trypanosoma
cruzi, Ureaplasma urealyticum, Vibrio cholerae, Vibro
parahaemolyticus, Pbro vulnificus, Wigglesworthia brevipalpis,
Wolbachia endosymbiont of Drosophilia melanogaster, W01inella
succinogenes, Xanthomonas axonopodis pv. Citri, Xanthomonas
campestris pv. Campestris, Xylella fastidiosa, or Yersinia
pestis.
[0069] In one embodiment, the pathogenic effect, infection or
combination thereof is mediated by a parasite. In one embodiment,
the parasite is a worm. In one embodiment, the parasitic worm is a
helminth, Acanthocephala, Clonorchis sinensis (the Chinese liver
fluke), Dracunculiasis (Guinea Worm Disease), or Enterobius
vermicularis (pinworm). In another embodiment, the parasite is a
fish, which is, in one embodiment, a Candiru (Vampire fish of
Brazil). In another embodiment, the parasite is a fungi, which is,
in one embodiment, a Tinea (ringworm). In one embodiment, the
parasite is a protist. In one embodiment, the protist parasite is a
Plasmodium (malaria), Balantidium coli, or Giardia lamblia. In one
embodiment, the parasite is Hirudinea (leech), Phthiraptera (lice),
Siphonaptera (fleas), or Acarina (ticks).
[0070] In another embodiment, the parasite is an intracellular
bacterial parasite. In one embodiment, the intracellular bacterial
parasite is Rickettsias, while in another embodiment, it's
Mycobacterium leprae. In one embodiment, the intracellular
bacterial parasite is Rickettsia prowazekii, while in another
embodiment, it's Rickettsia rickettsii (Rocky mountain spotted
fever).
[0071] In one embodiment, the methods of the present invention may
be used to treat a pathogenic infection acquired via zoonotic
transmission. In one embodiment, the methods of the present
invention may be used to treat pathogenic infections acquired from
avian, swine, bovine, or bat. In another embodiment, the methods of
the present invention may be used to treat Menangle, Hendra,
Australian Bat Lyssavirus, Nipah, or Tioman. In another embodiment,
the methods of the present invention may be used to diminish
pathogen reservoirs in animal species. In another embodiment, the
methods of the present invention may be used to treat a human
infected with a pathogen
HIV
[0072] In another embodiment, the viral pathogenic effect described
hereinabove is mediated by Human Immunodeficiency Virus (HIV). In
another embodiment, the infection described hereinabove is mediated
by HIV.
[0073] In one embodiment, the methods of the present invention
comprise treating secondary complications of HIV infection. In
another embodiment, the methods comprise treating opportunistic
infections, neoplasms, neurologic abnormalities, or progressive
immunologic deterioration. In another embodiment, the methods
comprise treating acquired immunodeficiency syndrome. In another
embodiment, the methods comprise treating a decline in the number
of CD4.sup.+ T lymphocytes.
[0074] In another embodiment, methods comprise treating HIV
transmitted by direct sexual contact, either homosexual or
heterosexual; by blood or blood products; or from an infected
mother to infant, either intrapartum, perinatally, or via breast
milk.
[0075] In one embodiment, the methods of the present invention may
be used to treat HIV or related infections that were acquired via
zoonotic transmission. In one embodiment, the methods of the
present invention may be used to treat simian immunodeficiency
virus.
[0076] In one embodiment, methods of treating infection comprise
treating Clade A, B, C, D, A/E, F, G, H, J, or K. In another
embodiment, the viral pathogenic effect, infection or combination
thereof is mediated by HIV-1, while in another embodiment, it's
mediated by HIV-2. In one embodiment, it's mediated by the M group
of HIV-1, in another embodiment, it's mediated by the O group of
HIV-1, while in another embodiment, it's mediated by the N group of
HIV-1. In one embodiment, it's mediated by the A clade (or subtype)
of the M group of HIV-1, in another embodiment, it's mediated by
the B clade of the M group of HIV-1, in another embodiment, it's
mediated by the C clade of the M group of HIV-1, in another
embodiment, it's mediated by the D clade of the M group of HIV-1,
in another embodiment, it's mediated by the A/E clade of the M
group of HIV-1, in another embodiment, it's mediated by the F clade
of the M group of HIV-1, in another embodiment, it's mediated by
the G clade of the M group of HIV-1, in another embodiment, it's
mediated by the H clade of the M group of HIV-1, in another
embodiment, it's mediated by the J clade of the M group of HIV-1,
in another embodiment, it's mediated by the K clade of the M group
of HIV-1, in another embodiment, it's mediated by the A/G/I clade
of the M group of HIV-1, while in another embodiment, it's mediated
by a circulating recombinant form (CRF) of any of the above
clades.
[0077] In one embodiment, methods of treating infection comprise
treating a macrophage-tropic strain of HIV, T cell-tropic strain of
HIV, or any combination thereof. In one embodiment, the compounds
for use in the present invention will treat infection mediated by a
macrophage-tropic strain of HIV. In another embodiment, the
compounds will treat infection mediated by a T cell-tropic strain
of HIV. In another embodiment, the compounds will treat infection
mediated by either a macrophage-tropic strain of HIV, a T
cell-tropic, or both. In another embodiment, the mechanism of
action of the compounds for use in the present invention differ
based on the tropism of HIV.
[0078] In another embodiment, this invention provides a method of
suppressing, inhibiting, preventing, or treating an HIV infection
in a subject comprising the step of administering to said subject
an effective amount of a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof. In one embodiment, the phospholipid moiety is
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer, is a glycosaminoglycan. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is heparin. In another
embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0079] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating an HIV infection
in a cell, comprising the step of contacting the cell with a
compound comprising a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0080] In one embodiment, the invention provides a method of
treating a subject suffering from an HIV infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from an HIV infection.
[0081] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with an
HIV infection.
[0082] In one embodiment, Lipid-conjugates of the present invention
suppress, inhibit, prevent, or treat HIV infection. In another
embodiment, Lipid-conjugates decrease HIV virus titer. This is
exemplified in FIG. 1.1 and represents an embodiment of this
invention. In another embodiment, Lipid-conjugates inhibit p24
production. This is exemplified in Tables 1.1-1.2 and represents an
embodiment of this invention. In another embodiment,
Lipid-conjugates inhibit fusion of HIV-infected cells to
non-HIV-infected cells. This is exemplified in Tables 1.3-1.4 and
represents an embodiment of this invention. In another embodiment,
Lipid-conjugates decrease V3 antibody binding. This is exemplified
in Table 1.5 and represents an embodiment of this invention. In one
embodiment, Compound XXII (see compound descriptions hereinbelow)
is useful to treat HIV infection. This is exemplified in Tables
1.1, 1.2, and 1.5 and represents an embodiment of this invention.
In another embodiment, Compound XXV (see compound descriptions
hereinbelow) is useful to treat HIV infection. This is exemplified
in Tables 1.1, 1.2, and 1.5 and represents an embodiment of this
invention. In another embodiment, Compound XXIII (see compound
descriptions hereinbelow) is useful to treat HIV infection. This is
exemplified in Tables 1.1-1.5 and represents an embodiment of this
invention. In another embodiment, Compound XXIV (see compound
descriptions hereinbelow) is useful to treat HIV infection. This is
exemplified in Tables 1.1-1.5 and represents an embodiment of this
invention.
Influenza
[0083] In one embodiment, the viral pathogenic effect described
hereinabove is mediated by influenza virus. In another embodiment,
the infection described hereinabove is mediated by influenza
virus.
[0084] Thus, in one embodiment, the methods of the present
invention include the treatment of symptoms of infection by
influenza virus comprising fever (usually high), headache,
tiredness (can be extreme), cough, sore throat, runny or stuffy
nose, body aches, diarrhea, vomiting, or a combination thereof.
[0085] In one embodiment, the methods of the present invention
treat secondary complications related to influenza infection, which
may comprise, inter alia, bacterial pneumonia, bronchitis,
dehydration, sinus infections, and ear infections. In another
embodiment, the methods of the present invention treat chronic
health problems that are exacerbated in a subject with influenza
infection which may comprise, inter alia, asthma.
[0086] In one embodiment, influenza viruses for treatment by the
methods of the present invention may be of type A or type B. In one
embodiment, the viral pathogenic effect, infection, or combination
thereof is mediated by Influenza Type A virus, in another
embodiment, it's mediated by Influenza Type B virus, while in
another embodiment, it's mediated by Influenza Type C virus. In one
embodiment, it's mediated by H1N1 strain of Influenza Type A, in
another embodiment, it's mediated by H2N2 strain of Influenza Type
A, in another embodiment, it's mediated by H3N2 strain of Influenza
Type A, while in another embodiment, it's mediated by H5N1 strain
of Influenza Type A. In one embodiment, it's mediated by any
combination of strains of the subtypes listed hereinabove.
[0087] In one embodiment, the methods of the present invention may
be used to treat influenza infections that were acquired via
zoonotic transmission. In one embodiment, the methods of the
present invention may be used to treat zoonotic avian influenza or
zoonotic swine influenza.
[0088] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating an influenza
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment, the phospholipid moiety
is dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0089] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating an influenza
infection of a cell, comprising the step of contacting the cell
with a compound comprising a lipid or phospholipid moiety bound to
a physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0090] In one embodiment, the invention provides a method of
treating a subject afflicted with an influenza infection,
including, inter alia, the step of administering to a subject an
effective amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject afflicted with an influenza
infection.
[0091] In another embodiment, the invention provides a use of a
lipid or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with an
influenza infection.
[0092] In one embodiment, Lipid-conjugates of the present invention
suppress, inhibit, prevent, or treat influenza infection. In
another embodiment, Lipid-conjugates decrease morphological changes
that result from cytotoxicity of influenza infection. This is
exemplified in Example 1.4 and represents an embodiment of this
invention. In another embodiment, Lipid-conjugates decrease cell
membrane permeability as is demonstrated by a neutral red dye
uptake assay. This is exemplified in Example 1.4 and represents an
embodiment of this invention. In one embodiment, Compound XXIV (see
compound descriptions hereinbelow) is useful to treat infection
with influenza, in another embodiment, influenza Type A, while in
another embodiment, influenza Type A, Strain H1N1. This is
exemplified in Tables 1.7 and 1.9 and represents an embodiment of
this invention.
Poxvirus
[0093] In one embodiment, the viral pathogenic effect described
hereinabove is mediated by poxviridae, while in another embodiment,
the viral pathogenic effect is mediated by chordopoxvirinae. In
another embodiment, the infection described hereinabove is mediated
by poxviridae while in another embodiment, the infection is
mediated by chordopoxvirinae.
[0094] In one embodiment, a range of pox viruses cause febrile
illnesses in man and animals with a prominent vesicular rash. In
one embodiment, "pox virus", "poxvirus" and "Poxviridae" refer to
the Poxviridae family of viruses.
[0095] In one embodiment, methods of the present invention comprise
treating secondary complications of infection, which may comprise
progressive necrosis at the site of infection, skin disorders such
as eczema, vesicular rash, neurological complications,
conjunctivitis, or a combination thereof.
[0096] In one embodiment, methods of the present invention comprise
treating variola major or variola minor. In another embodiment, the
methods of the present invention comprise treating ordinary,
modified, flat, and hemorrhagic types of variola major. In one
embodiment, the methods of the present invention may be used to
treat variola virus used as an agent of bioterrorism.
[0097] In another embodiment, the methods of the present invention
may be used to treat secondary complications of variola infection
comprising fever, malaise, head and body aches, vomiting, rash in
the tongue and mouth, rash on the skin, pustule formation,
scabbing, scarring, or a combination thereof.
[0098] In one embodiment, the methods of the present invention may
be used to treat poxvirus infections that were acquired via
zoonotic transmission. In one embodiment, the methods of the
present invention may be used to treat Molluscum contagiosum,
Cowpox, Monkey pox, pseudocowpox and orf. In one embodiment, the
methods of the present invention may be used to treat ulcerative or
non-ulcerating lesions (sometimes called "milkers nodules") on the
hands of dairy workers or to treat a papulo-vesicular lesion on the
hand, forearm or face of a subject.
[0099] In another embodiment, the viral pathogenic effect,
infection or combination thereof is mediated by Vaccinia virus. In
another embodiment, it's mediated by a poxvirus, while in another
embodiment, it's mediated by a chordopoxvirinae. In another
embodiment, it's mediated by Orf virus, Fowlpox virus, Sheep pox
virus, Myxoma virus, Swinepox virus, Molluscum contagiosum virus,
Yaba monkey tumor virus, Melolontha melolontha entomopoxvirus,
Amsacta moorei entomopoxvirus, or Chironomus luridus
entomopoxvirus.
[0100] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a vaccinia
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment, the phospholipid moiety
is dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0101] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a vaccinia
infection of a cell, comprising the step of contacting the cell
with a compound comprising a lipid or phospholipid moiety bound to
a physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0102] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a smallpox
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment, the phospholipid moiety
is dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0103] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a smallpox
infection of a cell, comprising the step of contacting the cell
with a compound comprising a lipid or phospholipid moiety bound to
a physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0104] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a poxvirus
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment the phospholipid moiety is
dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0105] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a poxvirus
infection of a cell, comprising the step of contacting the cell
with a compound comprising a lipid or phospholipid moiety bound to
a physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0106] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a chordopoxvirinae
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment, the phospholipid moiety
is dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0107] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a chordopoxvirinae
infection of a cell, comprising the step of contacting the cell
with a compound comprising a lipid or phospholipid moiety bound to
a physiologically acceptable monomer, dimer, oligomer, or polymer,
and/or a pharmaceutically acceptable salt or a pharmaceutical
product thereof.
[0108] In one embodiment, the invention provides a method of
treating a subject suffering from a vaccinia infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a vaccinia
infection.
[0109] In one embodiment, the invention provides a method of
treating a subject suffering from a smallpox infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a vaccinia
infection.
[0110] In one embodiment, the invention provides a method of
treating a subject suffering from a poxvirus infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a poxvirus
infection.
[0111] In one embodiment, the invention provides a method of
treating a subject suffering from a chordopoxvirinae infection,
including, inter alia, the step of administering to a subject an
effective amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a chordopoxvirinae
infection.
[0112] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
vaccinia infection.
[0113] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
smallpox infection.
[0114] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
poxvirus infection.
[0115] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
chordopoxvirinae infection.
[0116] In one embodiment, Lipid-conjugates of the present invention
suppress, inhibit, prevent, or treat vaccinia infection. In another
embodiment, Lipid-conjugates decrease vaccinia virus titer. This is
exemplified in Table 1.11 and represents an embodiment of this
invention. In one embodiment, Compound XXII (see compound
descriptions hereinbelow) is useful to treat vaccinia infection.
This is exemplified in Table 1.11 and represents an embodiment of
this invention. In another embodiment, Compound XXV (see compound
descriptions hereinbelow) is useful to treat vaccinia infection.
This is exemplified in Table 1.11 and represents an embodiment of
this invention. In another embodiment, Compound XXIII (see compound
descriptions hereinbelow) is useful to treat vaccinia infection.
This is exemplified in Table 1.11 and represents an embodiment of
this invention.
Chlamydia
[0117] In one embodiment, the bacterial pathogenic effect described
hereinabove is mediated by Chlamydia. In another embodiment, the
infection described hereinabove is mediated by Chlamydia.
[0118] In one embodiment, methods of the present invention treat
sexually transmitted diseases (STDs), pneumonia, conjunctivitis, or
a combination thereof due to Chlamydia infection.
[0119] In another embodiment, methods of the present invention
treat chlamydia infection of genitals, cervix, urethra, rectum or
throat. In one embodiment, the methods of the present invention may
be used to treat Chlamydia infections infecting mucosal membranes,
such as the cervix, rectum, urethra, throat, or conjunctiva. In
another embodiment, methods of the present invention treat
secondary complications of Chlamydia infection including inter
alia, abnormal vaginal discharge, a burning sensation when
urinating, lower abdominal pain, low back pain, nausea, fever, pain
during intercourse, or bleeding between menstrual periods, penile
discharge, or burning or itching around the opening of the penis.
In another embodiment, methods of the present invention treat
secondary complications of Chlamydia infection including pelvic
inflammatory disease (PID). In another embodiment, methods of the
present invention treat secondary complications of Chlamydia
infection including chronic pelvic pain, infertility, or
potentially fatal ectopic pregnancy in infected women. In another
embodiment, methods of the present invention treat secondary
complications of Chlamydia infection including pain, fever, or
sterility in infected men.
[0120] In another embodiment, methods of the present invention
treat arthritis that may be accompanied by skin lesions and
inflammation of the eye and urethra (Reiter's syndrome). In one
embodiment, methods of the present invention treat trachomoa or
inclusion conjunctivitis resulting directly or indirectly from
Chlamydia infection. In another embodiment, methods of the present
invention treat pneumonia or bronchopulmonary infections resulting
directly or indirectly from Chlamydia infection. In another
embodiment, methods of the present invention treat Lymphogranuloma
venereum due to Chlamydia trachomatis infection.
[0121] In one embodiment, the methods of the present invention may
be used to treat Chlamydia infections that are acquired via
zoonotic transmission, which may include inter alia, Chlamydia
psittaci an avian form of Chlamydia. In one embodiment, Chlamydia
psittaci is referred to as Psittacosis, Parrot Fever or
chlamydiosis.
[0122] In one embodiment, the bacterial pathogenic effect,
infection or combination thereof is mediated by Chlamydia. In one
embodiment, the bacterial pathogenic effect, infection or
combination thereof is mediated by Chlamydia trachomatis, in
another embodiment by Chlamydia muridarum in another embodiment by
Chlamydophila caviae, in another embodiment by Chlamydia psittaci,
in another embodiment by Chlamydia puerorum, and in another
embodiment by Chlamydia pneumoniae. In one embodiment, the
bacterial pathogenic effect is mediated by any of Chlamydia
trachomatis serovars (serologically variant strains). In one
embodiment, it's mediated by Serovar A, in another embodiment by
Serovar B, in another embodiment by Serovar Ba, in another
embodiment by Serovar C, in another embodiment by Serovar D, in
another embodiment by Serovar E, in another embodiment by Serovar
F, in another embodiment by Serovar G, in another embodiment by
Serovar H, in another embodiment by Serovar I, in another
embodiment by Serovar J, in another embodiment by Serovar K, in
another embodiment by Serovar L1, in another embodiment by Serovar
L2, and in another embodiment by Serovar L3.
[0123] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a Chlamydia
infection in a subject comprising the step of administering to said
subject an effective amount of a lipid or phospholipid moiety bound
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, and/or a pharmaceutically acceptable salt or a
pharmaceutical product thereof. In one embodiment, the phospholipid
moiety is phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is a
glycosaminoglycan. In another embodiment, the phospholipid moiety
is dipalmitoyl phosphatidylethanolamine and the physiologically
acceptable monomer, dimer, oligomer, or polymer is heparin. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is chondroitin sulfate. In
another embodiment, the phospholipid moiety is dipalmitoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid. In another
embodiment, the phospholipid moiety is dimyristoyl
phosphatidylethanolamine and the physiologically acceptable
monomer, dimer, oligomer, or polymer is hyaluronic acid.
[0124] In another embodiment, the invention provides a method of
suppressing, inhibiting, preventing, or treating a pathogenic
effect on a cell due to Chlamydia, comprising the step of
contacting the cell with a compound comprising a lipid or
phospholipid moiety bound to a physiologically acceptable monomer,
dimer, oligomer, or polymer, and/or a pharmaceutically acceptable
salt or a pharmaceutical product thereof.
[0125] In one embodiment, the invention provides a method of
treating a subject suffering from a Chlamydia infection, including,
inter alia, the step of administering to a subject an effective
amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a Chlamydia
infection.
[0126] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
Chlamydia infection.
[0127] In one embodiment, Lipid-conjugates of the present invention
suppress, inhibit, prevent, or treat chlamydia infection. In
another embodiment, Lipid-conjugates prevent infection by chlamydia
virus. This is exemplified in FIG. 2.1 and represents an embodiment
of this invention. In another embodiment, Lipid-conjugates prevent
apoptosis induced by chlamydia virus. This is exemplified in FIG.
2.2 and represents an embodiment of this invention. In one
embodiment, Compound XXII (see compound descriptions hereinbelow)
is useful to treat chlamydia infection. This is exemplified in
FIGS. 2.1-2.2 and represents an embodiment of this invention. In
another embodiment, Compound XXIII (see compound descriptions
hereinbelow) is useful to treat chlamydia infection. This is
exemplified in FIGS. 2.1-2.2 and represents an embodiment of this
invention. In another embodiment, Compound XXV (see compound
descriptions hereinbelow) is useful to treat chlamydia infection.
This is exemplified in FIGS. 2.1-2.3 and represents an embodiment
of this invention. In another embodiment, Compound XXIV (see
compound descriptions hereinbelow) is useful to treat chlamydia
infection. This is exemplified in FIGS. 2.2-2.3 and represents an
embodiment of this invention.
Other Pathogen-Mediated Diseases and Conditions
Obstructive Respiratory Disease
[0128] In one embodiment, the methods of the present invention
treat obstructive respiratory disease, which in one embodiment, can
be caused or exacerbated by microbial infections. In one
embodiment, obstructive respiratory disease is a disease of luminal
passages in the lungs, which in one embodiment, is marked by
dyspnea, tachypnea, or ausculatory or radiological signs of airway
obstruction. In one embodiment, the methods of the present
invention treat obstruction of air flow due to constriction of
airway lumen smooth muscle, accumulation of infiltrates in and
around the airway lumen, or a combination thereof.
[0129] In one embodiment, microbial-induced respiratory diseases
may include influenza infection, which may, in one embodiment,
exacerbate chronic asthma. In another embodiment, microbial-induced
respiratory diseases may include Chlamydia infection, of which
certain strains, in one embodiment target the respiratory tract. In
one embodiment, microbial-induced respiratory diseases may include
tuberculosis (TB), as is described hereinbelow.
[0130] In one embodiment, the invention provides a method of
treating a subject suffering from obstructive respiratory disease,
including, inter alia, the step of administering to a subject an
effective amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from obstructive respiratory
disease.
[0131] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject suffering from
obstructive respiratory disease.
[0132] In one embodiment, obstructive respiratory disease is due to
a pathogenic effect, while in another embodiment, it's due to a
pathogenic infection. In another embodiment, it is due to a
microbial infection, in another embodiment, it's due to a viral
infection, while in another embodiment, it's due to a bacterial
infection. In one embodiment, it's due to influenza, tuberculosis,
schistosomiasis, chronic bronchitis, pneumonia, SARS, respiratory
syncitial virus, Empyema Thoracis, whooping cough, or a combination
thereof.
[0133] In one embodiment, the bacterial pathogenic effect described
hereinabove is mediated by tuberculosis. In another embodiment, the
infection described hereinabove is mediated by tuberculosis.
[0134] In one embodiment, the microbial-induced obstructive
respiratory disease is tuberculosis (TB; Mycobacterium
tuberculosis). In another embodiment, the methods of the present
invention may be used to treat tuberculosis infections that are
acquired via zoonotic transmission, which may include inter alia
Mycobacterium bovis. In another embodiment, the invention provides
a method of suppressing, inhibiting, preventing, or treating a
pathogenic effect on a cell due to tuberculosis, comprising the
step of contacting the cell with a compound comprising a lipid or
phospholipid moiety bound to a physiologically acceptable monomer,
dimer, oligomer, or polymer, and/or a pharmaceutically acceptable
salt or a pharmaceutical product thereof. In another embodiment,
the invention provides a method of suppressing, inhibiting,
preventing, or treating a tuberculosis infection in a subject
comprising the step of administering to said subject an effective
amount of a lipid or phospholipid moiety bound to a physiologically
acceptable monomer, dimer, oligomer, or polymer, and/or a
pharmaceutically acceptable salt or a pharmaceutical product
thereof.
[0135] In one embodiment, the invention provides a method of
treating a subject suffering from a tuberculosis infection,
including, inter alia, the step of administering to a subject an
effective amount of a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer,
thereby treating the subject suffering from a tuberculosis
infection.
[0136] In one embodiment, the invention provides a use of a lipid
or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject afflicted with a
tuberculosis infection.
Cytokine Overproduction
[0137] In one embodiment, the methods of the present invention
treat cytokine overproduction, which in one embodiment, can be
caused or exacerbated by microbial infections. In another
embodiment, the methods of the present invention treat secondary
complications including, inter alia, tissue damage. In one
embodiment, cytokine overproduction is due to blood bourne bacteria
(septicemia) or to the pulmonary condition known as acute (or
adult) respiratory distress syndrome (ARDS). In one embodiment, the
methods of the present invention prevent monocytic phagocytes and
leukocytes from adhering to endothelial surfaces or undergoing a
respiratory burst. In another embodiment, the methods prevent
oxidant injury or release of chemokines such as Gro .alpha.,
ENA-78, CX3X and MCP-1, leukotrienes, thromboxanes, prostaglandins,
or a combination thereof. In another embodiment, the methods
prevent the release of oxidants, mediators, or degradative enzymes,
in another embodiment prevent endothelial cell damage or release of
lysosomal enzymes by leukocytes. In one embodiment, the methods of
the present invention treat vaginal bacterial infection in which
cytokine overproduction plays a role.
[0138] In one embodiment, the invention provides a method for
treating a subject with an infection marked by unchecked
inflammation, inappropriate cytokine response, or a combination
thereof, including inter alia, influenza, tuberculosis,
schistosomiasis, chronic bronchitis, pneumonia, SARS, respiratory
syncitial virus, Empyema Thoracis, whooping cough, etc.
[0139] In one embodiment, the invention provides a method for
treating a subject requiring anti-TNF therapy, including, inter
alia, the step of administering to a subject an effective amount of
a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer, thereby treating
the subject requiring an anti-TNF therapy. In one embodiment, the
invention provides a use of a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, in the preparation of a pharmaceutical composition for
treating a subject requiring an anti-TNF therapy.
Sepsis
[0140] In one embodiment, the methods of the present invention
treat sepsis, which in one embodiment, can be caused or exacerbated
by microbial infections. In one embodiment, sepsis refers to
sepsis, septicemia or septic shock. In one embodiment, sepsis
syndrome and shock are triggered by the interactions of various
microbial products in the blood, which in one embodiment are
gram-negative endotoxins, with host mediator systems. In one
embodiment, the methods of the present invention prevent activation
of host mediators, including inter alia, cytokines, tumor necrosis
factor-.alpha. (TNF), Gro .alpha., ENA-78, CX3X and MCP-1,
NF.kappa..beta. transcription factor, lysosomal enzymes, oxidants
from leukocytes, products of the metabolism of arachidonic acid, or
a combination thereof.
[0141] In one embodiment, the invention provides a method of
treating a subject suffering from sepsis, including, inter, alia,
the step of administering to a subject an effective amount of a
lipid or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, thereby treating the subject
suffering from sepsis. In one embodiment, the invention provides a
use of a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer, in the preparation
of a pharmaceutical composition for treating a subject suffering
from sepsis. In one embodiment, sepsis is due to a pathogenic
effect. In another embodiment, it's due to a pathogenic infection,
in another embodiment, a viral infection, in another embodiment, a
bacterial infection. In one embodiment, the compounds for use in
the present invention may protect against bacterial or viral
induced septic shock.
Hemolysis
[0142] In one embodiment, the methods of the present invention
treat hemolysis, which in one embodiment, can be caused or
exacerbated by microbial infections. In one embodiment, hemolysis
is red blood cell lysis, which in one embodiment may be an acquired
disorder. In one embodiment the methods of the present invention
may be used to treat anemia, iron deficiency, or jaundice
associated with hemolysis. In one embodiment, the methods of the
present invention may be used to treat membrane anomalies, which in
one embodiment, are due to infectious agents, including inter alia,
viral, bacterial and parasitic etiologies. In one embodiment, the
pathogen causing hemolysis is malaria, while in another embodiment,
it's hemorrhagic fevers.
[0143] In one embodiment, the invention provides a method of
treating a subject with hemolysis, including, inter alia, the step
of administering to a subject an effective amount of a lipid or
phospholipid moiety bonded to a physiologically acceptable monomer,
dimer, oligomer, or polymer, thereby treating the subject with
hemolysis. In one embodiment, the invention provides a use of a
lipid or phospholipid moiety bonded to a physiologically acceptable
monomer, dimer, oligomer, or polymer, in the preparation of a
pharmaceutical composition for treating a subject with hemolysis.
In one embodiment, hemolysis is due to a pathogenic effect. In
another embodiment, it's due to a pathogenic infection, in another
embodiment, a viral infection, in another embodiment, a bacterial
infection. In one embodiment, the compounds for use in the present
invention may protect against cytopathic effects due to infection
or cell to cell spread.
Oxidative Injury
[0144] In one embodiment, the methods of the present invention
treat oxidative injury, which in one embodiment, can be caused or
exacerbated by microbial infections. In one embodiment, oxidative
injury refers to the effect of peroxidation and free radical
production on body tissues. In one embodiment, peroxide production
is produced by the body in response to pathogenic infections, such
as viral or bacterial infections. In one embodiment, free radicals
are unpaired electrons that can damage cell proteins, DNA and
lipids that may be formed as biological weapons against viruses,
bacteria and cancer cells. In one embodiment, the methods of the
present invention treat oxidative injury to membrane components or,
in another embodiment, to blood proteins.
[0145] In one embodiment, the invention provides a method of
treating a subject requiring anti-oxidant therapy, including, inter
alia, the step of administering to a subject an effective amount of
a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer, thereby treating
the subject requiring an anti-oxidant therapy. In one embodiment,
the invention provides a use of a lipid or phospholipid moiety
bonded to a physiologically acceptable monomer, dimer oligomer, or
polymer, in the preparation of a pharmaceutical composition for
treating a subject requiring an anti-oxidant therapy. In one
embodiment, oxidative tissue damage is due to a pathogenic effect.
In another embodiment, it's due to a pathogenic infection, in
another embodiment, a viral infection, in another embodiment, a
bacterial infection. In one embodiment, the compounds for use in
the present invention may protect against tissue damage induced by
viruses, bacteria or a combination thereof.
Central Nervous System Insult
[0146] In one embodiment, the methods of the present invention
treat Central Nervous System Insult, which in one embodiment, can
be caused or exacerbated by microbial infections. In one embodiment
"Central Nervous System Insult" refers to neurological injury that
may result from infection, ischemic stroke, trauma, cancer
metastases, degenerative disease, or a combination thereof. In one
embodiment, the methods of the present invention treat
physiological responses to stress resulting from tissue injury. In
one embodiment, the methods prevent the release of or treat the
damage caused by chemical substances released by support
tissue.
[0147] In one embodiment, central nervous system (CNS) tissue
insult is due to a pathogenic effect. In another embodiment, it's
due to a pathogenic infection, in another embodiment, a viral
infection, in another embodiment, a bacterial infection. In one
embodiment, CNS insult is due to viral meningitis, Encephalitis,
Poliomyelitis, bacterial meningitis, subdural empyema, CNS
helminthic infections or any combination thereof.
[0148] In one embodiment, the invention provides a method of
treating a subject suffering from central nervous system tissue
insult, including, inter alia, the step of administering to a
subject an effective amount of a lipid or phospholipid moiety
bonded to a physiologically acceptable monomer, dimer, oligomer, or
polymer, thereby treating the subject suffering from a central
nervous system insult. In one embodiment, the invention provides a
use of a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer, in the preparation
of a pharmaceutical composition for treating a subject suffering
from central nervous system insult.
Conjunctivitis
[0149] In one embodiment, the methods of the present invention
treat conjunctivitis, which in one embodiment, can be caused or
exacerbated by microbial infections, which in one embodiment are
viral and in another embodiment are bacterial. In one embodiment it
is an infection of the conjunctiva (the outer-most layer of the eye
that covers the sclera).
[0150] In one embodiment, the methods of the present invention
treat symptoms of viral conjunctivitis including inter alia
conjunctival injection, tearing, serous discharge, edematous
eyelids, pinpoint subconjunctival hemorrhages, pseudomembrane
formation and palpable preauricular lymph nodes. In some
embodiment, they treat conjunctival desiccation, which may cause
scarring and symblepharon formation (adherence of the bulbar and
palpebral conjunctivas), in one embodiment.
[0151] In one embodiment the methods of the present invention treat
symptoms of conjunctivitis caused by epidemic keratoconjunctivitis
(EKC) or pharyngoconjunctival fever (PCF). In one embodiment,
symptoms of PCF comprise fever, sore throat and follicular
conjunctivitis. In one embodiment, the pathogen causing PCF is
adenovirus type 3, adenovirus type 4, or adenovirus type 7. In one
embodiment, symptoms of EKC comprise bilateral, inferior,
palpebral, follicular conjunctivitis, with epithelial and stromal
keratitis or subepithelial corneal infiltrates. In one embodiment,
the pathogen causing EKC is adenovirus type 8 or adenovirus type
19.
[0152] In one embodiment, the methods of the present invention
treat symptoms of bacterial conjunctivitis, which, in one
embodiment, is thick mucopurulent discharge, photophobia or
discomfort. In one embodiment, the bacteria causing conjuctivitis,
in one embodiment, is staphylococcus or streptococcus. In another
embodiment, the pathogen is Staphylococcus aureus, Haemophilus
influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa,
Neisseria gonorrhoeae or Corynebacterium diptheroides. In one
embodiment, the methods of the present invention treat penetration
of an intact cornea.
[0153] In one embodiment, a subject is afflicted with
conjunctivitis from a pathogenic source. In one embodiment,
conjunctivitis is mediated by Chlamydia trachomatis, in another
embodiment it's mediated by Streptococcus pneumoniae, in another
embodiment it's mediated by Haemophilus influenze, in another
embodiment it's mediated by Corynebacterium diptheroides, in
another embodiment it's mediated by Pseudomonas aeruginosa, while
in another embodiment it's mediated by Staphlococcus aureus. In one
embodiment, conjunctivitis is mediated by Neisseria gonorrhoeae,
while in another embodiment, it's mediated by herpes simplex virus,
while in another embodiment, while in another embodiment, it's
mediated by an Adenovirus, while in another embodiment, it's
mediated by an Enterovirus.
[0154] In one embodiment, the invention provides a method of
treating a subject afflicted with conjunctivitis, including, inter
alia, the step of administering to a subject an effective amount of
a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer, thereby treating
the subject afflicted with conjunctivitis. In one embodiment, the
invention provides a use of a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer, in the preparation of a pharmaceutical composition for
treating a subject afflicted with conjunctivitis.
[0155] Administration of the Lipid-conjugates in a diversity of
animal and cell models of disease invoked remarkable, and
unexpected, cytoprotective effects, which, as exemplified herein,
are useful in the treatment of diseases related to pathogenic
infection. Lipid-conjugates reduce HIV titre, inhibit p24
production, suppress cell fusion, block V3 antibody binding in a
cell model of HIV infection; block cytopathic effects in a cell
model of influenza infection; and prevent infection of cells by
vaccinia virus (as exemplified in Example 1). Lipid-conjugates also
reduced infection and apoptosis in a cell model of chlamydia
infection (as exemplified in Example 2). The compounds for the use
in the present invention also prevent smooth muscle airway
constriction, reduce sPLA2 expression in rat lung, reduce cysteinyl
leukotrienes, reduce NO production, prevent airway remodeling, and
reduce tumor necrosis factor-.alpha. (TNF-.alpha.) in animal and
cell models of obstructive respiratory disease (as exemplified in
Example 3). Lipid-conjugates also increased survival of septic
rats, reduced TNF-.alpha. and IL-6 mRNA and protein levels, reduced
sPLA2-IIA and iNOS mRNA, and reduced ICAM-1 protein in cell and
animal models of sepsis (as exemplified in Example 4).
Lipid-conjugates also stabilized red blood cell membranes in a cell
model of hemolysis (as exemplified in Example 5). Lipid-conjugates
are also effective in stabilizing biological membranes, preventing
GAG degradation, and protecting against copper-induced oxidation in
cell models of oxidative injury (as exemplified in Example 6).
Lipid-conjugates also inhibit NO production, PGE2, sPLA2, oleic
acid, dopamine, and serotonin release from PC12 and glial cells in
cell models of CNS injury (as exemplified in Example 7).
[0156] Thus, in one embodiment, the invention provides a treatment
method that utilizes a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer.
These Lipid-conjugates display a wide-range combination of
cytoprotective pharmacological activities. These compounds may in
some embodiments, interfere with bacterial and viral spread and
signs of infection, alleviate airway obstruction, attenuate
oxidative damage to tissue proteins and cell membranes, reduce
intracellular levels of chemokines and cytokines after exposure to
bacterial endotoxins, and protect CNS cells by reducing release of
neurotoxic agents.
[0157] In one embodiment of the present invention, the useful
pharmacological properties of the Lipid-conjugates, some of which
are described hereinabove, may be applied for clinical use, and
disclosed herein as methods for the prevention or treatment of a
disease. The biological basis of these methods may be readily
demonstrated by standard cellular and animal models of disease, for
example, as described in the Examples 1-7, hereinbelow.
[0158] In one embodiment, the pharmacological activities of
Lipid-conjugates, including membrane stabilization,
anti-inflammation, anti-oxidant action, and attenuation of
chemokine levels, may contribute to a Lipid-conjugate-treated
cell's resistance to pathogenic infection, such as HIV, influenza,
vaccinia, smallpox, poxvirus, chordopoxvirinae, and Chlamydia
infection. In one embodiment, cell membrane stabilization may
ameliorate or prevent tissue injury arising in the course of a
pathological disease state. In another embodiment, anti-oxidant
action may limit oxidative damage to cell and blood components
arising in the course of a pathological disease state. In another
embodiment, attenuation of chemokines levels may attenuate
physiological reactions to stress that arise in the course of a
pathological disease state.
[0159] In one embodiment, the present invention provides for use of
a lipid moiety bonded to a physiologically acceptable monomer,
dimer, oligomer, or polymer, in the preparation of a pharmaceutical
composition for treating a subject afflicted with a pathogenic
infection, which in one embodiment is a viral infection, and in
another embodiment, a bacterial infection. In another embodiment,
the use of the compounds is for treating symptoms or secondary
complications related to the pathogenic infection.
[0160] In one embodiment, the methods of the present invention
include a composition comprising the compounds as described and may
be formulated for administration by topical, oral, nasal, aerosol,
intravenous, intraocular, intra-arterial, subcutaneous, or
suppository routes as will be described hereinbelow.
[0161] In one embodiment of the invention, the Lipid-conjugates
described herein can be used to treat disease, through
amelioration, or prevention, of tissue injury arising in the course
of pathological disease states by stabilizing cell membranes;
limiting oxidative damage to cell and blood components; or
attenuating physiological reactions to stress, as expressed in
elevated chemokine levels.
[0162] The medicinal properties of the compounds for use in the
present invention are readily exemplified using animal models of
particular diseases of interest. The patients to whom the lipid or
phospholipid conjugates should be administered are those that are
experiencing symptoms of disease or who are at risk of contracting
the disease or experiencing a recurrent episode or exacerbation of
the disease. Thus, the lipid or phospholipid conjugates of the
present invention may be used to treat an individual with a disease
or disorder or to prevent an individual from contracting a disease
or developing a disorder.
[0163] The combination of lipids, such as, but not limited to
phosphatidylethanolamine and phosphatidylserine, with additional
monomer or polymer moieties, is thus a practical route to the
production of new drugs for medical purposes, provided that the
resultant chemical composition displays the desired range of
pharmacological properties. In one embodiment, the Lipid-conjugates
of this invention possess a combination of multiple and potent
pharmacological effects in addition to the ability to inhibit the
extracellular form of the enzyme phospholipase A2. While the
pharmacological activity of the Lipid-conjugates described herein
may be due in part to the nature of the lipid moiety, the multiple
and diverse combination of pharmacological properties observed for
the Lipid-conjugates emerges from the ability of the compound
structure to act essentially as several different drugs in one
chemical entity.
[0164] In the cases described herein, the diversity of biological
activities and the effectiveness in disease exhibited by the
compounds for use in the present invention far exceed the
properties anticipated by use of the starting materials themselves,
when administered alone or in combination. However, the
phospholipid conjugate compounds, alone or in combination, are
valuable when used in the methods of treating diseases and
conditions specifically described herein.
[0165] In one embodiment, methods of the present invention involve
treating a subject by inter alia controlling the expression,
production, and activity of phospholipases such as PLA2;
controlling the production and/or action of lipid mediators, such
as eicosanoids, platelet activating factor (PAF) and
lyso-phospholipids; amelioration of damage to cell surface
glycosaminoglycans (GAG) and proteoglycans; controlling the
production of oxidants, oxygen radicals and nitric oxide;
protection of cells, tissues, and plasma lipoproteins from damaging
agents, such as reactive oxygen species (ROS) and phospholipases;
controlling the expression, production, and activity of cytokines,
chemokines and interleukins; anti-oxidant therapy; anti-endotoxin
therapy or any combination thereof.
[0166] In one embodiment of the invention, the term "controlling"
refers to inhibiting the production and action of the above
mentioned factors in order to maintain their activity at the normal
basal level and suppress their activation in pathological
conditions.
[0167] In one embodiment of the invention, infection is
characterized by the presence of damaging agents, which comprise,
inter alia, phospholipases, reactive oxygen species (ROS), free
radicals, lysophospholipids, fatty acids or derivatives thereof,
hydrogen peroxides, phospholipids, oxidants, cationic proteins,
streptolysins, proteases, hemolysins, or sialidases.
Dosages and Routes of Administration
[0168] As used herein, the term "pharmaceutically acceptable"
refers to any formulation which is safe, and provides the
appropriate delivery for the desired route of administration of an
effective amount of at least one compound for use in the present
invention. As such, all of the above-described formulations of the
present invention are hereby referred to as "pharmaceutically
acceptable." This term refers to the use of buffered formulations
as well, wherein the pH is maintained at a particular desired
value, ranging from pH 4.0 to pH 9.0, in accordance with the
stability of the compounds and route of administration.
[0169] In one embodiment, a Lipid-conjugate used in the methods of
this invention may be administered alone or within a composition
comprising a Lipid-conjugate. In another embodiment, compositions
comprising Lipid-conjugates in admixture with conventional
excipients, i.e. pharmaceutically acceptable organic or inorganic
carrier substances suitable for parenteral, enteral (e.g., oral) or
topical application which do not deleteriously react with the
active compounds may be used. Suitable pharmaceutically acceptable
carriers include but are not limited to water, salt solutions,
alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene
glycols, gelatine, carbohydrates such as lactose, amylose or
starch, magnesium stearate, talc, silicic acid, viscous paraffin,
white paraffin, glycerol, alginates, hyaluronic acid, collagen,
perfume oil, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid esters, hydroxy methylcellulose,
polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
coloring, flavoring and/or aromatic substances and the like which
do not deleteriously react with the active compounds. They can also
be combined where desired with other active agents, e.g.,
vitamins.
[0170] In one embodiment, the therapeutic composition of the
instant invention comprises a Lipid-conjugate and additional
compounds effective in preventing or treating pathogenic
infections. In one embodiment, the additional compounds comprise
nucleotide analogs, interferons, or immunoglobulins. In another
embodiment, the nucleotide analogs comprise acyclovir, ganciclovir,
or ribavirin and interferons comprise alpha-, beta-, or
gamma-interferons. In one embodiment, any one of the abovementioned
additional compounds is administered with one or more
Lipid-conjugates to treat a viral infection, which in one
embodiment is influenza, in another embodiment, it's HIV, in
another embodiment, it's poxvirus, in another embodiment, it's
smallpox, while in another embodiment, it's vaccinia. In another
embodiment, the additional compounds comprise nucleoside reverse
transcriptase inhibitors, non-nucleoside reverse transcriptase
inhibitors, protease inhibitors, or fusion and attachment
inhibitors. In one embodiment, any one of the abovementioned
additional compounds is administered with one or more
Lipid-conjugates to treat a viral infection, which is in one
embodiment HIV. In another embodiment, the additional compounds
comprise Zidovudine, Didanosine, Zalcitabine, Stavudine,
Lamivudine, Abacavir, Tenofovir, Nevirapine, Delavirdine,
Efavirenz, Saquinavir, Ritonavir, Indinavir, Nelfinavir,
Amprenavir, Lopinavir, Atazanavir, Fosamprenavir, or Enfuvirtide.
In one embodiment, any one of the abovementioned additional
compounds is administered with one or more of the Lipid-conjugates
to treat a viral infection, which is in one embodiment HIV.
[0171] In another embodiment, the additional compounds comprise
Lactam Antibiotics, Aminoglycosides, Macrolides, Lincomycin,
Clindamycin, Tetracyclines, Quinolones, Polypeptides, Sulfonamides,
Trimethoprim-Sulfametlioxazole, or antimicrobial Chemoprophylaxis.
In another embodiment, the additional compound is Erythromycin. In
one embodiment, any one of the abovementioned additional compounds
is administered with one or more of the Lipid-conjugates to treat a
bacterial infection, which is in one embodiment chlamydia. In
another embodiment, the additional compounds comprise Rifampicin,
Pyrazinamid, Isoniazid, or Ethambutol. In one embodiment, any one
of the abovementioned additional compounds is administered with one
or more of the Lipid-conjugates to treat a bacterial infection,
which is in one embodiment tuberculosis.
[0172] In another embodiment, the additional compounds comprise
albendazole, mebendazole, pyrantel pamoate, thiabendazole,
chloroquine, mefloquine, quinine, atovaquone-proguanil, quinidine,
pyrimethamine, doxycycline, or a combination thereof. In one
embodiment, any one of the abovementioned additional compounds is
administered with one or more of the Lipid-conjugates to treat a
parasitic infection.
[0173] In another embodiment, the additional compounds comprise
analgesics, cytokines, growth factors, or a combination thereof.
Compositions of the present invention may comprise any one of the
compounds listed hereinabove or any combination thereof for use in
the methods of this invention.
[0174] While the examples provided herein describe use of the
phospholipid conjugates in subcutaneous, intraperitoneal or topical
administration, the success described affords good evidence to
suppose that other routes of administration, or combinations with
other pharmaceutical preparations, would be at least as successful.
In one embodiment, the route of administration may be parenteral,
enteral, or a combination thereof. In another embodiment, the route
may be subcutaneous, intraperitoneal, intravenous, intra-arterial,
topical, transdermal, intradermal, vaginal, rectal, intra-ocular,
conjunctival, inhalation, nasal aspiration (spray), sublingual,
oral, or a combination thereof. In one embodiment, the dosage
regimen will be determined by skilled clinicians, based on factors
such as exact nature of the condition being treated, the severity
of the condition, the age and general physical condition of the
patient, etc.
[0175] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories and enemas. Ampoules are convenient unit
dosages. Such a suppository may comprise any agent described
herein, and, in one embodiment, may be used to treat Chlamydia.
[0176] For application by inhalation, particularly for treatment of
airway obstruction or congestion, solutions or suspensions of the
compounds mixed and aerosolized or nebulized in the presence of the
appropriate carrier suitable. Such an aerosol may comprise any
agent described herein and, in one embodiment, may be used to treat
diseases or conditions caused by airborne pathogens, which may be
in one embodiment, influenza or tuberculosis.
[0177] For topical application, particularly for the treatment of
skin diseases such as contact dermatitis or psoriasis, admixture of
the compounds with conventional creams, lotions, or delayed release
patches is acceptable. Such a cream or lotion may comprise any
agent described herein, and, in one embodiment, may be used to
treat Chlamydia. In another embodiment, compounds for use in the
present invention may be used to coat condoms, or any intravaginal
or intraanal device. According to this embodiment, a compound of
the invention may act as a lubricant, prevent infection by
pathogens, such as HIV, or a combination thereof.
[0178] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, or capsules. A syrup, elixir, or the like
can be used when a sweetened vehicle is employed.
[0179] Sustained or directed release compositions can be
formulated, e.g., liposomes or those wherein the active compound is
protected with differentially degradable coatings, e.g., by
microencapsulation, multiple coatings, etc. It is also possible to
freeze-dry the new compounds and use the lyophilisates obtained,
for example, for the preparation of products for injection.
[0180] Thus, in one embodiment, the route of administration may be
directed to an organ or system that is affected by the pathogenic
infection. For example, compounds may be administered in aerosol
form to treat infections by airborne pathogens. In another
embodiment, the route of administration may be directed to a
different organ or system than the one that is affected by the
pathogenic infection. For example, compounds may be administered
parenterally to treat infections by airborne pathogens.
[0181] Thus, the present invention provides for the use of
Lipid-conjugates in various dosage forms suitable for
administration using any of the routes listed hereinabove.
[0182] In general, the doses utilized for the above described
purposes will vary, but will be in an effective amount to exert the
desired anti-disease effect. As used herein, the term
"pharmaceutically effective amount" refers to an amount of a
compound of formulae A and I-XXI as described hereinbelow, which
will produce the desired alleviation in symptoms or signs of
disease in a patient. The doses utilized for any of the
above-described purposes will generally be from 1 to about 1000
milligrams per kilogram of body weight (mg/kg), administered one to
four times per day, or by continuous IV infusion. When the
compositions are dosed topically, they will generally be in a
concentration range of from 0.1 to about 10% w/v, administered 1-4
times per day.
[0183] In one embodiment of the invention, the concentrations of
the compounds will depend on various factors, including the nature
of the condition to be treated, the condition of the patient, the
route of administration and the individual tolerability of the
compositions.
[0184] It will be appreciated that the actual preferred amounts of
active compound in a specific case will vary according to the
specific compound being utilized, the particular compositions
formulated, the mode of application, and the particular conditions
and organism being treated. Dosages for a given host can be
determined using conventional considerations, e.g., by customary
comparison of the differential activities of the subject compounds
and of a known agent, e.g., by means of an appropriate,
conventional pharmacological protocol.
[0185] In one embodiment, the compounds of the invention may be
administered acutely for acute treatment of temporary conditions,
or may be administered chronically, especially in the case of
progressive, recurrent, or degenerative disease. In one embodiment,
one or more compounds of the invention may be administered
simultaneously, or in another embodiment, they may administered in
a staggered fashion. In one embodiment, the staggered fashion may
be dictated by the stage or phase of the disease.
[0186] In one embodiment, the present invention offers methods for
the treatment of disease based upon administration of lipids
covalently conjugated through their polar head group to a
physiologically acceptable chemical moiety, which may be of high or
low molecular weight.
[0187] The present invention has been illustrated in terms of the
anti-disease activity of Lipid-conjugates and methods of their use
as pharmaceutical compositions in the treatment of disease. The
following sections present some examples of the therapeutic
Lipid-conjugate compounds for use in the present invention and
their chemical preparation.
Compounds
[0188] In one embodiment, the compounds for use in the present
invention comprise a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer. In
one embodiment, the lipid compounds (Lipid-conjugates) for use in
the present invention are described by the general formula:
[phosphatidylethanolamine-Y]n-X [phosphatidylserine-Y]n-X
[phosphatidylcholine-Y]n-X [phosphatidylinositol-Y]n-X
[phosphatidylglycerol-Y]n-X [phosphatidic acid-Y]n-X
[lyso-phospholipid-Y]n-X [diacyl-glycerol-Y]n-X
[monoacyl-glycerol-Y]n-X [sphingomyelin-Y]n-X [sphingosine-Y]n-X
[ceramide-Y]n-X wherein Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; and X is a physiologically
acceptable monomer, dimer, oligomer or polymer; and n is the number
of lipid molecules bound to a molecule of X, wherein n is a number
from 1 to 1000.
[0189] In one embodiment, the invention provides low-molecular
weight Lipid-conjugates, previously undisclosed and unknown to
possess pharmacological activity, of the general formula described
hereinabove. In another embodiment, wherein the general formula
described hereinabove describes low-molecular weight
Lipid-conjugates, X is a mono- or disaccharide, carboxylated
disaccharide, mono- or dicarboxylic acids, a salicylate, salicylic
acid, aspirin, lactobionic acid, maltose, an amino acid, glycine,
acetic acid, butyric acid, dicarboxylic acid, glutaric acid,
succinic acid, fatty acid, dodecanoic acid, didodecanoic acid, bile
acid, cholic acid, cholesterylhemmisuccinate, a di- or tripeptide,
an oligopeptide, a trisacharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid.
[0190] In one embodiment of this invention, X is salicylate,
salicylic acid, aspirin, a monosaccharide, lactobionic acid,
maltose, an amino acid, glycine, carboxylic acid, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemmisuccinate, a dipeptide, a disaccharide, a
trisaccharide, an oligosaccharide, an oligopeptide, or a di- or
trisaccharide monomer unit of heparin, heparan sulfate, keratin,
keratan sulfate, chondroitin, chondroitin-6-sulfate,
chondroitin-4-sulfate, dermatin, dermatan sulfate, dextran, or
hyaluronic acid, a glycosaminoglycan, polygeline (`haemaccel`),
alginate, hydroxyethyl starch (hetastarch), polyethylene glycol,
polycarboxylated polyethylene glycol, chondroitin-6-sulfate,
chondroitin-4-sulfate, keratin, keratin sulfate, heparan sulfate,
dermatin, dermatan sulfate, carboxymethylcellulose, heparin,
dextran, or hyaluronic acid.
[0191] In one embodiment of this invention, n is a number from 1 to
1000. In another embodiment, n is a number from 1 to 500. In
another embodiment, n is a number from 1 to 100. In another
embodiment, n is a number from 2 to 100. In another embodiment, n
is a number from 2 to 200. In another embodiment, n is a number
from 3 to 300. In another embodiment, n is a number from 10 to 400.
In another embodiment, n is a number from 50 to 500. In another
embodiment, n is a number from 100 to 300. In another embodiment, n
is a number from 300 to 500. In another embodiment, n is a number
from 500 to 800. In another embodiment, n is a number from 500 to
1000.
[0192] In one embodiment, the set of compounds comprising
phosphatidylethanolamine covalently bound to a physiologically
acceptable monomer or polymer, is referred to herein as the
PE-conjugates. In another embodiment, related derivatives, in which
either phosphatidylserine, phosphatidylcholine,
phosphatidylinositol, phosphatidic acid or phosphatidylglycerol are
employed in lieu of phosphatidylethanolamine as the lipid moiety
provide equivalent therapeutic results, based upon the biological
experiments described below for the Lipid-conjugates and the
structural similarities shared by these compounds.
[0193] In another embodiment, the lipid or phospholipid moiety is
phosphatidic acid, an acyl glycerol, monoacylglycerol,
diacylglycerol, triacylglycerol, sphingosine, sphingomyelin,
chondroitin-4-sulfate, chondroitin-6-sulfate, ceramide,
phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine,
phosphatidylinositol, or phosphatidylglycerol, or an ether or alkyl
phospholipid derivative thereof.
[0194] Other Lipid-conjugate derivatives relevant to this invention
are Lipid-conjugates wherein at least one of the fatty acid groups
of the lipid moieties at position C1 or C2 of the glycerol backbone
are substituted by a long chain alkyl group attached by either
ether or alkyl bonds, rather than ester linkages.
[0195] As defined by the structural formulae provided herein for
the Lipid-conjugates, these compounds may contain between one to
one thousand lipid moieties bound to a single physiologically
acceptable polymer molecule.
[0196] In the methods, according to embodiments of the invention,
the Lipid-conjugates administered to the subject are comprised from
at least one lipid moiety covalently bound through an atom of the
polar head group to a monomeric or polymeric moiety (referred to
herein as the conjugated moiety) of either low or high molecular
weight. When desired, an optional bridging moiety can be used to
link the Lipid-conjugates moiety to the monomer or polymeric
moiety. The conjugated moiety may be a low molecular weight
carboxylic acid, dicarboxylic acid, fatty acid, dicarboxylic fatty
acid, acetyl salicylic acid, cholic acid, cholesterylhemisuccinate,
or mono- or di-saccharide, an amino acid or dipeptide, an
oligopeptide, a glycoprotein mixture, a di- or trisaccharide
monomer unit of a glycosaminoglycan such as a repeating unit of
heparin, heparan sulfate, hyaluronic acid, chondroitin-sulfate,
dermatan, keratan sulfate, or a higher molecular weight peptide or
oligopeptide, a polysaccharide, polyglycan, protein,
glycosaminoglycan, or a glycoprotein mixture. The composition of
phospholipid-conjugates of high molecular weight, and associated
analogues, are the subject of U.S. Pat. No. 5,064,817.
[0197] In one embodiment of the invention, when the conjugated
carrier moiety is a polymer, the ratio of lipid moieties covalently
bound may range from one to one thousand lipid residues per polymer
molecule, depending upon the nature of the polymer and the reaction
conditions employed. For example, the relative quantities of the
starting materials, or the extent of the reaction time, may be
modified in order to obtain Lipid-conjugate products with either
high or low ratios of lipid residues per polymer, as desired.
[0198] In one embodiment, the term "moiety" means a chemical entity
otherwise corresponding to a chemical compound, which has a valence
satisfied by a covalent bond.
[0199] In one embodiment, examples of polymers which can be
employed as the conjugated moiety for producing Lipid-conjugates
for use in the methods of this invention may be physiologically
acceptable polymers, including water-dispersible or -soluble
polymers of various molecular weights and diverse chemical types,
mainly natural and synthetic polymers, such as glycosaminoglycans,
hyaluronic acids, heparin, heparin sulfates, chondroitin sulfates,
chondroitin-6-sulfates, chondroitin-4-sulfates, keratins, keratin
sulfates, dermatins, dermatan sulfates, dextrans, plasma expanders,
including polygeline ("Haemaccel", degraded gelatin polypeptide
cross-linked via urea bridges, produced by "Behring"),
"hydroxyethylstarch" (Hetastarch, HES) and extrans, food and drug
additives, soluble cellulose derivatives (e.g., methylcellulose,
carboxymethylcellulose), polyaminoacids, hydrocarbon polymers
(e.g., polyethylene), polystyrenes, polyesters, polyamides,
polyethylene oxides (e.g. polyethyleneglycols,
polycarboxyethyleneglycols, polycarboxylated polyethyleneglycols),
polyvinylpyrrolidones, polysaccharides, alginates, assimilable gums
(e.g., xanthan gum), peptides, injectable blood proteins (e.g.,
serum albumin), cyclodextrin, and derivatives thereof.
[0200] In one embodiment, examples of monomers, dimers, and
oligomers which can be employed as the conjugated moiety for
producing Lipid-conjugates for use in the methods of the invention
may be mono- or disaccharides, trisaccharides, oligopeptides,
carboxylic acids, dicarboxylic acids, fatty acids, dicarboxylic
fatty acids, salicylates, slicyclic acids, acetyl salicylic acids,
aspirins, lactobionic acids, maltoses, amino acids, glycines,
glutaric acids, succinic acids, dodecanoic acids, didodecanoic
acids, bile acids, cholic acids, cholesterylhemisuccinates, and di-
and trisaccharide unit monomers of glycosaminoglycans including
heparins, heparan sulfates, hyaluronic acids, chondroitins,
chondroitin sulfates, chondroitin-6-sulfates,
chondroitin-4-sulfates, dermatins, dermatan sulfates, keratins,
keratan sulfates, or dextrans.
[0201] In some cases, according to embodiments of the invention,
the monomer or polymer chosen for preparation of the
Lipid-conjugate may in itself have select biological properties.
For example, both heparin and hyaluronic acid are materials with
known physiological functions. In the present invention, however,
the Lipid-conjugates formed from these substances as starting
materials display a new and wider set of pharmaceutical activities
than would be predicted from administration of either heparin or
hyaluronic acid which have not been bound by covalent linkage to a
phospholipid. It can be shown, by standard comparative experiments
as described below, that phosphatidylethanolamine (PE) linked to
hyaluronic acid. (Compound XXII), to heparin (Compound XXIV), to
chondroitin sulfate A (Compound XXV), to carboxymethylcellulose
(Compound XXVI), to Polygeline (haemaccel) (Compound XXVII), or to
hydroxyethylstarch (Compound XXVIII), are far superior in terms of
potency and range of useful pharmaceutical activity to the free
conjugates (the polymers above and the like). In fact, these latter
substances are, in general, not considered useful in methods for
treatment of most of the diseases described herein, including the
treatment of pathogenic infections. Thus, the combination of a
phospholipid such as phosphatidylethanolamine, or related
phospholipids which differ with regard to the polar head group,
such as phosphatidylserine (PS), phosphatidylcholine (PC),
phosphatidylinositol (PI), and phosphatidylglycerol (PG), results
in the formation of a compound which has novel pharmacological
properties when compared to the starting materials alone.
[0202] The biologically active Lipid-conjugates described herein
can have a wide range of molecular weight, e.g., above 50,000 (up
to a few hundred thousands) when it is desirable to retain the
Lipid conjugate in the vascular system and below 50,000 when
targeting to extravascular systems is desirable. The sole
limitation on the molecular weight and the chemical structure of
the conjugated moiety is that it does not result in a
Lipid-conjugate devoid of the desired biological activity, or lead
to chemical or physiological instability to the extent that the
Lipid-conjugate is rendered useless as a drug in the method of use
described herein.
[0203] In one embodiment, the compound according to the invention
is represented by the structure of the general formula (A):
##STR1## wherein [0204] L is a lipid or a phospholipid; [0205] Z is
either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0206] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0207] X is a physiologically acceptable
monomer, dimer, oligomer, or polymer; and [0208] n is a number from
1 to 1000; [0209] wherein any bond between L, Z, Y and X is either
an amide or an esteric bond.
[0210] In one embodiment, X is a glycosaminoglycan.
[0211] In one embodiment, L, is phosphatidyl, Z is ethanolamine,
wherein L and Z are chemically bonded resulting in
phosphatidylethanolamine, Y is nothing, and X is hyaluronic acid,
wherein any bond between the phosphatidylethanolamine and the
hyaluronic acid is an amide bond. In one embodiment, the
phosphatidylethanolamine moiety is dipalmitoyl
phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is dimyristoyl
phosphatidylethanolamine.
[0212] In another embodiment, L is phosphatidyl, Z is ethanolamine,
wherein L and Z are chemically bonded resulting in
phosphatidylethanolamine, Y is nothing, and X is chondroitin
sulfate, wherein any bond between the phosphatidylethanolamine and
the chondroitin sulfate is an amide bond. In one embodiment, the
phosphatidylethanolamine moiety is
dipalmitoyl-phosphatidyl-ethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is
dimyristoyl-phosphatidyl-ethanolamine.
[0213] In another embodiment, L is phosphatidyl, Z is ethanolamine,
wherein L and Z are chemically bonded resulting in
phosphatidylethanolamine, Y is nothing, and X is heparin, wherein
any bond between the phosphatidylethanolamine and the heparin is an
amide bond. In one embodiment, the phosphatidylethanolamine moiety
is dipalmitoyl-phosphatidyl-ethanolamine. In another embodiment,
the phosphatidylethanolamine moiety is
dimyristoyl-phosphatidyl-ethanolamine.
[0214] In another embodiment, L is phosphatidyl, Z is ethanolamine,
wherein L and Z are chemically bonded resulting in
phosphatidylethanolamine, Y is nothing, and X is polygeline,
wherein any bond between the phosphatidylethanolamine and the
polygeline is an amide bond. In one embodiment, the
phosphatidylethanolamine moiety is
dipalmitoyl-phosphatidyl-ethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is
dimyristoyl-phosphatidyl-ethanolamine.
[0215] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(I): ##STR2## wherein [0216] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0217] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0218] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms; and
[0219] X is either a physiologically acceptable monomer, dimer,
oligomer or a physiologically acceptable polymer; and [0220] n is a
number from 1 to 1,000; [0221] wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond
and if Y is a spacer, the spacer is directly linked to X via an
amide or an esteric bond and to the phosphatidylethanolamine via an
amide bond.
[0222] Preferred compounds for use in the methods of the invention
comprise one of the following as the conjugated moiety X: acetate,
butyrate, glutarate, succinate, dodecanoate, didodecanoate,
maltose, lactobionic acid, dextran, alginate, aspirin, cholate,
cholesterylhemisuccinate, carboxymethyl-cellulose, heparin,
hyaluronic acid, polygeline (haemaccel), polyethyleneglycol, and
polycarboxylated polyethylene glycol. The polymers used as starting
material to prepare the PE-conjugates may vary in molecular weight
from 1 to 2,000 kDa.
[0223] Examples of phosphatidylethanolamine (PE) moieties are
analogues of the phospholipid in which the chain length of the two
fatty acid groups attached to the glycerol backbone of the
phospholipid varies from 2-30 carbon atoms length, and in which
these fatty acids chains contain saturated and/or unsaturated
carbon atoms. In lieu of fatty acid chains, alkyl chains attached
directly or via an ether linkage to the glycerol backbone of the
phospholipid are included as analogues of PE. According to the
present invention, a most preferred PE moiety is
dipalmitoyl-phosphatidyl-ethanolamine. In another preferred
embodiment of the present invention, the PE moiety is
dimyristoyl-phosphatidyl-ethanolamine.
[0224] Phosphatidyl-ethanolamine and its analogues may be from
various sources, including natural, synthetic, and semisynthetic
derivatives and their isomers.
[0225] Phospholipids which can be employed in lieu of the PE moiety
are N-methyl-PE derivatives and their analogues, linked through the
amino group of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE
derivatives and their analogues linked through the amino group of
the N,N-dimethyl-PE by a covalent bond, phosphatidylserine (PS) and
its analogues, such as palmitoyl-stearoyl-PS, natural PS from
various sources, semisynthetic PSs, synthetic, natural and
artifactual PSs and their isomers. Other phospholipids useful as
conjugated moieties in this invention are phosphatidylcholine (PC),
phosphatidylinositol (PI), phosphatidic acid and
phosphoatidylglycerol (PG), as well as derivatives thereof
comprising either phospholipids, lysophospholipids, phosphatidyic
acid, sphingomyelins, lysosphingomyelins, ceramide, and
sphingosine.
[0226] For PE-conjugates and PS-conjugates, the phospholipid is
linked to the conjugated monomer or polymer moiety through the
nitrogen atom of the phospholipid polar head group, either directly
or via a spacer group. For PC, PI, and PG conjugates, the
phospholipid is linked to the conjugated monomer or polymer moiety
through either the nitrogen or one of the oxygen atoms of the polar
head group, either directly or via a spacer group.
[0227] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(II): ##STR3## wherein [0228] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0229] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0230] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0231] X is a physiologically acceptable monomer, dimer, oligomer
or polymer; and [0232] n is a number from 1 to 1000; [0233] wherein
if Y is nothing, the phosphatidylserine is directly linked to X via
an amide bond and if Y is a spacer, the spacer is directly linked
to X via an amide or an esteric bond and to the phosphatidylserine
via an amide bond.
[0234] In one embodiment, the phosphatidylserine may be bonded to
Y, or to X if Y is is nothing, via the COO.sup.- moiety of the
phosphatidylserine.
[0235] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(III): ##STR4## wherein [0236] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0237] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0238] Z is either
nothing, inositol, choline, or, glycerol; [0239] Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms;
[0240] X is a physiologically acceptable monomer, dimer, oligomer,
or polymer; and [0241] n is a number from 1 to 1000; [0242] wherein
any bond between the phosphatidyl, Z, Y and X is either an amide or
an esteric bond.
[0243] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(IV): ##STR5## wherein [0244] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0245] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0246] Z
is either nothing, inositol, choline, or glycerol; [0247] Y is
either nothing or a spacer group ranging in length from 2 to 30
atoms; [0248] X is a physiologically acceptable monomer, dimer,
oligomer, or polymer; and [0249] n is a number from 1 to 1000;
[0250] wherein any bond between the phospholipid, Z, Y and X is
either an amide or an esteric bond.
[0251] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(V): ##STR6## wherein [0252] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0253] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0254] Z
is either nothing, inositol, choline, or glycerol; [0255] Y is
either nothing or a spacer group ranging in length from 2 to 30
atoms; [0256] X is a physiologically acceptable monomer, dimer,
oligomer, or polymer; and [0257] n is a number from 1 to 1000;
[0258] wherein any bond between the phospholipid, Z, Y and X is
either an amide or an esteric bond.
[0259] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(VI): ##STR7## wherein [0260] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0261] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0262] Z
is either nothing, inositol, choline, or glycerol; [0263] Y is
either nothing or a spacer group ranging in length from 2 to 30
atoms; [0264] X is a physiologically acceptable monomer, dimer,
oligomer, or polymer; and [0265] n is a number from 1 to 1000;
[0266] wherein any bond between the phospholipid, Z, Y and X is
either an amide or an esteric bond.
[0267] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(VII): ##STR8## wherein [0268] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0269] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0270] Z
is either nothing, inositol, choline, or glycerol; [0271] Y is
either nothing or a spacer group ranging in length from 2 to 30
atoms; [0272] X is a physiologically acceptable monomer, dimer,
oligomer, or polymer; and [0273] n is a number from 1 to 1000;
[0274] wherein any bond between the phospholipid, Z, Y and X is
either an amide or an esteric bond.
[0275] In one embodiment of the invention, phosphatidylcholine
(PC), phosphatidylinositol (PI), phosphatidic acid (PA), wherein Z
is nothing, and phosphatidylglycerol (PG) conjugates are herein
defined as compounds of the general formula (III).
[0276] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(VIII): ##STR9## wherein [0277] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0278] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0279] Z
is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0280] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0281] X is a physiologically acceptable
monomer, dimer, oligomer, or polymer; and [0282] n is a number from
1 to 1000; [0283] wherein any bond between the phospholipid, Z, Y
and X is either an amide or an esteric bond.
[0284] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(IX): ##STR10## wherein [0285] R.sub.1 is either hydrogen or a
linear saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0286] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0287] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0288] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0289] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer; and [0290] n is a
number from 1 to 1000; [0291] wherein any bond between the
phospholipid, Z, Y and X is either an amide or an esteric bond.
[0292] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(IXa): ##STR11## wherein [0293] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0294] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0295] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0296] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0297] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer; and [0298] n is a
number from 1 to 1000; [0299] wherein any bond between the
phospholipid, Z, Y and X is either an amide or an esteric bond.
[0300] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(IXb): ##STR12## wherein [0301] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0302] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0303] Z is either nothing, ethanolamine, serine, inositol,
choline, or glycerol; [0304] Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; [0305] X is a physiologically
acceptable monomer, dimer, oligomer, or polymer; and [0306] n is a
number from 1 to 1000; [0307] wherein any bond between the
phospholipid, Z, Y and X is either an amide or an esteric bond.
[0308] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(X): ##STR13## wherein [0309] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0310] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0311] Z
is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; [0312] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0313] X is a physiologically acceptable
monomer, dimer, oligomer, or polymer; and [0314] n is a number from
1 to 1000; [0315] wherein any bond between the ceramide phosphoryl,
Z, Y and X is either an amide or an esteric bond.
[0316] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XI): ##STR14## wherein [0317] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0318] Y is either nothing or a
spacer group ranging in length from 2 to 30 atoms; [0319] X is a
physiologically acceptable monomer, dimer, oligomer or polymer; and
[0320] n is a number from 1 to 1000; [0321] wherein if Y is nothing
the sphingosyl is directly linked to X via an amide bond and if Y
is a spacer, the spacer is directly linked to X and to the
sphingosyl via an amide bond and to X via an amide or an esteric
bond.
[0322] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XII): ##STR15## wherein [0323] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0324] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0325] Z is either
nothing, ethanolamine, serine, inositol, choline, or glycerol;
[0326] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0327] X is a physiologically acceptable monomer,
dimer, oligomer or polymer; and [0328] n is a number from 1 to
1000; [0329] wherein any bond between the ceramide, Z, Y and X is
either an amide or an esteric bond.
[0330] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XIII): ##STR16## wherein [0331] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0332] R.sub.2 is a linear,
saturated, mono-unsaturated, or poly-unsaturated, alkyl chain
ranging in length from 2 to 30 carbon atoms; [0333] Z is either
nothing, choline, phosphate, inositol, or glycerol; [0334] Y is
either nothing or a spacer group ranging in length from 2 to 30
atoms; [0335] X is a physiologically acceptable monomer, dimer,
oligomer or polymer; and [0336] n is a number from 1 to 1000;
[0337] wherein any bond between the diglyceryl, Z, Y and X is
either an amide or an esteric bond.
[0338] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XIV): ##STR17## wherein [0339] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0340] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0341] Z
is either nothing, choline, phosphate, inositol, or glycerol;
[0342] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0343] X is a physiologically acceptable monomer,
dimer, oligomer or polymer; and [0344] n is a number from 1 to
1000; [0345] wherein any bond between the glycerolipid, Z, Y and X
is either an amide or an esteric bond.
[0346] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XV): ##STR18## wherein [0347] R.sub.1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in
length from 2 to 30 carbon atoms; [0348] R.sub.2 is either hydrogen
or a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0349] Z
is either nothing, choline, phosphate, inositol, or glycerol;
[0350] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0351] X is a physiologically acceptable monomer,
dimer, oligomer or polymer; and [0352] n is a number from 1 to
1000; [0353] wherein any bond between the glycerolipid, Z, Y and X
is either an amide or an esteric bond.
[0354] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XVI): ##STR19## wherein [0355] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0356] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0357] Z
is either nothing, choline, phosphate, inositol, or glycerol;
[0358] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0359] X is a physiologically acceptable monomer,
dimer, oligomer or polymer; and [0360] n is a number from 1 to
1000; [0361] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0362] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XVII): ##STR20## wherein [0363] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0364] R.sub.2
is a linear, saturated, mono-unsaturated, or poly-unsaturated,
alkyl chain ranging in length from 2 to 30 carbon atoms; [0365] Z
is either nothing, choline, phosphate, inositol, or glycerol;
[0366] Y is either nothing or a spacer group ranging in length from
2 to 30 atoms; [0367] X is a physiologically acceptable monomer,
dimer, oligomer or polymer; and [0368] n is a number from 1 to
1000; [0369] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0370] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XVIII): ##STR21## wherein [0371] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0372] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0373] Z is either nothing, choline, phosphate, inositol, or
glycerol; [0374] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0375] X is a physiologically acceptable
monomer, dimer, oligomer or polymer; and [0376] n is a number from
1 to 1000; [0377] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0378] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XIX): ##STR22## wherein [0379] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0380] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0381] Z is either nothing, choline, phosphate, inositol, or
glycerol; [0382] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0383] X is a physiologically acceptable
monomer, dimer, oligomer or polymer; and [0384] n is a number from
1 to 1000; [0385] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0386] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XX): ##STR23## wherein [0387] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0388] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0389] Z is either nothing, choline, phosphate, inositol, or
glycerol; [0390] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0391] X is a physiologically acceptable
monomer, dimer, oligomer or polymer; and [0392] n is a number from
1 to 1000; [0393] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0394] In another embodiment, the compound according to the
invention is represented by the structure of the general formula
(XXI): ##STR24## wherein [0395] R.sub.1 is either hydrogen or a
linear, saturated, mono-unsaturated, or poly-unsaturated, alkyl
chain ranging in length from 2 to 30 carbon atoms; [0396] R.sub.2
is either hydrogen or a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; [0397] Z is either nothing, choline, phosphate, inositol, or
glycerol; [0398] Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; [0399] X is a physiologically acceptable
monomer, dimer, oligomer or polymer; and [0400] n is a number from
1 to 1000; [0401] wherein any bond between the lipid, Z, Y and X is
either an amide or an esteric bond.
[0402] For any or all of the compounds represented by the
structures of the general formulae: (A), (I), (II), (III), (IV),
(V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI), (XII),
(XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI),
and (XXII) hereinabove. In one embodiment, X is a
glycosaminoglycan.
[0403] In one embodiment of the invention, the glycosaminoglycan
may be, inter alia, hyaluronic acid, heparin, heparan sulfate,
chondroitin sulfate, keratin, keratan sulfate, dermatan sulfate or
a derivative thereof.
[0404] In another embodiment, the glycosaminoglycan is a polymer of
disaccharide units. In another embodiment, the number of the
disaccharide units in the polymer is m. In another embodiment, m is
a number from 2-10,000. In another embodiment, m is a number from
2-500. In another embodiment, m is a number from 2-1000. In another
embodiment, m is a number from 50-500. In another embodiment, m is
a number from 2-2000 In another embodiment, m is a number from
500-2000. In another embodiment, m is a number from 1000-2000. In
another embodiment, m is a number from 2000-5000. In another
embodiment, m is a number from 3000-7000. In another embodiment, m
is a number from 5000-10,000. In another embodiment, a disaccharide
unit of a glycosaminoglycan may be bound to one lipid or
phospholipid moiety. In another embodiment, each disaccharide unit
of the glycosaminoglycan may be bound to zero or one lipid or
phospholipid moieties. In another embodiment, the lipid or
phospholipid moieties are bound to the --COOH group of the
disaccharide unit. In another embodiment, the bond between the
lipid or phospholipid moiety and the disaccharide unit is an amide
bond.
[0405] In another embodiment, the chondroitin sulfate may be, inter
alia, chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative
thereof.
[0406] In one embodiment of the invention Y is nothing. Non
limiting examples of suitable divalent groups forming the optional
bridging group (spacer) Y, according to embodiments of the
invention, are straight or branched chain alkylene, e.g., of 2 or
more, preferably 4 to 30 carbon atoms, --CO-alkylene-CO,
--NH-alkylene-NH--, --CO-alkylene-NH--,
--NH-alkylene-NHCO-alkylene-NH--, an amino acid, cycloalkylene,
wherein alkylene in each instance, is straight or branched chain
and contains 2 or more, preferably 2 to 30 atoms in the chain,
--(--O--CH(CH.sub.3)CH.sub.2--).sub.x-- wherein x is an integer of
1 or more.
[0407] According to embodiments of the invention, in addition to
the traditional phospholipid structure, related derivatives for use
in this invention are phospholipids modified at the C1 or C2
position to contain an ether or alkyl bond instead of an ester
bond. In one embodiment of the invention, the alkyl phospholipid
derivatives and ether phospholipid derivatives ate exemplified
herein.
[0408] In one embodiment of the invention, the sugar rings of the
glycosaminoglycan are intact. In another embodiment, intact refers
to closed. In another embodiment, intact refers to natural. In
another embodiment, intact refers to unbroken.
[0409] In one embodiment of the invention, the structure of the
lipid or phospholipid in any compound according to the invention is
intact. In another embodiment, the natural structure of the lipid
or phospholipids in any compound according to the invention is
maintained.
[0410] In one embodiment, the compounds for use in the present
invention are biodegradable.
[0411] In one embodiment, the compound according to the invention
is phosphatidylethanolamine bound to aspirin. In one embodiment,
the compound according to the invention is phosphatidylethanolamine
bound to glutarate.
[0412] In some embodiments, the compounds for use are as listed in
Table 1 below. TABLE-US-00001 TABLE 1 Com- Phospholipid Spacer
Polymer (m.w.) pound PE None Hyaluronic acid XXII (2-2000 kDa)
Dimyristoyl-PE None Hyaluronic acid XXIII PE None Heparin XXIV
(0.5-110 kDa) PE None Chondroitin sulfate A XXV PE None
Carboxymethylcellulose XXVI (20-500 kDa) PE Dicarboxylic Polygeline
(haemaccel) XXVII acid + Diamine (4-40 kDa) PE None
Hydroxyethylstarch XXVIII PE Dicarboxylic Dextran XXIX acid +
Diamine 1-2,000 kDa) PE None Aspirin XXX PE Carboxyl amino
Hyaluronic acid XXXI group (2-2000 kDa) PE Dicarboxyl group
Hyaluronic acid XXXII (2-2000 kDa) PE Dipalmitoic acid Hyaluronic
acid XXXIII (2-2000 kDa) PE Carboxyl amino Heparin XXXIV group
(0.5-110 kDa) PE Dicarboxyl group Heparin XXXV (0.5-110 kDa) PE
Carboxyl amino Chondroitin sulfate A XXXVI group PE Dicarboxyl
group Chondroitin sulfate A XXXVII PE Carboxyl amino
Carboxymethylcellulose XXXVIII group (20-500 kDa) PE Dicarboxyl
group Carboxymethylcellulose XXXIX (20-500 kDa) PE None Polygeline
(haemaccel) XL (4-40 kDa) PE Carboxyl amino Polygeline (haemaccel)
XLI group (4-40 kDa) PE Dicarboxyl group Polygeline (haemaccel)
XLII (4-40 kDa) PE Carboxyl amino Hydroxyethylstarch XLIII group PE
Dicarboxyl group Hydroxyethylstarch XLIV PE None Dextran XLV
(1-2,000 kDa) PE Carboxyl amino Dextran XLVI group (1-2,000 kDa) PE
Dicarboxyl group Dextran XLVII (1-2,000 kDa) PE Carboxyl amino
Aspirin XLVIII group PE Dicarboxyl group Aspirin XLIX PE None
Albumin L PE None Alginate LI (2-2000 kDa) PE None Polyaminoacid
LII PE None Polyethylene glycol LIII PE None Lactobionic acid LIV
PE None Acetylsalicylate LV PE None Cholesteryl- LVI hemmisuccinate
PE None Maltose LVII PE None Cholic acid LVIII PE None Chondroitin
sulfates LIX PE None Polycarboxylated LX polyethylene glycol
Dipalmitoyl-PE None Hyaluronic acid LXI Dipalmitoyl-PE None Heparin
LXII Dipalmitoyl-PE None Chondroitin sulfate A LXIII Dipalmitoyl-PE
None Carboxymethylcellulose LXIV Dipalmitoyl-PE None Polygeline
(haemaccel) LXV Dipalmitoyl-PE None Hydroxyethylstarch LXVI
Dipalmitoyl-PE None Dextran LXVII Dipalmitoyl-PE None Aspirin
LXVIII Dimyristoyl-PE None Heparin LXVIX Dimyristoyl-PE None
Chondroitin sulfate A LXX Dimyristoyl-PE None
Carboxymethylcellulose LXXI Dimyristoyl-PE None Polygeline
(haemaccel) LXXII Dimyristoyl-PE None Hydroxyethylstarch LXXIII
Dimyristoyl-PE None Dextran LXXIV Dimyristoyl-PE None Aspirin LXXV
PS None Hyaluronic acid LXXVI PS None Heparin LXXVII PS None
Polygeline (haemaccel) LXXVIII PC None Hyaluronic acid LXXIX PC
None Heparin LXXX PC None Polygeline (haemaccel) LXXXI PI None
Hyaluronic acid LXXXII PI None Heparin LXXXIII PI None Polygeline
(haemaccel) LXXXIV PG None Hyaluronic acid LXXXV PG None Heparin
LXXXVI PG None Polygeline (haemaccel) LXXXVII
[0413] In one embodiment of the invention, the compounds
administered are Compound XXII, Compound XXIII, Compound XXIV,
Compound XXV, Compound XXVI, Compound XXVII, Compound XXVIII,
Compound XXIX and Compound XXX, or pharmaceutically acceptable
salts thereof, in combination with a physiologically acceptable
carrier or solvent. According to embodiments of the invention,
these polymers, when chosen as the conjugated moiety, may vary in
molecular weights from 200 to 2,000,000 Daltons. In one embodiment
of the invention, the molecular weight of the polymer as referred
to herein is from 200 to 1000 Daltons. In another embodiment, the
molecular weight of the polymer as referred to herein is from 200
to 1000 Daltons. In another embodiment, the molecular weight of the
polymer as referred to herein is from 1000 to 5000 Daltons. In
another embodiment, the molecular weight of the polymer as referred
to herein is from 5000 to 10,000 Daltons. In another embodiment,
the molecular weight of the polymer as referred to herein is from
10,000 to 20,000 Daltons. In another embodiment, the molecular
weight of the polymer as referred to herein is from 10,000 to
50,000 Daltons. In another embodiment, the molecular weight of the
polymer as referred to herein is from 20,000 to 70,000 Daltons. In
another embodiment, the molecular weight of the polymer as referred
to herein is from 50,000 to 100,000 Daltons. In another embodiment,
the molecular weight of the polymer as referred to herein is from
100,000 to 200,000 Daltons. In another embodiment, the molecular
weight of the polymer as referred to herein is from 200,000 to
500,000 Daltons. In another embodiment, the molecular weight of the
polymer as referred to herein is from 200,000 to 1,000,000 Daltons.
In another embodiment, the molecular weight of the polymer as
referred to herein is from 500,000 to 1,000,000 Daltons. In another
embodiment, the molecular weight of the polymer as referred to
herein is from 1,000,000 to 2,000,000 Daltons. Various molecular
weight species have been shown to have the desired biological
efficacy, as shown in the section below.
[0414] In one embodiment of this invention, low molecular weight
phosphatidylethanolamine (PE)-conjugates are defined hereinabove as
the compounds of formula (I) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0415] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemmisuccinate, a di- or tripeptide, an
oligopeptide, a trisacharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0416] In one embodiment of this invention, low molecular weight
phosphatidylserine (PS)-conjugates are defined hereinabove as the
compounds of formula (II) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0417] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemisuccinate, a di- or tripeptide, an
oligopeptide, a trisaccharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0418] In one embodiment of this invention, Phosphatidylcholine
(PC), Phosphatidylinositol (PI), and Phosphatidylglycerol (PG)
conjugates are hereinabove defined as the compounds of formula
(III) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Z is either nothing, inositol, choline, or glycerol;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0419] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemisuccinate, a di- or tripeptide, an
oligopeptide, a trisaccharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0420] Examples of suitable divalent groups forming the optional
bridging group Y are straight- or branched-chain alkylene, e.g., of
2 or more, preferably 4 to 18 carbon atoms, --CO-alkylene-CO,
--NH-alkylene-NH--, --CO-alkylene-NH--, cycloalkylene, wherein
alkylene in each instance, is straight or branched chain and
contains 2 or more, preferably 2 to 18 carbon atoms in the chain,
--(--O--CH(CH.sub.3)CH.sub.2--).sub.x-- wherein x is an integer of
1 or more.
[0421] In another embodiment, in addition to the traditional
phospholipid structure, related derivatives for use in this
invention are phospholipids modified at the C1 or C2 position to
contain an ether or alkyl bond instead of an ester bond. These
derivatives are exemplified hereinabove by the general formulae
(VIII) and (IX) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Z is either nothing, ethanolamine, serine, inositol, choline, or
glycerol;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0422] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemmisuccinate, a di- or tripeptide, an
oligopeptide, a trisaccharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0423] In another embodiment, related low molecular weight
derivatives for use in this invention are exemplified hereinabove
by the general formulae (X), (XI) and (XII) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Z is either nothing, ethanolamine, serine, inositol, choline, or
glycerol;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0424] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemmisuccinate, a di- or tripeptide, an
oligopeptide, a trisaccharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0425] In another embodiment, related low molecular weight
derivatives for use in this invention are exemplified hereinabove
by the general formulae (XIII) wherein:
R.sub.1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
R.sub.2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms;
Z is either nothing, choline, phosphate, inositol, or glycerol;
Y is either nothing or a spacer group ranging in length from 2 to
30 atoms;
[0426] X is a mono- or disaccharide, carboxylated disaccharide,
mono- or dicarboxylic acids, a salicylate, salicylic acid, aspirin,
lactobionic acid, maltose, an amino acid, glycine, acetic acid,
butyric acid, dicarboxylic acid, glutaric acid, succinic acid,
fatty acid, dodecanoic acid, didodecanoic acid, bile acid, cholic
acid, cholesterylhemisuccinate, a di- or tripeptide, an
oligopeptide, a trisaccharide, or a di- or trisaccharide monomer
unit of heparin, heparan sulfate, keratin, keratan sulfate,
chondroitin, chondroitin-6-sulfate, chondroitin-4-sulfate,
dermatin, dermatan sulfate, dextran, or hyaluronic acid; and
n is the number of lipid moiety molecules bound to a molecule of X
wherein n is a number from 1 to 1000.
[0427] In one embodiment of the invention, x is covalently
conjugated to a lipid. In another embodiment, x is covalently
conjugated to a lipid via an amide bond. In another embodiment, x
is covalently conjugated to a lipid via an esteric bond. In another
embodiment, the lipid is phosphatidylethanolamine. In another
embodiment, the GAG may be, inter alia, chondroitin sulfate. In
another embodiment, the GAG may be, inter alia, heparin. In another
embodiment, the GAG may be, inter alia, hyaluronic acid. In another
embodiment, the conjugate is biodegradable.
[0428] In one embodiment, the invention provides glycosaminoglycan
(GAG) compounds covalently conjugated to a lipid to obtain a
compound having preferred therapeutic properties. In another
embodiment, the GAG compound is covalently conjugated to a lipid
via an amide bond. In another embodiment, the GAG compound is
covalently conjugated to a lipid via an esteric bond. In another
embodiment, the lipid may be, inter alia, phosphatidylethanolamine.
In another embodiment, the GAG may be, inter alia, chondroitin
sulfate. In another embodiment, the GAG may be, inter alia,
heparin. In another embodiment, the GAG may be, idler aria,
hyaluronic acid. In another embodiment, the conjugate is
biodegradable.
[0429] Cell surface GAGs play a key role in protecting cells from
diverse damaging agents and processes, such as reactive oxygen
species and free radicals, endotoxins, cytokines, invasion
promoting enzymes, and agents that induce and/or facilitate
degradation of extracellular matrix and basal membrane, cell
invasiveness, white cell extravasation and infiltration,
chemotaxis, and others. In addition, cell surface GAGs protect
cells from bacterial, viral and parasitic infection, and their
stripping exposes the cell to interaction and subsequent
internalization of the microorganism. Enrichment of cell surface
GAGs would thus assist in protection of the cell from injurious
processes. Thus, in one embodiment of the invention, PLA2
inhibitors are conjugated to GAGs or GAG-mimicking molecules. In
another embodiment, these Lipid-conjugates provide wide-range
protection from diverse injurious processes, and are effective in
amelioration of diseases that requires cell protection from
injurious biochemical mediators.
[0430] In another embodiment, a GAG-mimicking molecule may be,
inter alia, a negatively charged molecule. In another embodiment, a
GAG-mimicking molecule may be, inter alia, a salicylate derivative.
In another embodiment, a GAG-mimicking molecule may be, inter alia,
a dicarboxylic acid.
[0431] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
pathogenic effect, including a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0432] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
viral infection, including a lipid or phospholipid moiety bonded to
a physiologically acceptable monomer, dimer, oligomer, or polymer;
and a pharmaceutically acceptable carrier or excipient.
[0433] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from an
HIV infection, including a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer;
and a pharmaceutically acceptable carrier or excipient.
[0434] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from an
influenza infection, including a lipid or phospholipid moiety
bonded to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0435] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
poxvirus infection, including a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0436] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
chordopoxvirinae infection, including a lipid or phospholipid
moiety bonded to a physiologically acceptable monomer, dimer,
oligomer, or polymer; and a pharmaceutically acceptable carrier or
excipient.
[0437] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
vaccinia infection, including a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0438] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
smallpox infection, including a lipid or phospholipid moiety bonded
to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0439] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
bacterial infection, including a lipid or phospholipid moiety
bonded to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0440] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
Chlamydia infection, including a lipid or phospholipid moiety
bonded to a physiologically acceptable monomer, dimer, oligomer, or
polymer; and a pharmaceutically acceptable carrier or
excipient.
[0441] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
pathogenic effect, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII), or any combination thereof.
[0442] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
viral infection, including any one of the compounds for use in the
present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0443] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from an
HIV infection, including any one of the compounds for use in the
present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0444] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from an
influenza infection, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0445] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
poxvirus infection, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0446] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
chordopoxvirinae infection, including any one of the compounds for
use in the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae: as described hereinbelow (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0447] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
vaccinia infection, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0448] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
smallpox infection, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa) (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0449] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
bacterial infection, including any one of the compounds for use in
the present invention or any combination thereof, and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
[0450] In another embodiment, the invention provides a
pharmaceutical composition for treating a subject suffering from a
Chlamydia infection, including any one of the compounds for use in
the present invention or any combination thereof; and a
pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include,
inter alia, the compounds represented by the structures of the
general formulae as described hereinbelow: (A), (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (XI),
(XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX),
(XXI), (XXII) or any combination thereof.
Preparation of Compounds for Use in the Present Invention
[0451] The preparation of some high molecular weight
Lipid-conjugates is the subject of U.S. Pat. No. 5,064,817, which
is incorporated herein by reference. These synthetic methods are
considered to be applicable as well to the preparation of low
molecular weight Lipid-conjugates, i.e. Lipid-conjugates comprising
monomers and dimers as the conjugated moiety, with appropriate
modifications in the procedure as would be readily evident to one
skilled in the art. The preparation of some low molecular weight
Lipid-conjugates may be conducted using methods well known in the
art or as described in U.S. patent application Ser. No. 10/952,496,
which is incorporated herein by reference.
[0452] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limiting the remainder of the disclosure in
any way whatsoever.
EXAMPLES
The abbreviations used in the examples below are:
PE=phosphatidyl-ethanolamine
HA=hyaluronic acid
Cpd=Compound
Compound XXII=dipalmitoyl-PE conjugated to HA
Compound XXIII=dimyristoyl-phosphatidyl-ethanolamine linked to
HA
Compound XXIV=PE conjugated to heparin
CSA=chondroitin sulfate A
Compound XXV=PE conjugated to CSA
CMC=carboxymethyl cellulose
Compound XXVI=PE conjugated to CMC
Compound XXVII=PE conjugated to Polygeline (haemaccel)
The compounds used in the examples below were prepared as described
in U.S. patent application Ser. No. 10/952,496, which is
incorporated herein by reference
Example 1
Viral Infection
[0453] The Lipid-conjugates are effective in the prophylaxis and
treatment of viral infection, particularly infections due to the
human immunodeficiency virus (HIV), human influenza virus and
vaccinia virus. This is demonstrated for HIV in Experiments 1.1-1.3
below, for human influenza virus in Experiment 1.4-1.5, and for
vaccinia in Experiment 1.6 below.
[0454] Viral infection is the cause of a number of human and animal
diseases throughout the world. The process of viral infection
comprises several stages, including attachment, penetration,
uncoating, replication, maturation, release and reinfection. In
order to assess the ability of Lipid-conjugates to prevent viral
infection, human cell lines were incubated with a preparation of a
viral agent, and the ability of the virus to infect cells is
compared in the presence and absence of Lipid-conjugate.
[0455] Experiment 1.1: To demonstrate that the Lipid-conjugates are
capable of preventing HIV infection of target cells, whole blood
units were mixed with HIV and a Lipid-conjugate (50 .mu.M Compound
XXIV, 30 .mu.M Compound XXII) for 30 min. The cells were then spun
and the supernatant was examined for HIV infectivity on HT4-1022
cells as described by Margolis-Nunno et al. (Transfusion, 36,
743-750, 1996). FIG. 1.1 demonstrates the ability of
Lipid-conjugates to prevent HIV infection of cells.
[0456] Experiment 1.2: Inhibition of HIV-1.sub.IIIB Infection
[0457] Tables 1.1-1.2 demonstrate the capacity of the
Lipid-conjugates to inhibit HIV replication, as expressed by the
production of the nucleocapsid p24 antigen, which is produced in
the host cell upon its infection by HIV virus. .sup.31MT-2 cells
(10.sup.4) in 96-well plates were infected with a dose of HIV-1
sufficient to accomplish a multiplicity of infection of 0.0045 in
200 .mu.l of RPMI 1640 medium supplemented with 10% (v/v) fetal
bovine serum (FBS), in the absence (control) and presence of the
indicated Lipid-conjugate. After 1 h, half of the culture medium
was changed and replaced by fresh medium (with/without
Lipid-conjugate) and after 24 h, the second half of the culture
medium was changed and replaced by fresh medium (with/without
Lipid-conjugate). On the fourth day after incubation at 37.degree.
C., 100 .mu.l of culture supernatants were collected from each well
and an equal volume of fresh medium was added to the wells. The
collected supernatants were mixed with an equal volume of 5% (v/v)
Triton X-100 and assayed for p24 antigen using an ELISA kit from
Coulter Immunology (Hialeah, Fla.). TABLE-US-00002 TABLE 1.1
Inhibition of p24 production Compound IC.sub.50 (mean .+-. SD)
.mu.g/ml IC.sub.90 (mean .+-. SD) .mu.g/ml Compound XXII 207.0 .+-.
18.0 384.3 .+-. 79.3 Compound XXIII 118.0 .+-. 16.8 296.3 .+-.
104.0 Compound XXIV 10.0 .+-. 2.3 19.3 .+-. 4.5 Compound XXV 72.5
.+-. 8.0 106.0 .+-. 10.3 Compound XXVII 375.8 .+-. 119.5
>500
[0458] TABLE-US-00003 TABLE 1.2 Inhibition of p24 production
Compound IC.sub.50 (.mu.M) IC.sub.90 (.mu.M) Compound XXIII 1.77
4.44 Compound XXII 3.11 5.76 Compound XXIV 0.70 1.35 Compound XXV
1.45 2.12
[0459] Experiment 1.3 demonstrates the ability of Lipid-conjugates
to inhibit fusion between HIV-1-infected and HIV-uninfected cells.
In this assay, HIV-1.sub.IIIB-uninfected H9 cells were labeled with
BCECF
(2',7'-bis(2-carboxyethyl)-5-6-carboxyfluorescein-acetoxymethyl-ester,
Molecular Probes, Eugene, Oreg.) according to the manufacturer's
instructions. BCECF-labeled H9/HIV-1 IIIB cells (10.sup.4) were
mixed with 1.times.10.sup.5 uninfected MT-2 cells. After incubation
in a 96-well plate at 37.degree. C. for 2 h, the fused and unfused
labeled cells were counted under an inverted fluorescence
microscope at .times.160 magnification. At least 200 BCECF-labeled
cells were counted and the proportion of fused cells was
determined. Fusion tests were carried out in the presence and
absence of graded quantities of the tested Lipid-conjugates. Data
are presented as the IC.sub.50 and IC.sub.90 of the lipid
conjugates tested (Table 1.3). The IC.sub.50 represents the
concentration of a drug that is required to achieve 50% inhibition.
Similarly, the IC.sub.90 represents the concentration of a drug
that is required to achieve 90% inhibition. TABLE-US-00004 TABLE
1.3 Inhibition of fusion between HIV-infected and uninfected cells.
Compound IC.sub.50 (mean .+-. SD) .mu.g/ml IC.sub.90 (mean .+-. SD)
.mu.g/ml Compound XXII >500 >500 Compound XXIII 122.8 .+-.
14.8 219.8 .+-. 10.6 Compound XXIV 7.9 .+-. 1.3 15.3 .+-. 3.9
Compound XXV >500 >500 Compound XXVII >500 >500
[0460] In another experiment, whole blood units were mixed with HIV
and Lipid-conjugates (between 30 .mu.M and 50 .mu.M) for 30 min.
Cells were spun, and supernatant was examined for HIV infectivity
on HT4-1022 cells (Table 1.4). TABLE-US-00005 TABLE 1.4 Inhibition
of fusion between HIV-infected and uninfected cells. Compound
IC.sub.50 (.mu.M) IC.sub.90 (.mu.M) Compound XXII >7.5 >7.5
Compound XXIII 1.83 3.30 Compound XXIV 0.55 1.07 Compound XXV
>10 >10
[0461] Table 1.5 further demonstrates the ability of
Lipid-conjugates to inhibit HIV infection. V3 antibody binding is
an assay that uses an antibody that binds to the V3 (third
variable) domain of the human immunodeficiency virus type 1 (HIV-1)
envelope glycoprotein gp120. Anti-V3 domain antibodies may provide
an indicator of the presence and amount of HIV. V3 antibody binding
was determined by standard ELISA. TABLE-US-00006 TABLE 1.5 Effect
of Lipid-conjugates on V3 antibody binding Compound IC.sub.50
(.mu.M) Compound XXII 45 Compound XXIII 3 Compound XXIV 140
Compound XXV 0.2
[0462] These experiments demonstrate that administration of
Lipid-conjugates is an effective therapy in the treatment HIV,
including prevention of infection, replication and fusion.
[0463] Experiment 1.4: The effect of Lipid-conjugate treatment on
human influenza virus infection in vitro.
[0464] Virus and cell lines. Each virus was obtained from the
source described in Table 1.6. Kidney cell lines were obtained from
American Type Culture Collection (ATCC). The cells were grown in
minimal essential medium (Gibco-BRL, Gaithersburg, Md.)
supplemented with 0.1% NaHCO.sub.3 and 5 to 9% fetal bovine serum
(HyClone Laboratories, Logan, Utah). When performing antiviral
assays, serum was reduced to 2% and 50 .mu.g gentamicin (Sigma
Chemical Company, St. Louis, Mo.) per ml was added to the medium
TABLE-US-00007 TABLE 1.6 Description of viruses used in a screen of
some Lipid-conjugates Virus Strain Source Cell line Influenza type
A A/New Center for Disease Madin Darby Caledonia/ Control and
canine kidney 20/99 (H1N1) Prevention [CDC] (MDCK) cells A/Panama/
CDC Madin Darby 2007/99 canine kidney (H3N2) (MDCK) cells Influenza
type B B/Hong CDC Madin Darby Kong/330/02 canine kidney (MDCK)
cells Pichinde virus An 4763 Dr. J. D. Gangemi, African green Univ.
of South monkey kidney Carolina School of (BS-C-1) cells Medicine,
Columbia, SC Punta Toro virus Adames U.S. Army Rhesus monkey
Medical Research kidney Institute for (LLC-MK2) Infectious cells
Diseases, Fort Detrick, Frederick, MD Respiratory A2 ATCC African
green syncytial virus monkey kidney (MA-104) cells
1. Inhibition of Viral Cytopathic Effect (CPE)
[0465] A. Visual Observation
[0466] A viral CPE assay was performed as described (Barnard D L et
al. Antivir Chem Chemother. 2001 July;12(4):241-250).
[0467] Compounds were evaluated using four log 10 dilutions of each
test compound (e.g., 1000, 100, 10, 1 .mu.g/ml) (Tables 1.7 and
1.8) with an additional concentration of 2000 .mu.g/ml for some
experiments (Tables 1.9 and 1.10). Viruses (Influenza type A Strain
H1N1, Influenza type A Strain H3N2, Influenza type B, Pichinde
virus, Punta Toro virus, and Respiratory syncytial virus) were used
at a multiplicity of infection (MOI) of 0.001 to 0.010. The MOIs
used were virus dependent and chosen for each strain such that 100%
of the cells in the virus controls showed cytopathic effects (CPE)
within 5 to 7 days. Cell were grown to an 18 h monolayer (80-100%
confluent) in 96-well tissue culture plates and were incubated with
various concentrations of each compound as described above. Within
5 minutes of compound incubation, a volume of virus equal to that
of the compound was added to the cells. The plates were then sealed
and incubated at 37.degree. C. for approximately 72 to 120 hr until
the cells in the virus control wells showed complete viral CPE as
observed by light microscopy.
[0468] Each concentration of drug was assayed for virus inhibition
in triplicate. Three wells were set aside as uninfected, untreated
cell controls per test and three wells per test compound receive
untreated, virus-infected cells and represented positive controls
for virus replication. Ribavirin, used as a positive control drug,
was evaluated in parallel with compounds for each virus.
[0469] The 50% effective concentrations (EC.sub.50) were calculated
by regression analysis of the means of the CPE ratings as compared
to untreated, uninfected controls for each concentration. Cells
were rated based on changes in enlargement, granularity, ragged
edges, filmy appearance, rounding, detachment from the surface of
the well, and other changes Morphological changes results from
cytotoxicity of a compound were graded on a scale of 0-5; 0=no
toxicity, 1=partial toxicity--slight, 2=partial toxicity, 3=partial
toxicity--heavy, 4=partial toxicity--very heavy, and 5=complete
cytotoxicity, based on the degree of cytotoxicity observed. The CPE
results were then quantified spectrophotometrically by neutral red
(NR) uptake assay (see below).
[0470] B. Increase in Neutral Red (NR) Dye Uptake
[0471] A Neutral Red Dye Uptake assay was performed as described
previously (McManus, N H, Appl. Environment. Microbiol. 31:35-38,
1976) to verify the inhibitory activity and cytotoxicity that was
observed in the CPE inhibition assay. Briefly, medium was removed
from each well of a plate scored for CPE from a CPE inhibition
assay, 0.034% NR in Sorenson's citrate buffer (pH 4.0) was added to
each well of the plate and the plate incubated for 2 h at
37.degree. C. in the dark. The NR solution was removed from the
wells. After rinsing and aspirating to dryness, the remaining dye
was extracted for 30 min, at room temperature in the dark, from the
cells using absolute ethanol buffered with Sorenson's citrate
buffer. The percentage of NR uptake, indicating viable cells, was
read on a microplate autoreader (Bio-Tek EL 1309; Bio-Tek
Instruments, Winooski, Vt., USA) at dual wavelengths of 405 and 540
nm. The difference between the two readings were calculated to
eliminate background. Absorbance values were expressed as
percentages of untreated controls, and EC50 values were calculated
as described above.
2. Cytotoxicity Assay
[0472] A. Visual Observation
[0473] Uninfected cells were treated with each concentration of
test compound in duplicate and run in parallel with the infected,
treated wells in the CPE inhibition tests described above. The
toxicity control cells (uninfected and treated) were examined under
a light microscope for changes in cell appearance compared to
control cells (uninfected, untreated) on the same plate as
described above. The 50% cell inhibitory (cytotoxic) concentrations
(IC.sub.50) were calculated by regression analysis.
[0474] B. Neutral Red Uptake
[0475] The toxicity control cells (uninfected and treated)
described in the previous section were further examined for neutral
red dye uptake compared to control cells (uninfected, untreated) on
the same plate. Neutral red was added to the toxicity control
wells, and the degree of color intensity was determined
spectrophotometrically as described above. A neutral red IC50 (NR
IC50) was subsequently determined. Absorbance values were expressed
as percentages of uninfected, untreated controls, and IC.sub.50
values were calculated as described above.
3. Data Analysis
[0476] Each test compound's antiviral activity was expressed as a
selectivity index (SI), which is the IC.sub.50 divided by the
EC.sub.50. Generally, an SI of 10 or greater is indicative of
positive antiviral activity, although other factors, such as a low
SI for the positive control, are also taken into consideration.
[0477] Tables 1.7 and 1.8 demonstrate the capacity of the
Lipid-conjugates evaluated at low concentration to prevent
infection of target cells by influenza virus.
[0478] Nine compounds were evaluated for in vitro antiviral testing
against influenza A (H1N1 strain) virus, influenza A (H3N2 strain)
virus, influenza B virus, respiratory syncitial virus (RSV), Punta
Toro virus, and Pichinde virus using various kidney cell lines
described in Table 1.6. Two series of Lipid-conjugate dosages were
used as described in the methods hereinabove.
[0479] Using a lower range of doses, Compound XXIV had significant
anti-viral activity against influenza A (H1N1 strain) virus (Table
1.7). The EC50 vs this virus was 5 .mu.g/ml by visual assay and 2.5
.mu.g/ml by neutral red assay, with an IC50 (cytotoxicity)>100
.mu.g/ml. Against the influenza A (H3N2 strain) virus, the EC50 was
35 .mu.g/ml by visual assay and 45 .mu.g/ml by neutral red assay
with the same IC50 as above. Compound XXIV was also efficacious vs
RSV, with an EC50 of 4 .mu.g/ml using visual assay only, but was
not active by neutral red assay. Compound XXIV did not display a
virus inhibitory effect against Punta Toro virus at the
concentrations tested.
[0480] Compound XXV was less active, with EC50 values vs the
influenza A (H1N1 strain) virus of 50 .mu.g/ml by visual assay and
35 .mu.g/ml by neutral red assay and an IC50>100 .mu.g/ml (Table
1.8). This compound did not demonstrate a virus inhibitory effect
against influenza A (H3N2 strain), influenza B, or RSV at the
concentrations tested. Compound XXV did not display a virus
inhibitory effect against Punta Toro virus. TABLE-US-00008 TABLE
1.7 Antiviral activity of Compound XXIV (dipalmitoyl-phosphatidyl-
ethanolamine conjugated to heparin) at low concentrations EC.sub.50
SI Virus (.mu.g/ml) (IC.sub.50/EC.sub.50) Visual Observation Assay
Punta Toro A >100 0 Respiratory Syncytial A 4 25 Influenza A
(H1N1 strain) 5 20 Influenza A (H3N2 strain) 35 2.9 Neutral Red
Uptake Assay Punta Toro A >100 0 Respiratory Syncytial A >100
0 Influenza A (H1N1 strain) 2.5 40 Influenza A (H3N2 strain) 45 2.2
SI--selectivity index. Generally, an SI .gtoreq. 10 is indicative
of positive antiviral activity, although other factors such as a
low SI for the positive control are also taken into consideration.
IC.sub.50 for Compound XXIV was >100 .mu.g/ml for all
viruses.
[0481] TABLE-US-00009 TABLE 1.8 Antiviral activity of Compound XXV
(dipalmitoyl-phosphatidyl- ethanolamine (PE) conjugated to
chondroitin-sulfate A) at low concentrations EC.sub.50 SI Virus
(.mu.g/ml) (IC.sub.50/EC.sub.50) Visual Observation Assay Punta
Toro A >100 0 Influenza A (H1N1 strain) 50 2 Neutral Red Uptake
Assay Punta Toro A >100 0 Influenza A (H1N1 strain) 35 2.9
SI--selectivity index. IC.sub.50 for Compound XXV was >100
.mu.g/ml for all viruses.
[0482] Using higher concentrations of Lipid-conjugates, the results
demonstrate a strong effect of Compound XXIV (100) against
infection with Influenza A virus H1N1 strain (Tables 1.9 and 1.10).
In addition, Compound XXIII (170) and Compound XXIII (80) showed
antiviral activity against the H3N2 strain of Influenza A virus in
the visual test but not the neutral red assay (Table 1.10).
TABLE-US-00010 TABLE 1.9 Antiviral activity of Compound XXIV
(dipalmitoyl-phosphatidyl- ethanolamine conjugated to heparin;
MK-610) at high concentration IC.sub.50 EC.sub.50 SI Virus
(.mu.g/ml) (.mu.g/ml) (IC.sub.50/EC.sub.50) Visual Observation
Assay Influenza A (H1N1 strain) 35 400 11 Influenza A (H3N2 strain)
100 200 2 Influenza B 90 350 3.9 Neutral Red Uptake Assay Influenza
A (H1N1 strain) 64 1000 15.6 Influenza A (H3N2 strain) 110 900 8.2
Influenza B 220 450 2
[0483] TABLE-US-00011 TABLE 1.10 Antiviral activity of
Lipid-Conjugates against Influenza A (H1N1 and H3N2 strains) and
Influenza B viruses at high concentration. Compound (phosphate
Influenza A Influenza A content) (H1N1 strain) (H3N2 strain)
Influenza B Name Visual NR Visual NR Visual NR Ribavirin 22 25 56
36 22 19 Compound XXIV 11 15.6 2 8.2 3.9 2 (100) Compound XXII 3.6
0 25 0 10 0 (170) Compound XXV 2.5 0 0 0 0 0 (60) Compound XXIII 0
0 10 6.5 0 0 (80) Compound XXV 0 0 6.7 7.2 0 0 (230) Compound XXII
0 0 2.5 0 0 0 (8.5) Compound XXIV 0 0 0 0 0 0 (50) Compound XXII 0
0 0 0 0 0 (40) Compound XXV 0 0 0 0 0 0 (100) SI--selectivity index
(IC.sub.50/EC.sub.50); Visual = Visual Observation Assay; NR =
Neutral Red Uptake Assay
[0484] Experiment 1.5 demonstrates the effect of Lipid-conjugate
treatment on human influenza virus infection in vivo. We use young
adult (18-21 g) female BALB/c mice infected intranasally with
either influenza A/NWS/33 (H1N1), A/PR8/34 (H1N1), A/New
Calcdonia/20/99 (H1N1), A/Victoria/3/75 (H3N2), A/Port
Chalmers/1/73 (H3N2), B/Hong Kong/5/72, B/Lee/40, B/Sichuan/379/99,
or A/Duck/MN/1525/81 (H5N1) virus at sufficient dose to render
death in approximately 90% of the mice, with the mean day to death
being 6-10 days. The animals are monitored for arterial oxygen
saturation levels using a pulse oximeter on days 3 through 11 (the
infection usually induces major declines in these levels by about
day 9-10 due to lung consolidation). We also sacrifice mice on days
1, 3, 6, and 9 for assay of lung score, lung weight increase, and
lung virus titer. We usually use 22 infected mice for each dosage
of test compound, and 35 infected mice treated with placebo. Three
uninfected mice are included as toxicity controls, these are
treated in parallel to the above, and weight loss or gain is
determined during the period of treatment. A group of normal
controls are also run in parallel to ascertain their weight gain
during the study as well as the normal arterial oxygen saturation
levels. Some of these animals are also killed to determine normal
lung parameters.
[0485] If the test compound is considered to be an immunomodulator,
we would inject mice with the compound intraperitoneally every
other day for a total of 4 treatments beginning 24 h prior to virus
exposure. If the material is considered to be antiviral, a twice
daily for 5 days treatment schedule is recommended, with therapy
beginning 4 h pre-virus exposure. We generally try to select three
dosages varying 2-fold or 1/2 log 10 from each other, with the high
dose being approximately the maximum tolerated dose.
[0486] Ribavirin is usually included at a single dose as a known
positive control.
[0487] Experiment 1.6 demonstrates the effect of Lipid-conjugate
treatment on vaccinia virus infection in vitro.
[0488] BS-C-1 cell monolayers (3.times.10.sup.6 cells), in 3 cm
diameter plastic dishes, were infected with a dilution of a crude
stock of vaccinia virus (WR strain), to give a m.o.i. of 1 PFU per
10 cells. After adsorption for 1 hr, the cells were washed and 2 ml
of Dulbecco's MEM, supplemented with 2% fetal calf serum,
containing 1:10 dilution of the compound to be tested, were added.
The cultures were incubated for 2 days at 37.degree. C. and then
harvested. Control infected cultures that were not treated with the
compounds, were harvested at 0 time and at 48 hr. The virus titer
in all cultures was determined, after three cycles of freezing and
thawing, by plaque assay in BS-C-1 cells.
[0489] Table 1.11 demonstrates the capacity of the Lipid-conjugates
to prevent infection of target cells by vaccinia virus. Compounds
XXII, XXIII, and XXV inhibited viral infection in culture by
62-99%. TABLE-US-00012 TABLE 1.11 Antiviral activity of
Lipid-Conjugates against Vaccinia virus Time (PFU/culture) Compound
% (hr) tested Virus titer inhibition 0 -- less than 10.sup.4 48 --
8.6 .times. 10.sup.7 0% 48 Compound 3.3 .times. 10.sup.6 96.2%
XXII-40* 48 Compound 2.3 .times. 10.sup.7 73.3% XXII-80* 48
Compound 7.7 .times. 10.sup.4 99.9% XXIII 48 Compound 3.2 .times.
10.sup.7 62.8% XXV *The number expresses the amount of nmoles lipid
conjugated to 1 mg of polymer
[0490] These experiments demonstrate that administration of
Lipid-conjugates is effective therapy in the prevention and
treatment of viral infection, including HIV, influenza and vaccinia
viruses.
Example 2
Treatment of Chlamydia Infection
[0491] Intracellular bacterial parasites are one of the most
prevalent forms of sexually transmitted disease and are frequently
intractable to conventional antibiotic therapy. Infection of the
female genital tract with chlamydia species is a salient example.
Experiment 2.1 demonstrates the ability of Lipid-conjugate
treatment to prevent infection of HeLa cells by Chlamydia. Human
cervical adenocarcinoma cell line, HeLa 229 (ATCC, Manassas,
Calif.), were cultured and incubated with the phospholipid
conjugates (20 micromolar) for 30 min, then incubated with
Chlamydia psittaci (an avian form of Chlamydia trachomatis) (guinea
pig inclusion conjunctivitis serologically variant strains
(serovars)) for 24 hr. Infected cells were detected by
cytofluorometry (FACS) using FITC-conjugated anti-Chlamydia
antibody (FIG. 2.1A).
[0492] FIG. 2.1B depicts the dose response of the Lipid-conjugates'
inhibitory effect on infection of HeLa cells by Chlamydia. HeLa
cells were treated with the Lipid-conjugates at the indicated
concentration, and infected with Chlamydia as described above.
[0493] Experiment 2.2 demonstrates the ability of Lipid-conjugates
to inhibit Chlamydia-induced cell apoptosis. HeLa cells were
treated with Lipid-conjugates and infected with Chlamydia psittaci
as in Experiment 2.1. For determination of apoptosis,
detergent-permeabilized cells were stained with propidium iodide,
and their fluorescence was measured by cytofluorometry (FIG.
2.2).
[0494] The Lipid-conjugates are effective in the prophylaxis and
treatment of infection with intracellular bacterial parasites,
particularly infections due to chlamydial species. Taken together,
the data presented here demonstrate the Lipid-conjugate capacity to
ameliorate bacterial toxicity.
Example 3
Obstructive Respiratory Disease
[0495] In asthma, the impeded airflow is due to airway obstruction
which is the result of constriction and obstruction of luminal
vessels of the lungs. In order to determine the effect of
Lipid-conjugates on obstructive respiratory disease, contraction of
smooth muscle preparations isolated from airways was induced in the
presence and absence of Lipid-conjugates. This is a widely-accepted
experimental system to investigate airway constriction.
[0496] A muscle preparation (tracheal rings) was isolated from rats
(Experiments 3.1-3.3) and from guinea pigs (Experiments 3.4-3.5).
Muscle contraction was measured by attachment of the muscle to a
pressure transducer, which works much like a spring. Administration
of asthmatogenic substances such as endothelin-1 (ET) and
acetylcholine (AcCh) induces muscle contraction. Endothelins are
released upon vascular endothelial injury, and they activate
macrophages and act as strong chemo-attractants for circulating
monocytes. Endothelins affect vascular smooth muscle fibroblast
proliferation; help regulate vascular, airway, and intestinal
smooth muscle tone; increase the activity of bone alkaline
phosphatase; stimulate release of atrial natriuretic peptide (ANP)
from atrial cardiocytes; inhibit the release of renin from
glomeruli and modulate norepinephrine at sympathetic nerve termini.
Opposing vasomotor effects are regulated through the binding of
distinct endothelin receptors for vasoconstriction and
vasodilation. Endothelins have been linked to atherosclerosis and
various cardiovascular disease states. ET-1 is important in
congestive heart failure, renal failure, pulmonary hypertension,
hyperlipidemia and metastatic prostate cancer. On the other hand,
AcCh is one of the three main receptors on the bronchi of the
lungs, which are basically tubes with muscular walls. Stimulation
of bronchial AcCh receptor induces muscle contraction and decreased
airflow through the bronchi. Thus, ET and AcCh were used to
experimentally induce muscle contraction in the lungs as a model of
obstructive respiratory disorders.
[0497] Experiment 3.1: Effect of post-treatment of rat tracheal
rings with Compound XXII on endothelin-1 (ET)-induced contraction.
Isolated rat tracheal rings (in a linear array) were bathed in
Krebs-Hanselet buffer (pH=7.4), and linked to a tension transducer.
ET-1 was added to a final concentration as indicated, and the
tracheal ring contraction was determined by the change in the force
applied to the tension transducer (FIG. 3.1A). Subsequently, the
highest ET concentration was used in testing the Lipid-conjugates
to inhibit smooth muscle contraction (FIG. 3.1B). Rat tracheal
rings were incubated with 0-3.5 .mu.M of Compound XXII for 1 hr.
ET-1 was then added to a final concentration of 1 .mu.M, and ring
contraction was determined as in Experiment 3.1A. Data are
presented as mean.+-.S.D. of four separate experiments (4
rats).
[0498] Experiment 3.2: Effect of pretreatment of rat tracheal rings
with Compound XXII and HA on ET-1 induced contraction. Rat tracheal
rings were incubated with either 3 .mu.M Compound XXII or
hyaluronic acid (HA) for 1 hr. ET-1 was then added to a final
concentration of 1 .mu.M (empty bars) or 10 .mu.M (full bars) and
the tracheal ring contraction was determined as in Experiment 3.1
(FIG. 3.2).
[0499] Experiment 3.3: Effect of pretreatment of rat tracheal rings
with Compound XXII and HA on AcCh-induced contraction. The
experiment was performed as in Experiment 3.2, except that the
tracheal ring contraction was induced by 10 .mu.M AcCh, as shown in
FIG. 3.3.
[0500] Experiment 3.4: Guinea pig tracheal rings (in a linear
array), immersed in a ringer bath, were connected to an apparatus
measuring the length of the ring chain. Compound XXIV or Compound
XXVI was added to the bath 1 h prior to the stimulation of
contraction by either a snake venom PLA2 (Crotalus atrox type II)
enzyme, histamine or endothelin-1 as indicated (Table 3.1). Human
airways have both Histamine (H)-1 and H2 receptors. H1 receptors,
which mediate bronchoconstriction, predominate. Histamine
application in experimental models produces signs and symptoms of
asthma, such as narrowing of the airways, mucus secretion,
wheezing, and coughing. TABLE-US-00013 TABLE 3.1 Inhibition of
Tracheal Ring Contraction by Compound XXVI and Compound XXIV
Stimulant Lipid-conjugate % Inhibition Phospholipase (0.5 .mu./ml)
Compound XXVI 100 .+-. 0.3 (crotalus atrox type II) (10 .mu.M)
Histamine (20 .mu.M) Compound XXVI 69 .+-. 0.1 (10 .mu.M) Histamine
(20 .mu.M) Compound XXIV 56 .+-. 0.05 (15 .mu.M) Endothelin-1 (100
nM) Compound XXVI 92 .+-. 1.1 (10 .mu.M)
[0501] Experiment 3.5: Guinea pig tracheal rings were incubated
with or without Compound XXVI for 30 minutes prior to stimulation.
The medium was collected after 30 minutes, and PGE.sub.2 and
TXB.sub.2 levels were determined by radioimmunoassay (Table 3.2).
PGE.sub.2 and TXB.sub.2 are metabolites of arachidonic acid
produced during inflammatory response. TABLE-US-00014 TABLE 3.2
Inhibition of Tracheal Tissue PGE.sub.2 and TBX.sub.2 Production by
Compound XXVI PGE.sub.2 TXB.sub.2 Stimulant (ng/ml) (ng/ml)
Histamine (40 .mu.M) 5.1 5.6 Histamine (40 .mu.M) + Compound n.d.
1.75 XXVI (10 .mu.M) (n.d. = below limit of detection.)
[0502] Another widely-accepted test of anti-asthma drug action is
to study asthma in vivo in an animal model. Asthma is present in
animals which have been sensitized to an antigen, and can be
monitored for exacerbation and recovery from asthmatic breathing
using a body plethysmography. Experiments 3.6-3.8 demonstrate the
ability of Lipid-conjugates to exert their pharmacological effect
in live animals. The following procedures were applied in these
experiments:
[0503] Subjects: Inbred Brown Norway male rats (4 weeks old)
obtained from Harlan, USA, were used in this study. The Hebrew
University Animal Welfare Committee approved all protocols.
[0504] Induction of asthma: Asthma was induced in rats by
sensitization with ovalbumin (OVA, Sigma--Rehovot, Israel)
according to a previously described protocol (Offer et al. Am J.
Physiol Lung Cellular and Molecular Physiology 288:L523-L529,
2005): On day 0, rats received a single subcutaneous injection of 1
mg OVA+aluminum-hydroxide (200 mg/ml in 0.9% NaCl) (Sigma--Rehovot,
Israel) and an intraperitoneal injection of 1 ml containing
6.times.10.sup.9 heat-killed Bordetella Pertussis bacteria (Pasteur
Marieux, France). Repeated bronchial allergen challenge was
performed from day 14 every other day for 1 month by inhalation of
OVA (1 mg/ml in 0.9% Normal Saline) for 5 minutes each time in a 20
L box connected to an ultrasonic nebulizer (LS 230 System
Villeneuve Sur Lot, France).
[0505] Treatments: Rats were divided into 4 treatment groups: 1. No
sensitization and no treatment, used as control (Naive). 2.
Sensitization+challenge with OVA and placebo treatment with 1 ml
saline before each challenge, used as positive control (OVA). 3.
Sensitization+challenge with OVA and treatment with Lipid-conjugate
(Compound XXII), either by subcutaneous (SC) injection or
inhalation, before every challenge (OVA/Compound XXII). 4.
Sensitization+challenge with OVA and treatment with SC injection of
dexamethasone 300 .mu.g before each challenge (OVA/Dx) (only in
select experiments).
[0506] One of two modes of Compound XXII treatments were employed:
1. The rats received an SC injection of 1 ml saline containing 15
mg Compound XXII (to obtain about 1 mg/ml body fluid=20 .mu.M). 2.
The rats, placed unrestrained in a 20 litre box connected to an
ultrasonic nebulizer, inhaled Compound XXII as follows: 5 ml of 1
mg/ml Compound XXII was aerosolized into the 20 L cage, thus
diluting Compound XXII to 0.25 .mu.g/ml aerosol. The rat
respiratory rate was 120 breaths/min, with a tidal volume of about
1 ml, thus reaching ventilation of 120 ml/minute. If all of the
Compound XXII inhaled in 5 min was absorbed (600 ml), the maximal
Compound XXII absorbed was 150 .mu.g.
[0507] In mode 1, all groups (5 rats in each) were treated and
challenged as described above on day 14, 16, 18 and 20, and
pulmonary function (Penh) was assessed on day 20 before and 5 min
after challenge (EAR).
[0508] In mode 2, each group (10 rats in each) were treated and
challenged from day 14, every other day, until day 45. Pulmonary
function (Penh) was assessed on day 20 before and 5 min and 8 h
after challenge, corresponding to early and late asthmatic reaction
(EAR and LAR, respectively).
[0509] Assessment of broncho-constriction: Unrestrained conscious
rats were placed in a whole-body plethysmograph (Buxco Electronics
Inc., Troy, N.Y., USA) connected to a pneumotach (EMKA
Technologies, Type 0000) at one end, and to a 10 ml bottle at the
other end. The pneumotach was connected to a preamplifier (model
MAX2270, Buxco Electronics), Analogue signals from the amplifier
were converted to a digital signal by an AD card (LPM-16 National
Instruments, Austin, Tex., USA). Broncho-constriction measures were
expressed as the enhanced pause (Penh).
Penh=(PEF/PIF)*((Te-Tr)/Tr), where PEF=Peak Expiratory Flow,
PIF=Peak Inspiratory Flow, Te=Expiratory Time, Tr=Relaxation
Time=time of the pressure decay to 36% of total box pressure during
expiration.
[0510] Broncho-alveolar lavage (BAL): On day 45, the rats were
sacrificed by bleeding through the abdominal aorta under
anaesthesia with intra-peritoneal injection of sodium pentobarbital
(100 mg/kg). The rats were tracheotomized and incannulated through
the trachea. Bronco-alveolar lavage (BAL) was collected by repeated
washing of the lungs with 5 ml saline to a total of 50 ml.
[0511] Assessment of airway pathology: Subsequent to collection of
BAL, lungs were removed and inflated with 4% buffered formaldehyde
under pressure of 20 cm H.sub.2O. The lungs were sliced
longitudinally and embedded in paraffin. Three .mu.m histological
sections were cut and stained with hematoxylin and eosin for
assessments of interstitial and peri-bronchial inflammation and
airway smooth muscle thickening. Other slides were stained with
Tri-chrome for assessment of sub-epithelial fibrosis (basal
membrane) and with PAS for epithelial cell mucus metaplasia.
[0512] Histological morphometry of airway structural changes was
performed using the "ImageJ" computer program (NIH Bethesda USA) on
3 randomly selected slides from each mouse. Quantification of
peribronchial cellular infiltrate in airway tissue was achieved by
counting the numbers of these cells in the 50 .mu.m region beneath
the epithelium of the airway in hematoxylin and eosin stained
sections. Cells were expressed as number per millimeter of airway
basal lamina length, which was measured by tracing the basal lamina
in calibrated digital images (Kuhn III, C et al. Am. J. Respir.
Cell Mol. Biol. 2000; 22(3):289-295). Morphometric analysis of ASM
and the basal membrane mass as indices of their thickening were
performed as previously described (Panettieri R A Jr et al. Am. J.
Physiol. Lung Cell. Mol. Physiol. 1998; 274:L417-L424). Briefly,
measurements of the airway were obtained by tracing the digitalized
images of interest. The outlines of the airway structures were
subsequently measured. All airways were evaluated for the following
morphometric dimensions: length of the airway basement membrane of
the epithelium (Lbm) and area of the ASM in the eosin hematoxylin
stained slides and the blue stain of the basal membrane of the
Tri-chrome stained slides. ASM cells or the basal membrane
thickening were normalized to the square of the Lbm (in
.mu.m.sup.2) to correct for differences in airway size. Only large
(>2,000 .mu.m Lbm) and medium size airways (1,000-2,000 .mu.m
Lbm) were selected as it was shown that the most significant
pathological changes occur in these airways.
[0513] Protein expression of sPLA2 in lung tissue: Proteins were
identified in homogenized lung tissue (100 .mu.g protein) using
standard Western blot. A specific polyclonal antibody against
Anti-sPLA2 antibody (Santa Cruz) was diluted 1:500 (v/v) in TBST
buffer+0.1% BSA. The immune reaction was detected by enhanced
chemiluminescence (ECL).
[0514] Cysteinyl Leukotriene (CysLT): CysLT levels were measured in
BAL using a kit for direct enzyme immunoassay (EIA), according to
manufacturer's instructions (Amersham Pharmacia Biotech U.K). The
specificity of the kit was 100% for LTC.sub.4, 100% for LTD.sub.4,
and 70% for LTE.sub.4 Result range was between 0 and 48 .mu.g.
[0515] Cell culture: Cells were isolated from the BAL and suspended
in DMEM medium supplemented with 10% fetal calf serum (FCS) and
plated in a 96-well plate at 106 cells/well. The cells were
incubated for 2 hours in 37.degree. C., then non-adherent cells
were removed by washing with PBS. The adherent cells were
re-suspended in DMEM supplemented with 10% FCS at 106 cells/well
and incubated for 48 hours. The culture medium was then collected
and assayed for determination of biochemical markers.
[0516] Nitric Oxide (NO) production: NO production by the BAL
cultured macrophages was determined by measuring their level in the
culture medium using the photometric method of Griess (Green, L.
C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S.,
and Tannenbaum, S. R. 1982. Analysis of nitrate, nitrite, and
[15N]nitrate in biological fluids. Anal. Biochem. 126:131-138).
[0517] TNF.alpha. production: TNF.alpha. production by the BAL
cultured macrophages was determined in the culture medium using
radio-immunoassay (RIA) kits (Amersham-Pharmacia, UK).
[0518] Statistical Analysis: All data are expressed as mean.+-.SEM.
One way ANOVA was used to compare treatment groups. Pair-wise
comparisons were performed by the Tukey-Kramer HSD test (p=0.05).
Where necessary, data were log transformed before analysis to
stabilized variances. In all analyses P<0.05 was considered
statistically significant
[0519] Statistics: Statistical analysis was performed using
statistical software (GB-STAT, Dynamic Microsystem, Silver Spring,
Md., USA. Analysis of variance (ANOVA) was used to assess
difference of the results of the treatment groups. A Tukey test was
used to compare between each one of the treatment groups. A value
of p<0.05 was considered as a significant difference.
[0520] Experiment 3.6: SC administration of Lipid-conjugates
considerably ameliorates OVA-induced broncho-constriction (FIG.
3.4). Bronchoconstriction was induced in OVA-sensitized rats by
inhalation of OVA, and expressed by the difference in Penh measured
before and 5 min after allergen challenge. Data are presented as
mean.+-.SEM for 10 rats. Statistical significance: a--P<0.01
between columns marked "a"; b, c--P<0.05 between columns marked
"b" and "c", respectively. SC administration of Lipid-conjugates
also reduced the expression of secretory phospholiapse (FIG. 3.5).
The figure depicts Western blot and corresponding densitometry of
sPLA.sub.2 in lung homogenates of rats with naive, OVA-induced
asthma, and OVA-induced asthma treated with Compound XXII. For
densitometric analysis, the density values for each enzyme were
normalized to Naive values. Lipid-conjugates also prevented the
production of the broncho-constricting lipid mediators cysteinyl
leukotrienes (FIG. 3.6). Broncho-alveolar lavage (BAL) was
collected upon sacrifice and CysLT levels were determined by EIA,
as described in Methods. Data are presented as mean.+-.SEM for 10
rats. Statistical significance: a, b--P<0.01. There was no
significant difference between OVA/Compound XXII-treated and Naive
rats.
[0521] Experiment 3.7: Treatment of asthmatic rats with
Lipid-conjugates administered by aerosol protects the rats from
sensitization to OVA. Lipid-conjugates markedly reduced OVA-induced
broncho-constriction in both the early and late asthmatic reaction
(FIG. 3.7). Bronchoconstriction, expressed as the percent change of
Penh, was induced in OVA-sensitized rats by inhalation of OVA, and
measured before allergen challenge, and 5 min and 8 h after
allergen challenge. Data are presented as mean.+-.SEM for 10 rats.
Two experiments were performed for early asthmatic reation (EAR). 5
rats were included in each group in the first experiment. The same
experiment was repeated with 10 rats in each group, which were
further used for determination of late asthmatic reation (LAR). A
combined statistical test for EAR yielded p<0.01 between
Asthmatic (OVA/OVA) and Compound XXII-treated (OVA/OVA+Compound
XXII). There was no significant difference between the Compound
XXII-treated and the Naive or Dx-treated groups. For LAR, p<0.01
between Asthmatic and Compound XXII-treated and no significant
difference between the Compound XXII-treated and the Naive or
Dx-treated groups.
[0522] Lipid-conjugates administered in aerosol form also inhibited
the production of CysLT in OVA-sensitized rats (FIG. 3.)
Broncho-alveolar lavage (BAL) was collected upon sacrifice of rats,
and CysLT levels were determined by EIA. Data are presented as
mean.+-.SEM for 10 rats. P<0.01 between Asthmatic and Compound
XXII-treated rats, and no significant difference between Compound
XXII-treated and Naive rats.
[0523] Lipid-conjugates administered in aerosol form also inhibited
the production of nitric oxide (NO), a characteristic constrictor
of smooth muscle cells (FIG. 3.9). Macrophages, collected from the
BAL of the different groups, were cultured without further
treatment with Compound XXII or Dx, and NO production was
determined as described in Methods. Data are presented as
mean.+-.SEM for 10 rats. NO level was reduced compared to asthmatic
and naive rats by both Compound XXII, p<0.001 and p<0.001
respectively and by Dx p<0.001 and p<0.001, respectively.
[0524] These treatments also prevented asthma-associated
inflammation, as expressed by prevention of inflammatory cell
infiltration and airway remodeling (FIG. 3.10-3.11). Rats were
subjected to OVA inhalation every other day for 30 days. Rats were
administered aerosolized Compound XXII for 5 min before every
allergen inhalation. The rats were sacrificed on Day 45.
A--Staining with hematoxylin eosin for detection of inflammatory
cell infiltration and changes in smooth muscle cell (ASM)
thickness. B--Staining of connective tissue (collagen) with
Mason-Trichrom, for detection of changes in basal membrane
thickness. C--Staining with Periodic Acid Schiff (PAS) for
detection of mucus metaplasia of respiratory epithelial cells. 1,
2, 3 and 4 depict tissues of Naive, Asthmatic, Compound
XXII-treated and Dx-treated rats, respectively.
[0525] Lipid-conjugates further prevent production of TNF.alpha. by
lung macrophages (FIG. 3.12). Macrophages, collected from the BAL
of the different groups, were cultured without further treatment
with Compound XXII or Dx, and TNF.alpha. production was determined
as described. Data are presented as mean.+-.SEM for 10 rats.
p<0.001 between Asthmatic and Compound XXII-treated rats. There
were no significant differences between Compound XXII-treated,
Naive and Dx-treated rats.
[0526] Experiment 3.8: Treatment of Asthmatic rats with
Lipid-conjugates administered by aerosol sensitized to OVA.
Compound XXII is effective in preventing allergen-induced
broncho-constriction in already asthmatic subjects when inhaled
before allergen (OVA) challenge (FIG. 3.13). OVA-sensitized
asthmatic rats inhaled nebulized Compound XXII (1 mg/ml) for 5
minutes, or nebulized normal saline. 30 minutes later, all groups
were challenged by inhalation of OVA (1 mg/ml) for 5 minutes. Penh
was measured before the treatments (baseline), and 5 minutes after
each inhalation. Data are presented as mean.+-.SEM for 5 rats. *,
**, P<0.05). Lipid-conjugates also reverse broncho-constriction
(induce broncho-dilation) when inhaled after allergen challenge
(FIG. 3.14) OVA-sensitized asthmatic rats challenged by inhalation
of OVA (1 mg/ml) for 5 minutes. Thirty minutes later, they were
treated by inhalation of nebulized Compound XXII (1 mg/ml) or with
normal saline for 5 minutes. Penh was measured before challenge
(baseline), and after challenge and treatment. Data are presented
as mean.+-.SEM for 5 rats. *, P<0.05.
[0527] These experiments demonstrate that the Lipid-conjugates may
be used for the treatment of obstructive respiratory disease,
alleviating airway narrowing by a plurality of mechanisms,
including inhibition of contraction and reduction of airway
obstructing infiltrates. Additional support for the utility of the
Lipid-conjugates in treating obstructive respiratory disease is
provided by the results of Experiments 7.1-7.3 in U.S. application
Ser. No. 10/627,981, incorporated herein by reference,
demonstrating that the Lipid-conjugates are effective in inhibiting
smooth muscle cell proliferation, which is a major cause of
morbidity in chronic asthma. Anti-inflammatory effects of
Lipid-conjugates may be useful in treating lung infections
accompanied by excessive, uncontrolled inflammation such as
influenza, tuberculosis, schistosomiasis, chronic bronchitis,
pneumonia, SARS, respiratory syncitial virus, Empyema Thoracis,
whooping cough, and other respiratory infectious disease.
Example 4
Sepsis
[0528] Sepsis is characterized by enhanced levels of cytokines such
as Tumor necrosis factor (TNF.alpha.) and interleukin (IL)-1, IL-6
and IL-8, and endothelial cell adhesion molecules, such as ICAM-1
and E-Selectin. These molecules are involved in the pathogenesis of
septic shock, and are released both locally and systemically to
produce noxious and irreversible effects on tissue integrity and
systemic hemodynamics. Exposure of cells to the bacterial
lipopolysaccharide (LPS) and Lipoteichoic acid (LTA) immunogens
comprises a commonly-used model system for assaying the response of
these agents to septicemic conditions. It should be noted that
bacterial LPS has both endotoxic and immunogenic components. LPS
toxicity is associated with the lipid component (Lipid A) and
immunogenicity is associated with the polysaccharide
components.
[0529] Experiment 4.1 demonstrates the ability of the
Lipid-conjugates to inhibit elaboration of TNF-.alpha. in human
tissue. Fresh heparinized (12.5 U/ml) human venous blood from
healthy blood donors was diluted 1:3 with RPMI-1640 medium
supplemented with 200 mM glutamine, 200 U/ml penicillin and 200
U/ml streptomycin. Fractions (300 .mu.l) of 1:3 diluted blood were
distributed in 24 well Multidisk plates (Nunclon). Blood samples
were pre-incubated (30 min at 37.degree. C.) in a humidified
atmosphere of 6% CO.sub.2 with 100 .mu.l of compound or solvent
before being stimulated by the addition of 100 .mu.l of
lipopolysaccharide E. coli 026:B6 (LPS) at a final concentration of
100 ng/ml. After a 6 h incubation, the 24 well plates were spun
down (20,000 rpm) and assayed for cytokine content by ELISA. The
various Compound XXIIs differed in their phosphate content (FIGS.
4.1-I and 4.1-II).
[0530] Experiment 4.2 demonstrates the capacity of Lipid-conjugates
to ameliorate sepsis in an in vivo rat model. Endotoxins
administered to animals produce cardiovascular and multiorgan
disorders that are similar to clinical sepsis. Thus, in the present
study, a rat model was developed to test possible Lipid-conjugate
effects on mediator production and mortality in endotoxin-induced
Sepsis. Rats were intraperitoneally (i.p.) or intravenously (i.v.)
injected with a Lipid-conjugate (Compound XXII, 100 mg/kg)
dissolved in sterile saline or with sterile saline alone as
placebo. After 3 hours, all rats received LPS (15 mg/kg i.p.;
Escherichia coli 111:B44 LPS, Sigma, Deisenhofen, Germany). In rats
that were pretreated with Compound XXII, LPS was injected together
with a second dose of Compound XXII (50 mg/kg). The effect of
Compound XXII on LPS injected rats was observed over a time period
of 48 hours. As show in FIG. 4.2, treatment with Compound XXII
markedly reduced the mortality rate among septic rats.
[0531] Experiment 4.3 demonstrates the ability of Lipid-conjugates
to suppress LPS-induced increases of cytokines TNF-.alpha. and IL-6
in serum. Rats were administered 100 mg/kg i.p. Compound XXII 3 h
prior to an i.p. injection of LPS (7.5 mg/kg). Blood samples were
collected 1, 6, 12, and 24 hours after LPS injection to assess
cytokine concentrations (FIG. 4.3A). In another experiment, rats
were pretreated with a priming dose of Lipid-conjugates (Compound
XXII or Compound XXV) 0, 3, 6, or 12 hours before LPS
administration or did not receive Lipid-conjugates. Thereafter, the
animals received LPS (7.5 mg/kg) i.p. alone or together with
Compound XXII (150 mg/kg) or Compound XXV (50 mg/kg). Blood samples
were collected after LPS injection. A group of rats that was not
treated with Lipid-conjugates or LPS served as negative controls
(FIG. 4.3B). All cytokines were measured in separated serum by
ELISA Immunoassays (R&D Systems GmbH, Wiesbaden, Germany)
according to the instructions of the manufacturer. FIG. 4.3
demonstrates that cytokine levels in the serum of septic rats are
markedly reduced by treatment with Lipid-conjugates.
[0532] In Experiment 4.4, Compound XXII was given i.v. at the same
time as LPS was given i.p. As demonstrated in FIG. 4.4,
endotoxin-induced cytokine production was suppressed equally well
by co-administration of i.v. Compound XXII with LPS as with
pretreatment with i.p. Compound XXII.
[0533] In Experiment 4.5, sepsis was induced by LPS (gram-positive
endotoxin, 5 mg/kg) and lipoteichoic acid (LTA, gram-negative
endotoxin, 5 mg/kg; Staph. aureus LTA, Sigma, Germany). FIG. 4.5
demonstrates that 150 mg/kg Compound XXII is effective in
suppressing cytokine production induced by 5 mg/kg i.p. LPS as well
as by a combined treatment with 5 mg/kg i.p. LPS and 5 mg/kg i.p.
LTA 1 and 6 hours after LPS or LPS+LTA treatment.
[0534] Experiment 4.6 demonstrates that Lipid-conjugates inhibit
endotoxin-induced cytokine mRNA expression. For RNase protection
assay (RPA), rat lung and kidney were removed from
Lipid-conjugate-treated or untreated rats 24 hours after induction
of sepsis for total RNA isolation using Trizol reagent (Gibco BRL,
Eggenstein, Germany). The concentration of RNA in each sample was
assessed spectrophotometrically. To evaluate specific RNA levels in
rat lung and kidney, a multiprobe RPA kit was used (riboQuant,
PharMingen, Heidelberg, Germany) according to manufacturer's
instructions. Briefly, a set of .sup.32P-labeled RNA probes
synthesized from DNA templates using T7 polymerase was hybridized
with 7 .mu.g of total RNA, after which free probes and
single-stranded RNA were digested with RNase. Undigested probes and
digested samples were loaded on to a 5% denaturing polyacrylamide
gel, dried and exposed to a Kodak X-apart film. The expression of
each specific mRNA was related to two housekeeping genes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32, to
exclude differences in the amount of RNA that was hybridized. The
following templates for rat cytokines were used in the present
study: IL-1-.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6,
IL-10, TNF-.alpha., TNF-.beta., IFN-.gamma., L32 and GAPDH. As
shown in FIG. 4.6, treatment with Lipid-conjugate inhibited
endotoxin-induced cytokine gene expression. This supports the
findings of FIG. 4.3, which demonstrates a decrease in IL-6
protein.
[0535] Experiment 4.7 demonstrates the effect of Lipid-conjugates
on the RNA expression of iNOS and secretory PLA.sub.2 Type II
(sPLA.sub.2II) in the kidney and lung of rats with LPS-induced
sepsis. RNA expression was measures using Polymerase Chain Reaction
(PCR). Total RNA isolated from rat lung and kidney was subjected to
DNAse digestion (Gibco BRL, Eggenstein, Germany) to remove possible
contaminations of genomic DNA. 1 .mu.g of total RNA was
reverse-transcribed to cDNA using SuperScript.TM. II
Preamplification System (Gibco BRL, Eggenstein, Germany),
essentially as recommended by the manufacturer's instructions. PCR
amplification of 0.5 .mu.l cDNA was performed in a total volume of
25 .mu.l containing 19.6 pmol of each primer, 5 mM dNTPs, 2.5 U Taq
Polymerase, 10 mM Tris HCl, 7.5 mM KCl, 1.5 mM MgCl.sub.2. PCR
reactions were initiated at 94.degree. C. for 3 min, followed by
varying cycles of amplification, each consisting of denaturation at
94.degree. C. for 1 min, annealing at 60.degree. C. for iNOS and
65.degree. C. for sPLA.sub.2-IIA and primer extension 72.degree. C.
for 2 min. At the end of the amplification cycles, the products
were incubated for 10 min at 72.degree. C. In each set of PCR
reactions, two control reactions were included. In one control
reaction, reverse transcriptase was omitted, while in the other
control reaction, cDNA was omitted. PCR products were separated on
a 1% agarose gel. FIG. 4.7 demonstrates the ability of
Lipid-conjugates to suppress the endotoxin-induced gene expression
of sPLA.sub.2 IIA and iNOS
[0536] Experiment 4.8 demonstrates Lipid-conjugate inhibition of
adhesion molecule expression: For immunohistochemical determination
of ICAM-1 expression in rat tissue, cryostat sections of pulmonal
and renal tissue were analyzed by an indirect immunoperoxidase
technique. Briefly, ethanol-fixed sections were incubated with
primary antibody against ICAM-1 for 1 hour, washed and incubated
with peroxidase-conjugated secondary rat IgG antibody for 30 min.
The reaction was developed with ABC solution Vectastain (Wertheim,
Germany) and terminated by washing with TBS. Sections were
counterstained with hematoxylin-eosin, dehydrated and analyzed.
FIG. 4.8 demonstrates the inhibitory effect of the Lipid-conjugates
on endotoxin-induced adhesion molecule expression in septic rat
tissue.
[0537] The results presented in FIGS. 4.1-4.8 demonstrate the
capacity of Lipid-conjugates to ameliorate the endotoxin-induced
mortality among septic rats (FIG. 4.2); reduce arachidonic acid
release induced by hydrogen peroxide and PLA2 (FIG. 4.1); reduce
the blood level of the cytokines TNF.alpha. and IL-6 when induced
by LPS given either i.p. (FIG. 4.3), or i.v. (FIG. 4.4), and by
LPS+LTA (FIG. 4.5); suppress the mRNA expression of TNF.alpha.,
IL-1 and IL-6 (FIG. 4.6), and of secretory phospholipase A)
(sPLA.sub.2-IIA) and the inducible nitric oxide synthase (iNOS) in
the lung and kidney of the septic rats (FIG. 4.7); and suppress the
expression of the adhesion molecule ICAM-1 in lung and kidney of
the septic rats (FIG. 4.8). Additional support for Lipid-conjugate
protection from bacterial toxicity is provided in U.S. application
Ser. No. 10/627,981, incorporated herein by reference. These
results clearly demonstrate the therapeutic capacity of the
Lipid-conjugates in the treatment of sepsis, bacteremia-induced
shock, septic shock, or septicemia. Further, the efficacy of
Lipid-conjugates in protecting against septic shock may contribute
to their usefulness in treating pathogenic bacterial
infections.
Example 5
Hemolysis
[0538] Hemolysis, the breakdown of red blood cells (RBC), may be
either a primary disease in itself, or a syndrome associated with
another disease or physiological insult. In order to determine the
effect of Lipid-conjugates on hemolysis, red blood cells were
incubated in the presence of known membrane destabilizing agents
and the release of hemoglobulin into the extracellular medium was
detected.
[0539] Experiment 5.1 demonstrates that the Lipid-conjugates serve
to maintain the stability of human red blood cells exposed to
membrane-destroying agents. Human RBC were washed in saline and
suspended in Hanks buffer (pH 7.4). Hemolysis was induced in the
absence or presence of 10 .mu.M Lipid-conjugates by treatment with
either 5 U/ml streptolysin O (SLO), 25 U/ml streptolysin S (SLS),
or 5 .mu.g/ml lysophosphatidylcholine (lyso-PC) for 20 min. The
cell membranes were spun and the hemoglobin content in the
supernatant was determined by measuring the O.D. at 540 nm (Table
5.1). TABLE-US-00015 TABLE 5.1 Prevention of Hemolysis by Compound
XXII, Compound XXVI and Compound XXIV HEMOLYSIS (O.D. AT 540 nm)
Lipid-conjugate SLO SLS Lyso-PC None 1.000 1.000 1.000 HA 1.000
1.000 1.875 Compound XXII-30* 0.650 0.750 0.335 Compound XXII-60*
0.012 0.005 0.017 Compound XXII-110* 0.005 0.002 0.012 Compound
XXIV 0.002 1.100 0.002 Compound XXVI-60* 0.012 0.005 0.002 Compound
XXVI-110* 0.002 0.002 *The number expresses the amount of nmoles
lipid conjugated to 1 mg of polymer.
[0540] These experiments demonstrate that the Lipid-conjugates are
effective therapy in the treatment of hemolysis and of value as
preservatives in blood product storage. Thus Lipid-conjugates are
demonstrated to have utility in maintaining hematocrit and in
blood-banking. Further, the efficacy of Lipid-conjugates in
protecting against membrane destabilization may contribute to their
usefulness in treating infections. For example, Lipid-conjugates
may protect against cytopathic effects due to infection or cell to
cell spread.
Example 6
Anti-Oxidant Therapy
[0541] The noxious effect of peroxide free radicals on living
tissue is known as oxidative damage. When cell membranes are the
targets for this damaging process, membrane dysfunction and
instability result. Oxidative damage to blood proteins,
particularly blood lipid proteins, results in their
over-accumulation in cells lining the vasculature, thus
contributing to atherogenesis. In fact, oxidative cell damage is a
major mechanism attributed to the process of aging or
senescence.
[0542] In order to determine the effect of Lipid-conjugates on
oxidative damage to proteins or cell membranes, tissue was exposed
to hydrogen peroxide (H.sub.2O.sub.2) produced by (a) the enzyme
glucose oxidase (GO) in the absence or presence of additional
membrane destabilizing agents such as PLA.sub.2 or (b) by exposure
to divalent cations, such as copper.
[0543] Experiments 6.1-6.3 demonstrate the ability of
Lipid-conjugates to preserve cells from oxidative damage, as judged
by the cells' retention of both arachidonic acid and of low
molecular weight intracellular substances.
[0544] Experiment 6.1: Confluent BGM (green monkey kidney
epithelial) cells were labeled with .sup.3H-arachidonic acid. The
cells were treated with Compound XXVI for 30 min prior to treatment
with GO and PLA.sub.2 (0.5 U/ml) (FIG. 6.1).
[0545] Experiment 6.2: BGM cells were labeled with .sup.35SO.sub.4
overnight. The cells were washed with DMEM (containing 10 mg/ml
BSA) 4 times with PBS. The cells were then incubated in DMEM
supplemented with GO (an H.sub.2O.sub.2 generator) for 90 min. and
the culture medium was collected and counted for .sup.35S
radioactivity. For treatment with Compound XXVI, cells were
incubated with 3 or 10 .mu.M Compound XXVI for 30 min prior to
introduction of GO. Data are presented as mean.+-.SEM for 5
replications. *p<0.005; **p<0.001 (FIG. 6.2).
[0546] Experiment 6.3 demonstrates the ability of Lipid-conjugates
to inhibit the oxidation of blood lipoprotein. Low density
lipoprotein (LDL; 0.1 .mu.M) and or hydroperoxides (LOOH) were
incubated in the absence and presence of various concentrations of
Compound XXII or HA at 37.degree. C. At time zero, 5 .mu.M
CuCl.sub.2 was added to the dispersions, and the mixtures were
continuously monitored for oxidation products at 245 nm (FIG. 6.3).
The absorbance at 245 (OD units) is depicted as a function of time
(Shnitzer et al., Free Radical Biol Med 24; 1294-1303, 1998).
[0547] Additional support for the anti-oxidant capacity of the
Lipid-conjugates is provided by Experiment 7.4 in U.S. application
Ser. No. 10/627,981, incorporated herein by reference, showing the
inhibitory effect of Lipid-conjugates on
ischemia/reperfusion-induced activation of white cells.
[0548] These experiments demonstrate that administration of
Lipid-conjugates is an effective therapy to prevent tissue damage
induced by oxidative stress (associated with free radical and
hydrogen peroxide production) by a plurality of mechanisms,
including inhibiting the oxidation of lipoprotein, inhibiting
arachidonic acid release, and preserving the integrity of cell
membranes (inhibiting GAG degradation), including red blood cell
membranes, as described below. The efficacy of Lipid-conjugates in
protecting against tissue damage induced by oxidative stress may
contribute to their usefulness in treating pathogenic
infections.
Example 7
Central Nervous System (CNS) Insult
[0549] Infection, ischemic stroke, trauma, cancer metastases, and
degenerative disease exemplify physiological insults in which brain
tissue injury may be severe and irreversible. Tissue injury
typically evokes a myriad of physiological responses to stress,
which in the central nervous system take the form of chemical
substances released by support tissue. However, an excess of one or
more of these potentially neurotoxic "wound" chemicals may serve to
further disrupt the healing process and contribute to the brain
tissue damage. Commonly accepted models for assessing the
neuroprotective ability of a new drug employ preparations of brain
matrix cells (e.g., glial cells), neurotransmitter-releasing cells
(e.g., PC12 cells), and migratory blood cells (macrophages and
lymphocytes) which are typically recruited to the sites of damaged
brain tissue. Tissue injury in the CNS is frequently compounded by
local disruption of the blood brain barrier and subsequent passage
of migratory blood cells which may exacerbate the effects of the
original insult and lead to more extensive tissue damage.
[0550] In response to substances associated with stress and
impending injury, such as the immunogen lipopolysaccharide (LPS),
the cytokine TNF.alpha. or the neurotoxin pardaxin, cells of the
central nervous system activate a myriad of wound-response
substances, such as sPLA.sub.2, prostaglandin (PGE.sub.2),
thromboxane (TXB.sub.2), 5-HETE, oxygen radicals, nitric oxide, or
dopamine. When expressed in excess, these substances are either
themselves neurotoxic or indicative of cotemporal neurotoxicity,
thus their suppression is a frequently chosen target for developing
neuroprotective drugs.
[0551] Experiments 7.1-7.2 demonstrate Lipid-conjugate inhibition
of prostaglandin (PGE.sub.2) release.
[0552] Experiment 7.1: Glial cell media was replaced with fresh
media prior to all experiments, supplemented with 10 .mu.g/ml LPS
Lipid-conjugates were added 30 minutes before exposure to LPS. The
tissue cultures were further incubated at 37.degree. C. for 24 h.
Then the medium was collected and the cells were incubated in fresh
medium containing LPS and Lipid-conjugate. After an additional 24
h, supernatants were taken for determination of PGE.sub.2 content
by ELISA (FIG. 7.1).
[0553] Experiment 7.2: Rat adrenal pheochromocytoma (PC12) cells
were incubated with the indicated Lipid-conjugate and then washed
and then stimulated with pardaxin (PX) for 30 minutes. The amount
of PGE.sub.2 released to the medium was determined by ELISA (FIG.
7.2).
[0554] Experiments 7.3 and 7.4 demonstrate suppression of nitric
oxide production by the Lipid-conjugates. Glial cell media was
replaced with fresh media, supplemented with 10 .mu.g/ml LPS.
Lipid-conjugates were added 30 minutes before exposure to LPS. The
tissue cultures were further incubated at 37.degree. C. for 24-48
h. Supernatants were taken after 24 h for determination of NO by
colorimetric measurement using the Griess reagent (FIG. 7.3).
Alternately, primary mouse peritoneal macrophages were treated with
Lipid-conjugates at the indicated concentration for 30 minutes
(FIG. 7.4). Then LPS (1 .mu.g/ml) was added to the culture either
directly or after washing of the Lipid-conjugates. Nitric oxide was
determined by the Griess calorimetric method.
[0555] Experiment 7.5 demonstrates Lipid-conjugate-induced
inhibition of soluble phospholipase A.sub.2 (sPLA.sub.2) release
from glial cells (FIG. 7.5). Glial cell media was replaced with
fresh media, supplemented with 10 .mu.g/ml LPS. Lipid-conjugates
were added 30 minutes before exposure to LPS. The tissue cultures
were further incubated at 37.degree. C. for 24-48 h. Culture medium
samples were taken after 24 h for determination of PLA.sub.2
activity by the hydrolysis of radioactively labeled E. coli
membranes. The radioactive free fatty acid released in this
reaction was counted in a radioactivity scintillation counter.
[0556] Experiments 7.6-7.7 demonstrate the ability of the
Lipid-conjugates to suppress the activation of endogenous
phospholipase A.sub.2, measured as fatty acid release. PC12 cells
were metabolically labeled with .sup.3H-arachidonic acid (AA) or
.sup.3H-oleic acid for at least 6 h, then washed and incubated with
Lipid-conjugate as indicated for 30 minutes. The cells were then
washed, stimulated with PX for 30 minutes and the amount of
.sup.3H-fatty acid released to the medium was determined in a
scintillation counter (FIG. 7.6). For release of oleic acid from
macrophages, murine P388D.sub.1 cells were metabolically labeled
with radioactive oleic acid, and the release of radioactive oleic
acid was determined in the presence (full circles) and absence
(empty circles) of LPS following pre-treatment with the indicated
concentration of the Lipid-conjugate, as shown in FIG. 7.7.
[0557] Experiment 7.8 demonstrates the ability of Lipid-conjugates
to suppress dopamine (DOPA) release. PC12 cells (at confluence)
were loaded with radioactive DOPA for 4 h, then washed in the
presence of an antioxidant. The cells were then incubated with the
indicated Lipid-conjugate for 30 min, then washed and stimulated
with PX for 15 min. The amount of labeled DOPA released to the
culture medium was determined in a scintillation counter (FIG.
7.8).
[0558] Experiment 7.9 demonstrates Lipid-conjugate suppression of
5-HETE release. PC12 cells, under identical conditions to those in
Experiment 7.8, were incubated with the indicated Lipid-conjugate,
followed by PX stimulation. The amount of 5-HETE released was
determined by ELISA (FIG. 7.9).
[0559] Experiment 7.10 demonstrates the potency of Lipid-conjugates
in inhibiting cell permeation through endothelial cell barrier.
Using the T cell transendothelial migration assay (FIG. 7.10),
primary pig brain endothelial cells (PBEC) were plated onto a
collagen-coated filter, separating between the upper and lower
chambers. Human peripheral blood T cells were prepared as described
in Cabanas and Hogg (1993, PNAS 90: 5838-5842). The T cells were
maintained in recombinant human IL-2 for up to 12 days prior to
use. Approximately 10.sup.5 T-cells were added to the upper chamber
of the Transwells above the confluent PBEC monolayer and incubated
at 37.degree. C. for 5 h. Compounds for testing were also added on
the PBEC monolayer at the same time as the T cells. Electrical
resistance values were measured over this period at hourly
intervals. At 5 hours the Transwells were briefly rinsed in warm
medium and fixed in paraformaldehyde. The number of T cells which
had migrated to the underside of the filter (i.e., through the PBEC
monolayer) was counted as described.
[0560] These experiments demonstrate that the Lipid-conjugates are
potent neuroprotective agents and effective against neurotoxic
agents. Lipid-conjugates can prevent tissue damage following
physiological insult to the central nervous system and are thus
useful when administered as therapy for the treatment of brain
injury in settings such as infection, stroke, tumor, trauma, and
degenerative disease. Additional support for the efficacy of
administering Lipid-conjugates as neuroprotective agents is found
in the results of Experiment 7.4 in U.S. application Ser. No.
10/627,981, incorporated herein by reference, demonstrating the
efficacy of administering Lipid-conjugates for the treatment of
ischemia/reperfusion injury. The efficacy of Lipid-conjugates in
protecting against tissue injury in the CNS by decreasing
inflammatory activators may contribute to their usefulness in
treating or altering symptoms of CNS infectious disorders, such as
viral meningitis, Encephalitis, Poliomyelitis, bacterial
meningitis, subdural empyema, and CNS helminthic infections.
Example 8
Toxicity Tests
[0561] Toxicity is a measure to the degree to which a compound or
substance is deleterious to an organism. Toxicological effects are
generally dose-dependent. A therapeutic compound that is non-toxic,
even at high doses, would have an advantage over other
compounds.
[0562] In Experiment 8, the Lipid-conjugates Compound XXII,
Compound XXIV, Compound XXV and Compound XXVI were evaluated for
toxicity. Toxicity was evaluated in mice (3/group) one week after a
single i.p. dose of 1000, 500 or 200 mg/kg of Lipid-conjugates.
Mortality rate, body weight, blood count (red and white cells),
hematocrit, and internal organ histology after sacrifice were
assessed. These parameters were compared in Lipid-conjugate-treated
and in control, untreated mice. Treatment with Lipid-conjugates did
not alter the parameters described above, with the exception of
Compound XXIV, which induced hemorrhage.
[0563] Tables 8.1 and 8.2 depict the non-toxicity of Compound XXII
as demonstrated in acute (Table 8.1) and long-term (Table 8.2)
toxicity tests. TABLE-US-00016 TABLE 8.1 Results of acute (7 day)
toxicity test Dose of Com- pound XXI (mg/kg body Body weight (g)
RBC WBC Hematocrit weight) Start Final .times.10.sup.6
.times.10.sup.3 % 0 21.9 .+-. 0.2 22.6 .+-. 0.3 10.7 .+-. 0.4 9.3
.+-. 0.3 45.0 .+-. 0.5 (control) 250 22.1 .+-. 0.4 23.1 .+-. 0.6
11.4 .+-. 0.1 7.7 .+-. 0.2 43.3 .+-. 0.7 500 21.4 .+-. 0.3 22.3
.+-. 0.4 11.5 .+-. 0.3 8.1 .+-. 1.3 44.7 .+-. 2.3 1000 21.7 .+-.
0.2 22.1 .+-. 0.2 10.9 .+-. 0.4 7.4 .+-. 0.6 40.3 .+-. 0.7 RBC =
red blood cells; WBC = white blood cells. Data are presented as
mean .+-. SEM.
[0564] For the long-term toxicity test, a group of 6 mice received
an i.p. injection of 100 mg Lipid-conjugate (Compound XXII)/kg body
weight 3 times a week for 30 weeks (e.g., 180 mg total to a mouse
weighing 20 g). Toxicity was evaluated as for Table 8.1. The
results of the long-term toxicity test are depicted in Table 8.2.
There were no incidents of mortality and no significant changes in
body weight, red or white blood cell count, or hematocrit induced
by this treatment compared to control, untreated mice.
TABLE-US-00017 TABLE 8.2 Results of long-term (30 weeks) toxicity
test Dose of Compound XXII (mg/kg body weight, 3 times/week for 30
Body weight (g) RBC WBC weeks) Final .times.10.sup.6
.times.10.sup.3 Hematocrit % 0 39.5 .+-. 3.1 10.9 .+-. 0.8 9.3 .+-.
0.6 45.0 .+-. 0.8 (control) 100 39.0 .+-. 2.7 11.7 .+-. 0.7 8.1
.+-. 15 43.4 .+-. 4.9
[0565] Thus, the Lipid-conjugates have very low toxicity, as
indicated in short and long-term toxicity tests.
[0566] It will be appreciated by persons skilled in the art that
the present invention is not limited by what has been particularly
shown and described herein above and that numerous modifications,
all of which fall within the scope of the present invention, exist.
Rather, the scope of the invention is defined by the claims which
follow:
* * * * *