WO1999045916A2 - Novel methods for treating disorders in which docosahexaenoic acid (dha) levels are affected - Google Patents
Novel methods for treating disorders in which docosahexaenoic acid (dha) levels are affected Download PDFInfo
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- WO1999045916A2 WO1999045916A2 PCT/US1999/004722 US9904722W WO9945916A2 WO 1999045916 A2 WO1999045916 A2 WO 1999045916A2 US 9904722 W US9904722 W US 9904722W WO 9945916 A2 WO9945916 A2 WO 9945916A2
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- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Definitions
- Cystic Fibrosis is the most prevalent autosomal recessive disorder in the Caucasian population (Gorelick (1991) Gastroenterology 103:681-693). Approximately 1 in 2000 live births are afflicted with CF and 5% of Caucasians in the United States are carriers of the abnormal CF gene. CF individuals rarely survive past their mid-thirties, and most mortalities are a result of recurrent pulmonary infection and, ultimately, pulmonary failure. Two other major clinical manifestations of CF are pancreatic dysfunction and male infertility.
- the CF gene had been cloned and was found to code for a chloride channel.
- Activation of the channel in the normal pancreas activates the chloride/bicarbonate exchanger, resulting in a net secretion of bicarbonate into the lumenal space and alkalinization of the pancreatic juice.
- Mutations in the chloride channel like those found in CF result in a reduced chloride conductance and a reduced ability of ductal cells to secrete bicarbonate into the lumenal space. This results in the formation of inspissated plugs within the ducts leading to obstruction of the pancreatic ducts.
- pancreatic insufficiency appears to be a result of defects in membrane recycling with obstruction of the ducts occurring as a secondary event.
- cystic fibrosis transmembrane regulator CFTR
- CF gene mutations lead to three important clinical events: 1) increased mucus secretion; 2) increased inflammatory response; and 3) decreased immune function.
- Previous reports have indicated that arachidonic acid (AA) levels are increased in CF patients, i.e., an increase in AA has been demonstrated in lavage fluid from the lungs of CF patients. However, this was thought to be secondary to infection and not a primary process.
- Our research demonstrates that there is an increase in archidonic acid (AA) while levels of docosahexaenoic acid (DHA) are significantly decreased. Thus, the ratio of DHA to AA is affected. It appears that the ratio of docosahexaenoic acid (DHA) to AA is affected. Similar findings are made in chronic inflammatory diseases.
- RDS respiratory distress syndrome
- an object of the invention is to provide a method for treating a disorder in which the serum, tissue or membrane levels and/or ratios of DHA and AA are affected.
- Another object of the invention is to provide a method of treating cystic fibrosis in a subject by administering to the subject a therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds.
- a still other object of the invention is to provide a method of treating a chronic inflammatory disease, e.g., ulcerative colitis, Crohn's disease, chronic pancreatitis, asthma, rheumatoid arthritis or chronic gastritis.
- a further object of the invention is to provide a method for treating hypertrophy of small intestine in a subject suffering from a disorder in which DHA levels are affected.
- An additional object of the invention is to provide a method of restoring normal morphology to a cell or tissue affected with a disorder in which DHA levels are affected.
- a still further object of the invention is to provide a method for diagnosing in a subject a disorder in which DHA levels are affected.
- Another object of the invention is to provide a method of ameliorating affects of cystic fibrosis in a newborn.
- a still additional object of the invention is to provide a method for increasing surfactant levels in a fetus.
- the invention features a novel method of treating a subject suffering from a disorder in which DHA levels are affected.
- the method includes administering to the subject a therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds, (e.g., DHA and DHA precursors, e.g., docosapentaenoic acid, tetracosapentaenoic acid and tetracosahexaenoic acid).
- the dosage may be of the fatty acid itself or in a form more easily incorporated into the tissue such as a derivative or a structured lipid.
- This method is particularly useful in treating subjects suffering from a disorder characterized by a defect in the CF gene, e.g., cystic fibrosis; or a chronic inflammatory disorder, e.g., ulcerative colitis, Crohn's disease, chronic pancreatitis, asthma, rheumatoid arthritis or chronic gastritis.
- the method is also useful in treating a disorder characterized by reduced levels of fetal surfactant, e.g., by abrogation of fetal surfactant biosynthesis.
- Proper levels of treatment can be determined in a variety of ways, e.g., measurement of the DHA levels in plasma which are raised to at least about 170 ⁇ g/ml. Preferably, the DHA levels are raised to the range of about 200 ⁇ g/ml to about 500 ⁇ g/ml.
- Omega 3 fatty acids comprising 22-24 carbon atoms and 5 or more double bonds may be administered in different modes, e.g., as a part of a nutritional additive or total formulation. If a total formulation is used, it can be as a part of an enteral nutrition solution, an emulsion or as a solid formulation.
- the therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds is about 0.3-5% of the total caloric intake, preferably 0.5-3% of total caloric intake.
- the therapeutically effective amount of DHA should produce a pancreatic AA/DHA ratio between 0.2-1.5, a lung AA/DHA ratio between 0.1-1.6 and a blood AA/DHA ratio between 0.1-1.6.
- a preferred method of administration is as a structured lipid having at least one omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds, most preferably with the DHA located at R 2 position of the structured lipid.
- Preferred structured lipids also include medium chain or short chain fatty acids.
- the structured lipid has DHA (or the 22-24 carbon, 5 or more double bond omega 3 fatty acid) at two or even all three positions.
- the invention also features a method of increasing the level of DHA in one or more tissues of a subject having a disorder in which DHA levels are affected.
- the method includes administering to the subject a therapeutic composition comprising an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds in an amount effective to raise the levels of DHA in the subject's tissue.
- the therapeutic composition comprises an omega 3 fatty acid comprising 22-24 carbon atoms and 5 double bonds in the range of about 0.5-3% of the total caloric intake.
- the omega 3 fatty acid comprising 22-24 carbon atoms and 5 double bonds is DHA or a structured lipid containing DHA.
- the DHA levels are increased in affected tissue, e.g., plasma, pancreatic or lung tissue, most preferably, the DHA levels are increased in plasma to at least about 170 ⁇ g/ml.
- affected tissue e.g., plasma, pancreatic or lung tissue
- the DHA levels are increased in plasma to at least about 170 ⁇ g/ml.
- the disease in which DHA levels are affected is selected from the group consisting of a disorder characterized by an defect in the CF gene, e.g., cystic fibrosis, a chronic inflammatory disorder e.g., ulcerative colitis, Crohn's disease, chronic pancreatitis, asthma, rheumatoid arthritis, chronic gastritis, or lowered fetal surfactant levels.
- a disorder characterized by an defect in the CF gene e.g., cystic fibrosis
- a chronic inflammatory disorder e.g., ulcerative colitis, Crohn's disease, chronic pancreatitis, asthma, rheumatoid arthritis, chronic gastritis, or lowered fetal surfactant levels.
- the invention also pertains to a method of ameliorating the effects of cystic fibrosis in a newborn.
- the method includes administering a therapeutic composition comprising an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds to a mother during pregnancy such that the effects of cystic fibrosis in the newborn are ameliorated.
- the invention further pertains to a method of increasing surfactant levels in a fetus.
- the method includes administering a therapeutic composition comprising an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds to a pregnant woman who may or may not be at risk for premature delivery such that the surfactant levels in the fetus are increased.
- the invention features a method of treating hypertrophy of small intestine in a subject suffering from a disorder in which DHA levels are affected.
- the method includes administering to the subject a therapeutic composition comprising an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds in an amount effective to decrease hypertrophy of small intestine by at least about 25 percent.
- the invention features a method of restoring normal morphology of a cell or tissue which exhibits a disease morphology associated with disorder in which DHA levels are affected.
- the method includes contacting a cell, e.g., a pancreatic acinar cell or a lung cell, or a tissue, e.g., in vitro or in vivo, with a therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds such that the normal morphology of the cell or tissue is restored.
- a cell e.g., a pancreatic acinar cell or a lung cell
- a tissue e.g., in vitro or in vivo
- the invention also features a method of diagnosing a disorder in which DHA levels are affected.
- the method includes: a) obtaining a tissue biopsy from a subject; b) determining the ratio of AA to DHA in the tissue biopsy; and c) comparing the ratio in the tissue biopsy to a standard AA to DHA ratio. If the ratio in the tissue biopsy is at least about 20% higher than the standard AA to DHA ratio, this indicates that the subject is suffering from the disorder in which DHA levels are affected.
- the following description and non-limiting examples further elucidate the invention.
- Figure 1 is a bar graph depicting effects of DHA therapy on pancreatic luminal diameter in CFTR-/- mice (CF) and wild-type (WT) mice.
- Figure 2 is a bar graph depicting effects of DHA therapy on ileal height in CFTR-/- mice (CF) and wild-type (WT) mice.
- Figures 3 a and 3b are bar graphs depicting AA and DHA levels, respectively, in various organs in CFTR (-/-) mice (CF) and wild type mice (WT).
- Figures 4a and 4b are bar graphs depicting AA and DHA levels, respectively, in other organs from CFTR (-/-) mice (CF) and wild type mice (WT).
- Figures 5a and 5b are graphs showing the effects of oral DHA administration at various concentrations on AA and DHA levels in preparations of pancreatic acini and lung cells from CFTR (-/-) mice (CF) as compared with wild-type mice (WT).
- Figure 6 is a graph depicting DHA plasma levels in wild-type (WT) and CFTR (-/-) mice as a function of oral DHA dosing.
- Figure 7 is a bar graph depicting the pancreatic morphology from wild-type (WT), CFTR (-/-) mice (CF), and CFTR (-/-) mice treated orally with 40 mg per day of DHA for 7 days.
- Figure 8 is a bar graph depicting the ileal morphology from wild-type (WT), CFTR (-/-) (CF), and CFTR (-/-) mice treated orally with 40 mg per day of DHA for 7 days.
- Dietary fatty acids are classified according to their chain length. Long chain fatty acids contain 14 carbons or greater and can be further characterized by the number of double bonds contained in their structure into saturated, monounsaturated and polyunsaturated subgroups.
- the two fatty acids essential in human nutrition are linoleic acid (18:2n-6) and ⁇ -linolenic acid (18:3n-3) from which polyunsaturated fatty acids of the omega 6 (n-6) series and omega 3 (n-3) series, respectively, are formed through enzymatic desaturation and elongation by the liver.
- Arachidonic acid (AA, 20:4n-6), produced from essential fatty acids of the omega 6 pathway, is a recruiter of inflammatory cells and a stimulant of mucus secretion.
- DHA docosahexaenoic acid
- AA AA, 20:4n-6
- DHA docosahexaenoic acid
- DHA and AA precursors compete for the intracellular enzymes involved in their incorporation into membrane lipids. AA has profound effects on stimulating mucus secretion, recruiting inflammatory cells, and inhibiting immune function leading to recurrent infections. DHA has multiple effects on cell membrane function. This is supported by experiments in which rabbits with essential fatty acid deficiency (resulting in DHA deficiency) demonstrate lung disease mimicking that observed in CF (Harper et al, (1994) Am. Rev. Respir. Dis. 126:540-547). DHA has also recently been reported to activate CTP holine-phosphate citidyltransferase (Mallampalli et al, (1994) Am. J. Physiol.
- the inventors have now discovered a new lipid metabolic defect in cystic fibrosis (namely, a decrease in DHA and an increase in AA) that when corrected leads to a reversal of the clinical and histopathological manifestations of cystic fibrosis.
- This therapy results in an increase in DHA levels reaching plasma levels above 170 ⁇ g/ml and a profound decrease in AA approaching levels seen in normal mice.
- An equivalent amount of DHA for a man with an approximately 1500-2000 Kcal/day diet (on a calorie basis) would be approximately 5-7 or more g/day. Modifications to this dosage may be used depending on effectiveness of incorporation into tissue and the form in which the dosage is given. This therapy would also find wide applicability in other disorders in which DHA levels are affected, e.g., chronic inflammatory disorders and male infertility.
- the invention also features a method to restoring the ratio of AA/DHA by administration of DHA precursors (See Example 8).
- DHA levels are modified by administering DHA precursors (although not as effectively as administering DHA itself) which results in reversal of biochemical and morphological manifestations of the disease.
- administration of 40mg/day of docosapentaenoic acid (22:5n-3) to CFTR-/- mice resulted in 20% improvement in pancreatic morphology.
- administration of 40mg/day of ⁇ - linolenic acid (18:3n-3) resulted in increased intestinal villi height, increased acinar lumen dilation and plug formation (See Example 8).
- the invention features a method of treating a subject suffering from a disorder in which DHA levels are affected.
- the method includes administering to the subject a therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds, (e.g., DHA and DHA precursors, e.g., docosapentaenoic acid, tetracosapentaenoic acid and tetracosahexaenoic acid).
- derivatives of the omega fatty acids e.g., the salt or ester form of the fatty acids
- a structured lipid containing the omega 3 fatty acid can be used.
- the term "treating" includes a therapeutic treatment of an existing or established disorder in which DHA levels are affected or prevention of the symptoms in a subject at risk for a disorder in which DHA levels are affected.
- the term "subject” is intended to include human and non- human animals.
- the term “non-human animals” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
- the subject is a mammal, e.g., a primate, e.g., a human.
- a disorder in which DHA levels are affected refers to a disorder characterized by a defect in a cystic fibrosis (CF) gene, i.e., the cystic fibrosis transmembrane regulator (CFTR) gene, or a chronic inflammatory disorder.
- CF cystic fibrosis
- CFTR cystic fibrosis transmembrane regulator
- a disorder in which DHA levels are affected does not refer to a disorder in which the primary cause of the disease is a bacterial or a viral infection.
- chronic inflammatory disorder refers to a disorder characterized by an infiltration of inflammatory cells, e.g., lymphocytes and/or monocytes, within a target tissue resulting in recurring or prolonged disease symptoms.
- the infiltration of inflammatory cells also results in increased prostaglandin synthesis which results from increased arachidonic acid (AA) levels.
- AA arachidonic acid
- infiltration of monocytes and lymphocytes within a target tissue results in pain and impairment of the tissue's function.
- chronic inflammatory disorders include, but are not limited to, ulcerative colitis, Crohn's disease, chronic pancreatitis, asthma, rheumatoid arthritis and chronic gastritis.
- the term "a disorder in which DHA levels are affected” is also intended to refer to a disorder associated with the regulation of surfactant levels, such as biosynthesis via activation of CDP:cholinephosphate citidyltransferase.
- administering is intended to include routes of introducing to a subject DHA to perform its intended function. Administration can be carried out by any suitable route of administration. Preferred route of administration is oral administration.
- therapeutically effective amount refers to the quantity of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds necessary to prevent, to cure or at least partially arrest the symptoms of the disorder and its complications.
- the therapeutically effective amount of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds is the amount sufficiently large not only to increase DHA levels in an affected cell or tissue but also to decrease AA levels in the affected cell or tissue.
- the DHA levels are raised in plasma to at least about 170 ⁇ g/ml, preferably they are raised in the range of about 200 ⁇ g/ml to about 500 ⁇ g/ml, more preferably they are raised to at least about 250 ⁇ g/ml.
- Amounts of an omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds effective for use in the methods of the invention will, of course, depend on the severity of the disorder and the weight and general state of the patient.
- dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
- Effectiveness of the dosage can be monitored by measuring the AA to DHA ratio in affected cells or tissue, or in red blood cells as described herein.
- the therapeutically effective amount of an omega 3 fatty acid with 22-24 carbon atoms and 5 double bonds e.g.
- DHA is about 0.3-5% of the total calories, more preferably it is about 0.5-3% of total caloric intake.
- the omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds can be administered as a part of a nutritional formulation. Sources for these fatty acids include, but are not limited to, fish oils, e.g., salmon oil, and breast milk.
- DHA is commercially available from Martek, Inc. as a fungal derivative which contains 50% DHA.
- DHA-containing formulation Neuromins is commercially available from Nature's Way Products Inc., Springville, Utah.
- free DHA is also available from Sigma Chemical Corporation (St.
- the dosage may be given either as a loading dose provided as meal feeding, or continuous parenteral or enteral administration, for the initial therapy period. This allows sufficient time for the DHA to equilibrate within the tissues (e.g. 4- 6 weeks with meal feeding and about 2 days with continuous parenteral or enteral administration) which can be followed by a maintenance dose that is a small fraction of the loading dose.
- An omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds can also be administered in a form of an emulsion. Flavoring may also be added to the emulsion to make it more palatable for enteral use. Flavoring can be in a form of flavored extracts, volatile oils, chocolate flavoring, peanut butter flavoring, cookie crumbs, vanilla or any commercially available flavoring. While the nutritional formulation is preferably provided in a ready-to-feed form, it may also be concentrated by increasing the percent total solids of the formula or made in powder form, both procedures being well known to those skilled in the art. The concentrate or powder are then reconstituted for feeding by adding water (tap or deionized-sterilized water).
- An omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds can also be administered as a part of a structured lipid.
- the structured lipid has a glycerol backbone with three fatty acids linked thereto.
- the structured lipid has DHA located at the R 2 position.
- the structured lipid may include medium chain or short chain fatty acids, or have two or more DHA (or C22-24 omega 3 fatty acids with 5 or more double bonds) on the same backbone.
- the structured lipid may be manufactured by any conventional means such as transesterification but the use of blocking groups which allow positioning of the residues at specific locations is preferred.
- omega 3 fatty acid moiety e.g., DHA
- the preferred triglyceride improves absorption into the body as a whole and allows for ease of incorporation of DHA found at the R 2 position.
- An omega 3 fatty acid comprising 22-24 carbon atoms and 5 or more double bonds can also be administered as a solid formulation.
- solid formulation refers to an oral composition which generally includes in addition to an active compound, e.g., DHA, an inert diluent or an edible carrier.
- the active compound e.g., DHA
- the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
- Pharmaceutically compatible binding agents, and or adjuvant materials can be included as part of the composition.
- the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
- a lubricant such as magnesium stearate or Sterotes
- a glidant such as colloidal silicon dioxide
- the invention also pertains a method of treating hypertrophy of small intestine in a subject suffering from a disorder in which DHA levels are affected.
- the method includes administering to the subject a therapeutic composition comprising an omega 3 fatty acid with 22-24 carbon atoms and 5 double bonds, e.g. DHA, in an amount effective to decrease hypertrophy of small intestine.
- the treatment with DHA is effective to decrease hypertrophy of small intestine by at least about 25%, more preferably by at least about 50%, most preferably by at least about 70- 75%.
- the term "hypertrophy of small intestine” refers to a condition in which there is an increase in a number of intestinal cells, e.g., columnar mucosal cells, resulting in the increase in the villi size as compared to a normal small intestine morphology.
- the decrease or increase in the villi size can be measured using methods described below.
- the invention also pertains to a method of restoring normal morphology of a cell or tissue which exhibits a disease morphology associated with a disorder in which DHA levels are affected. The method includes contacting a cell or a tissue with a therapeutically effective amount of DHA such that the normal morphology of the cell or tissue is restored.
- the cell is a pancreatic acinar cell or a lung cell, e.g. , a serous cell within the submucosal gland or a type II alveolar cell.
- the tissue is lung, pancreas or small intestine. Contacting can occur both in vitro and in vivo.
- disease morphology refers to a morphology observed in an affected cell or tissue of a subject suffering from a disorder in which DHA levels are affected.
- diseases morphology observed in affected tissues or cells include, but are not limited to, hypertrophy of the small intestine, dilatation of pancreatic ducts, appearance of mucus plugs in the pancreatic duct system, appearance of mucus plugs in the lungs, lung inflammation and decrease in apical membrane recycling of acinar cells.
- the invention also features a method of diagnosing a disorder in which DHA levels are affected.
- the method includes: a) obtaining a tissue biopsy from a subject; b) determining the ratio of AA to DHA in the tissue biopsy; and c) comparing the ratio in the tissue biopsy to a standard AA to DHA ratio, wherein the ratio in the tissue biopsy is at least about 20% higher than the standard AA to DHA ratio, thereby indicating that the subject is suffering from the disorder in which DHA levels are affected.
- the ratio in the tissue biopsy is at least about 25%), more preferably at least about 30%, 35%, 40% or 50%) higher than the standard AA to DHA ratio.
- standard AA to DHA ratio is a ratio of AA to DHA found in a subject who is not affected with a disorder in which DHA levels are affected.
- the standard ratio can be determined from a tissue biopsy obtained from a subject who is not affected with a disorder in which DHA levels are affected.
- the standard ratio can be determined using techniques known in the art, e.g., mass spectrometry, gas chromatography, infrared spectrometry or high performance thin layer chromatography (HPTLC).
- HPTLC high performance thin layer chromatography
- the invention also pertains to a method of ameliorating effects of cystic fibrosis in a newborn. The method includes administering a therapeutic composition comprising DHA to a mother during pregnancy such that the effects of cystic fibrosis in the newborn are reduced.
- the invention further pertains to a method of increasing surfactant levels in a fetus.
- the method includes administering a therapeutic composition comprising DHA to a pregnant woman who may or may not be at risk for premature delivery such that the surfactant levels in the fetus are increased.
- the method includes administering a therapeutic composition comprising DHA to all pregnant women, preferably, starting between 24 and 30 weeks gestational age until delivery.
- the invention also features a method of upregulating fetal lung surfactant biosynthesis during pregnancy by increasing DHA levels in the fetal lung (See Example 9).
- Fetal lung surfactant is produced by type II alveolar cells in the fetal lung and its consists of approximately 90% phospholipid and 10% surfactant apoprotein (Farrel et al, (1975) Am. Rev. Resp. Dis. I l l :657-688). Surfactant biosynthesis in the human fetus starts at around 30 to 32 weeks of gestational age and the concentration in lung and amniotic fluid increases linearly until term. Several mechanisms have been implicated in the initiation of fetal lung surfactant biosynthesis during pregnancy.
- CDP holine-phosphate citidyltransferase a key enzyme in the biosynthesis of dipalmitoyl phosphatidycholine (DPPC), a critical component of fetal lung surfactant, comprising 90% of disaturated surfactant phosphatidylcholines
- DPPC dipalmitoyl phosphatidycholine
- Mallampalli et al. have recently reported that DHA, plays a critical role in the regulation of surfactant biosynthesis via activation of CDPxholinephosphate citidyltransferase (Mallampalli et al, (1994) Am. J. Physiol. 267:641-648).
- DHA dipalmitoyl phosphatidycholine
- Example 9 The data shows that DHA readily crosses the placenta in pregnant mice and is incorporated at significant levels in fetal lung and brain (See Example 9, Tables 8 and 9). The data also demonstrates that DHA therapy increases surfactant concentration in preterm amniotic fluid to levels normally found at term (See Example 9, Table 10). DHA therapy may also be used to accelerate brain and retina maturation in preterm newborns. Intraventricular hemorrhage due to brain immaturity is one of the main causes of morbidity and mortality in newborns before 32 weeks gestational age. DHA is one of the main lipid components of brain tissue and the increase of DHA during fetal development is correlated with brain maturation. Administration of 10 or 20 mg DHA/day to preterm wild-type pregnant mice results in a significant increase in DHA in fetal brain (See Example 9, Table 9).
- DHA biosynthesis pathway is outlined below.
- the AA pathway is an independent pathway known to compete for the same elongation and desaturation enzymes and for the site of esterification at the sn-2 position of lipids as the DHA pathway.
- the AA pathway is described below:
- the invention provides a method for restoring the ratio of AA/DHA by administration of DHA or its precursors or derivatives (See Example 8).
- DHA is known to down-regulate AA incorporation into membrane lipids and to play a major role in regulating membrane fluidity, membrane fusion, and membrane trafficking (Stillwell et al, (1997) Lipids 32:497-506; Ehringer et al, (1990) Chem. Phys. Lipids 54:79-88 and Stillwell et al, (1993) Lipids 28:103-108).
- AA levels in membrane lipids can be significantly decreased by the administration of DHA or eicosapentaenoic acid (EPA 20:5n-3), there is no known condition in which membrane-bound AA levels are increased above basal levels (Kuehl et al, (1980) Science 210:978-984).
- DHA and EPA are the most prominent members of the n-3 pathway. These fatty acids are the elongation and desaturation products of the essential fatty acid, ⁇ -linolenic acid (18:3n- 3).
- AA (20:4n-6) is the most prominent member of the n-6 pathway and is the elongation and desaturation product of the essential fatty acid, linoleic (18:2n-6). These pathways are independent from each other and are known to compete for the same elongation and desaturation enzymes and for the site of esterification at the sn-2 position of lipids.
- AA and DHA content in membrane lipids varies with cell type and plays an important role in regulating cell function. Disruption of this critical balance in AA and DHA content in cell membranes could lead to the development of pathological conditions.
- a primary defect in DHA biosynthesis could be explained by either an impairment in the enzymes involved in the elongation and desaturation of DHA precursors or by a defect in the translocation of DHA or its intermediates between different cell compartments.
- ⁇ -linolenic acid from the diet or from intracellular sources is metabolized to tetracosapentanoic acid (24:6n-3) via EPA by a series of chain elongation and desaturation reactions in the endoplasmic reticulum (Katz et al, (1996) Nutrition 12:334-339).
- Tetracosapentanoic acid (24:6n-3) is then translocated to the peroxisomes where it undergoes ⁇ -oxidation to produce DHA.
- DHA is then translocated from the peroxisomes to the endoplasmic reticulum where it is now ready to be incorporated into membrane phospholipids. Movement of 24:6n-3 and DHA between the endoplasmic reticulum, the peroxisomes, and cell membranes is mediated by specific fatty acid binding proteins.
- the biosynthesis of DHA requires multiple enzymatic steps and the translocation of omega 3 fatty acid intermediates between different cell compartments.
- CFTR in addition to its role in regulating chloride conductance in apical membrane of epithelial cells, has been described as a multifunctional protein involved in modulation of intracellular membrane trafficking (Jilling et al, (1997) Int. Rev Cytol 172:193-241). Therefore, an enzymatic impairment or defect in membrane lipid trafficking could potentially lead to a decrease in membrane-bound DHA.
- DHA biosynthesis is that the delta 6 desaturase, a key enzyme in DHA biosynthesis, is used in both the conversion of ⁇ -linolenic acid to octadecacatetraenoic acid (18:4n-3) and tetracosapentanoic acid (24:5n-3) to tetracosahexanoic acid (24:6n-3). This enzyme is also utilized in the conversion of 18:2n-6 to 18:3n-6, both AA precursors.
- Feeding of CFTR-/- mice with 40mg/day of 18:3n-3 was shown to result in a decrease in both AA and DHA because 18:3n-3 may "monopolize" the delta 6 desaturase slowing the rate of conversation of 24:5n-3 to 24:6n-3 through the delta 4-independent pathway and also the conversation of 18:2n-6 to 18:3n-6 in the AA biosynthetic pathway.
- the results of these experiments showed that administration of 40 mg of 18:3n-3 per day to CFTR-/- mice for one week resulted in a significant decrease in phospholipid-bound AA (p ⁇ 0.001) and a further decrease in phospholipid-bound DHA (p ⁇ 0.001).
- Eicosapentaenoic acid (20:5n-3), a DHA precursor downstream from 18:3n-3, is known to effectively compete with AA for the site of esterification at the sn- 2 position of membrane lipids.
- EPA Eicosapentaenoic acid
- CFTR-/- mice were fed with 40 mg/day of EPA (20:5n-3) for one week to down-regulate membrane-bound AA levels (See Example 8).
- Example 11 A comparison of the effects of DHA in reversing the pathological manifestation of cystic fibrosis in CFTR-/- mice by administration of n-3 fatty acids known to compete with AA and DHA biosynthesis and/or esterfication into membrane phospholipids and fish oil is shown in Example 11.
- Administration of ⁇ -linolenic acid resulted in a four- fold decrease in AA, a two-fold decrease in DHA, and a significant increase in ductal dilatation in pancreas indicative of worsening of the cystic fibrosis phenotype.
- Administration of EPA resulted in a four-fold decrease in AA and no significant changes in DHA levels or pancreas morphology.
- DHA therapy may also be efficacious in disorders where a membrane imbalance in AA and DHA levels may not exist; e.g., use of DHA for the treatment of adult respiratory distress syndrome (ARDS), or the use of DHA therapy to induce fetal lung maturation during pregnancy where there is no membrane lipid imbalance in AA and DHA levels in type II cells from the fetal lung.
- ARDS adult respiratory distress syndrome
- pancreas and ileum histopathology may be due to other DHA effects unrelated to down-regulation of phospholipid-bound AA or correction of the observed membrane lipid imbalance.
- administration of 40mg EPA/day to CFTR-/- mice decreased AA levels in pancreatic cells but did not have any significant effects on pancreas morphology and that (ii) administration of 40 mg DHA/day to CFTR-/- mice increased DHA levels in pancreas 3-fold above wild-type levels and corrected pancreas morphology.
- DHA therapy may be activating and/or inducing the expression of other chloride conductance pathways (e.g., calcium- activated chloride conductance) believed to compensate for the decreased CFTR- dependent chloride conductance in pancreas from CFTR-/- mice.
- other chloride conductance pathways e.g., calcium- activated chloride conductance
- DHA has been associated with the regulation of cell growth and the induction of apoptosis perhaps through the regulation of gene expression via cell cycle or early response genes. Similar processes may be operating in the ileum in response to DHA therapy (Clarke et al, (1994) Proc. Natl. Acad. Sci. USA 91 :479-483; Bruzzone et al, (1985) Biochem. J. 226:621-624; Alvarez et al, (1995) Mol Reprod. Dev. 42:334- 346).
- the invention further provides a diagnostic method of correlating plasma or red blood cell DHA or AA levels with other organs.
- the plasma or red blood cell levels of DHA or AA levels can be used as a diagnostic method to determine the effectiveness of treatment in the pancreas or other affected organs (See Example 10).
- CFTR-/- mice CF knockout mice
- DHA docosahexaenoic acid
- AA arachadonic acid
- brain, kidney, heart, spleen, and blood were sonicated in 0.3M sucrose buffer.
- Membrane lipids were extracted from cell and organ preparations with 20 volumes of chloroform-methanol 2:1 (v/v), transmethylated with 0.5 mL of 0.1 N methanolic- NaOH/BF 3 reagent, and the resulting fatty acid methyl esters analyzed by gas chromatography/mass spectrometry (GC/MS).
- lipids were fractionated by aminopropyl column chromatography into triglycerides, cholesterol esters, and phosphoglycerides and the fatty acid composition of each lipid class determined by a combination of enzymatic hydrolysis, transmethylation, and GC/MS.
- Cystic fibrosis (UNC strain) and wild type mice (12-20 grams body weight) were fed with a complete liquid enteral formulation (PeptamenTM, Nestle Clinical Nutrition, Inc.) supplemented with different quantities of the DHA-containing formulation Neuromins (Nature's Way Products Inc., Springville, Utah). DHA content in the Neuromins formulation was 100 mg per 500 mg of formulation.
- Mice were fed once a day at about 11 a.m., seven days a week with 20 ml of PeptamenTM supplemented with the various amounts of either Neuramins, free DHA (Sigma Chemical Co., MO) or DHA-ethyl ester (Nu-Check-Prep, Inc. MN) ranging from 0.5mg/day of DHA to 40mg/day ofDHA.
- mice were sacrificed between 9 and 10 a.m. and last fed between 3 and 4 p.m. on the day before
- Lung and acinar cells were isolated from a CF mouse fed with DHA formula (mouse- 1, 40 mg/day DHA).
- the pellets (0.5 mL for pancreas cell homogenate and 0.6 mL for lung cell homogenate) were extracted with 3 mL of chloroform-methanol 2:1 (v/v).
- the resulting emulsion was vortexed, centrifuged at 800g for 3 min., and the lower phase aspirated.
- the residual upper phase was partitioned again with 3 mL of chloroform-methanol 2:1 (v/v), the lower phase aspirated and both lower phases combined.
- Fractions were evaporated to dryness, processed for fatty acid methyl esters (FAME) and were analyzed by GC-MS analysis.
- FAME fatty acid methyl esters
- pancreas and intestine appeared normal after DHA treatment.
- alues are expressed in nmoles per total extract
- Lung and acinar cells were isolated from a CF and a wild-type (WT) mouse fed with DHA formula.
- the pellets (0.5 mL for pancreas cell homogenate and 0.6 mL for lung cell homogenate) were extracted with 3 mL of chloroform-methanol 2:1 (v/v).
- the resulting emulsion was vortexed, centrifuged at 800g for 3 min., and the lower phase aspirated.
- the residual upper phase was partitioned again with 3 mL of chloroform- methanol 2:1 (v/v), the lower phase aspirated and both lower phases combined. Fractions were evaporated to dryness, processed for FAME and analyzed by GC-MS.
- alues are expressed in nmoles per total extract
- mice-1 was started on DHA formulation (40 mg/day DHA) on day 30 and sacrificed on day 37
- mice-2 was started on DHA formulation (40 mg/day DHA) on day 24 and sacrificed on day 47.
- DHA formulation 40 mg/day DHA
- Lung and acinar cells were isolated from CF mice fed with different doses of DHA, i.e., 0.5 mg/day, 2.0 mg/day, 10 mg/day or 40 mg/day DHA.
- the pellets were extracted with 3 mL of chloroform-methanol 2:1 (v/v).
- the resulting emulsion was vortexed, centrifuged at 800g for 3 min., and the lower phase aspirated.
- the residual upper phase was partitioned again with 3mL of chloroform-methanol 2:1 (v/v), the lower phase aspirated and both lower phases combined. Fractions were evaporated to dryness, processed for FAME and analyzed by GC-MS. Aliquots of 30 ⁇ L of 17:0 in chloroform-methanol 2:1 (v/v) were added to the samples before methylation.
- CF and WT mice were fed as outlined in the method section above. Dilation of the pancreatic duct lumen is a hallmark feature of cystic fibrosis. Therefore, the luminal area was measured from a minimum of 30 images from CF knockout mice treated with PeptamenTM alone or PeptamenTM plus 40 mg/day of DHA as well as wild type (WT) mice treated with PeptamenTM alone beginning at the time of weaning. Animals were sacrificed at 37 days of age and each pancreas fixed in glutaraldehyde. Analysis was performed using NIH Image software. The results are outlined in Figure 1. Compared with wild-type mice, the luminal dilatation is significantly increased in CF mice.
- CF and WT mice were fed as outlined in the method section above. Heal height, i.e., the villi height in the ileum, was measured in CF knockout animals treated with either PeptamenTM alone, PeptamenTM + 40 mg per day DHA, or Wild Type (WT) animals treated with PeptamenTM alone. Animals were sacrificed at 50 days of age, ileum fixed in paraformaldehyde, and the height measured on micrographs at 100X. Villi height in the ileum was found to be significantly greater compared to wild-type mice. The results are outlined in Figure 2. Treatment of these CF mice with 40 mg DHA/day resulted in a decrease in villi height to values observed in wild-type animals.
- This example was designed to show the parallel between DHA and AA levels and the morphological effects on various tissue using DHA.
- CFTR-/- mice were prepared as described in Example 1. Both CFTR-/- mice and wild type mice were euthanized with carbon dioxide, and cell suspensions from pancreas and lung isolated. Pancreatic acini were prepared by collagenase and mechanical dissociation (Bruzzone et al, (1985) Biochem. J. 226:621-624). A total lung cell suspension was prepared by first flushing contaminating blood by repeated injection of lung with Krebs Henseleit buffer (KHB). Tissue was then extensively minced and incubated for 30 minutes at 37°C in 10 ml of KHB containing 1,000 units of collagenase (Worthington Biochemical, PA) 2,000 units of DNase, and 0.5 units of thermolysin (Sigma Chemical Co., St.
- KHB Krebs Henseleit buffer
- the resulting cell suspension was sedimented once through KHB containing 5% BSA and subsequently washed once in KHB, pH 7.4 containing 32 mM bicarbonate, 10 mM Hepes, and 0.5% BSA. Brain, kidney, and heart were removed and sonicated in 0.5 mL and 0.3M sucrose. A similar approach was used for duodenum and ileum except that mucosal scrapings were used for sonication.
- AA and DHA levels in cell preparations of these organs and in wild-type and CFTR-/- mice were quantitated by GC/MS analysis.
- Lipids were extracted from cell suspensions, tissue homogenates, and blood plasma with 6 volumes of chloroform- methanol (2:1, v/v). The resulting emulsions were centrifuged at 600 g for 3 min and the lower phase containing the lipids was collected. This procedure was repeated twice and the lower phases from the two extractions combined. Aliquots of 30 ⁇ L of heptadecanoic acid (17:0) (1 mg/ml) were added to the extracts and used as an internal standard.
- Fatty acid methyl ester peaks were identified by comparison of retention times of standard fatty acid mixtures and by mass spectrometry, and quantified using methylheptadecanoate as the internal standard.
- the different lipid classes including cholesterol esters, triglycerides, free fatty acids, and glycerolipids, were also fractionated from cell and tissue extracts by aminopropyl column chromatography and the corresponding fatty acid methyl esters prepared and analyzed (Alvarez et al, (1992) J. Chromatogr. B. 577:142-145).
- Morphometry For measurement of ileal villus height or pancreatic luminal diameter, multiple images were randomly taken at 200x using a Nikon Microphot-SA microscope equipped with a Diagnostic Instruments, Inc., Spot Digitizing System. Images were examined in a blinded fashion by two independent observers. Quantitation was performed using NIH Imaging Software. The differences between the means in the groups tested were evaluated using the Student's t-test.
- Phospholipid-bound AA is increased and phospholipid-bound DHA is decreased in pancreas, lung, and ileum from CFTR-/-mice AA and DHA levels in cell preparations of these organs from wild-type and
- pancreatic acini preparation contains acinar, centroacinar, and proximal intralobular duct cells, all of which are known to express CFTR, based on immunohistochemicical localization studies (Marino et al, (1991) J. Clin. Invest. 88:712-716 and Zeng et al, (1997) Am. J. Physiol. 273:C442-455).
- DHA was orally administered to CFTR-/- mice for 7 days at different concentrations.
- Figure 5 shows the effects of oral DHA administration on AA and DHA levels in preparations of pancreatic acini ( Figure 5a) and lung cells ( Figure 5b).
- mean AA and DHA levels from wild type mice (WT) are indicated by the dashed lines.
- the results show that administration of DHA at 0.5 and 2 mg per day for 7 days did not significantly alter AA levels in pancreatic acini preparations from CFTR-/-compared to wild-type mice (See Figure 5b).
- DHA 10 mg per day resulted in a significant decrease in AA levels in CFTR-/- mice below those seen in wild-type mice with further suppression as DHA was increased to 40 mg per day.
- the DHA levels in CFTR- /- mice were similar to those seen in wild-type mice, but administration of 2, 10 and 40 mg of DHA per day increased DHA levels to values higher than those observed in wild- type mice. Similar results were obtained for AA and DHA content in lung cell suspensions following administration of varying doses of DHA (See Figure 5a).
- Oral doses of 20 mg of DHA per day and higher resulted in a significant decrease in AA levels below those seen in wild-type mice.
- the decrease in DHA levels is not due to defects in intestinal absorption or hepatic biosynthesis
- Luminal diameter is approximately 9- fold greater in CFTR-/-mice.
- DHA doses used in those studies was significantly lower than the equivalent doses (based on total calorie usage) used in the present invention to treat CFTR-/-mice.
- the plasma levels of DHA reported in those studies were 40-50 ⁇ g/ml corresponding to the levels obtained in our CFTR-/- mice using 0.5 mg DHA/day.
- the data demonstrates that administration of 0.5 mg/day of DHA to CFTR-/- mice does not significantly change phospholipid-bound AA and DHA levels nor has any significant effect on pancreas or ileal morphology.
- DHA formulations used in those clinical trials also contained EPA, known to compete with DHA for esterification at the sn-2 position of phospholipids.
- mice were fed with 40 mg/day of either ⁇ linolenic acid or EPA (20:5n-3) for one week to magnify the accumulation of DHA precursors upstream from the putative block.
- PeptamenTM a complete liquid enteral formulation comprised of medium-chain triglycerides, carbohydrates and hydrolyzed protein
- PeptamenTM a complete liquid enteral formulation comprised of medium-chain triglycerides, carbohydrates and hydrolyzed protein
- the volume of PeptamenTM administered was measured on a daily basis using specific feeders.
- ⁇ - linolenic acid administered 40 mg/day to CFTR-/- mice for one week resulted in a significant decrease in phospholipid-bound AA (p ⁇ 0.001) and a further decrease in membrane-bound DHA (p ⁇ 0.001).
- administration of ⁇ - linolenic acid resulted in increased intestinal villi height and a significant increase in acinar lumen dilatation and plug formation in the exocrine pancreas as compared to CFTR-/-controls treated with PeptamenTM (data not shown).
- Eicosapentaenoic acid (EPA-20:5n-3), a DHA precursor downstream from ⁇ - linolenic acid, is known to effectively compete with AA for the site of esterification at the sn-2 position of membrane lipids.
- CFTR-/- mice were fed with 40 mg/day of EPA for one week to down-regulate membrane-bound AA levels. The results from these studies demonstrated that administration of EPA resulted in a significant increase in membrane-bound EPA, a significant decrease in membrane bound AA, and no significant changes in DHA/levels or pancreas morphology.
- DPA docosapentaenoic acid
- Wild-type and CFTR-/- mice were fed with 40 mg of DP A per day for 7 days and fatty acids in pancreas and lung analyzed by GC/MS.
- One to 2 mm pieces of pancreas from both wild-type and CFTR-/- were fixed for 1.5 h in 25 glutaraldehyde in 0.2M cacodylate buffer and processed for morphometric analysis.
- l values are expressed in nmoles of fatty acid per mg of protein and represent the mean of two experiments. Numbers in parenthesis correspond to values obtained in CFTR-/-control mice fed with PeptamenTM alone.
- the invention also provides a method for improving the amount of fetal lung surfactant in a mouse model. It appears that this is accomplished by upregulating fetal lung surfactant biosynthesis during pregnancy. This method again utilizes giving high doses of DHA which increases DHA and fetal surfactant levels in the fetal lung. Gestation in mice lasts about 19.5 days and previous reports have shown that surfactant levels in amniotic fluid are not detected until after day 16.5, equivalent to about 30 to 32 weeks gestational age in humans. Wild-type C57 mice were bred and identification of the cervical plug was used to determine the occurrence of pregnancy and gestational age.
- mice were placed on PeptamenTM ⁇ DHA for one week at different doses and sacrificed on day 16.5. Following sacrifice, amniotic fluid was aspirated from the amniotic sacs, and DPPC concentration determined by micro-HPTLC. In a different set of experiments, fetal lung and brain were removed, homogenized, lipids extracted with chloroform-methanol, and fatty acids transmethylated and analyzed by gas chromatography.
- DHA and AA levels in Blood Components Following DHA Therapy levels of DHA and AA in various blood components following administration of 40 mg DHA/day to mice was determined.
- 40 mg DHA was given to wild type mice and levels of DHA (Table 11) and AA (Table 12) were measured in plasma, red blood cells (RBC), and the pancreas.
- RBC red blood cells
- the results show that levels in the pancreas are paralleled in red blood cells and plasma. Accordingly, either plasma or red blood cell levels of DHA and AA (and their ratios) can be used as a diagnostic or to determine the effectiveness of treatment in the pancreas and other affected organs.
- CFTR-/- mice were fed 40 mg/day for one week of other n-3 fatty acids known to compete with AA and DHA biosynthesis and/or esterification into membrane phospholipids. AA and DHA levels and pancreas morphology were determined and compared to PeptamenTM alone, shown in Table 13.
- LNA ⁇ -linolenic acid
Abstract
Description
Claims
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AU29824/99A AU762660B2 (en) | 1998-03-10 | 1999-03-03 | Novel methods for treating disorders in which docosahexaenoic acid (DHA) levels are affected |
CA002322848A CA2322848A1 (en) | 1998-03-10 | 1999-03-03 | Novel methods for treating disorders in which docosahexaenoic acid (dha) levels are effected |
EP99911098A EP1061911A2 (en) | 1998-03-10 | 1999-03-03 | Novel methods for treating disorders in which docosahexaenoic acid (dha) levels are affected |
JP2000535331A JP2002506026A (en) | 1998-03-10 | 1999-03-03 | Novel method for treating disorders in which docosahexaenoic acid (DHA) levels are affected |
BR9908697-2A BR9908697A (en) | 1998-03-10 | 1999-03-03 | Methods for treating disorders in which levels of docosahexaenoic acid (dha) are affected |
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- 1999-03-03 AU AU29824/99A patent/AU762660B2/en not_active Ceased
- 1999-03-03 EP EP99911098A patent/EP1061911A2/en not_active Withdrawn
- 1999-03-03 JP JP2000535331A patent/JP2002506026A/en not_active Withdrawn
- 1999-03-03 CA CA002322848A patent/CA2322848A1/en not_active Abandoned
- 1999-03-03 WO PCT/US1999/004722 patent/WO1999045916A2/en not_active Application Discontinuation
-
2000
- 2000-11-03 US US09/706,404 patent/US6552081B1/en not_active Expired - Fee Related
-
2003
- 2003-04-08 US US10/410,511 patent/US20040127567A1/en not_active Abandoned
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004032918A1 (en) * | 2002-10-08 | 2004-04-22 | Chevis Agriservices & Consulting Pty Limited | Treatment of inflammatory conditions with ∝-3 polyunsaturated fatty acids |
WO2006054110A2 (en) * | 2004-11-22 | 2006-05-26 | Stanford Rook Limited | Immunotherapeutic agent |
WO2006054110A3 (en) * | 2004-11-22 | 2006-07-20 | Stanford Rook Ltd | Immunotherapeutic agent |
US7569213B2 (en) | 2004-11-22 | 2009-08-04 | Stanford Rook Limited | Immunotherapeutic agent |
EP1709961A1 (en) * | 2005-04-07 | 2006-10-11 | Bristol-Myers Squibb Company | Use of Docosahexaenoic Acid and/or Arachidonic Acid for preventing or treating respiratory infections in infants |
WO2007100560A2 (en) * | 2006-02-28 | 2007-09-07 | Bristol-Myers Squibb Company | Use of dha and ara in the preparation of a composition for inducing the expression of pulmonary surfactant protein-b |
WO2007100560A3 (en) * | 2006-02-28 | 2008-05-22 | Bristol Myers Squibb Co | Use of dha and ara in the preparation of a composition for inducing the expression of pulmonary surfactant protein-b |
AU2011201206B2 (en) * | 2011-03-17 | 2015-04-23 | Women's And Children's Health Research Institute | Methods and compositions for promoting the respiratory development of an infant |
RU2721555C2 (en) * | 2014-12-02 | 2020-05-20 | Эфиммьюн Лимитед | Compositions containing 15-hepe, and methods of treating or preventing fibrosis using said compositions |
Also Published As
Publication number | Publication date |
---|---|
EP1061911A2 (en) | 2000-12-27 |
AU762660B2 (en) | 2003-07-03 |
US20040127567A1 (en) | 2004-07-01 |
WO1999045916A3 (en) | 1999-11-11 |
US6552081B1 (en) | 2003-04-22 |
AU2982499A (en) | 1999-09-27 |
CA2322848A1 (en) | 1999-09-16 |
US6180671B1 (en) | 2001-01-30 |
JP2002506026A (en) | 2002-02-26 |
BR9908697A (en) | 2000-11-21 |
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