`
`(19) World Intellectual Property Organization
`
`International Bureau
`
`(43) International Publication Date
`13 February 2003 (13.02.2003)
`
`PCT
`
`(10) International Publication Number
`WO 03/011873 A2
`
`(51) International Patent Classification’:
`
`CO7F 9/02
`
`(21) International Application Number:
`
`PCT/CA02/01185
`
`(22) International Filing Date:
`
`29 July 2002 (29.07.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/307 ,842
`
`English
`
`English
`
`27 July 2001 (27.07.2001)
`
`US
`
`(71) Applicant (for all designated States except US): NEP-
`TUNE TECHNOLOGIES & BIORESSOURCESINC.
`[CA/CA]; 500, St-Martin Boulevard West, Suite 550,
`Laval, Québec H7M 3Y2 (CA).
`
`(74) Agents: SABET, Sohrabet al.; Smart & Biggar, 1000 de
`la Gauchetiére Ouest, Suite 3400, Montréal, Quebec H3B
`4W5 (CA).
`
`(81) Designated States (national): AE, AG, AL, AM,AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`GM,HR, HU,ID,IL, IN,IS, JP, KE, KG, KP, KR, KZ, LC,
`LK,LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, Mw,
`MX, MZ, NO, NZ, OM,PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VN, YU, ZA, ZM, ZW.
`.
`.
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,PT, SE, SK,
`TR), OAPIpatent (BF, BJ, CF, CG,CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`(72) Inventor; and
`Published:
`(75) Inventor/Applicant (for US only): SAMPALIS, Fotini
`[CA/CA]; 1348 Elizabeth Boulvard, Laval, Quebec H7W —_without international search report and to be republished
`3J8 (CA).
`upon receipt of that report
`
`(54) Title: NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING FLAVONOIDS, POLYUNSATURATED FATTY
`ACIDS AND THEIR APPLICATIONS
`
`[Continued on next page]
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`x 101 MINUTES
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`x102VOLTS
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`O03/011873A2
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`(57) Abstract: A phospholipid extract from a marine or aquatic biomass possesses therapeutic properties. The phospholipid extract
`comprises a variety of phospholipids, fatty acid, metals and a novel flavonoid.
`
`RIMFROST EXHIBIT 1013
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`RIMFROST EXHIBIT 1013 page 0001
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`page 0001
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`
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`WO 03/011873 A2_IMINIIITINAIUINMATTAATAAA
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andAbbreviations" appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
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`RIMFROST EXHIBIT 1013
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`RIMFROST EXHIBIT 1013 page 0002
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`page 0002
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`~d-
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`NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING FLAVONOIDS,
`
`POLYUNSATURATED FATTY ACIDS AND THEIR APPLICATIONS
`
`Cross-Reference to Related Application
`
`This application claims the benefit of United States
`
`Provisional Patent Application Serial No. 60/307,842, filed
`July 27, 2001, which is incorporated herein by reference in its
`
`entirety.
`
`Field of the Invention
`
`The present invention is directed to nutraceutical,
`
`10
`
`pharmaceutical or cosmetic compositions, particularly to
`
`phospholipid compositions derived from natural marine or
`
`aquatic sources.
`
`Background of the Invention
`
`United States Patent No. 5,434,183 issued on July 18,
`
`15
`
`1995 describes a phospholipid emulsion derived from marine
`
`and/or synthetic origin comprising polyunsaturated fatty acids
`
`and having anti-inflammatory and immunosuppressive effects and
`
`which promotes normal brain or retinal development and
`
`function. U.S. 5,434,183 does not disclose the presence of
`
`20
`
`flavonoids or nervonic acid (a mono-unsaturated fatty acid)
`
`in
`
`the composition.
`
`JP 2215351, published on August 28, 1990, discloses a
`
`method for extracting and purifying phospholipids from fresh
`
`krill. Krill is lyophilized and then extracted with ethanol to
`
`25
`
`produce an extract which is fractionated by absorption column
`
`chromatography to produce high purity phosphatidyl choline and
`
`phosphatidyl ethanolamine. There is no disclosure of a
`
`phospholipid extract comprising a flavonoid or nervonic acid.
`
`RIMFROST EXHIBIT 1013
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`RIMFROST EXHIBIT 1013 page 0003
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`page 0003
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`~2-
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`WO 00/23546, published on April 27, 2000, discloses
`methods for extracting lipid fractions from marine and aquatic
`animal material by acetone extractions.
`The resulting non-
`soluble and particulate fraction is further solvent extracted
`with ethanol or ethylacetate to achieve further lipid
`
`extractions.
`
`Summary of the Invention
`
`the invention provides novel
`In one aspect,
`phospholipids, wherein the two fatty acids chains of the
`phospholipid are occupied by eicosapentanoic acid (EPA) and
`docosahexanoic acid (DHA) simultaneously, within the same
`
`molecule, i.e.: a phospholipid of the general formula (I):
`
`H2C—O—C
`
`O
`b—o—tu
`
`0
`
`H¢—o-—o—x
`b
`
`(!)
`
`wherein X represents a moiety normally found in a phospholipid.
`
`In a further aspect,
`
`the invention provides a novel
`
`flavonoid compound (II):
`
`OH
`
`OH
`
`OH
`
`10
`
`15
`
`20
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`25
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`RIMFROST EXHIBIT 1013
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`page 0004
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`- 23-2
`
`The novel phospholipids and the novel flavonoid
`compound are derived from an extract from a marine or aquatic
`
`biomass.
`
`There is also provided a phospholipid extract
`
`comprising the above noted phospholipids and flavonoid compound
`derived from a marine or aquatic biomass.
`The extract and the
`
`in the prevention or treatment of a
`components are useful
`variety of disease states and for the aesthetic enhancement of .
`
`an animal,
`
`including human, body. Pharmaceutical,
`
`10
`
`nutraceutical and cosmetic compositions containing the extract
`
`and uses thereof are also within the invention, as are
`
`commercial packages contain the compositions of the invention.
`
`Detailed Description of the Invention
`
`1. Phospholipids
`
`15
`
`Phospholipids are complex lipids containing
`
`phosphorus.
`
`The phosphatides, known as phospholipids, are
`
`usually divided into groups on the basis of compounds from
`
`which they are derived.
`
`In addition to two chains of fatty
`
`acids they contain phosphoric acid, glycerol and nitrogenous
`
`20
`
`bases such as choline.
`
`Important phospholipids are
`
`phosphatidylcholine (PC), phosphatidylethanolamine (PE) and
`
`(PI). Their nature as amphophilic
`phosphatidylinositol
`molecules provides them with unique physicochemical properties.
`
`Their function as the principle components of cell membranes
`
`25
`
`makes phospholipids essential for all vital cell processes.
`
`They are widespread as secretory and structural components of
`
`the body and can mimic or enhance natural physiological
`
`processes.
`
`RIMFROST EXHIBIT 1013
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`page 0005
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`PCT/CA02/01185
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`HoC-——O—-P—-O—-CHp—CHs—N<—CH
`NN
`
`CH3
`
`CHg
`
`|
`
`o
`
`oO
`
`Phosphatidylcholine — common structure
`R, and R2 are fatty acid residues,
`different for each molecular species
`
`Oo
`
`wc—o—-—Rr,
`Ro—-—O-—CH
`
`Oo
`
`H»C——O——P——-O—-CHz—CH2—NH3
`
`O
`
`Phosphatidylethanolamine— common structure
`
`R, and Ro are fatty acid residues,
`different for each molecular species
`
`oO
`|
`H2C-——-O——-C—_R,
`
`HeC—-O—P—O
`
`Oo
`
`OH
`
`OH
`
`OH
`
`iO
`
`H
`
`HO
`
`Phosphatidylinositol— common structure
`R, and Ro are fatty acid residues,
`different for each molecular species
`
`10
`
`15
`
`20
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`RIMFROST EXHIBIT 1013
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`~ 5 -
`
`Phospholipid production may be either synthetic or
`
`through extraction from natural tissues.
`
`The chief source of
`
`commercial natural phospholipids are soybean, egg yolk and cows
`
`(brain and liver).
`
`Since an individual phospholipid may
`
`contain a variety of fatty acid residues, it may be described
`
`as pure only with this limitation in mind. Naturally occurring
`
`essential polyunsaturated fatty acids can contribute to the
`
`activation of cellular metabolism.
`
`The main fatty acid found
`
`in phospholipid products is linoleic acid (C18:2n6é), present in
`
`10
`
`soybean at more than 65%.
`
`The longest chain polyunsaturated
`
`fatty acids found in commercially available phospholipids
`
`either as preparations or individually are 20:4 among the
`eicosanoids, known as arachidonic acid, and 22:6 known as
`docosahexanoic acid.
`
`15
`
`Arachidonic acid is a fatty acid that is found as
`
`part of phospholipid membranes, generally as part of
`
`phosphatidylcholine and phosphatidylinositol. Adverse cellular
`
`stimuli will activate enzymes
`
`(phospholipase)
`
`that cleave
`
`arachidonic acid from the phospholipid backbone in the cell
`
`20
`
`membrane. Arachidonic acid, which serves as the precursor for
`
`prostaglandins and prostacyclin (PGs, PGI2) and thromboxane
`
`(TXs), can then be metabolized by one of two major pathways:
`
`the cyclooxygenase (COX) pathway or the lipoxygenase pathway.
`
`The COX pathway products, PGG, and PGH2,, can then be acted upon
`
`25
`
`by thromboxane synthase (in platelets) or prostacyclin synthase
`
`(in endothelium)
`
`to form TXs or PGI2, respectively. Arachidonic
`
`acid can also be acted upon by 5-lipoxygenase, primarily in
`
`leukocytes,
`
`to form leukotrienes (LTs).
`
`One or more of these
`
`metabolites can mediate all the signs and symptoms associated
`
`30
`
`with arachidonic acid, i.e.
`
`inflammatory disease and pain.
`
`Platelets,
`
`leukocytes, smooth muscle, and endothelium
`
`can produce vasoactive substances, products of arachidonic acid
`
`metabolsim such as prostaglandins (PGs), prostacyclin (PGI2),
`
`RIMFROST EXHIBIT 1013
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`~ 6 =
`
`leukotrienes (LTs), and thromboxanes
`
`(TXs). These substances
`
`can either act as vasodilators or as vasoconstrictors.
`
`PGI2 is
`
`essential in vascular function since it inhibits platelet
`
`adhesion to the vascular endothelium and has significant
`
`vasodilatation qualities. Damaged endothelial cells cannot
`produce PGIz, making the vessel more susceptible to thrombosis
`and vasospasm.
`Thromboxanes and leukotrienes serve a vascular
`
`function during inflammation, generally producing
`
`vasoconstriction. Prostaglandins have a vascular role during
`
`10
`
`inflammation, and also play a more subtle role in normal flow
`
`regulation, most notably as modulators of other control
`mechanisms.
`Prostaglandins have both vasoconstrictor and
`
`vasodilator activities. Leukotrienes and prostaglandins can
`
`also increase the endothelial membrane permeability thus
`
`15
`
`promoting edema during inflammation. Arachidonic acid is
`naturally present in most phospholipid mixtures or emulsions
`
`available today.
`
`Nervonic acid (C24:1)
`
`is also called selacholeic acid
`
`or tertracosenic acid. Nervonic acid is the predominant
`
`20
`
`nutrient of white matter in glucoside, which is quantitatively
`
`contained in nerve tissue and white matter.
`
`The absence of
`
`nervonic acid may result in cerebral lesion, fatigue,
`hypodynamia, amentia, and senile dementia. Nervonic acid,
`
`tertracosenic acid in another name,
`
`is monounsaturated, non-
`
`25
`
`oxidable/decomposed and absorptive.
`
`It is called a rare tonic
`
`as it is rare existent in nature.
`
`It may be obtained in small
`
`quantities by extracting from cerebral chrondriosome.
`
`Therefore,
`
`the substantance is far below the demand of human
`
`body.
`
`In foreign countries, nervonic acid mainly comes from
`
`30
`
`shark brain and oil.
`
`RIMFROST EXHIBIT 1013
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`PCT/CA02/01185
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`1.1 Phosphatidylinositol Clinical Applications
`
`_ 7
`
`-
`
`Recent advances in nutritional and biochemical
`
`research have documented inositol as an important dietary and
`
`cellular constituent. Functions of phosphatidylinositol in
`
`biological membranes include the regulation of cellular
`responses to external stimuli and/or nerve transmission as well
`as the mediation of enzyme activity through interactions with
`
`various specific proteins (1).
`
`Inositol has been identified as an important dietary
`
`10
`
`and cellular constituent. Biochemical functions:
`
`a. Regulation of cellular responses to external stimuli
`
`b. mediation of enzyme activity.
`
`Phosphoinositide composition of the central nervous
`
`15
`
`system cell membranes are fatty-acid enriched and consist
`primarily of phosphatidylinositol
`(PI), phosphatidylinositol-4-
`phosphate (PIP), and phosphatidylinositol-4,5-biphosphate
`
`(PIP2). Once the membrane is stimulated, phospholipase C is
`
`activated and consequently inositol triphosphate along with
`
`diacylglycerol is produced.
`PI is used as a precursor for
`phosphatidylinositol-3-phosphate and 3,4,5-triphosphate (2).
`
`20
`
`Active transport carriers, calcium pumps in the cell
`
`membrane itself, and in the endoplasmic reticulum, keep
`
`cytoplasmic calcium concentration very low. Usually the
`
`calcium concentration inside the cytoplasm is 5,000-10,000
`
`25
`
`times less than the concentration in the extracellular fluid.
`
`This endoplasmic store of calcium can be accessed upon
`
`stimulation by inositol.
`
`Inositol triphosphate is released
`
`from the cell membrane and travels through the cytoplasm until
`
`it reaches the endoplasmic reticulum. This inositol then
`
`30
`
`releases the sequestered calcium, which can go on to mediate
`
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`~-
`
`8
`
`-
`
`the release of neurotransmitters in response to depolarization
`
`(3).
`
`In addition to releasing endoplasmic reticulum
`
`calcium,
`inositol functions as the major central nervous system
`non-nitrogenous osmoregulator. Modulation of this inositol
`pool is regulated in response to states of high or low
`osmolalities.
`The inositol pool is supplied via a
`
`sodium/inositol transporter, a sodium dependent active
`transport system, and a passive low affinity transporter (4,5).
`
`10
`
`Numerous non-inositol receptors have been identified
`
`in the central nervous system that can potentially interact
`
`with the inositol signaling system. Most of these receptors
`
`are linked to the G proteins and produce inositol-1,4,5-
`
`triphosphate as second messengers. These receptors can be
`found in nearly every human organ system.
`The potential
`
`15
`
`interactions between these receptors and their agonists are
`
`responsible for regulation of the body on a day-to-day basis.
`In view of the complexity of these systems and their actions, a
`perfect balance is required for regulation of the signaling
`
`20
`
`systems.
`
`Theoretically, an imbalance of inositol concentration
`
`could potentially affect the development and function of one or
`all of these receptors. Cholinergic receptors are located in
`
`the liver, heart, stomach, and lungs. Serotonin and glutamine
`
`25
`
`receptors are found mostly in the central nervous system (CNS)
`tissues. Adrenergic receptors are present in various tissues
`
`including CNS, vascular tissues, and heart. Histaminergic
`
`receptors are predominantly found in the lungs and stomach.
`
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`Clinical Applications
`
`A change in CNS availability of inositol may produce
`altered brain signaling and eventually lead to the development
`
`of neurological disorders.
`
`a. Depression:
`
`10
`
`15
`
`20
`
`25
`
`The pathophysiology of depression is believed to be
`linked to a deficiency of neurotransmitters at post-synaptic
`receptor sites. According to the catecholamine theory,
`the
`deficiency is in the amount of norepinephrine;
`in the
`indolamine theory the deficiency is in the amount of serotonin.
`Receptors linked to the inositol signalling system include
`serotonin (5HT2a and 5HT2b) and norepinephrine (alpha la, 1b,
`
`and 1d).
`
`In 1978, Barkai et al demonstrated depressed patients
`had significantly decreased cerebospinal fluid (CSF)
`levels of
`inositol as compared to healthy patients (6).
`In 1993 this
`theory was expanded to conclude that administration of high-
`dose inositol could increase CSF levels by as much as 70
`
`(7). This led to the study of inositol for treatment
`percent
`of depression (8,9).
`In 1995 Levine et al completed a double-
`blind study for treatment of depression using inositol at a
`dose of 12 grams daily compared to placebo. Patients receiving
`inositol showed significant improvement in depression as ranked
`by the Hamilton Depression Rating Scale (33.4 +/- 6 versus .6
`+/- 10). Another important observation was the absence of
`manic episodes in the bipolar patients treated with inositol.
`This lack of manic episodes may suggest that when the
`signalling system is not overactive, addition of inositol will
`not increase the signalling system's activity (10,11).
`It can
`
`30
`
`be concluded that inositol is effective in managing the
`
`clinical manifestations of depression.
`
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`b.
`
`Panic Disorder:
`
`Benjamin et al expanded the clinical use of inositol
`
`by evaluating its effectiveness in panic disorder (12). This
`
`was an eight week double-blind, crossover study whereby
`
`patients were treated with inositol daily for four weeks and
`
`then crossed over to the other study arm.
`
`Improvement was
`
`assessed using patient diaries,
`
`the Marks-Matthews Phobia
`
`Scale,
`
`the Hamilton Anxiety Rating Scale, and the Hamilton
`
`Depression Scale.
`
`The frequency and severity of panic attacks
`
`10
`
`and the severity of agoraphobia declined significantly more
`
`after inositol than after placebo (a decrease from 10 attacks
`
`per week to 3 per week in the treated group compared to a
`
`decrease from 10 to 6 in the placebo group).
`
`The authors
`
`conclude inositol's efficacy and safety, and the fact that
`
`15
`
`inositol is a natural component of the human diet, make it a
`
`potentially attractive therapeutic agent for panic disorder.
`
`c. Obsessive Compulsive Disorder (OCD):
`
`Since the phosphatidylinositol cycle, as a second
`
`messenger is known to affect several neurotransmitters,
`
`20
`
`including serotonin receptors,
`
`inositol was studied for
`
`treatment in OCD in a double-blind, placebo controlled,
`
`crossover trial. Thirteen patients were treated for six weeks.
`
`There was a significant improvement at week six during the
`
`inositol period when compared to placebo period. There were no
`
`25
`
`side-effects reported during the study period (1).
`
`d. Alzheimer's Disease (AD):
`
`Although the role of aluminum in AD is still
`
`speculative at best,
`
`the presence of aluminosilicates at the
`
`core of senile plaques in diseased neurons is a consistent
`
`30
`
`feature found in the CNS of AD patients during autopsy.
`
`It is
`
`known that aluminum inhibits the incorporation of inositol into
`
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`- 11 -
`
`phospholipids and the hydrolysis of the phosphoinositides by
`binding to one of two specific phosphate groups. This binding
`
`of phosphate and aluminum affects the calcium releasing effects
`
`of the cell.
`
`The resulting profound disturbance of the
`
`phosphatidylinositol second messenger system may account for
`
`neuronal malfunction and eventual cell death (13).
`
`Since the potential role of aluminum as a causative
`
`agent for cell death may be affected by the deregulation of
`
`calcium concentration, possibly due to inositol depletion,
`
`10
`
`supplementation with inositol may produce positive CNS effects.
`
`Recent data suggests the loss of PI second messenger system
`
`target sites and IP3 receptors may add to cognitive impairment
`
`and the failure of conventional therapies in AD. Therefore,
`
`supplementation of inositol to replenish the diminished PI
`
`15
`
`system may be beneficial in the treatment of AD (13-20).
`
`In 1996 Barak et al completed a double-blind,
`
`controlled, crossover study of six grams inositol daily
`
`compared to placebo for 30 days in 11 Alzheimer's patients.
`
`Patients in the study were diagnosed with dementia of the AD
`
`20
`
`type as classified by DSM - IIIR and aged 65 years or older.
`
`The Cambridge Mental Disorder of the Elderly Examination
`
`(CAMDEX) was used as the basic assessment parameter and was
`
`administered upon admission into the study.
`
`Included in CAMDEX
`
`is part A: patient's present physical and mental state, part B:
`
`25
`
`Cognitive.Subscale of CAMDEX (CAMCOG), part C:
`
`interviewers
`
`observations, and part D: physical examination.
`
`CAMCOG was
`
`repeated at two, four, six, and eight weeks. Participants
`
`scored 80 or less on the CAMCOG examination and their symptoms
`
`of depression were not severe (21).
`
`30
`
`Patients were excluded from the study if they had a
`
`history of psychiatric, alcohol, and/or drug addiction
`
`disorders, or abnormalities in baseline laboratory values
`
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`-~ 12 -
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`(blood count, electrolytes,
`
`liver or kidney functions, VDRL, or
`
`CT scan) not consistent with AD. Patients with additional
`
`neurologic, metabolic, endocrinologic disorders, or presence of
`
`internal disease that grossly impaired brain functioning were
`
`also excluded.
`
`Subjects were given either three grams inositol or
`
`placebo in the morning and again in the evening. After four
`
`weeks patients were crossed over into the other arm (inositol
`
`or placebo) for an additional four weeks. Only benzodiazepines
`
`10
`
`were allowed during the study period (15 mg of oxazepam or
`
`equivalent), provided the patient was receiving it on study
`
`entry.
`
`Analysis of the improvement scores of all patients
`
`who completed the study showed inositol increased the total
`
`15
`
`CAMCOG score from a baseline of 31.36 +/- 20.90 to 40.09 +/-
`24.54, while the placebo group increased from baseline of 35.9
`+/- 25.96 to 39.27 +/- 25.
`The authors concluded only two of
`
`the eight subscales (language and orientation) showed
`
`;
`
`significant improvement with inositol.
`
`20
`
`Inositol's proposed mechanism of action in the CNS
`
`does not
`
`include direct manipulation with either pre- or post-
`
`receptors. However, it may indirectly affect the relationship
`
`between receptor and agonist.
`
`By mediating the physiochemical
`
`characteristics of the M1 pre-synaptic receptor (solubility,
`
`25
`
`osmolality, etc.),
`
`inositol may alter the binding site and
`
`influence the signaling that occurs as a result.
`
`1.2 Aging
`
`Phosphatidylcholine rich in polyunsaturated fatty
`
`acids is indispensable for cellular differentiation,
`
`30
`
`proliferation and regeneration.
`
`The physiologic functions of
`
`these phospholipids are related to the morphology of the
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`the incorporation of these molecules into
`biological membranes,
`membranes and thus the maintenance of intact cell membranes.
`
`The current study was designed to investigate the
`effects of Polyunsaturated phosphatidylcholine on age-related
`hearing loss by evaluating its ability to preserve
`mitochondrial function, protect mitochondrial DNA from
`oxidative damage and preserve auditory sensitivity (22).
`
`Harlan-Fischer 344 rats, 18-20 months of age, were
`
`used as the experimental subjects.
`
`10
`
`The subjects were caged individually and maintained
`at 21 to 22° C in a 12:12 light-dark cycle b.
`
`A dose of 300mg/kg/day of Polyunsaturated
`phosphatidylcholine was supplemented to each subject, by adding
`
`it to the oral diet.
`
`15
`
`The animals were divided randomly into two groups
`
`(n = 7 for each group). Group-1 served as the control, and
`
`group-2 as the experimental group.
`
`At the onset of the study, Auditory Brainstem
`Responses were obtained to measure baseline hearing thresholds
`
`20
`
`in all subjects.
`
`Age-associated changes in hearing sensitivities were
`then recorded at two-month intervals for six months.
`
`In order to assess age-related changes in
`
`mitochondrial function, mitochondrial membrane potentials were
`
`25
`
`studied using flow cytometry. For this purpose, peripheral
`blood was obtained from each subject at the beginning and at
`
`the end of the protocol.
`
`At the conclusion,
`
`the subjects were euthanized
`
`(according to NIH protocol), and tissue samples were obtained
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`from brain and cochlea (stria vascularis and auditory nerve)
`
`to
`
`study mitochondrial DNA deletion associated with aging. This
`
`was achieved by amplifying the specific common aging
`
`mitochondrial deletion by Polymerase Chain Reaction. DNA
`
`quantification was performed. The data obtained for each
`protocol was compared between the two groups and analyzed using
`
`ANOVA.
`
`The effects of Polyunsaturated phosphatidylcholine on
`
`age-related hearing loss demonstrate a gradual age-associated
`
`10
`
`decline in hearing sensitivities at all the frequencies tested
`
`(3, 6, 9,
`
`12 and 18 kHz).
`
`There was a statistically significant preservation of
`
`hearing noted in the treated subjects at all frequencies, which
`
`was observed at four and six months of treatment.
`
`15
`
`Overall,
`
`there was a continued decline in hearing in
`
`the control subjects and a statistically significant protective
`
`effect of Polyunsaturated phosphatidylcholine on the
`
`experimental subjects (p<.005).
`
`Mitochondrial membrane potentials were recorded by
`
`20
`
`flow cytometry as a measure of the uptake of Rhodamine 123 by
`
`mitochondria.
`
`The mean fluorescence intensity (MFI)
`
`in group-1
`
`subjects measured 3190 and 2100 at the beginning and end of the
`
`study, respectively.
`
`25
`
`This, approximately, 30% decline in membrane
`
`potential with time was statistically significant
`
`(p=0.003).
`
`Conversely,
`
`the MFI in the experimental group
`
`remained essentially unchanged at 2990 from 3165 at the
`
`beginning of the study.
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`This difference between the control and treated
`
`(p<0.05), demonstrating
`groups was statistically significant
`the protective effect of polyunsaturated phosphatidylcholine
`supplementation on mitochondrial membrane potential.
`
`Phospholipids are integral structural components of
`
`all biological membranes with polyunsaturated
`phosphatidylcholine and phosphatidylethanolamine being the
`
`predominant types, quantitatively. They constitute the
`phospholipid bilayer structure of cellular membranes, which is
`responsible for membrane stability and cellular function.
`
`10
`
`Polyunsaturated phosphatidylcholine maintains and promotes the
`
`activity of several membrane bound proteins and enzymes,
`
`including Na-K ATPase, adenylate cyclase and glutathione
`
`reductase. They are also known to be precursors of
`
`15
`
`cytoprotective agents such as eicosanoids, prostaglandins and
`
`antioxidants.
`
`The results of these studies suggest that
`
`polyunsaturated phosphatidylcholine and
`
`phosphatidylethanolamine may protect mitochondrial function by
`
`20
`
`preserving the age-related decline in mitochondrial membrane
`
`potentials and hence their activity. The observation that there
`
`was less mitochondrial DNA damage in the treated group may
`
`explain the effect of preservation of hearing loss associated
`
`with aging, by the ability of polyunsaturated
`
`25
`
`phosphatidylcholine and phosphatidylethanolamine to
`
`specifically up-regulate cochlear mitochondrial function. There
`
`are many studies demonstrating the effects of mitochondrial
`
`metabolites on cognition and aging (22-33). Additionally,
`
`recent work has shown that acetyl-L-carnitine and -lipoic acid
`
`30
`
`delay the progression of age-related hearing loss by protecting
`
`cochlear mitochondrial DNA from oxidative damage (34). These
`
`results support the membrane hypothesis of aging and provide
`
`further evidence to support this theory as a possible
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`explanation for age-related hearing loss. Thus,PPC may be one
`of many rational approaches to consider for the purpose of
`membrane preservation, enhanced mitochondrial function,
`reduction of age-associated mitochondrial DNA damage and
`
`slowing of some of the aging processes.
`
`1.3 Effect of phosphoglycolipid exract
`
`(NT factor) on normal
`
`and cancerous cells
`
`Reduced levels of phospholipids in normal cells can
`
`limit metabolic activity and limit available energy.
`
`10
`
`Phospholipids, as part of the membrane structure:
`
`i. maintain membrane integrity
`
`regulate enzyme activities and membrane transport
`ii.
`processes through changes in membrane fluidity (Spector 1981,
`
`1985)
`
`15
`
`20
`
`iii. Signal transduction utilizes phospatidylcholine and
`phosphatidylinositol for the production of diacyl-glycerol
`(DAG) by phospholipase C (Berridge 1989) and for the production
`of inositol triphosphate (IP3)
`(Ranan 1990, Michell 1988,
`
`Margolis 1990).
`
`iv. One of the choline phospholipids (l-alkyl-2 acetyl-SN-
`glycerol-3-phosphocholine)
`is the substrate for the synthesis
`of platelet activating factor (Synder 1989).
`
`v.
`The arachidonic acid found as part of the structure of
`choline or inositol phospholipid is utilized for the production
`of prostaglandin and leukotriene (Nordoy 1990).
`
`25
`
`The choline of phosphatidylcholine may be used in neural
`vi.
`tissue for the synthesis of acetylcholine (Blusztain 1987)
`
`vii. Phosphoglycolipid improves cell maintenance and metabolic
`
`activity of normal cells.
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`viii. Phosphatidylcholine derivatives disrupt cancer cells at
`concentrations that do not affect normal cells.
`
`ix. Phosphatidylcholine is selectively cytotoxic to cancer
`cells in vitro (Hoffman 1986, Harmann 1986, Berger 1984).
`
`Such compounds inhibit HL60 leukemic cells at a
`a.
`dosage that has no effect on normal human marrow cells,
`the
`tissue from which the leukemic cells are derived.
`
`b. Normal cells were able to tolerate 4 times higher
`dosage than the leukemic cells during 24 hours incubation with
`the phospholipid preparation (Berdel 1986).
`
`10
`
`c. There was up to a 5-fold difference in
`sensitivity between the normal and tumor cells with breast,
`ovarian, and lung cancer cells, as well as with mesothelioma
`
`cells (Namba 1993).
`
`15
`
`1.4
`
`Imaging
`
`Polyunsaturated phospholipids are known to be
`important with regard to the biological functions of essential
`fatty acids,
`for example,
`involving neural tissues such as the
`brain and retina. The NMR spectra of polyunsaturated bilayers
`are dramatically different from those of less unsaturated
`phospholipid bilayers. MD simulations can aid in interpreting
`the complex NMR spectra of polyunsaturated bilayers,
`in
`conjunction with electron density profiles determined from
`small-angle X-ray diffraction studies. This work clearly
`demonstrates preferred helical and angle-iron conformations of
`the polyunsaturated chains in liquid-crystalline bilayers,
`which favor chain extension while maintaining bilayer
`
`flexibility. The presence of relatively long, extended fatty
`acyl chains may be important for solvating the hydrophobic
`surfaces of integral membrane proteins, such as rhodopsin.
`
`In
`
`20
`
`25
`
`30
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`page 0019
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`the polyallylic DHA chains have a tendency to adopt
`addition,
`back-bended (hairpin-like) structures, which increase the
`interfacial area per lipid. Finally,
`the material properties
`have been analyzed in terms of the response of the bilayer to
`mechanical stress. Simulated bilayers of phospholipids
`containing docosahexaenoic acid were less sensitive to the
`applied surface tension than were saturated phospholipids,
`possibly implying a decrease in membrane elasticity (area
`elastic modulus, bending rigidity). The above features
`distinguish DHA-containing lipids from saturated or
`nonunsaturated lipids and may be important for their biological
`
`10
`
`modes of action.
`
`1.5
`
`In Summary
`
`The functions of the phospholipids are multiple and
`
`15
`
`different for each phospholipid:
`
`Sphingosine and carbohydrate containing lipids are mainly
`a.
`concentrated in nervous tissues.
`
`The hydrophilic and hydrophobic parts of the phospholipid
`b.
`molecule allow them to function as emulsifying agents in order
`to maintain the proper colloidal state of protoplasm.
`
`c. Phospholipids aid the transport of triglycerides through
`the liver, especially during mobilization from adipose tissue.
`
`d. Phospholipids and their metabolites play an important role
`in intracellular signalling, for example via
`phosphatidylinositol specific phospholipase C, phospholipase D
`or phosphatidylinositol-kinases.
`
`20
`
`25
`
`e. Through their concentration in cell membranes they may
`somehow be involved in the transport of hydrophobic
`
`constituents into and out of cells.
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`page 0020
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`£. Phospholipids affect brain function in two substantial
`ways:
`(Cohen B.M., Babb S.M.,