`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1069
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`
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`USSJBOJ48B2
`Page 2
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`OTHER PUBLICATIONS
`
`Rogers, S. et al. The efficacy and safety of donepezil in patients with
`Alzheimer’s disease:
`results of a US multicentre, randomized,
`double-blind, placebo-controlled trial. The donepezil study group.
`Dementia 1996; 7: 293-303 (Abstract Only).
`Schneider, L. et al. Potential role for estrogen replacement in the
`treatment ofAlzheimer’s dementia.Am. J. Med. 1997; 103: 468-508.
`Seidman, M. Polyunsaturated Phosphatidylcholine in NT FactorTM
`Improves Mitochondrial Function, Auditory Sensitivity and May
`Slow Some Aspects of the Aging Processes. Anti—Aging Medical
`News, Winter 2001.
`
`Suzuki, Y]. et al. Aipha-lipoic acid is a potent inhibitor of NFKb
`activation in human Tcells. Biochem. Biophys. Res. Commun. 1992;
`189: 1709-1715 (Abstract Only).
`Van Dyck, C. et al. The acetylcholine releaser linopiridine increases
`pariental regional cerebral blood flow in Alzheimer’s disease.
`Psychopharmacology 1997; 132: 217-226.
`Wiegand, R. et al. Phospholipid Molecular Species of Frog Rod
`Outer Segment Membranes. Exp. Eye Res. 1983; 37(2): 159-73.
`
`* cited by examiner
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`US 8,030,348 B2
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`1
`NATURAL MARINE SOURCE
`PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND
`THEIR APPLICATIONS
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`
`This application is the 371 National Phase of International
`Application No. PCT/CA02/01185, filed Jul. 29, 2002, which
`was published in English under PCT Article 21(2) as Inter-
`national Publication No. WO 03/011873. This application
`further claims the benefit of priority under 35 U.S.C. §1l9(e)
`of US. provisional application 60/307,842 filed, Jul. 27,
`2001. Each of the foregoing applications and the foregoing
`publication is hereby incorporated herein by reference in its
`entirety.
`
`FIELD OF THE INVENTION
`
`The present invention is directed to nutraceutical, pharma-
`ceutical or cosmetic compositions, particularly to phospho-
`lipid compositions derived from natural marine or aquatic
`sources.
`
`BACKGROUND OF THE INVENTION
`
`WO 92/21335 published on Dec. 10, 1992 and correspond-
`ing US. Pat. No. 5,434,183 issued on Jul. 18, 1995 describes
`a phospholipid emulsion derived from marine and/or syn-
`thetic origin comprising polyunsaturated fatty acids and hav-
`ing anti-inflammatory and immunosuppressive effects and
`which promotes-normal brain or retinal development and
`function. US. Pat. No. 5,434,183 does not disclose the pres-
`ence of flavonoids or nervonic acid (a mono -unsaturated fatty
`acid) in the composition.
`JP 2215351, published on Aug. 28, 1990, discloses a
`method for extracting and purifying phospholipids from fresh
`krill. Krill is lyophilized and then extracted with ethanol to
`produce an extract which is fractionated by absorption col-
`unm chromatography to produce high purity phosphatidyl
`choline and phosphatidyl ethanolamine. There is no disclo-
`sure of a composition comprising a flavonoid or nervonic
`acid.
`
`WO 00/23546, published onApr. 27, 2000, discloses meth-
`ods 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 extrac-
`tions.
`
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`Hosokawa et al. (35), published in 2000, discloses the
`conversion of docosahexanoic acid containing phosphatidyl-
`cholines (DHA-PC) from squid skin lecithin to docosahex-
`anoic acid containing phosphadylserines (DHA-PS) via
`transphosphatidylation with phospholipase D (PLD).
`According to Table 2 of this reference, the fatty acid compo-
`sition of the phospholipid includes important portions of
`eicosapentanoic acid. There is no disclosure concerning any
`pharmaceutical, nutraceutical, or cosmetic use of a composi-
`tion comprising a flavonoid.
`
`Henderson et al. (36), published in 1994, discloses lipid
`compositions ofthe pineal organ from rainbow trout compris-
`ing phospholipids.According to Table 4 ofthis reference, said
`phospholipids contain fatty acids corresponding to eicosap-
`entanoic and docosahexanoic acid. Similarly, Bell et al. (37),
`published in 1991, discloses ph6spholipid compositions
`derived from different organs of cod. Moreover, Wiegand et
`al. (38), published in 1983, discloses polyene derivatives of
`phosphatidylcholine as phospholipid molecular species of
`frog receptor membranes. However, there is no disclosure in
`any of these references concerning any pharmaceutical,
`nutraceutical, or cosmetic use of a composition comprising a
`flavonoid.
`
`WO 97/39759, published on Oct. 30, 1997, discloses (D'3
`fatty acids and (D'3 phosphatidylcholine in the treatment of
`bipolar disorder. The preferred (,0-3 phosphatidylcholine
`derivatives comprise eicosapentanoic and/or docosahexanoic
`acid. However, there is no disclosure concerning any phar-
`maceutical, nutraceutical, or cosmetic use of phospholipids
`beyond the treatment of bipolar disorder or the use of a
`composition comprising a flavonoid.
`
`EP 0609078 A1, published on Mar. 8, 1994, discloses a
`phospolipid comprising two different unsaturated fatty acids,
`wherein a preferred phospholipid contains both eicosapen-
`tanoic and docosahexanoic acid. Furthermore, the phospho-
`lipid can be used in the preparation of foods, skin care prepa-
`rations, or pharmaceutical agent. However,
`there is no
`disclosure concerning any pharmaceutical, nutraceutical, or
`cosmetic use of a composition comprising a flavonoid.
`
`SUMMARY OF THE INVENTION
`
`In one aspect, the invention provides novel 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):
`
`
`
`000007
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`US 8,030,348 B2
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`3
`wherein X represents a moiety normally found in a phospho-
`lipid.
`According to a further aspect ofthe present invention there
`is provided a composition, comprising:
`(a) a phospholipid of the general formula (I),
`
`4
`
`There is also provided a composition comprising the above
`noted phospholipid and flavonoid derived from a marine or
`aquatic biomass. The composition and the components are
`useful in the prevention or treatment of a variety of disease
`states and for the aesthetic enhancement of an animal, includ-
`
`
`
`(I)
`
`wherein X is iCHZCHzNH3, 4CH2CH2N(CH3)3 or
`OH
`
`OH
`
`HO
`
`, and
`
`OH
`
`(b) a flavonoid of the general formula (II),
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`(11)
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`30
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`ing human, body. Commercial packages containing the com-
`position are also within the invention.
`The novel phospholipids and the novel flavonoid com-
`pound 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
`components are useful in the prevention or treatment of a
`variety of disease states and for the aesthetic enhancement of
`an animal, including human, body. Pharmaceutical, nutraceu-
`tical 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
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`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 bases such
`as choline. Important phospholipids are phosphatidylcholine
`(PC), phosphatidylethanolamine (PE) and phosphatidyli-
`nositol (PI). Their nature as amphophilic molecules provides
`them with unique physicochemical properties. Their function
`as the principle components of cell membranes makes phos-
`pholipids essential for all vital cell processes. They are wide
`spread as secretory and structural components of the body
`and can mimic or enhance natural physiological process.
`
`
`
`In a further aspect, the invention provides a novel flavonoid
`compound (II):
`
`50
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`(11)
`
`
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`55
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`60
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`65
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`000008
`
`if
`HZC—O—C—Rl
`(ll
`I
`CH3
`RZ—C—O—CH
`0
`/
`I
`II
`HZC—O—P—O—CHz—CHz—N—CHg
`
`0-
`
`CH3
`
`Phosphatidylcholine%ommon structure
`R1 and R2 are fatty acid residues, different for each
`molecular species
`
`‘1?
`HZC—O—C—Rl
`(ll
`|
`Rz—C—O—CH
`o
`
`HZC—O—P—O—CHz—CHz—NHg
`
`O'
`
`Phosphatidylethanolamineicommon structure
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`000008
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`US 8,030,348 B2
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`5
`R1 and R2 are fatty acid residues, diffecular for each
`molecular species
`
`(ll
`IhC——O——C——R
`fi
`|
`&——C——o——cH
`fi
`HZC—O—P—O
`I
`o
`
`OH
`
`OH
`
`HO
`
`|OH
`
`OH
`
`Phosphatidylinositolicommon structure
`R1 and R2 are fatty acid residues, different for each
`molecular species
`Phospholipid production may be either synthetic or
`through extraction from natural tissues. The chief source of
`commercial natural pho spholipids 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 phospholipidproducts is linoleic acid (C18:2n6), present in
`soybean at more than 65%. The longest chain polyunsatu-
`rated fatty acids found in commercially available phospho-
`lipids either as preparations or individually are 20:4 among
`the eicosanoids, known as arachidonic acid, and 22:6 known
`as docosahexanoic acid.
`Arachidonic acid is a fatty acid that is found as part of
`phospholipid membranes, generally as part of phosphatidyl-
`choline and phosphatidylinositol. Adverse cellular stimuli
`will activate enzymes (phospholipase) that cleave arachi-
`donic acid from the phospholipid backbone in the cell mem-
`brane. Arachidonic acid, which serves as the precursor for
`prostaglandins and prostacyclin (PGs, PGIZ) and thrombox-
`ane (TXs), can then be metabolized by one of two major
`pathways: the cyclooxygenase (COX) pathway or the lipoxy-
`genase pathway. The COX pathway products, PGG2 and
`PGH2, can then be acted upon by thromboxane synthase (in
`platelets) or prostacyclin synthase (in endothelium) to form
`TXs or PGlz, 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 with arachi-
`donic 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
`(PGIZ), leukotrienes (LTs), and thromboxanes (TXs). These
`substances can either act as vasodilators or as vasoconstric-
`
`tors. PGl2 is essential in vascular function since it inhibits
`platelet adhesion to the vascular endothelium and has signifi-
`cant vasodilatation qualities. Damaged endothelial cells can-
`not produce PGlz, making the vessel more susceptible to
`thrombosis and vasospasm. Thromboxanes and leukotrienes
`serve a vascular function during inflammation, generally pro-
`ducing vasoconstriction. Prostaglandins have a vascular role
`during inflammation, and also play a more subtle role in
`normal flow regulation, most notably as modulators of other
`control mechanisms. Prostaglandins have both vasoconstric-
`tor and vasodilator activities. Leukotrienes and prostaglan-
`
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`dins can also increase the endothelial membrane permeability
`thus promoting edema during inflammation. Arachidonic
`acid is naturally present in most phospholipid mixtures or
`emulsions available today.
`Nervonic acid (C24:l) is also called selacholeic acid or
`tertracosenic acid. Nervonic acid is the predominant nutrient
`of white matter in glucoside, which is quantitatively c011-
`tained in nerve tissue and white matter. The absence of ner-
`
`vonic acid may result in cerebral lesion, fatigue, hypody-
`namia, amentia, and senile dementia. Nervonic acid,
`tertracosenic acid in another name, is monounsaturated, non-
`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. There-
`fore, the substantance is far below the demand ofhumanbody.
`ln foreign countries, nervonic acid mainly comes from shark
`brain and oil.
`
`l.l Phosphatidylinositol Clinical Applications
`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 vari-
`ous specific proteins (1).
`lnositol has been identified as an important dietary and
`cellular constituent. Biochemical functions:
`
`a. Regulation of cellular responses to external stimuli
`b. mediation of enzyme activity.
`Phosphoinositide composition of the central nervous sys-
`tem cell membranes are fatty-acid enriched and consist pri-
`marily of phosphatidylinositol (Pl), phosphatidylinositol-4-
`phosphate (PIP), and phosphatidylinositol-4,5-biphosphate
`(PlP2). 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 phos-
`phatidylinositol-3 -phosphate and 3,4,5-triphosphate (2).
`Active transport carriers, calcium pumps in the cell mem-
`brane itself, and in the endoplasmic reticulum, keep cytoplas-
`mic calcium concentration very low. Usually the calcium
`concentration inside the cytoplasm is 5,000-10,000 times less
`than the concentration in the extracellular fluid. This endo-
`
`plasmic store of calcium can be accessed upon stimulation by
`inositol. lnositol triphosphate is released from the cell mem-
`brane and travels through the cytoplasm until it reaches the
`endoplasmic reticulum. This inositol
`then releases the
`sequestered calcium, which can go on to mediate 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 trans-
`porter, a sodium dependent active transport system, and a
`passive low aflinity transporter (4,5).
`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-l,4,5-triphos-
`phate as second messengers. These receptors can be found in
`nearly every human organ system. The potential 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 bal-
`ance is required for regulation of the signaling systems.
`Theoretically, an imbalance ofinositol concentration could
`potentially affect the development and function of one or all
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`of these receptors. Cholinergic receptors are located in the
`liver, heart, stomach, and lungs. Serotonin and glutamine
`receptors are found mostly in the central nervous system
`(CNS) tissues. Adrenergic receptors are present in various
`tissues including CNS, vascular tissues, and heart. Histamin-
`ergic receptors are predominantly found in the lungs and
`stomach.
`
`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:
`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 ofnorepinephrine; 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 1a, lb, and Id).
`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 ofhigh-
`dose inositol could increase CSF levels by as much as 70
`percent (7). This led to the study of inositol for treatment of
`depression (8,9). In 1995 Levine et a1 completed a double-
`blind study for treatment ofdepressionusing 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 0.6+/—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 be concluded that inositol is effective in managing the
`clinical manifestations of depression.
`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 ofpanic attacks
`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 inosi-
`tol 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 messen-
`ger is known to affect several neurotransmitters, 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
`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 feature found in
`the CNS of AD patients during autopsy. It is known that
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`aluminum inhibits the incorporation of inositol into phospho-
`lipids 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 phos-
`phatidylinositol 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, supplemen-
`tation 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
`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 pla-
`cebo for 30 days in 11 Alzheimer’s patients. Patients in the
`study were diagnosed with dementia of the AD type as clas-
`sified by DSM7IIIR and aged 65 years or older. The Cam-
`bridge Mental Disorder of the Elderly Examination (CAM-
`DBX) 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: Cognitive.Subscale of CAMDEX (CAMCOG),
`part C: interviewers observations, and part D: physical exami-
`nation. 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).
`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 (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
`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
`CAMCOG score from a baseline of 31.36+/—20.90 to
`
`40.09+/—24.54, while the placebo group increased from base-
`line 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.
`Inositol’s proposed mechanism of action in the CNS does
`not include direct manipulation with either pre- or post-re-
`ceptors. However, it may indirectly affect the relationship
`between receptor and agonist. By mediating the physio-
`chemical characteristics of the M1 pre-synaptic receptor
`(solubility, 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, proliferation and
`regeneration. The physiologic functions of these phospholip-
`ids are related to the morphology of the biological mem-
`branes, the incorporation of these molecules into membranes
`and thus the maintenance of intact cell membranes.
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`9
`The current study was designed to investigate the effects of
`Polyunsaturated phosphatidylcholine on age-related hearing
`loss by evaluating its ability to preserve mitochondrial func-
`tion, protect mitochondrial DNA from oxidative damage and
`preserve auditory sensitivity (22).
`Harlan-Fischer 344 rats, 18-20 months ofage, were used as
`the experimental subjects.
`The subjects were caged individually and maintained at 21
`to 22° C. in a 12:12 light-dark cycle b.
`A dose of 300 mg/kg/day of Polyunsaturated phosphati-
`dylcholine was supplemented to each subject, by adding it to
`the oral diet.
`
`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 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 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 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-associ-
`ated decline in hearing sensitivities at all the frequencies
`tested (3, 6, 9, 12 and 18 kHz).
`There was a statistically significant preservation ofhearing
`noted in the treated subjects at all frequencies, which was
`observed at four and six months of treatment.
`
`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<0.005).
`Mitochondrial membrane potentials were recorded by 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.
`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.
`This difference between the control and treated groups was
`statistically significant (p<0.05), demonstrating the protec-
`tive effect of polyunsaturated phosphatidylcholine supple-
`mentation on mitochondrial membrane potential.
`Phospholipids are integral structural components of all
`biological membranes with polyunsaturated phosphatidyl-
`choline and phosphatidylethanolamine being the predomi-
`nant types, quantitatively. They constitute the phospholipid
`bilayer structure of cellular membranes, which is responsible
`for membrane stability and cellular function. Polyunsaturated
`phosphatidylcholine maintains and promotes the activity of
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`several membrane bound proteins and enzymes, including
`Na-K ATPase, adenylate cyclase and glutathione reductase.
`They are also known to be precursors ofcytoprotective agents
`such as eicosanoids, prostaglandins and antioxidants.
`The results of these studies suggest that polyunsaturated
`phosphatidylcholine and phosphatidylethanolamine may
`protect mitochondrial function by preserving the age-related
`decline in mitochondrial membrane potentials and hence
`their activity. The observation that there was less mitochon-
`drial DNA damage in the treated group may explain the effect
`of preservation of hearing loss associated with aging, by the
`ability of polyunsaturated phosphatidylcholine and phos-
`phatidylethanolamine to specifically up-regulate cochlear
`mitochondrial function. There are many studies demonstrat-
`ing the effects of mitochondrial metabolites on cognition and
`aging (22-33). Additionally, recent work has shown that
`acetyl-L-carnitine and -lipoic acid 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 explanation for age-related
`hearing loss. Thus,PPC may be one of many rational
`approaches to consider for the purpose of membrane preser-
`vation,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 Nor-
`mal and Cancerous Cells
`
`Reduced levels of phospholipids in normal cells can limit
`metabolic activity and limit available energy. Phospholipids,
`as part of the membrane structure:
`i. maintain membrane integrity
`ii. regulate enzyme activities and membrane transport pro-
`cesses through changes in membrane fluidity (Spector
`1 981 , 1 985)
`iii. Signal transduction utilizes phospatidylcholine and phos-
`phatidylinositol
`for the production of diacyl-glycerol
`(DAG) by phospholipase C (Berridge 1989) and for the
`production of inositol triphosphate (1P3) (Ranan 1990,
`Michell 1988, Margolis 1990).
`iv. One of the choline phospholipids (1-alkyl-2 acetyl-SN-
`glycerol-3 -phosphocholine) is the substrate for the synthe-
`sis 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 produc-
`tion of prostaglandin and leukotriene (Nordoy 1990).
`vi. The choline of phosphatidylcholine may be used in neural
`tissue for the synthesis of acetylcholine (Blusztain 1987)
`vii. Phosphoglycolipid improves cell maintenance and meta-
`bolic activity of normal cells.
`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
`1 984).
`a. Such compounds inhibit HL60 leukemic cells at a dos-
`age 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).
`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
`1 993).
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`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 dra-
`matically different from those of less unsaturated phospho-
`lipid bilayers. MD simulations can aid in interpreting the
`complex NMR spectra of polyunsaturated bilayers, in con-
`junction with electron density profiles determined from
`small-angle X-ray diffraction studies. This work clearly dem-
`onstrates preferred helical and angle-iron conformations of
`the polyunsaturated chains in liquid-crystalline bilayers,
`which favor chain extension while maintaining bilayer flex-
`ibility. The presence of relatively long, extended fatty acyl
`chains may be important for solvating the hydrophobic sur-
`faces of integral membrane proteins, such as rhodopsin. In
`addition, the polyallylic DHA chains have a tendency to adopt
`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 con-
`taining 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 distin-
`guish DHA-containing lipids from saturated or nonunsat-
`urated lipids and may be important for their biological modes
`of action.
`
`1.5 In Summary
`The functions of the phospholipids are multiple and differ-
`ent for each phospholipid:
`a. Sphingosine and carbohydrate containing lipids are mainly
`concentrated in nervous tissues.
`
`b. The hydrophilic and hydrophobic parts ofthe phospholipid
`molecule allow them to function as emulsifying agents in
`order to maintain the proper col