(12) United States Patent
`Sampalis
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,030,348 B2
`Oct. 4, 2011
`
`1JS008030348B2
`
`(54) NATURAL MARINE SOURCE
`PHOSPHOLIPIDS COMPRISING
`POLYUNSATURATED FATTY ACIDS AND
`THEIR APPLICATIONS
`
`(75)
`
`Inventor: Fotini Sampalis, Laval (CA)
`
`(73) Assignee: Neptune Technologies &
`Bioressources, Inc., Laval (CA)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. l54(b) by 871 days.
`
`(21) Appl. No.:
`
`10/485,094
`
`(22) PCT Filed:
`
`Jul. 29, 2002
`
`PCT No.:
`
`(86)
`
`PCT/CA02/01185
`
`§ 371 (C)(1),
`(2), (4) Date:
`
`Jul. 13, 2004
`
`(87) PCT Pub. No.: WO03/011873
`
`PCT Pub. Date: Feb. 13, 2003
`
`(65)
`
`Prior Publication Data
`
`US 2004/0234587 A1
`
`Nov. 25, 2004
`
`Related U.S. Application Data
`
`Provisional application No. 60/307,842, filed on Jul.
`27, 2001.
`
`Int. Cl.
`
`(2006.01)
`A61K 31/215
`(2006.01)
`A61K 31/661
`U.S. Cl.
`........................................ .. 514/506; 514/75
`Field of Classification Search ...................... .. None
`
`See application file for complete search history.
`
`References Cited
`
`(60)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`U.S. PATENT DOCUMENTS
`2003/0083238 A1*
`5/2003 Toda et al.
`...................... .. 514/9
`
`EP
`W0
`W0
`W0
`
`FOREIGN PATENT DOCUMENTS
`0 609 078 A1
`8/1994
`W0 92 21335 A
`12/1992
`W0 97 39759 A
`10/1997
`W0 00 23546 A
`4/2000
`
`OTHER PUBLICATIONS
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`2-(3,4-
`“4H-1-Benzopyran-4-one,
`ACS Chemical Registry,
`dihydroxyphenyl)-6,8-di-[3-D-glucopyranosyl-5,7-dihydroxy-,
`”,
`Nov. 16, 1984, 1 page.*
`Aureli , M. et al. Aging brain: effect ofacetyl L-carnitine treatment on
`rat brain energy and pho spholipid metabolism. A study by 3 1P and 1H
`NMR spectroscopy. Brain Research. 1990; 526(1): 108-112.
`Barak, Y. et al. Inositol treatment of Alzheimer’s disease : a double
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`cross-over
`placebo
`controlled
`trial.
`Prog.
`Neuropsychopharmacol. Biol. Psychiatry 1996; 20: 729-735.
`Bast, A. et al. Interlay between lipoic acid and glutathione in the
`protection against microsomal lipid peroxidation. Biochem. Biophys.
`Acta. 1988; 963: 558-561.
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`000001
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`Bell, M.V. et al. Molecular Species Composition ofthe Maj or Diacyl
`Glyceropho spholipids from Muscle, Liver, Retina and Brain of Cod
`(Gadus morhua). Lipids 1991: 26(8): 563-73.
`Benjamin, J. et al. Double-blind, placebo-controlled, crossover trial
`of inositol treatment for panic disorders. Am. .1. Psychiatry 1995; 15:
`1084-1086.
`Birchall, J. et al. Aluminum, chemical physiology, and Alzheimer’s
`disease. Lancet 1988; 29:1008-1010.
`Caprioli, A. et al. Age-dependent deficits in radial maze performance
`in the rats: effect of chronic treatment with acetyl L-carnitine. Prog.
`In Neuropsychopharmacol. Biol. Psychiatr. 1990; 14(3): 3 59-369.
`Cheng, D. et al. A novel promising acetylcholinesterase inhibitor.
`Neuroreport 1996; 8: 97-101.
`Devasagayam, T.P. et al., Prevention of Singlet Oxygen-Induced
`DNA Damage by Lipoate. Chemical Biological Interactions 1993;
`86 : 79-92.
`Ghirardi, O. et al. Effects of acetyl L-carnitine chronic treatment on
`discrimination models in aged rats. Pysiol. Behav. 1988; 44(6): 769-
`773.
`Gill, D.L. et al. Calcium signalling mechanisms in endoplasmic
`reticulum activated by inositol 1, 4, 5 triphosphate and GTP. Cell
`Calcium 1989; 10: 363-374.
`Henderson, R.J. et al. Lipid Composition fo the Pineal Organ from
`Rainbow Trout (Oncorhynchus mykiss). Lipids 1994; 29(5): 311-17.
`Hosokawa, M. et al., Conversion to Docosahexaenoic Acid-Contain-
`ing Phosphatidylserine from Squid Skin Lecithin by Phospholipase
`D-Mediated Transphosphataidylation. Journal ofAgricultural and
`Food Chemistry 20000; 48(10): 4550-54.
`Imperato. A. et al. L-carnitine enhances acetylcholine release in the
`striatum and hippocampus of
`awake
`freely moving rats.
`Neuroscience Letters. 1989: 107(1-3): 251-255.
`Kagan, V.E. et al. Dihydrolipoic acid: A universal antioxidant bot11 in
`the membrane and in the aqueous phase. Biochem. Pharmacol. 1992;
`44: 1637-1649.
`Kawakami, Y. et al. The rationale for E2020 as a potent
`acetylcholinesterase inhibitor. Bioorg. Med Chem. 1996; 4: 1429-
`1446.
`Kitamura, H. et al. Inhibition of myo-inositol transport causes acute
`renal failure wit11 selective medullary injury in the rat. Kidney Int.
`1998; 53: 146-153.
`Knopman, D. et al. Long term tacrine (Cognex) treatment: effects on
`nursing home placement and mortality, tacrine study group. Neurol-
`ogy 1996; 47: 166-167.
`Levine, J. et al. Double-blind, controlled trial of inositol treatment of
`depression. Am. .1. Psychiatry 1995; 152: 792-794 (Abstract Only).
`Levine, J. et al. Inositol treatment raises CSF inositol levels. Brain
`Research 1993; 627 : 168-169.
`inositol
`Levine,
`J. Controlled trials of
`Neuropsychopharmacol. 1997; 7: 147-155.
`Colodny, L. et al. Altern. Med Rev. 1998; 3(6): 432-447.
`Mohr, E. et al. Treatment of Alzheimer’s disease wit11 sabeluzole :
`functional and structural correlates. Clin. Neuropharmacol. 1997;
`20: 338-345 (Abstract Only).
`Paradies, G. et al. Carnitine-acylcarnitine translocase activity in car-
`diac mitochondria from aged rats: the effect of acetyl-L-carnitine.
`Mech. ofAging andDevelop. 1995; 84(2): 103-112.
`Parthasarathy, L. et al. Biochemical and molecular properties of
`lithium-scnsitivc myo-inositol monophosphatasc. Life Sci. 1994; 54:
`1 127-1142.
`
`in psychiatry. Eur.
`
`(Continued)
`
`Primary Examiner — James Anderson
`Assistant Examiner — Gregg Polansky
`
`(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 fiavonoid.
`
`21 Claims, 4 Drawing Sheets
`
`Petition for Inter Partes Review
`Of US. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1069
`
`000001
`
`

`
`US 8,030,348 B2
`Page 2
`
`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: 46S-50S.
`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
`
`000002
`
`000002
`
`

`
`U.S. Patent
`
`Oct. 4, 2011
`
`Sheet 1 of4
`
`US 8,030,348 B2
`
`8.00
`
`6.00
`
`2.00
`
`.
`.
`.
`.
`.
`.
`.
`,
`.
`.
`_.,,,_,__,,,,__,_‘_,_,- _,_......................................... ....,!.....................-
`
`0.00
`1.00
`2.00
`3.00
`4.00
`
`x 101 MINUTES
`
`Fig. 1
`
`000003
`
`000003
`
`

`
`U.S. Patent
`
`Oct. 4, 2011
`
`Sheet 2 of4
`
`US 8,030,348 B2
`
`Fig.2
`
`0
`
`000004
`
`000004
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`

`
`U.S. Patent
`
`Oct. 4, 2011
`
`Sheet 3 of4
`
`US 8,030,348 B2
`
`Fig.3
`
`% 4
`
`000005
`
`000005
`
`

`
`U.S. Patent
`
`Oct. 4, 2011
`
`Sheet 4 of4
`
`US 8,030,348 B2
`
`asaa¢amm=9”emuac“am_ca.am.=._.
`.1...“3....__:.__.::._3.“_///r//\.293amgm:5mmmmflan.E/3/ammm“on
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`
`.3
`
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`=52H32.22
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`000006
`
`
`
`

`
`US 8,030,348 B2
`
`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 U.S. 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 U.S. 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. U.S. 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-
`umn 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 fro1n marine and aquatic
`animal material by acetone extractions. The resulting non-
`soluble and particulate fraction is further solvent extracted
`witl1 ethanol or ethylacetate to achieve further lipid extrac-
`tions.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`2
`
`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 u)-3
`fatty acids and 00-3 phosphatidylcholinc in thc trcatmcnt of
`bipolar disorder. The preferred u)-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 sa1ne molecule, i.e.: a
`phospholipid of the general formula (I):
`
`
`
`000007
`
`000007
`
`

`
`US 8,030,348 B2
`
`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-
`
`
`
`(1)
`
`wherein X is —CH2CH2NH3, —CH2CH2N(CH3)3 or
`OH
`
`OH
`
`HO
`
`, and
`
`OH
`
`(b) a flavonoid of the general formula (II),
`
`20
`
`25
`
`(11)
`
`30
`
`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
`
`35
`
`40
`
`45
`
`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 arnphophilic 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
`
`(11)
`
`
`
`55
`
`60
`
`65
`
`000008
`
`if
`H2C—O—C—R1
`(lf
`I
`CH3
`R2—C—O—CH
`0
`/
`I
`ll
`H2C—O—P—O—CH2—CH2—N—CH3
`
`0-
`
`CH3
`
`Phosphatidylcholine—common structure
`R1 and R2 are fatty acid residues, different for each
`molecular species
`
`if
`H2C—O—C—R1
`cfi
`I
`R2—C—O—CH
`o
`
`H2C—O—P—O—CH2—CH2—NH3
`
`O.
`
`Phosphatidylethanolamine—common structure
`
`000008
`
`

`
`US 8,030,348 B2
`
`5
`R1 and R2 are fatty acid residues, diffecular for each
`molecular species
`
`ll
`IfiC——O——C——R
`fi
`I
`m——C——O——cH
`fi
`H2C—O—P—O
`I
`O
`
`OH
`
`OH
`
`HO
`
`IOH
`
`OH
`
`Phosphatidylinositol—common 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 (Cl 8:2n6), 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.
`Arachidomc 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, PGI2) 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 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 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
`(PGI2), leukotrienes (LTs), and thromboxanes (TXs). These
`substances can either act as vasodilators or as vasoconstric-
`
`tors. PGI2 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 PGI2, 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-
`
`000009
`
`6
`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 con-
`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.
`In foreign countries, nervonic acid mainly comes from shark
`brain and oil.
`
`1 .1 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).
`Inositol 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 (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 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. Inositol 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 sodiun1/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 glutarnine
`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 indolarnine 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, 1b, 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 al 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
`rar1ked 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 ann. 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 DSM—IIIR and aged 65 years or older. The Cam-
`bridge Mental Disorder of the Elderly Examination (CAM-
`DEX) 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 oxazeparn 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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`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 pr