(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`13 February 2003 (13.02.2003)
`
`PCT
`
`(10) International Publication Number
`WO 03/011873 A2
`
`(51) International Patent Classification7:
`
`C07F 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:
`
`English
`
`English
`
`(30) Priority Data:
`60/307,842
`
`27 July 2001 (27.07.2001) US
`
`(71) Applicant (for all designated States except US): NEP(cid:173)
`TUNE TECHNOLOGIES & BIORESSOURCES INC.
`[CA/CA]; 500, St-Martin Boulevard West, Suite 550,
`Laval, Quebec H7M 3Y2 (CA).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): SAMPALIS, Fotini
`[CA/CA]; 1348 Elizabeth Boulvard, Laval, Quebec H7W
`3J8 (CA).
`
`iiiiiiii
`
`(74) Agents: SABET, Sohrab et al.; Smart & Biggar, 1000 de
`la Gauchetiere Ouest, Suite 3400, Montreal, 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), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`[Continued on next page]
`
`(54) Title: NATURAL MARINE SOURCE PHOSPHOLIPIDS COMPRISING FLAVONOIDS, POLYUNSATURATED FATTY
`ACIDS AND THEIR APPLICATIONS
`
`-iiiiiiii
`= !!!!!!!!
`------
`== -iiiiiiii
`iiiiiiii ----
`
`8.00
`
`6.00
`
`Cl)
`!:::;
`c::J
`~ 4.00
`b
`
`><
`
`2.00
`
`~
`l"
`QO
`,....i
`,....i
`Q
`..........
`~
`2.00
`Q
`x 101 MINUTES
`0
`> (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.
`
`0.00
`
`1.00
`
`3.00
`
`4.00
`
`RIMFROST EXHIBIT 1013 page 0001
`
`

`

`WO 03IO118 7 3 A2
`
`I lllllllllllll II llllll lllllllll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`RIMFROST EXHIBIT 1013 page 0002
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 1
`
`-
`
`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
`
`5 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 page 0003
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 2 -
`
`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(cid:173)
`
`soluble and particulate fraction is further solvent extracted
`
`5 with ethanol or ethylacetate to achieve further lipid
`
`extractions.
`
`Summary of the Invention
`
`In one aspect, the invention provides novel
`
`phospholipids, wherein the two fatty acids chains of the
`
`10
`
`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):
`
`15
`
`0
`II
`H2C-O-C
`
`~
`
`I
`C-0-CH
`
`0
`II
`H C-0-P-O-X
`I
`2
`o-
`
`(I)
`
`wherein X represents a moiety normally found in a phospholipid.
`
`In a further aspect, the invention provides a novel
`
`20
`
`flavonoid compound (II) :
`
`HO
`
`OH
`
`OH
`
`OH
`
`(II)
`
`25
`
`OH OH
`
`RIMFROST EXHIBIT 1013 page 0004
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 3 -
`
`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
`
`5
`
`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,
`
`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
`
`phosphatidylinositol (PI) . Their nature as amphophilic
`
`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 page 0005
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 4 -
`
`5
`
`10
`
`15
`
`20
`
`Phosphatidylcholine- common structure
`R1 and R2 are fatty acid residues,
`different for each molecular species
`
`~
`
`0
`II
`H2C-O-C-R1
`I
`R2-C-O-CH
`I
`W
`H2C-o-1-o-CH2-CH2-NH3
`o-
`
`Phosphatidylethanolamine- common structure
`
`R1 and R2 are fatty acid residues,
`different for each molecular species
`
`~
`
`0
`II
`H2C--O-C--R1
`I
`R2-C-O-CH
`W
`I
`H2C--O-P--O
`b-
`
`OH
`
`Phosphatidylinositol- common structure
`R 1 and R2 are fatty acid residues,
`different for each molecular species
`
`RIMFROST EXHIBIT 1013 page 0006
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 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
`
`5
`
`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 (Cl8:2n6), 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, PGI 2 ) 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, PGG2 and PGH2 , can then be acted upon
`by thromboxane synthase (in platelets) or prostacyclin synthase
`
`25
`
`(in endothelium) to form TXs or PGI 2 , 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 (PGI 2 ),
`RIMFROST EXHIBIT 1013 page 0007
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 6
`
`leukotrienes (LTs), and thromboxanes (TXs) . These substances
`
`can either act as vasodilators or as vasoconstrictors.
`
`PGI 2 is
`essential in vascular function since it inhibits platelet
`
`adhesion to the vascular endothelium and has significant
`
`5 vasodilatation qualities. Damaged endothelial cells cannot
`
`produce PGI 2 , making the vessel more susceptible to thrombosis
`and vasospasrn. 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 page 0008
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`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
`
`5
`
`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
`
`system cell membranes are fatty-acid enriched and consist
`
`15 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
`
`20 phosphatidylinositol-3-phosphate and 3,4,5-triphosphate (2).
`
`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
`
`RIMFROST EXHIBIT 1013 page 0009
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 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
`
`5
`
`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
`
`15
`
`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 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.
`
`RIMFROST EXHIBIT 1013 page 0010
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`Clinical Applications
`
`- 9 -
`
`A change in CNS availability of inositol may produce
`
`altered brain signaling and eventually lead to the development
`
`of neurological disorders.
`
`5
`
`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 of norepinephrine; in the
`
`10
`
`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, lb,
`
`and ld) .
`
`In 1978, Barkai et al demonstrated depressed patients
`
`15
`
`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(cid:173)
`
`dose inositol could increase CSF levels by as much as 70
`
`percent (7) . This led to the study of inositol for treatment
`
`20
`
`of depression (8,9).
`
`In 1995 Levine et al completed a double(cid:173)
`
`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
`25 +/- 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.
`
`RIMFROST EXHIBIT 1013 page 0011
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`b. Panic Disorder:
`
`- 10 -
`
`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
`
`5 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 plagues 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
`
`RIMFROST EXHIBIT 1013 page 0012
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 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
`
`5 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
`
`RIMFROST EXHIBIT 1013 page 0013
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 12 -
`
`(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
`
`5
`
`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
`
`15
`
`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 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(cid:173)
`
`receptors. However, it may indirectly affect the relationship
`
`between receptor and agonist. By mediating the physiochemical
`
`characteristics of the Ml 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
`
`RIMFROST EXHIBIT 1013 page 0014
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 13 -
`
`biological membranes, the incorporation of these molecules into
`
`membranes and thus the maintenance of intact cell membranes.
`
`The current study was designed to investigate the
`
`effects of Polyunsaturated phosphatidylcholine on age-related
`
`5
`
`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° Cina 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
`RIMFROST EXHIBIT 1013 page 0015
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 14 -
`
`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
`
`5 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.
`
`RIMFROST EXHIBIT 1013 page 0016
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 15 -
`
`This difference between the control and treated
`
`groups was statistically significant (p<0.05), demonstrating
`
`the protective effect of polyunsaturated phosphatidylcholine
`
`supplementation on mitochondrial membrane potential.
`
`5
`
`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
`
`10
`
`responsible for membrane stability and cellular function.
`
`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
`
`RIMFROST EXHIBIT 1013 page 0017
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 16 -
`
`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
`
`5
`
`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
`
`ii.
`
`regulate enzyme activities and membrane transport
`
`processes through changes in membrane fluidity (Spector 1981,
`
`1985)
`
`15
`
`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).
`
`20
`
`iv. One of the choline phospholipids (l-alkyl-2 acetyl-SN(cid:173)
`
`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
`
`25 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 metabolic
`
`activity of normal cells.
`
`RIMFROST EXHIBIT 1013 page 0018
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 17 -
`
`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).
`
`5
`
`a. Such compounds inhibit HL60 leukemic cells at 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
`
`10
`
`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 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
`
`20
`
`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
`
`25
`
`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
`
`30
`
`surfaces of integral membrane proteins, such as rhodopsin. In
`
`RIMFROST EXHIBIT 1013 page 0019
`
`

`

`WO 03/011873
`
`PCT/CA02/01185
`
`- 18 -
`
`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
`
`5 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
`
`10 distinguish DHA-containing lipids from saturated or
`
`nonunsaturated lipids and may be important for their biological
`
`modes of action.
`
`1.5
`
`In Summary
`
`The functions of the phospholipids are multiple and
`
`15 different for each phospholipid:
`
`a. Sphingosine and carbohydrate containing lipids are mainly
`
`concentrated in nervous tissues.
`
`b. The hydrophilic and hydrophobic parts of the phospholipid
`
`molecule allow them to function as emulsifying agents in order
`
`20
`
`to maintain the proper colloidal state of protoplasm.
`
`c. Phospholipids aid the transport of triglycerides through
`
`the liver, especially during mobilization from adipose