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`256
`
`lnternat. .T. Vit. Nutr. Res. 62
`(1992) 256-260
`Received for publication
`November 12, 1991
`
`S. NozAKI et al: Effects of Purified EPA on Plasma Lipoproteins 1
`
`I
`
`Eicosapentaenoic acid
`Hypercholesterolemia
`Cholesteryl ester transfer protein
`Low density lipoprotein
`High density lipoprotein
`
`Effects of Purified Eicosapentaenoic Acid Ethyl Ester on Plasma
`Lipoproteins in Primary Hypertholesterolemia
`
`Shuichi NozAKI, Yuji MATSUZAWA, Ken-ichi HIRANO, Naohiko SAKAI, Masaharu KuBo,
`Seiichiro TARUI
`
`The Second Department of Internal Medicine, Osaka University Medical School, Fukushima, Fuskushima-ku,
`Osaka 553, Japan
`
`Summary: We investigated the effects of puri(cid:173)
`fied eicosapentaenoic acid (EPA) ethyl ester
`capsules (90% purity), which are free from
`cholesterol, saturated fatty acids and docosa(cid:173)
`hexaenoic acid (DHA), on plasma lipoproteins
`and cholesteryl ester transfer protein (CETP)
`activity. We administered 2.7 g of EPA per day
`as capsules for 6 months to 14 primary hyper(cid:173)
`cholesterolemic subjects. Total cholesterol,
`triglyceride and low density lipoprotein (LDL)(cid:173)
`cholesterol levels in plasma were significantly
`reduced. The LDL cholesterol!apoB ratio and
`LDL particle size did not change. The ratio of
`high density lipoprotein (HDL)2/HDL3 chol(cid:173)
`esterol increased from 1.04 to 1.35 (p<0.05),
`while the HDL cholesterol level did not change.
`CETP activity was significantly reduced. The
`reduction of CETP activity may explain the in(cid:173)
`crease in the HDL2/HDL3 cholesterol ratio.
`These results suggest that purified EPA not
`only reduces LDL cholesterol levels but also
`acts on HDL metabolism in patients with
`hypercholesterolemia and therefore will be use(cid:173)
`! ul for the treatment of hypercholesterolemia.
`
`Abbreviations used: Cholesteryl ester transfer protein,
`CETP; docosahexaenoic acid, DHA; eicosapentaenoic
`acid, EPA; low density lipoprotein, LDL; high density
`lipoprotein, HDL; hepatic triglyceride lipase, HTGL; very
`low density lipoprotein, VLDL.
`
`Introduction
`
`Since it has been suggested [1] that high content
`of eicosapentaenoic acid (EPA) in diet is linked
`to low incidence of coronary heart disease in
`Greenland Eskimos, many studies reported that
`EPA has a variety of beneficial effects protect(cid:173)
`ing against atherosclerotic disease [2-8]. The
`effect of EPA on plasma lipoproteins has been
`studied [9] since lipoprotein abnormalities are
`an important factor in the development of
`atherosclerosis.
`In previous studies, fish oil has been used
`since purified EPA has not been available.
`These studies have confirmed the reduction of
`low density
`triglyceride and very
`serum
`lipoprotein (VLDL) concentrations by fish oil
`(9, 10]. There is still controversy concerning the
`effects of fish oil on low density lipoprotein
`(LDL) and high density lipoprotein (HDL)
`cholesterol levels [9]. Fish oil conventionally
`used contains at most 25% EPA in addition to
`various amount of other fatty acids including
`docosahexaenoic acid (DHA) and cholesterol
`[2].
`The insufficient purity of fish oil may be one
`of the reasons for inconsistent data about the
`effects of fish oil on LDL and HDL cholesterol
`levels. Furthermore, it is suggested that EPA
`and DHA have different properties against
`lipoprotein metabolism [11]. In our previous
`study, we reported the effect of a capsule con(cid:173)
`taining 80% pure EPA on platelet and plasma
`
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`s. NozAKI et al: Effects of Purified EPA on Plasma Lipoproteins
`
`257
`
`> lipids, showing a substantial reduction in total
`plasma cholesterol level [12]. However even this
`capsule still contained much DHA and we
`could not differentiate the effects of EPA and
`DHA.
`This report describes the effect of purified
`EPA ethylester, recently developed in Japan, on
`concentrations and composition of plasma
`Jipoproteins in primary hypercholesterolemia.
`
`Materials and Methods
`
`Subjects: Fourteen patients (11 females and 3 males) with
`primary hypercholesterolemia were studied as outpatients.
`The patients were selected from the Lipid Clinic of Osaka
`University Medical School. Familial hypercholesterolemia
`, was excluded. We further excluded patients with diabetes
`mellitus, renal disease, and liver disease. Their ages ranged
`from 36 to 65 years old (mean±SD, 55 ± 8.8 years). Their
`body mass indexes averaged 22.9 ± 2.2 kg/m2
`• Patients did
`not take any medicine affecting lipid metabolism for at least
`2 months before the start of this study. Eight of the patients
`had type Ila hyperlipidemia and 6 of them had type Ilb.
`Their mean total cholesterol levels and triglyceride levels
`were 277±30 and 165±25 mg/di respectively. The mean
`HDL cholesterol level was 52± 13 mg/di.
`
`Study protocol: The subjects were instructed to take 20%
`fat diet on the entry of this study. It was confirmed that
`their serum lipids levels were plateau at least for 2 months
`before the onset of treatment with EPA ethyl ester. They
`were also instructed to maintain this diet throughout the
`stndy. The subjects took 2.7 g of EPA ethyl ester daily for
`6 months. The encapsulated EPA ethyl ester was provided
`by Mochida Pharmaceutical Company. The EPA used in
`this study was purified from sardine oil. Each capsule con(cid:173)
`tained over 90% EPA ethyl ester. The content of DHA
`(C22:6) was less than 1 % and no cholesterol was present.
`This EPA capsule contains 0.2% vitamin E to inhibit
`, peruxidizatiun in capsule and in vivo. Venous blood sam(cid:173)
`ples were obtained before and during the study period. All
`' patients gave informed consent to participate in the investi(cid:173)
`gation.
`
`Lipoprotein analyses: Venous blood was drawn in a glass
`tube with EDTA 3.5 mM (final concentration) from patients
`after overnight fasting. Total cholesterol and triglycerides
`were measured by an enzymatic method (Kyowa Medex Co.,
`1 Tokyo, Japan). HDL cholesterol was measured by the hepa(cid:173)
`rin manganese precipitation method [13]. Total protein was
`measured in the LD L fraction [14]; it was assumed to consist
`entirely of apoBIOO. Very low density lipoprotein (VLDL,
`d < 1.006 g/ml), intermediate density lipoprotein (IDL,
`1.006<d<1.019 g/ml), low density lipoprotein (LDL,
`1.019< d< 1.063 g/ml), high density lipoprotein 2 (HDL2,
`1.063,d < 1.125 g/ml) and high density lipoprotein 3 (HDL3,
`
`1.125 < d g/ml) were separated by sequential preparative
`ultracentrifugation according to the method of Havel [15]
`as we previously reported [16]. LDL size was estimated
`using 2-16% polyacrylamide gradient gel electrophoresis
`according to the method of KRAUSS [17]. Size was deter(cid:173)
`mined by scanning each lane with a densitometer and com(cid:173)
`paring this value to a standardized curve of compounds of
`known size as we previously reported [18]. Cholesteryl ester
`transfer protein (CETP) activity was measured by ALBERS'
`method [19] as we previously reported [20]. Briefly, the
`activity in the patient's plasma is assayed by examining the
`lransfcr of carbon-14 cholesteryl ester from HDL3 to
`VLDL and LDL. The CETP activity was expressed as the
`percentage of donor cholesteryl ester transferred to LDL.
`
`Fatty acids analyses: Total plasma fatty acids were extracted
`with chloroform methanol solution (2/1,v/v). The fatty
`acids were analyzed as their methyl esters by gas chromato(cid:173)
`graphy using Yokogawa Hewlett Packard HP-5731 before
`and 6 months after EPA administration. Plasma fatty acids
`composition was calculated as percentage of total mass.
`
`Plasma peroxide analysis: Plasma peroxide levels were
`measured before and after 6 months of EPA treatment by
`using Determiner J ,PO kit (Kyowa Medex Co., Tokyo,
`Japan), which depends on the method of 0HISHI and YAGI
`et al [21].
`
`Statistical analysis: The significance of differences between
`mean values was determined by paired t-test.
`
`Results
`
`EPA concentrations of serum fatty acids are in(cid:173)
`creased in all subjects (data non shown) and the
`mean plasma EPA concentration of 14 subjects
`was significantly increased from 2.8% to 9.7%
`after EPA administration as shown in Table I.
`The change of plasma
`levels of total
`cholesterol, triglyceride and HDL cholesterol
`before and after EPA administration are shown
`in Table IL Total cholesterol levels were signifi(cid:173)
`cantly reduced by 10%. The triglyceride levels
`were also significantly decreased by 16%. The
`HDL cholesterol levels did not significantly
`change.
`Lipoprotein fractionation was done before
`and after EPA administration (Tab. III). Mean
`VLDL triglyceride levels decreased by 18%. The
`levels of IDL cholesterol and triglyceride were
`not changed. A significant reduction in LDL
`cholesterol was noted (p < 0.05). The LDL
`apolipoprotein (apo) BlOO levels were signifi(cid:173)
`cantly reduced (p < 0,05). The LDL cholesterol
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`258
`
`S. NozAKI et al: Effects of Purified EPA on Plasma Li po proteins
`
`Table I: Changes of serum fatty acids compositions after
`EPA administration
`
`Before
`
`After EPA
`
`c 14:0
`c 16:0
`C 16:1 n-7
`c 18:0
`C 18:1 n-9
`C 18:2 n-6
`C 18:3 n-3
`c 20:0
`C 20:1 n-9
`C 20:2 n-6
`C 20:3 n-9
`C 20:4 n-6
`c 22:0
`C 20:5 n-3
`C 22:5 n-3
`C 22:6 n-3
`
`1.15 ± 0.39
`24.9 ±2.42
`4.17± 1.06
`5.35 ± 0.46
`20.3 ±2.05
`30.0 ±4.09
`0.88 ± 0.36
`0.11 ±0.06
`0.16±0.06
`0.18 ±0.04
`0.79±0.26
`4.68±1.10
`0.08±0.06
`2.88 ± 1.47
`0.50±0.14
`3.86±0.90
`
`I .OJ± 0.36
`23.7 ±2.74
`3.60± 1.35*
`5 .90 ± 0.64**
`18.2 ± 2.82**
`25 .3 ± 4.30**
`0.84±0.36
`0.13 ±0.04
`0.16± 0.05
`0.13 ± 0.02**
`0.51 ±0.16**
`4.96 ± 1.01
`0.12±0.07
`9.75 ± 3.13**
`1.36 ± 0.37**
`4.26± 1.06
`
`Each value shows percent of total fatty acids and presented
`as mean±SD.
`*: p < 0.05, **: p < 0.01, Significantly different from before
`EPA administration
`
`Table II: Effects of EPA administration on plasma lipids
`
`Before
`
`After EPA administration
`
`Total cholesterol mg/di 277 ± 30 249 ± 28* (-10%)
`Triglyceride
`mg/di 165 ± 25 139 ± 27* (-16%)
`52 ± 13
`50 ± 12
`HDL-cholesterol mg/di
`
`Values are presented as mean±SD
`* Significantly different from before EPA administration
`at p < 0.05 (student's t-test; paired)
`
`Table !IL· Changes of lipid levels, ratio of high density
`lipoprotein subfractions and CETP activities before and
`after EPA administration
`
`Before
`
`After
`
`p value
`
`25.4 ± 18.1
`VLDL CH (mg/di) 29.8 ± 14.2
`ns
`87.0 ±45.0 <0.05
`TO (mg/di) 106.5 ±42.6
`11.8 ± 8.3
`IDL CH (mg/di) 10.8 ± 7.9
`ns
`TO (mg/di)
`9.8 ± 6.3
`9.3 ± 5.7
`ns
`LDL CH (mg/di) 185.4 ±49.5 156.2 ±43.2 <0.01
`TO (mg/di) 37.6 ± 10.5
`34.0 ± 13.0
`ns
`apoB (mg/di) 142.2 ±27.9 122.5 ±27.9 <0.05
`HDL2 CH (mg/di) 25.2 ±11.2
`28.8 ± 11.5
`ns
`HDL3 CH (mg/di) 26.1 ± 6.9
`21.9 ± 4.4
`ns
`HDL2-CH/
`HDL3-CH
`
`1.35± 0.53 <0.05
`
`1.04± 0.6
`
`CETP activity (%) 29.0 ± 6.3
`
`24.2 ± 5.5 <0.05
`
`Values are presented as mean±SD.
`Abbreviations: CH, cholesterol; TO, triglyceride; CETP,
`cholesteryl ester transfer protein activity; ns, not significant
`
`to apo B ratio did not change (1.2±0.11 vs.
`1.19±0.18, before and after treatment of EPA,
`respectively. There was no statistically signifi(cid:173)
`cant relationship between the increase of EPA
`the decrease of LDL
`concentration and
`cholesterol level.
`There was an increase in HDL2 cholesterol
`(from 25.2±11.2 mg/dl to 28.8±11.5) and de(cid:173)
`crease in HDL3 cholesterol (from 26.1±6.9 mg/
`dl to 21.9±4.4), although the changes did not
`reach statistical significance. There was a sig(cid:173)
`nificant
`increase
`in
`the HDL2/HDL3
`cholesterol ratio (from 1.04±0.6 to 1.35±0.53,
`p<0.05).
`LDL fractions were analyzed by 2-16% PAG
`gradient electrophoresis. EPA treatment did
`not cause any change in the electrophoretic
`mobility of LDL before (267 A± 8.1) and during
`EPA (268A±8.8), suggesting the size of LDL
`particles had not changed.
`CETP activities were significantly decreased
`from 29.0±6.3%
`to 24.2±5.5%, p<0.05,
`(Tab. III).
`The weight percent chemical compositions of
`LDL before and after EPA administration is
`shown in Figure 1. No statistically significant
`changes could be observed.
`There was no significant increase of plasma
`peroxide level before and after 6 months of EPA
`treatment (mean± SD: 1.2 ± 0.24 to 1.1±0.31
`nmol/ml, before and after).
`
`50
`
`40
`
`ti)
`
`~ 30
`E
`'+-
`0
`~ 20
`
`10
`
`0
`
`FC
`
`CE
`
`PL
`•:before
`
`~:EPA
`
`TG
`PR
`free cholesterol
`FC
`CE cholesterol ester
`PL phosphollpid
`triglyceride
`TG
`PR protein
`
`Figure 1: Chemical compositions of LDL before and after
`EPA administration.
`
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`s. NozAKI et al: Effects of Purified EPA on Plasma Lipoproteins
`
`259
`
`> Discussion
`
`1
`
`We demonstrated that purified EPA reduced
`plasma LDL cholesterol levels in primary
`hypercholesterolemic patients.
`It has been confirmed that n-3 fatty acids
`' consistently reduce serum triglyceride levels [9,
`IO]. However, the results from the various
`studies regarding the effect of n-3 fatty acids
`on serum cholesterol levels have been inconsis(cid:173)
`tent [9]. The inconsistency of the results among
`· the reports concerning the effect of fish oil on
`LDL levels might be, in part, relative to the fact
`that the subjects with different LDL composi(cid:173)
`tion show different responses to fish oil [22].
`Fish oil had been used as a preparation contain(cid:173)
`ing much n-3 fatty acids since purified EPA had
`( not been available. While, fish oil contains
`, much cholesterol and fatty acids other than
`EPA. It also contains much DHA, another
`major n-3 fatty acid. Furthermore, the quality
`of fish oil is different in each study. This hetero(cid:173)
`geneity of fish oil used in those studies may be
`another cause of the inconsistency of the results
`., regarding the effect of fish oil on LDL levels.
`(, We previously reported the reduction of LDL
`cholesterol by administration of the capsules
`which contain 80% EPA, but as this capsule
`also contained much DHA, we could not
`ascribe this effect to EPA alone [12].
`In the present study, EPA itself reduced
`VLDL and LDL levels and the reduction in
`LDL cholesterol levels was associated with the
`reduction in apo BlOO levels. The cholesterol/
`apo BlOO ratio of LDL did not change. These
`results suggest that the number of LDL parti-
`i' cles is reduced by EPA treatment.
`'
`The present study also demonstrated that
`EPA treatment resulted in the increase of
`HDL2/HDL3 cholesterol ratio. We also meas(cid:173)
`ured CETP activity as an important factor in
`HDL and LDL metabolism and found a reduc-
`·1 tion in CETP activity. This finding is well in
`~ accordance with the recent observations made
`in a study where fish oil was used as a supple(cid:173)
`ment [23]. Patients with absent or markedly
`reduced CETP activities have increased HDL
`cholesterol, mainly associated with HDL2
`cholesterol [24]. Patients with heterozygous
`CETP deficiency have CETP levels between
`
`(
`
`those of normal persons and those with
`homozygous deficiency and also have an in(cid:173)
`creased HDL2/HDL3-cholesterol ratio [20],
`indicating that even a partial reduction in
`CETP activity is associated with changes in dis(cid:173)
`tribution of HDL subfractions. Lipoprotein
`lipase (LPL) and hepatic triglyceride lipase
`{HTOL) are well known to play an important
`role in HDL metabolism. We and other re(cid:173)
`searchers [25, 26] reported no change of LPL
`and HTOL activity in either hypertriglyceride(cid:173)
`mia or normolipidemia using fish oil, suggest(cid:173)
`ing LPL and HTOL might not be responsible
`for the increase in the HDL2/HDL3 cholesterol
`ratio. As these studies were done using fish oil,
`further study of the effect of pure EPA on LPL
`and HTOL will be needed. Increased catabol(cid:173)
`ism of VLDL particles due to a change in
`VLDL composition may also be related to the
`increase of the HDL2/HDL3 cholesterol ratio
`[27, 28]. In the patients studied, there was an
`increase in TO content and a decrease in the CE
`content of LDL although these changes did not
`reach statistical significance. The reduction of
`CETP activity may explain, in part, the change
`of LDL composition since LDL from CETP
`deficient patients is TO rich and CE poor as we
`reported [29]. There is, recently, much concern
`about feeding large amounts of highly unsatu(cid:173)
`rated fatty acids such as EPA because this may
`increase the risk of lipid peroxidation. In order
`to reduce the problem of excess peroxidation in
`vivo, the capsule used in this study contains
`0.2% vitamin E. This amount of vitamin E did
`not produce any increase of peroxide at least
`for 1 year in the capsule. Further, as studied in
`this study, the plasma peroxide levels were not
`increased. From these observations, we believe
`this EPA capsule is safe for use even for rela(cid:173)
`tively long period, 6 months.
`Regarding to the study design, although this
`study was not placebo-controlled study, their
`serum lipids levels were confirmed to be plateau
`before the entry (data not shown) and their
`good compliance to the EPA capsule adminis(cid:173)
`tration was ascertained by the increase of
`plasma EPA levels (Tab. III). These observa(cid:173)
`tions suggest that the results obtained in this
`study was due to the effect of EPA administra(cid:173)
`tion.
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`

`260
`
`S. NozAKI et al: Effects of Purified EPA on Plasma Li po proteins
`
`In conclusion, the reduction in LDL chol(cid:173)
`esterol level and the change in HDL subfrac(cid:173)
`tions by EPA will provide clinical benefits and
`pure EPA capsules will be useful in the treat(cid:173)
`ment of primary non-familial hyperchol(cid:173)
`esterolemia.
`
`References
`
`1. DYERBERG, J., BANG, D. H. o. and HJORNE, N. (1975)
`Am. J. Clin. Nutr. 28, 958-966.
`2. CONNOR, w. E., CONNOR, s. L. (1990) Adv. Intern. Med.
`35, 139-172.
`3. KROMHOUT, D., BosSCHIETER, E. B. and DE LEZENNE
`CouLANDER, C. (1985) N. Engl. J. Med. 312, 1205-1209.
`4. DAVIS, H. R., BRIDENSTINE, R. T., VESSEL!NOVITCH, D.
`and WISSLER, R. W. (1987) Arteriosclerosis 7, 441-449.
`5. LICHTENSTEIN, A.H. and CHOBANIAN, A. v. (1990)
`Arteriosclerosis 10, 597-606.
`6. DYERBERG, J. and BANG, H. 0. (1979) Lancet 2,
`433-435.
`7. GOODNIGHT, s. H. Jr., HARRIS, w. s. and CONNOR,
`W. E. (1981) Blood 58, 880-885.
`8. LEE, T. K., HOOVER, R. L., WILLIAMS, J. D., SPERLING,
`R. I., RAVALESE III, J., SPUR, B. W., ROBINSON, D.R.,
`COREY, E. J., LEWIS, R. A. and AUSTEN, K. F. (1985) N.
`Engl. J. Med. 312, 1217-1224.
`9. HARRIS, W. S. (1989) J. Lipid Res. 30, 785-807.
`10. PHILLIPSON, B. E., ROTHROCK, D. W., CONNOR, W. E.,
`HARRIS, w. s. and ILLINGWORTH, D.R. (1985) N. Engl.
`J. Med. 312, 1210-1216.
`11. KoBATAKE, Y., KuRoDA, K., J1NNOUCHI, H., N1sHmE,
`E. and INNAMI, S. (1984) J. Nutr. Set. Vitaminol. 30,
`357-372.
`12. KAMIDO, H., MATSUZAWA, Y. and TARur, s. (1988)
`Lipids 23, 917-923.
`
`13. WARNICK, G. R., CHEUNG, M. c. and ALBERS, J. J.
`(1979) Clin. Chem. 25, 596-604.
`14. LowRY, 0. H., RosEBROUGH, N. J., FARR, A. L. and
`RANDALL, R. F. (1951) J. Biol. Chem. 193, 265-275.
`15. HAVEL, R. J., EDER, H. A. and BRAGDON, J. H. (1955)
`J. Clin. Invest. 34, 1345-1352.
`16. NOZAKI, s., KUBO, M., Suno, H., MATSUZAWA, Y. and
`TARur, S. (1986) Metabolism 35, 53-58.
`17. KRAUSS, R. M. and BURKE, D. (1982) J. Lipid Res. 23,
`97-102.
`18. NozAKI, S., VEGA, G. L., HADDOX, R. J., DoLAN, E.T.
`and GRUNDY, S. M. (1990) Atherosclerosis 84, 101-110.
`19. ALBERS, J. J., TOLLEFSON, J. H., CHEN, C.-H. and STEIN(cid:173)
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`Arteriosclerosis and thrombosis 11, 71-79.
`
`Dr. Shuichi Nozaki, The Second department of Internal Medicine, Osaka University Medical School, Fukushima,
`Fukushima-ku, Osaka 553, Japan
`
`ICOSAPENT DFNDTS00006594
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1015, p. 7 of 7
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