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`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1038
`
`

`

`FFA Formation and pH of Triglyceride Emulsions
`
`was by direct titration to neutrality with 0.01 N potassium
`hydroxide solution standardized using potassium hydrogen
`phthalate, with phenolphthalein as indicator (USP XXII).
`The IUPAC method (10) specifies ethanolzdiethyl ether (95:
`5) as the solvent of choice. However, for safety reasons, the
`solvent was changed to ethanolzchloroform (2:1), each titra-
`tion being preceded by bringing the solvent to neutrality with
`0.0] N potassium hydroxide solution.
`
`Thermal Stress
`
`Thermal stress was applied by filling heating block
`chambers (Dry Baths, Fisher Scientific, Itasca, IL; 60 cham-
`bers per block, each 12 mm diameter and 50 mm deep) with
`oil and immersing the 2-mL arnpoules containing the emul-
`sion at the desired temperature, covering the blocks with
`aluminum foil to minimize thermal fluctuation. Tempera-
`tures were determined with calibrated mercury—in—glass ther-
`mometers placed at random in the block chambers.
`In general, all ampouled emulsion samples were initially
`sterilized at 121°C (F0 = 18) using a Getinge BioFOOE au-
`toclave. Unautoclaved controls were stored at 5°C prior to
`evaluation.
`
`RESULTS AND DISCUSSION
`
`775
`
`Logk
`
`2.4
`
`2.6
`
`2.8
`
`3
`
`a2
`
`1/T x 1000
`
`Fig. 2. The rate of pH change in a 20% soy oil emulsion stabilized
`with egg lecithin or saturated phospholipids and sparged with nitro-
`gen or oxygen before sterilization and storage over the range 25—
`90°C. Control: egg phospholipids, nitrogen sparged (estimated slope
`= —5.93), ——O——; saturated phospholipids, nitrogen sparged
`(estimated slope = —5.30), I; egg phospholipids, oxygen sparged
`(estimated slope = — 6.11), »—-—.----.
`
`The rate of pH change and the rate of FFA formation
`are compared in Fig. 1 for a model emulsion prepared with
`the pharmaceutical grade of soy oil. Results obtained using
`the “superrefined” grade of oil were similar. It is evident
`that the rate of formation of FFA is slower than the rate at
`which the pH drops, suggesting that some other factors are
`involved. As shown in Fig. 2, emulsions prepared with un-
`saturated or saturated acyl groups on the phospholipid moi-
`eties and sparged with nitrogen prior to sterilization and stor-
`age changed pH at almost identical rates. However, it should
`be noted that these two emulsions had markedly different
`hydrolysis rates, (3), which suggested that the lowering of
`
`pH was due to some other factor not necessarily associated
`with phospholipid hydrolysis. As noted earlier, free fatty
`acids can also arise by hydrolysis of triglycerides but the ’
`total (titratable) FFA in the system, irrespective of its
`source, is clearly unable to account for the relatively rapid
`lowering of pH.
`When sparged with oxygen, the pH change in the con-
`trol emulsion was considerably increased (Fig. 2), and this
`observation suggests that the effect of oxygen on the emul-
`sion pH was more pronounced than that produced by the
`heat-induced hydrolytic degradation reaction.
`The solubility of gases in liquids is described by Henry’s
`law (11):
`
`LogkFormation .4
`
`2.5
`
`275
`
`3
`
`3.25
`
`3.5
`
`1/Tx1000
`
`Fig. l. The rate of FFA formation (by KOH titration) and the fall of
`pH (glass electrode) in a 20% soy oil emulsion stabilized with egg
`lecithin after sterilization and storage over the range 25—90°C. pH
`(slope = —3.42),—I—; FFA (slope = —- 1.47), -—-.———. K, rate
`of formation (slope of change against time); T, absolute temperature
`(kelvins).
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`PA=XAKa
`
`where PA is the vapor pressure of a solution containing sol-
`ute A, XA is the mole fraction of A, and K2, is Henry’s law
`constant. This, intuitively, since the K2‘ for gases in non-
`aqueous solvents is generally higher than the corresponding
`value in water, it would appear that the oxygen is likely to
`preferentially dissolve in the oil phase of the emulsion. At-
`kins (11) discussed this issue in relation to benzene at stan-
`dard temperature and pressure and observed that, in all
`cases, the gas was more soluble in the benzene than in the
`water.
`
`Based on this consideration, it seems feasible to suggest
`that residual oxygen may remain dissolved in the triglyceride
`oil phase after preparation and manipulation of the emulsion.
`Some of the triglycerides contain unsaturated acyl centers,
`which could, therefore, become partially oxidized, although
`not necessarily to the point where they would be titratablc
`with alkali and estimated as FFA. These moieties would
`affect the pH of the unbuffered system, in addition to the
`effects produced by the FFA resulting from hydrolysis of
`phospholipid and triglyceride entities. The evident complex-
`ity of the physical and chemical structure of phospholipid-
`
`

`

`776
`
`Herman and Groves
`
`J. M. A. Kemps and D. J. A. Crommelin. Chemical stability of
`phospholipids in pharmaceutical preparations: Hydrolysis of
`phospholipids in an aqueous medium. Pharm. Weekblad.
`123:355—363 (1988).
`C. J. Herman and M. J. Groves. Hydrolysis kinetics of phos—
`pholipids in thermally stressed intravenous lipid emulsion for—
`mulations. J. Pharm. Pharmacol. 44:539—542 (1992).
`. M. Grit, J. H. de Smidt. A. Struijke, and D. J. A. Crommelin.
`Hydrolysis of phosphatidylcholine in aqueous liposome disper-
`sions. Int. J. Pharm. 50:1—6 (1989).
`. D. M. Lidgate, R. C. Fu, and J. S. Fleitman. Using a microflu-
`idizer to manufacture emulsions. BioPharm. 45:28—33 (1989).
`G. Lee, D. Dick, E. V. Vasquez, and K. Werner. pH measure-
`ments of suspensions. In M. H. Rubenstein (ed), Pharmaceu-
`tical Technology: Drug Stability. Ellis Horwood, Chichester,
`UK, pp.
`ll3—1l7.
`C. Paquot. Determination of the acid value and the acidity. In
`Standard Methods for the Analysis of Oils, Fat, and Deriva—
`tives, Pergamon, New York, 1979. pp. 52—55.
`F. W. Atkins. Physical Chemistry, W. H. Freeman, New York,
`1986.
`
`10.
`
`11
`
`stabilized emulsions makes this suggestion difficult to con‘
`firm at present.
`
`ACKNOWLEDGMENTS
`
`Our joint thanks are due to Mr. Peter Cade and his
`colleagues at Croda (US) Inc. for partial support on this
`project.
`
`REFERENCES
`
`1. P. K. Hansrani, S. S. Davis, and M. I. Groves. The preparation
`and properties of sterile intravenous emulsions. J. Parent. Sci.
`Technol. 37:145—150 (1983).
`2. I. Hakansson. Physictrchemical changes in artificial fat emul—
`sions during storage. Acta Chem. Scand. 202267—2281 (1966).
`3. C. J. Herman. The Influence of Thermal Stress on the Proper-
`ties of Phospholipid Stabilized Emulsions, Ph.D. thesis, Uni-
`versity of Illinois at Chicago, Chicago, 1992.
`4. C.‘Washington and S. S. Davis. Ageing effects in parenteral fat
`emulsions: The role of fatty acids. Int. J. Pharm. 39:33-37 (1987).
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