Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects

Definitions

• This invention lies in the field of lipids and the phase transitions of certain lipids from the liquid crystalline phase to the gel phase.
• this invention addresses the problem of leakage of internal substances through the membranes of biological cells and liposomes as these bodies pass the phase transition temperature.
• thermotropic phase transitions between a gel phase and a liquid crystalline phase upon cooling down to temperatures close to but not at or below the freezing temperature. Included among such substances are plant and animal cells, bacteria and liposomes. Cooling of these materials to this phase transition region is a practical and useful means of preservation for purposes such as storage and shipping, since freezing is destructive of the cell structure and dehydration is impractical in many circumstances.
• Liposomes are vesicles formed of concentrically ordered phospholipid bilayers encapsulating an aqueous phase.
• functional molecules such as pharmaceuticals, imaging agents, skin care agents and other useful substances as solutes in the encapsulated aqueous phase, researchers have developed liposomes as useful carriers of these substances. Liposome formulations are thus of interest for such industries as the cosmetics industry and the pharmaceuticals industry, and the leakage problem is a potential obstacle to their stability during storage, shipping and handling.
• FIG. 1 is a plot showing the leakage of a marker compound from liposomes as temperature is lowered through the thermotropic transition temperature. Liposomes treated in accordance with the invention are compared with untreated liposomes.
• FIG. 2 is a plot similar to that of FIG. 1, but additionally showing the effect of varying the amount of treatment agent used in the treatment of the liposomes.
• FIG. 3 is a plot similar to that of FIG. 1, but additionally showing the effect of using a heat-denatured treatment agent in comparison to one which had not been denatured.
• FIG. 4 shows two calorimetric scans, one taken of liposomes treated in accordance with the invention and the other of untreated liposomes.
• FIG. 5 is a plot showing the leakage of a marker compound from a different type of liposomes, through both a warming past the thermotropic transition temperature and a cooling back to the initial low temperature.
• FIG. 6 is a plot showing the leakage of the marker compound from the same liposomes as FIGS. 1 through 4 as the temperature is raised, rather than lowered, through the thermotropic transition temperature.
• antifreeze proteins The existence of naturally-occurring macromolecular species known as "antifreeze proteins,” “thermal hysteresis proteins,” “antifreeze glycoproteins,” and “antifreeze polypeptides” is well known and widely reported in the literature.
• antifreeze glycoproteins for example, was first reported by DeVries, A.L., et al . , in “Freezing Resistance in Some Antarctic Fishes," Science 163:1073-1075 (7 March 1969) . DeVries, et al .
• AFPs antifreeze polypeptides or proteins
• Antifreeze proteins and glycoproteins have been isolated from a wide variety of sources, and these sources and the structures of the various proteins obtained from them have been reported extensively in the literature.
• the sources include both fish species and non-fish species, and are listed in Tables I and II below. TABLE I THERMAL HYSTERESIS PROTEINS OF FISH SPECIES
• AFGPs Pagothe i a borchgrev I nk; contain alanine, Trematomus borc grevinki Antarctic cod threonine and Gal-GalNAc Trematomus bernac n disaccharide: Dissostichus mawsom
• Gadus agac Greenland cod Gadus morhua Atlantic cod Microgadus tomcod Atlantic tomcod Boreogadus sat da Arctic polar cod Eligenus gract lis Saffron cod
• Myoxycepha lus scorp.us Shorthorn sculpin Myoxycepha lus aenaeus Grubby sculpin Myoxycepha 1 us scorp l odes Arctin sculpin
• AFPs Type II: Hemitnpterus amencanus Sea raven cysteine-rich; Osmerus mordex Smelt homologous to C-type Clupea harengus harengus Herring lectins;
• AFPs Type III: Macrozoarces amencanus Ocean pout no cysteines, and not rich Rh; goph I 1 a dearborn i Antarctic eel pout in alanines; Lycodes polans Arctic eel pout
• Coccinellidae Coccinella novemnotata Scolytidae Ips acu inatus
• proteins which have been the most extensively studied, and which are the preferred proteins for use in the practice of the present invention, are those isolated from fish species. As indicated in Table I, these proteins include both glycosylated proteins (AFGPs) and non-glycosylated proteins (AFPs) , and the latter fall within three general categories, designated Type I, Type II, and Type III.
• AFGPs glycosylated proteins
• AFPs non-glycosylated proteins
• the AFGPs generally consist of a series of repeats of the tripeptide unit alanyl-threonyl-alanyl, with the disaccharide ⁇ -D-galactosyl - (1 ⁇ 3) - ⁇ -N-acetyl- D-galactosamine attached to the hydroxyl group of the threonine residue, although variations exist.
• AFGPs of relatively low molecular weight contain proline and arginine residues in place of some of the alanine and threonine residues, respectively.
• Chromatographic studies of the AFGPs from representative fish species have revealed eight major molecular weight fractions, as indicated in Table III. TABLE III
• Preferred AFGPs for purposes of the present invention are those of Fraction No. 8.
• the AFPs differ from one another to a larger degree than do the AFGPs. As indicated in Table I, the three types of AFPs differ from each other in their residue content. Type I AFPs are rich in alanine residues (about 65%) , with most of the remainder consisting of polar residues such as aspartic acid, glutamic acid, lysine, serine and threonine. The molecular weight ranges from about 3,300 to about 6,000. Type II AFPs are considered to be rich in cysteine (actually half-cysteine) residues, and are homologous to C-type lectins.
• Type II AFPs from the sea raven contain 7.6% cysteine, 14.4% alanine, 19% total of aspartic and glutamic acids, and 8% threonine. The molecular weight ranges from about 14,000 to about 16,000.
• Type III AFPs are devoid of cysteine residues and not rich in alanine residues. No conspicuous dominance of any particular amino acid is evident, and the amino acid content is evenly divided between polar and non-polar residues. The molecular weight ranges from about 5,000 to about 6,700. All percents referred to in this paragraph are on a mole basis.
• Antifreeze proteins from insects are primarily AFPs of Type II, and typical compositions in terms of amino acid residues are those of the Choristoneura fumiferana (spruce budworm) and Tenebrio moli tor (beetle) . These are listed in Table IV, which also includes the amino acid composition of the sea raven for comparison.
• Antifreeze proteins and glycoproteins can be extracted from the sera or other bodily fluids of fish or insects by conventional means. Isolation and purification of the proteins is readily achievable by chromatographic means, as well as by absorption, precipitation, and evaporation. Other methods, many of which are described in the literature, will be readily apparent to those skilled in the art. Thermal hysteresis proteins may also be produced synthetically, either by conventional chemical synthesis methods or by methods involving recombinant DNA. The DNA coding sequences of the genes which form these proteins have been elucidated and are extensively reported. See, for example, DeVries, A.L., e ⁇ al., J. Biol . Chem.
• Liposomes are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
• the choice of a particular lipid is generally based on such factors as the desired size and stability of the resulting liposomes in the bloodstream or other intended mode of administration.
• a commonly used lipid component in the liposomes is phosphatidylcholine.
• Phosphatidylcholines to which a variety of acyl chain groups of varying chain length and degree of saturation have been bonded are commercially available or may be isolated or synthesized by well-known techniques.
• the more common phosphatidylcholines are those containing saturated fatty acids with carbon chain lengths in the range of C 14 to C 22 , although phosphatidylcholines formed from mono- or diunsaturated fatty acids and from mixtures of saturated and unsaturated fatty acids are of use as well.
• This invention also extends to liposomes formed from phosphonolipids in which the fatty acids are linked to glycerol via ether linkages rather than ester linkages; liposomes formed from sphingomyelin or phospholipids with head groups other than choline, such as ethanolamine, serine, glycerol and inositol; and liposomes formed from cholesterol , dig1yce r i de s , ceramides, phosphatidylethanolamine-polyoxyethylene conjugates and phosphatidic acid-polyoxyethylene conjugates.
• a sterol such as cholesterol
• the mole ratio of sterol to phospholipid is generally from about 0.1 to 1.0.
• liposome compositions are distearoyl- phosphatidylcholine/cholesterol, dipalmitoylphosphatidyl- choline/cholesterol, and sphingomyelin/cholesterol.
• Liposomes may be prepared by a variety of methods described in the literature. Descriptions appear for example in Szoka, et al . , Ann . Rev. Biophy ⁇ . Bioeng. 9:467 (1980); U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028; the text Liposomes, Marc J. Ostro, ed. , Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, et al . , Chem . Phys . Lip . 40:89 (1986), all of which are incorporated herein by reference.
• One method involves dissolving the vesicle-forming lipids in a suitable organic solvent or solvent system and drying the solution under vacuum or an inert gas to form a thin lipid film.
• the liposomes produced by this method are multilamellar vesicles which are heterogeneous in size.
• the film can be redissolved in a suitable solvent such as t-butanol, then lyophilized, covered with an aqueous buffered solution and allowed to hydrate.
• Liposomes can be sized by a variety of known techniques. One method is sonication, specifically bath or probe sonication, resulting in a progressive size reduction. Another method is homogenization by the use of shearing energy to fragment large liposomes into smaller ones. A third method is the extrusion of liposomes through a small-pore polycarbonate membrane or an asymmetric ceramic membrane.
• Functional compounds such as drugs, cosmetics, imaging agents, and the wide variety of other materials supplying biological utility of some kind can be incorporated into the liposome interior by conventional means.
• the most common such means are encapsulation and transmembrane potential loading.
• Encapsulation of a drug or other functional agent can be achieved by dissolving the agent and the liposome components in an organic solvent in which all species are miscible, then concentrating the resulting solution and evaporating the solvent to a dry film. A buffer is then added to the film and liposomes are formed with the agent incorporated into the vesicle walls.
• the agent can be dissolved in a buffer and added to a dry film formed solely from the lipid components.
• the buffer can be any biologically compatible buffer solution. Examples are isotonic saline, phosphate buffered saline, and other low ionic strength buffers.
• the buffer method will result in liposomes with the agent encapsulated in the aqueous interior of the liposome. In either method, the agent will constitute from about 0.01 ng/mL to about 50 mg/mL of the liposome suspension.
• the liposomes with the agent incorporated in the aqueous interior or in the membrane are then optionally sized as
• Transmembrane potential loading has been described in detail in U.S. Patent No. 4,885,172, U.S. Patent No. 5,059,421, and U.S. Patent No. 5,171,578, the contents of which are incorporated herein by reference.
• the method can be used to load any conventional drug which can exist in a charged state when dissolved in an appropriate aqueous medium.
• the potential is established across the bilayers of the liposomes by producing liposomes having different internal and external media such that a concentration gradient of one or more charged species (such as Na + , K + and/or H + ) is imposed across the bilayers.
• a liposome created with an inside potential which is positive relative to the outside potential is used.
• Biological cells to which this invention is applicable include a wide range of living cells that undergo thermotropic phase transitions. This includes both animal cells and plant cells. Among animal cells, mammalian cells are of particular interest, as well as mammalian tissues, organs and organisms. Examples of mammalian cells to which the invention is applicable are mammalian oocytes, hepatocytes, erythrocytes and leukocytes. Examples of tissues and organs are tissue of livers, hearts, and kidneys, and the organs themselves. Examples of organisms are embryos, and self-sustaining whole animals. Plant cells to which the present invention is applicable include cells from a wide variety of plants. The cells which will benefit from the invention are those which undergo a thermotropic phase transition in temperature regions above the freezing point.
• the phenomenon observed in these plant materials is cold shock, or loss through the membrane of low molecular weight constituents.
• These plant materials include fruits, vegetables, grains, and other food-source plants, and the type and form of cells which exhibit this behavior range from seeds to germinated seedlings to mature plants, including portions of plants such as leaves, fruits, vegetables, stalks and roots.
• the cells, tissues or liposomes can be treated with the antifreeze proteins and glycoproteins in accordance with this invention in a variety of ways.
• a convenient method is the incubation of the cells or liposomes as a suspension in an aqueous solution of the treatment agent.
• the antifreeze proteins or glycoproteins will be present in an amount preferably ranging from about 0.3 mg/mL to about 30 mg/mL of the suspension, more preferably from about 1 mg/mL to about 20 mg/mL, and most preferably from about 3 mg/mL to about 10 mg/mL.
• the incubation will be performed at a temperature above the phase transition temperature, and the cells or liposomes can be maintained in the suspension until ready for use or concentrated or recovered from the suspension, provided that they are maintained in an environment which will prevent outward diffusion of the antifreeze proteins or glycoproteins.
• Other means of contacting the cells or liposomes with antifreeze proteins or glycoproteins will be readily apparent to those skilled in the handling of cells, tissues or liposomes.
• This example illustrates the effect of antifreeze glycoproteins and antifreeze proteins in . inhibiting leakage from dielaidoylphosphatidylcholine liposomes during a phase transition.
• Liposomes were prepared from dielaidoyl- phosphatidylcholine (DEPC) vesicles in a conventional manner, except that carboxyfluorescein was included in the forming solution at a concentration of 200 mM and accordingly trapped inside the resulting liposomes as a marker. Once formed, liposomes were sized by extrusion through polycarbonate filters, using the commercial apparatus produced by Avestin, Inc., Ottawa, Ontario, Canada. Excess carboxyfluorescein not trapped by the liposomes was removed by passing the liposomes through a Sephadex column. The resulting liposome suspensions had a liposome concentration of 20 mg/mL.
• DEPC dielaidoyl- phosphatidylcholine
• Antifreeze glycoproteins obtained from Trematomus borchgrevinki including combined chromatographic fractions 1-8 as well as subcombinations including fractions 2-6, 5-7 and 3-4 were used.
• antifreeze proteins Type I obtained from Pseudopleuronectus americanus were used.
• the AFPs and AFGPs were tested against a control of untreated liposomes, and comparisons were also made against other potential treatment agents. These included alanine, galactose, N-acetyl galactosamine, glycerol, proline, Rock Fish blood serum and ovotransferrin. Alanine, galactose and N-acetyl galactosamine were included because they are prominent components of AFPGs.
• the treatment agent was added to the aqueous liposome suspension to achieve a range of final concentrations as shown below.
• the liposomes, both treated and control were placed in a fluorometer and cooled from 20°C to 0°C in a temperature controlled cuvette at a rate of 0.5°C/minute. Leakage was assayed by the increase in fluorescence observed continuously as carboxyfluorescein leaked into the external medium. The results are listed in Table V below, where the experiments are arranged in increasing order of percent leakage.
• AFGP AFGP fraction
• EXAMPLE 2 This example presents further test results on carboxyfluorescein-marked DEPC liposomes, with emphasis on the differences between AFGP fractions, constituent subunits of AFGPs and denatured AFGPs .
• Example 1 The procedures of Example 1 were followed, with bovine serum albumin (BSA) and fractions 6, 7 and 8 of the AFGPs denatured at 80°C for 30 minutes as additional comparative treatment agents. The results are listed in Table VI below.
• BSA bovine serum albumin
• AFGP AFGP fraction
• AFGP 8 inverted triangles ( ⁇ ) represent the sample treated with 2 mg/mL
• triangles (A) represent the sample treated with 10 mg/mL.
• FIG. 3 The effect of heat denaturation is shown in FIG. 3.
• the AFGP fractions used in the data shown in this Figure are Fractions 2-4 combined. The circles represent these fractions used after heat denaturation at 80°C for thirty minutes, while the squares represent the same fractions used without heat denaturation.
• FIG. 4 is a calorimetric scan of the liposomes whose test data appears in Table II and in FIGS. 2 and 3. Two scans are shown, the upper scan performed on liposomes treated with AFGP fractions 2-6 from Dissostichus mawsoni and the lower scan on liposomes not treated with any treatment agent.
• the peak seen at approximately 12°C is a melting endotherm which occurs as the hydrocarbon chains melt . The fact that the peak is present in both scans and occurs at the same location leads to the conclusion that the AFGPs do not achieve their leakage inhibition effect by any effect on the phase transition of the liposomes.
• This example illustrates the leakage occurring during a liposome phase transition in the opposite direction, i.e., with an increase in temperature, and the lack of effect of antifreeze glycoproteins on the leakage.
• DEPC liposomes were again used in this study. These liposomes were prepared at approximately 23°C, which is above their phase transition temperature. The liposomes were then cooled rapidly through the phase transition. AFGPs Fractions 5-7 from Tremato ⁇ ius Jbernachii were then added at a concentration of 1 mg/mL. The liposomes were incubated at 4°C for one hour, and then rewarmed to approximately 27°C at a rate of 0.5°C/min. The percent leakage was recorded at two-degree intervals by fluorometer. A parallel test was performed on control liposomes which had not been treated with the AFGPs.
• EXAMPLE 4 This example presents a study of dimyristoyl- phosphatidylcholine (DMPC) liposomes, showing the phase transitions occurring during warming, and comparing the results obtained with and without antifreeze proteins present.
• Liposomes were prepared from DMPC at about 4°C in a conventional manner, with carboxyfluorescein as a marker, following the procedure described in Example 1.
• the antifreeze proteins used for treatment were AFGPs Fractions 2-6 from Dissostichusus mawsoni , added to the liposomes at a concentration of 1 mg/mL while the liposomes were still at the low temperature.
• the vesicles were warmed to 28°C at a rate of 0.5°C/min while the percent leakage was recorded at one- or two-degree intervals by fluorometer. This was followed by cooling the vesicles back down to 3°C, again at 0.5°C/min while leakage measurements were recorded by fluorometer. The results are shown in FIG. 6, where the circles represent the control liposomes and the squares the AFGP-treated liposomes.

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