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WO1989006977A1 - Formulation et utilisation de retinoides dans le traitement du cancer et d'autres maladies - Google Patents

Formulation et utilisation de retinoides dans le traitement du cancer et d'autres maladies Download PDF

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Publication number
WO1989006977A1
WO1989006977A1 PCT/US1989/000435 US8900435W WO8906977A1 WO 1989006977 A1 WO1989006977 A1 WO 1989006977A1 US 8900435 W US8900435 W US 8900435W WO 8906977 A1 WO8906977 A1 WO 8906977A1
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Prior art keywords
retinoid
phospholipid
liposomes
retinoic acid
retinoids
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PCT/US1989/000435
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English (en)
Inventor
Kapil Mehta
Roman Perez-Soler
Gabriel Lopez-Berestein
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Board Of Regents, The University Of Texas System
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Priority to AU30475/89A priority Critical patent/AU622890B2/en
Publication of WO1989006977A1 publication Critical patent/WO1989006977A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

  • the present invention relates to therapeutic usage of retinoids encapsulated in liposomes.
  • retinoids the family of molecules comprising both the natural and synthetic analogues of retinol (vitamin A)
  • vitamin A analogues of retinol
  • retinoids can suppress the process of carcinogenesis in vivo in experimental animals (for reviews, see e.g. , Bollag, Cancer Chemother. Pharmacol., 2:207-215, 1979, and Sporn et al. , In Zedeck et a-1. (eds.), Inhibition of Tumor induction and development, pp. 71-100.
  • Retinoids have also been shown to be effective in "the treatment of cystic acne (see e.g. , Peck, et al. , New Engl. J. Med., 300:329-333, 1979).
  • retinoid therapy has been shown to be effective in gram-negative folliculitis, acne fulminans, acne conglobata, hidradenitis suppuritiva, dissecting cellulitis of the scalp, and acne rosacea (see e.g. , Plewig et al. , J. Am. Acad. Dermatol., 6_:766-785, 1982).
  • retinoids may have access to the surrounding normal tissues which might be the basis of their profound toxicity to liver, central nervous system and skeletal tissue.
  • the liposomal format is a useful one for controlling the topography of drug distribution in vivo. This, in essence, involves attaining a high concentration and/or long duration of drug action at a target (e.g. a tumor) site where beneficial effects may occur, while maintaining a low concentration and/or reduced duration at other sites where adverse side effects may occur (Juliano, et al. , In: Drug Delivery Systems, Juliano ed., Oxford Press, N.Y., pp 189-230, 1980). Liposome-encapsulation of drug may be expected to impact upon all the problems of controlled drug delivery since encapsulation radically alters the pharmacokinetics, distribution and metabolism of drugs.
  • the present invention involves a method for thera ⁇ Chamberic administration of retinoid to an animal.
  • This method comprises the basic steps of: preparing liposomes comprising phospholipid and retinoid; and administering a quantity of the resultant liposomes to the animal, said quantity containing a therapeutically effective amount of the retinoid.
  • the retinoids may be administered parenterally, topically, orally or intraperitoneally.
  • the liposomes may bear a tumor impeded by retinoids and the administering step serve to impede growth of said tumor or the animal may have a derma- tological disorder, opthalmic disease, rheumatic disease or vitamin deficiency responsive to retinoids wherein the administering step results in clinical improvement.
  • the most preferred retinoid is all-trans retinoic acid although other retinoic acids may prove useful.
  • the retinoid may be retinol, particularly all trans-retinol.
  • the phospholipids of the present invention may be one or more of phosphatidylcholine, phosphatidylserine. phosphatidylglycerol, sphingomyelin and phosphatidic acid. These phospholipids, their derivatives, and those of analogous structure and hydropathic properties may be used to prepare the liposome-encapsulated retinoids of the present invention as would be apparent to one skilled in the relevant arts upon examination of the present d scriptions.
  • the liposomes may also comprise a sterol component, for example, cholesterol.
  • the phospholipids of these retinoid-containing liposomes in a preferred embodiment, comprise dimyrist ⁇ yl- phosphatidylcholine and dimyristoylphosphatidylglycerol, still more preferably in about a 7:3 ratio.
  • the processes of the present invention are particularly useful as a method for therapy or prophylaxis of an animal afflicted with cancer.
  • Such a method may comprise: identifying ' an animal afflicted with cancer; preparing liposomes comprising phospholipid and a retinoid; and parenterally administering a quantity of said liposomes to the animal, said quantity containing a therapeutically effective amount of the retinoid.
  • the present invention may also comprise a method of inducing cellular differentiation.
  • Such induced differentiation may be useful to impede proliferation of undifferentiated neoplastic cells or to promote the differentiation of normal cells having the potential differentiated capacity to attack neoplastic cells.
  • the liposome-encapsulated retinoids of the present invention may, for example, be used for inducing the in. vivo differentiation of peripheral blood monocytes.
  • This particular method comprises the steps of: preparing liposomes comprising phospholipid and retinoid; and parenterally administering a quantity of said liposomes to an animal, said quantity containing an amount of the retinoid effectively inducing peripheral blood monocyte differentiation.
  • the present invention further includes a process for producing a powder which forms liposomes comprising a retinoid upon suspension.of the powder in an aqueous solution.
  • This process comprises the steps of: dissolving retinoid in t-butanol to form a first solution, mixing the fir.st solution and a dry phospholipid to form a second solution, and lyophilizing the second solution to produce a powder.
  • the phospholipids are defined as a phospholipid film.
  • the solution or powder preferably has a ratio of retinoid to phospholipid between about 1:5 and about 1:50, more preferably between about 1:10 and about 1:15.
  • a composition of matter produced essentially by this process is also an object of this invention.
  • a reconstituted liposomal retinoid preparation may be produced by simple agitation of the above powder in an aqueous solution. Such a reconstituted liposomal retinoid preparation may be used for therapy or prevention by parenteral administration.
  • liposomes as used herein means man-made lipid vesicles which may include plurilamellar lipid vesicles, stable plurilamellar vesicles, small sonicated multilamellar vesicles, reverse phase evaporation vesicles, large multilamellar lipid vesicles, and so forth.
  • Figure 1 shows a time profile of liposomal retinoic acid (L-RA) stability in the presence ( ) and absence (Q ) of serum.
  • L-RA liposomal retinoic acid
  • FIG. 2 shows human red blood cell (RBC) lysis as a function of time with RA (#) and L-RA ( ⁇ ).
  • Figure 3 shows RBC lysis as a function of retinoic acid (RA) concentration ( ⁇ ) and L-RA concentration ( ⁇ ).
  • Figure 4 shows tne inhibition of THP-1 cell growth as a function of RA concentration ( ⁇ ), L-RA concentration (Q) or empty liposome concentration (A).
  • FIG. 5 shows the induction of transglutaminase (TGase) in human monocytic THP-1 cells as a function of treatment with RA or L-RA ( ) .
  • Figure 6 shows the inhibition of human histiocytic U-937 cell growth as a function of RA concentration (0 ) , L-RA concentration ( ) an( 2 empty liposome concentration ( A) -
  • FIG.7 shows the time course of accumulation of tissue TGase activity ⁇ in cultured human peripheral blood monocytes (HPBM) .
  • HPBM peripheral blood monocytes
  • HPBM were fractionated into small [ Q ) and large (0) subpopulations by centrifugal elutriation, and they were cultured in 35-mm-well tissue culture plates as described in Materials and Methods. At the indicated time points the cells were washed, sonicated, and assayed for TGase activity. Values are the means of six deter- minations from two dishes.
  • Figure 8 shows dose-dependent effects of recombinant interferon-gamma (rIFN-g) on induction of tissue TGase activity in HPBM subpopulations.
  • Small ( ) and large C0) monocytes were cultured in serum containing medium alone or medium containing increasing concentrations of rIFN-g. After 72 hr, the cells were harvested and the cell lysates assayed for tissue TGase activity. The results shown represent mean + SD of three determinations from an individual donor.
  • Figure 9 shows effects of retinol (ROH) and RA on induction of tissue TGase activity in cultured HPBM.
  • ROH retinol
  • Figure 10 shows effects of free- and liposome- encapsulated RA on induction of tissue TGase in HPBM.
  • A The cells were cultured in tissue culture dishes in presence of serum-containing medium alone ( , ) 500 nm liposomal RA ( ⁇ ), or medium containing 500 nM free-RA ( ) , or "empty liposomes" ( ) for indicated periods of time. Both the liposomal RA and "empty liposomes" contained 200 ug/ml lipid. At the end of each time point, the cultures were washed and cell lysates assayed for TGase activity. Values shown are the mean + SD of six determinations from two independent experiments.
  • Figure 11 shows effect of free and liposome- encapsulated ROH on induction of tissue TGase in HPBM.
  • HPBM monolayers were cultured in serum-containing medium alone ( ⁇ ) or medium containing 1 uM of free- ( Q-) or liposomal-ROH (A) for 72 hr. Then the cultures were washed and the cell lysates assayed for enzyme activity as described in Materials and Methods.
  • the present invention relates to liposome-encapsulated retinoids.
  • the lipid membranes of liposomes are formed of a bimolecular layer of one or more naturally occurring and/or synthetic lipid compounds having polar heads and nonpolar tails.
  • the present inventors have shown that the encapsulation of retinoic acid in liposomes decreases the toxicity observed with use of the free drug.
  • Suitable compounds for forming liposomes useful in the present invention are phosphatidylcholine, both naturally occurring and synthetically prepared, phosphatidic acid, phosphatidyl- serine, phosphatidylglycerol, sphingolipids, sphingomyelin, cardiolipin, glycolipids, gangliosides, cerebrosides, phosphatides, sterols, and the like.
  • More particularly useful phospholipids include di yristoylphosphatidylcholine and dimyristoylphospha- tidylglycerol.
  • the following compounds may be suitable: egg phosphatidylcholine, dilauryloyl- phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, 1-myristoyl-2-palmitoyl ⁇ phosphatidylcholine, l-palmitoyl-2-myristoyl phosphatidylcholine, l-palmitoyl-2-stearoyl phosphatidylcholine, l-stearoyl-2-palmitoyl phosphatidylcholine, dioleoylphosphatidylcholine, dilauryloylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglyce
  • lipids such as steroids and cholesterol may be intermixed with the phospholipid components to confer certain desired and known properties on the resultant liposomes.
  • Suitable therapeutic agents for encapsulation may include various retinoids. Although retinoic acid, more particularly trans-retinoic acid and retinol, more particularly, all-trans-retinol, are preferred, it also believed that the following compounds, may be successfully encapsulated: all-trans-retinoic acid, retinoic acid methyl ester, retinoic acid ethyl ester, phenyl analog of retinoic acid, etretinate, retinol, retinyl acetate, retinaldehyde, and 13-cis-retinoic acid.
  • retinoids such as retinol
  • liposomes permit their direct delivery to the intracellular sites and thus circumvents the require- ment for cell surface receptors. This may be of particular significance, for example, in therapy of tumors which lack the cell surface receptors for serum retinol binding protein but possess intracellular receptors for retinoic acid.
  • Encapsulation of retinoids within liposomes allows an intravenous administration of the drug. There has been no acceptable vehicle available to permit intravenous administration of retinoids. Due to the highly lipohilic character of retinoids the use of liposomes to deliver retinoids is an attractive approach to reduce their toxicity. Using multilameller liposomes, retinoids can be efficiently encapsulated without losing their activity while reducing their toxicity by at least 15-fold.
  • vitamin A and its analogues in the prevention and treatment of human cancer represents a relatively new direction in oncologic therapeutics.
  • Recent laboratory investigations have documented that the retinoids can block phenotypic expression of cancer, whether initiated by chemical, viral, physical, or biologic carcinogens.
  • the retinoids have been shown to cause regression of premalignant lesions and leukoplakia.
  • the use of retinoids has been associated with both short term toxicity such as central nervous system alterations, as well as chronic intoxication such as skin and mucous membrane dryness and liver impairment, which eventually become irreversible.
  • a considerable amount of effort has been devoted to develop vitamin A derivatives with an improved therapeutic/toxicity ratio. The effort has met with very little success.
  • HPBM Human peripheral blood monocytes
  • TGase tissue transglutaminase
  • the two subpopulations were equally responsive to the augmenting effect of recombinant interferon-gam a (rIFN-g on expression of tissue TGase.
  • rIFN-g recombinant interferon-gam a
  • the retinoid- induced expression of tissue TGase was the result of increased accumulation of the enzyme peptide and not activation of preexisting enzyme. Maturation of HPBM, induced by in vitro culture or treatment with rIFN-g, appeared to be associated with acquisition of cell surface receptors for serum retinol-binding protein.
  • liposomal retinoids may be used as anti-inflammatory reagents.
  • serum retinoids vitamin A and analogs
  • LPS gamma-interferon-lipopolysaccharide
  • Retinoid induced suppression of macrophage activation is associated with induction and accumulation of a protein cross-linking enzyme, tissue transglutaminase.
  • TNF-alpha tumor necrosis factor
  • TNF-alpha can serve as an endogenous substrate for tissue TGase. Therefore, retinoid-induced expression of tissue TGase may cause inter- or intra-molecular cross-linking of TNF, thereby inactivating it or inhibiting its secretion into the extracellular environment. Since factors such as tumor necrosis factor and interleukin-1 (IL-1) (both are released by activated macrophages) are the main mediators of inflammation (Nawroth, et al. , J. Exp. Med.', 163:1363- 1375, 1986), by inhibiting the release of such mediators from macrophages, it may be possible to inhibit the whole cascade leading to inflammation.
  • IL-1 interleukin-1
  • retinoids have been shown to be effective as anti-inflammatory agents (Hensby, Agents and Actions, 21:238, 1987). Also, TNF has been shown to induce the release of IL-1 by endothelial cells (Dinarello, et al. , J. Exp. Med., 163:1433-1439, 1986). In addition, certain retinoids (Etretinate and Isotretinoin) have been reported to inhibit neutrophil and monocyte migration in patients with dermatological disorders. Retinoids have also been shown to inhibit other discrete polymorphonuclear leukocyte functions in vitro. It has been suggested that retinoids may exert their anti-inflammatory effects by interacting with neutrophil membranes to inhibit a variety of responses, such as lysosomal enzyme release and superoxide generation.
  • retinoids have proven effective in treating a wide variety of dermatological diseases, including all types of acne, and recent studies- have shown that topical administration of retinoids may be effective in reversing UV-induced aging of the skin.
  • Retinoids have also been used to treat rheumatic diseases, as immunomodulators (against cancer, infectious diseases, and parasitic diseases), as eye drops or ointments for preventing certain eye diseases, for treatment of vitamin A deficiency disorder, and as dietary supplements. It is contemplated that the liposome encapsulated retinoids of the present invention will prove as effective in treating these diseases as the free retinoids, but will not have the associated toxicity. Therefore, use of liposomal encapsulated retinoids should prove advantageous for treating these disorders and is considered to be within the scope of the present invention.
  • a solution of retinoic acid in t-butanol (1-5 mg/ l) was added to a dry lipid film containing dimyristolyphos- phatidyl choline (DMPC) and dimyristoylphosphatidyl glycerol (DMPG) at a 7:3 molar ratio.
  • the phospholipids were solubilized in the t-butanol containing the all-trans retinoic acid and the solution was freeze-dried overnight.
  • a powder containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) , and all-trans retinoic acid was obtained.
  • the lipid:drug ratio used was from 10:1 to 15:1.
  • Reconstitution of liposomal retinoic acid from the lyophilized powder was done as follows.
  • the lyophilized powder was mixed with normal saline at room temperature to form multilamellar liposomes containing all trans-retinoic acid.
  • This reconstitution method required mild hand- shaking for 1 min to obtain a preparation devoid of any aggregates or clumps.
  • the reconstituted preparation contained multilamellar liposomes of a close size range. No aggregates or drug clumps were identified in the liposomal preparation in three different experiments.
  • Encapsulation efficiency and size distribution of the liposomal all-trans retinoic acid preparation were determined as follows.
  • the liposomal all-trans retinoic acid preparation was centrifuged at 30,000 x g for 45 minutes. A yellowish pellet containing the retinoic acid and the lipids was obtained. By light microscopy, the pellet was composed of liposomes with no crystals or drug aggregates. The encapsulation efficiency was calculated to be greater than 90% by measuring the amount of free retinoic acid in the supernatant by UV spectrophotometry. Liposomes were sized in a Coulter-Counter and Channelizer. The size distribution was as follows: 27% of liposomes less than 2 micrometers (um), 65% between 2 um and 3 um, 14% between 3 um and 5 um, 1% more than 5 um. The method used for encapsulation of retinoids was simple, reproducible and could be used for large scale production, for.example, for clinical trials.
  • DMPC:DMPG at ratios between 7:3 and 9:1 gave superior encapsulation efficiencies.
  • Liposomal all-trans retinol was prepared by the methods described above for L-RA with DMPC:DMPG, 7:3.
  • Liposomal 3H-retmoic acid (L-3H-RAJ was prepared with DMPC:DMPG, 7:3 as described in Example 1. Samples of 3 the L- H-RA were incubated with either phosphate-buffered saline (PBS) or PBS with 20% (by volume) fetal calf serum
  • Lysis of human red blood cells was quantitated by measuring the release of hemoglobin in the supernatants by observation of increases in optical density at 550 nanometers (nm), as described previously (Mehta, et al. , Biochem. Biophys, Acta., Vol. 770-, pp 230-234 (1984). Free-RA dissolved in dimethyl formamide (DMFA), was added to the RBCs. Results with appropriate solvent controls, empty liposomes, and empty liposomes plus free-drug were also noted. Release of hemoglobin by hypotonic lysis of the same number of human RBCs by water was taken as a 100% positive control, while cells treated with PBS were taken as negative controls.
  • DMFA dimethyl formamide
  • Free all-trans retinoic acid was prepared as an emulsion in normal saline containing 10% DMSO and 2% Tween 80 at a concentration of 3 to 5 mg/ml.
  • Liposomal all-trans retinoic acid was prepared using a lipid:drug ratio of 15:1. The final concentration of all-trans retinoic acid in the liposomal preparation was 3 mg/ml.
  • Empty liposomes of the same lipid composition (DMPC;DMPG 7:3) were also tested at doses equivalent to 80 mg/kg, 100 mg/kg, and 120 mg/kg of liposomal-all trans retinoic acid.
  • Normal saline containing 10% DMSO and 2% Tween 80 was also tested as a control at a dose equivalent to 50 mg/kg of free all-trans retinoic acid. All drugs tested were injected intra ⁇ venously via tail vein as a single bolus. The injected volumes of free and liposomal-all-trans retinoic acid were the same for each dose.
  • Table 3 shows data obtained from these acute toxicity experiments.
  • the maximum non-toxic dose of free all-trans retinoic acid was 10 mg/kg. Higher doses caused seizures immediately after injection.
  • the acute LD 50 (deaths occurring up to 72 hours after injection) of free all- trans retinoic acid was 32 mg/kg.
  • the cause of death was cardiopulmonary arrest after seizures for 1-2 minutes in all animals. No seizures or deaths were observed in the animals treated with liposomal all-trans retinoic acid at a dose of 120 mg/kg (maximum non-toxic dose and LE> CQ greater than 120 mg/kg). Higher doses were not tested. No seizures were observed in the animals treated with empty liposomes or normal saline with 10% DMSO and 2% Tween 80.
  • Liposomal all-trans retinoic acid (L-RA) was prepared as described in Example 1.
  • Cells of the human monocytic cell line THP-1 and of the human histiocytic cell line U-937 were inoculated at about 20,000 cells per cell in aliquots of eucaryotic cell culture, medium contained in wells of a 96 well microtiter plate. The medium in various wells contained different amount of free RA or L-RA (DMPC:DMPG 7:3). The cells were incubated for 72 hr at 37 % C and cell growth determined and compared to that of controls without any form of retinoic * acid.
  • Figure 4 shows the inhibition of THP-1 cell growth by increasing concentrations of free RA or L-RA (DMPC:DMPG 7:3). At concentrations of less than 1 ug RA/ml, both preparations inhibited cell growth by over 90%.
  • the human monocytic leukemia THP-1 cells after a 72 hr incubation with either free RA or L-RA at a concentra ⁇ tion of 0.3 ug RA/ml, were observed to have lost their generally ovate form and to have a more flattened and spread morphological appearance often associated with cellular differentiation. The generally ovate form was retained when the cells were cultured in the absence of any free or liposomal retinoic acid.
  • THP-1 cells After incubation for 24 hr with 0.3 ug/ml or 0.6 ug/ml RA or L-RA in another experiment, THP-1 cells had increased levels of tissue transglutaminase enzymic activity, or marker for monocytic cell differentiation. As shown in Figure 5, THP-1 cells, at 4 x 10 cells/ml, showed about 50% greater transglutaminase activity when incubated with L-RA as compared to free RA at equivalent retinoic acid concentrations.
  • DMPC liposomal-all trans retinoic acid
  • DMPG liposomal-all trans retinoic acid
  • Liposomal all-trans retinoic acid was shown, therefore, to have antitumor activity at a dose well below the maximum non-toxic dose, against a ceil line (M5076) which was resistant to free retinoic acid in in vitro studies.
  • Circulating blood monocytes are the precursors of macrophages which accumulate at the sites of tumor rejection [2], delayed hypersensitivity [25], chronic inflammation [6], and at the site of damaged tissue as a part of the healing processes [11] (see reference citations in section D) .
  • peripheral blood monocytes acquire new functional and biochemical charac ⁇ teristics that are associated with the maturation or differentiation process. To understand clearly the mechanisms involved in differentiation, it is necessary to manipulate the extracellular environment and assess precisely a variety of cellular functions and biochemical activities.
  • Vitamin A and its analogues have been shown to exert a profound effect on the differentiation of monocytic cells. Both normal [19] and leukemic [7,17,28] monocytic cells differentiate in response to retinoids which might suggest that retinoids play a role in regulating the differentiation of these cells. According to recent reports, the cellular activity of trans ⁇ glutaminase (TGase), an enzyme that catalyzes the covalent cross-linking of proteins, may be directly linked to the retinoid's action [4,15,21,23,35,39,39].
  • TGase trans ⁇ glutaminase
  • Terminal differen ⁇ tiation of human monocytic leukemia cells (THP-1) induced by phorbol ester and retinoic acid was associated with induction and accumulation of tissue TGase (17], suggesting that the induction of tissue TGase was a marker of monocytic cell differentiation.
  • the present invention involves further definition of the role of retinoids in differentiation and maturation of HPBM and comprises studies of culture conditions that inhibit or facilitate the internalization of retinoids by HPBM on expression of tissue TGase.
  • HPBM isolated into two subpopulations
  • rIFN-g recombinant interferon gamma
  • RPMI-1640 medium supplemented with L-glutamine and human AB serum were from Gibco Laboratories (Grand Island, NY); Escherichia coli-derived human recombinant g- interferon (rIFN-g) was kindly supplied by Genentech Inc. (South San Francisco, CA) ; and all-trans retinol (ROH) and all-trans retinoic acid (RA) were purchased from Sigma Chemical Co. (St. Louis, MO).
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • HPBM Pure populations of HPBM were obtained by counter- current centrifugal elutriation of mononuclear leukocytre-rich fractions obtained from normal donors who were undergoing routine plateletapheresis [12]. HPBM were isolated into two subpopulations according to size with a Coulter ZBI counter and C-1000 channelizer (Coulter Electronics, Hialeah, FL) . The median volume of small
  • Tissue TGase activity in cell extracts was measured as a Ca 2+, dependent incorporation of [3H] putrescine into dimethylcasein.
  • cultured HPBM were washed three times in Tris-buffered saline (20 mM Tris-HCl, 0.15 M NaCl, pH 7.6) and scraped from the dish in a minimal "volume of the same buffer containing 1 mM EDTA and 15 mM Beta-mercaptoethanol.
  • the cells were lysed by sonication, and TGase activity in the lysates was determined as described previously [13,20].
  • the protein content in cell lysates was determined by Lowry's method [14] with bovine gamma globulin as standard.
  • the enzyme activity was expressed as nanomoles of putrescine incorporated into dimethyl-casein per hour per milligram of cell protein.
  • the cell lysates were solubilized in 20 mm Tris-HCl (pH 6.8) containing 1% sodium dodecyl sulfate (SDS), 0.75 M Beta- mercaptoethanol, 2.5% sucrose and 0.001% bromophenol blue. Solubilized extracts were fractionated by electrophoresis on a 6.5% discontinuous polyacrylamide gel and electro- blotted onto nitrocellulose paper. The paper was neutralized with 5% bovine serum albumin and treated with iodinated anti-tissue TGase antibody; the preparation, characterization and properties of this antibody have been described elsewhere (24].
  • the unbound antibody was removed by washing the paper in Tris-HCl buffer (50 mM, pH 7.5) containing 200 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, and 0.25% gelatin, and the paper was dried and autoradiographed as described earlier [20,24].
  • Multilamellar vesicles containing DMPC and DMPG at a molar ratio of 7:3 were prepared as described [16,18]. All-trans ROH or RA were encapsulated by adding the required amount of the drug (predissolved in ethanol) in lipid-containing organic solvents before vacuum drying. The dried lipid-drug film was dispersed by agitation in sterile saline solution. Retinoids up to a 1:10 drug:lipid ratio could be completely encapsulated within the liposomes and were highly stable. The stability and encapsulation efficiency of the liposome preparations were studied by using radiolabelled retinol and showed that only 5% +.2% of the incorporated radio ⁇ activity leaked out in the supernatant after 24-hr incubation at 37 V C.
  • HPBM onolayers were washed twice in ice cold medium and resuspended in 0.5 ml of prechilled reaction mixture containing 5.0 microcuries (uCi)/ml
  • the culture of HPBM in the presence of serum- containing medium for up to 10 days was associated with a marked induction of tissue TGase activity in both small and large HPBM (Fig. 7), the increase in enzyme activity being more rapid after about 4 days of culture.
  • small monocytes showed a 93-fold increase in enzyme activity (from 0.44 to 41.1 nmol/hr/mg), whereas large HPBM accumulated about 103-fold increase in the enzyme activity (from 0.36 to 37.4 nmol/hr/mg).
  • Small and large HPBM mixed together and cultured under similar conditions showed no significant difference in the rate and amount of accumulation of tissue TGase activity compared with that of individual HPBM fractions (data not shown) .
  • rIFN-g The inductive effect of rIFN-g on tissue TGase activity was evidence at 5 U/ml and pretreatment of HPBM cultures with rIFN-g (100 U/ml) followed by washing and subsequent culture in medium alone did not enhance the expression of tissue TGase.
  • HPBM cultured in the presence of 500 nM RA for 24 hr accumulated at least three-fold higher enzyme activity than did the control cells cultured in medium along (Fig. 9).
  • Continuous exposure to RA caused a rapid and linear increase in the enzyme activicy, whereas in the control cells no signi ⁇ ficant change in the level of tissue TGase activity was observed for up to 2 days -of culture.
  • Liposome-encapsulated RA was more effective in inducing tissue TGase expression than was free RA at an equimolar concentration. After 24-hr culture, the amount of tissue TGase activity in HPBM induced by free or liposomal RA at an equimolar concentration of 500 nM was not significantly different (3.4 and 3.7 nmol/hr/mg,
  • liposomal RA- treated cells accumulated at least 50% more enzyme activity than did free RA-treated cells (Fig. 10A) . That increase in enzyme activity by liposome-encapsulated RA was a specific effect of RA and not of lipids was demonstrated by the fact that a culture of HPBM in the presence of "empty liposomes," and containing equivalent amount of lipids did not induce enzyme activity throughout the incubation period. "Empty liposomes,” as reported earlier [20], inhibited serum-induced expression of tissue TGase after 72 hr of culture (Fig 10A) .
  • the free or liposomal RA-induced increase in enzyme activity was caused by an increased amount of the enzyme peptide, as revealed by Western-blot analysis of cell lysates using a iodinated antibody to tissue TGase (Fig. 10B) .
  • the increase in enzyme activity was proportional to the increase in enzyme peptide and not caused by activation of preexisting enzyme.
  • Retinol which in its free form was unable to enhance the expression of tissue TGase in HPBM, became active when presented in liposomal form.
  • Liposome-encapsulated ROH caused a rapid and linear increase in tissue TGase activity with time in culture (Fig 11A) .
  • liposomal-ROH caused a nine-fold increase in enzyme activity (7.1 nmol/hr/mg) when compared to that of control cells exposed to free ROH under similar conditions (0.8 nmol/hr/mg).
  • Liposomal ROH-induced expression of tissue TGase resulted from increased accumulation of the enzyme peptide as demonstrated by Western-blot analysis (Fig. 11B).
  • HPBM were cultured in serum-containing medium alone or medium containing 50 U/ml rIFN-g for indicated periods of time.
  • Binding of tritiated ROH during different periods of culture was determined as described in Materials and Methods.
  • HPBM isolated into two populations based on their size and density, have equal potential to differentiate into mature macrophages.
  • the in. vitro maturation of HPBM to macrophages was associated with enhanced binding and uptake of retinol, presumably as a result of the acquisition of cell surface receptors for serum retinol- binding protein.
  • serum retinoids The factors in serum responsible for induction and accumulation of tissue TGase in cultured HPBM and macro- phages have been shown to be endogenous retinoids and serum retinol-binding protein [21]. Extraction of retinoids by delipidization or depletion of retinol- binding protein from the serum completely abolished its enzyme-inducing ability [19,21]. Serum retinol-binding protein is believed to be responsible for intravascular transport and delivery of retinol to specific target tissues [8,9,29,31]. Receptors for serum retinol-binding protein present on the surface of target cells are responsible for the specificity of the delivery process [9,31].
  • retinoids play an important role in the differentiation process of HPBM support the idea that retinoids are the important regulators of monocyte/macrophage functions.
  • Roberts, A.B., and Sporn, M.B. Cellular biology and biochemistry of the retinoids. In The Retinoids, Vol. 2 (Sporn, M.B., Roberts A.B., and Goodman, D.S., Eds.) New York: Academic Press, p. 209, 1984.

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Abstract

La présente invention concerne un procédé d'administration thérapeutique de rétinoïde à un animal. Ce procédé, dans un mode préféré de réalisation, comprend les étapes de base de préparation de liposomes contenant des phospholipides et des rétinoïdes, et d'administration de manière parentérale d'une quantité des liposomes résultants à l'animal, ladite quantité contenant une quantité thérapeutiquement efficace du rétinoïde. L'animal auquel on a administré les liposomes peut présenter une tumeur stoppée par des rétinoïdes, l'étape d'administration servant à empêcher la croissance de ladite tumeur. Le rétinoïde préféré est l'acide allo trans-rétinoïque, bienque d'autres rétinoïdes peuvent s'avérer utiles. Dans certains cas, le rétinoïde peut être du rétinol, notamment de l'allo trans-rétinol. Les phospholipides de la présente invention peuvent être un ou plusieurs phosphatidylcholines, phosphatidylsérines, phosphatidylglycérols, sphingomyélines et acides phosphatidiques. On peut utiliser ces phospholipides, leurs dérivés et ceux de structure et de propriétés hydrophatiques analogues, afin de préparer les rétinoïdes à liposomes encapsulés de la présente invention comme cela serait apparent pour un homme de l'art après examen des présentes decriptions. Les phospholipides de ces liposomes contenant des rétinoïdes comprennent, dans un mode de réalisation préféré, du dimyristoylphosphatidylcholine et du dimyristoylphosphatidylglycérol, de préférence dans un rapport d'environ 7:3.
PCT/US1989/000435 1988-02-04 1989-02-03 Formulation et utilisation de retinoides dans le traitement du cancer et d'autres maladies WO1989006977A1 (fr)

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Cited By (21)

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WO1993013751A1 (fr) * 1992-01-16 1993-07-22 Board Of Regents, The University Of Texas System Formulation a base de carotenoides et utilisation de ces derniers dans le traitement du cancer
WO1994013786A1 (fr) * 1992-12-15 1994-06-23 Board Of Regents, The University Of Texas System Nouveau facteur de croissance de cellules precurseurs granulocytiques et procede associe
WO1995007089A1 (fr) * 1993-09-09 1995-03-16 Imutec Corporation Compositions immunomodulatrices tirees de la bile
US5417978A (en) * 1993-07-29 1995-05-23 Board Of Regents, The University Of Texas System Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
EP0689426A1 (fr) * 1993-03-22 1996-01-03 Betatene Limited Agent therapeutique pour le traitement de melanomes
EP0689427A1 (fr) * 1993-03-22 1996-01-03 Betatene Limited Composes therapeutiques pouvant se disperser dans l'eau
EP0708648A1 (fr) * 1993-05-17 1996-05-01 Research Development Foundation Inhibition de la production d'oxyde nitrique et du facteur de necrose des tumeurs au moyen de l'acide retinoique
WO1997002030A1 (fr) * 1995-06-30 1997-01-23 Khodor Ammar Composition cosmetique antimycosique pour applications cutanees, et composition pharmaceutique pour le traitement des cellules tumorales et des affections vesicales ou nerveuses
WO1997013499A1 (fr) * 1995-10-11 1997-04-17 The University Of British Columbia Formulations de liposomes a base de mitoxantrone
US5811119A (en) * 1987-05-19 1998-09-22 Board Of Regents, The University Of Texas Formulation and use of carotenoids in treatment of cancer
US5855911A (en) * 1995-08-29 1999-01-05 Board Of Regents, The University Of Texas System Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides
US6013278A (en) * 1996-05-14 2000-01-11 Burzynski Research Institute Liposomal antineoplaston therapies with markedly improved antineoplastic activity
US6551623B1 (en) 1993-09-09 2003-04-22 Lorus Therapeutics Inc. Immunomodulating compositions from bile
EP1307220A1 (fr) * 2000-03-31 2003-05-07 Aronex Pharmaceuticals, Inc. Interferon alfa et acide all-trans retinoique liposomal encapsule combines, preparation et utilisation
US6977244B2 (en) 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7285288B1 (en) 1997-10-03 2007-10-23 Board Of Regents, The University Of Texas System Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7704962B1 (en) 1997-10-03 2010-04-27 Board Of Regents, The University Of Texas System Small oligonucleotides with anti-tumor activity
US8709379B2 (en) 2006-03-29 2014-04-29 Scitech Development, Llc Liposomal nanoparticles and other formulations of fenretinide for use in therapy and drug delivery
WO2017176916A1 (fr) * 2016-04-05 2017-10-12 The Research Foundation For The State University Of New York Compositions contenant de la phosphosérine pour l'induction d'une tolérance immunitaire
CN115089506A (zh) * 2022-05-16 2022-09-23 北京化工大学 应用超重力技术制备视黄醇棕榈酸酯脂质体的方法及所得视黄醇棕榈酸酯脂质体
WO2024174011A1 (fr) * 2023-02-24 2024-08-29 Rossi Bergmann Bartira Liposome, procédé de préparation d'un liposome, composition intranasale comprenant un liposome, procédé de préparation d'une composition intranasale, kit et utilisation de la composition

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EP0274174A1 (fr) * 1985-12-06 1988-07-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Composition de dérivé anthraquinone-liposome et sa préparation
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Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5811119A (en) * 1987-05-19 1998-09-22 Board Of Regents, The University Of Texas Formulation and use of carotenoids in treatment of cancer
US6200597B1 (en) * 1987-05-19 2001-03-13 Board Of Regents, The University Of Texas System Formulation and use of carotenoids in treatment of cancer
WO1993013751A1 (fr) * 1992-01-16 1993-07-22 Board Of Regents, The University Of Texas System Formulation a base de carotenoides et utilisation de ces derniers dans le traitement du cancer
WO1994013786A1 (fr) * 1992-12-15 1994-06-23 Board Of Regents, The University Of Texas System Nouveau facteur de croissance de cellules precurseurs granulocytiques et procede associe
EP0689427A4 (fr) * 1993-03-22 1998-04-15 Betatene Ltd Composes therapeutiques pouvant se disperser dans l'eau
EP0689426A4 (fr) * 1993-03-22 1998-07-29 Betatene Ltd Agent therapeutique pour le traitement de melanomes
EP0689426A1 (fr) * 1993-03-22 1996-01-03 Betatene Limited Agent therapeutique pour le traitement de melanomes
EP0689427A1 (fr) * 1993-03-22 1996-01-03 Betatene Limited Composes therapeutiques pouvant se disperser dans l'eau
US5897871A (en) * 1993-03-22 1999-04-27 Betatine Limited Therapeutic agent for the treatment of melanomas
EP0708648A1 (fr) * 1993-05-17 1996-05-01 Research Development Foundation Inhibition de la production d'oxyde nitrique et du facteur de necrose des tumeurs au moyen de l'acide retinoique
EP0708648A4 (fr) * 1993-05-17 1998-01-14 Res Dev Foundation Inhibition de la production d'oxyde nitrique et du facteur de necrose des tumeurs au moyen de l'acide retinoique
US5417978A (en) * 1993-07-29 1995-05-23 Board Of Regents, The University Of Texas System Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
WO1995007089A1 (fr) * 1993-09-09 1995-03-16 Imutec Corporation Compositions immunomodulatrices tirees de la bile
US6551623B1 (en) 1993-09-09 2003-04-22 Lorus Therapeutics Inc. Immunomodulating compositions from bile
US6280774B1 (en) 1993-09-09 2001-08-28 Lorus Therapeutics Inc. Immunomodulating compositions from bile
WO1997002030A1 (fr) * 1995-06-30 1997-01-23 Khodor Ammar Composition cosmetique antimycosique pour applications cutanees, et composition pharmaceutique pour le traitement des cellules tumorales et des affections vesicales ou nerveuses
US6319957B1 (en) 1995-06-30 2001-11-20 Khodor Ammar Method for treating skin
US5855911A (en) * 1995-08-29 1999-01-05 Board Of Regents, The University Of Texas System Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides
US6042846A (en) * 1995-08-29 2000-03-28 Board Of Regents, University Of Texas System Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides
US7176302B2 (en) 1995-08-29 2007-02-13 Board Of Regents, The University Of Texas System Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides
US7754872B2 (en) 1995-08-29 2010-07-13 Board Of Regents, The University Of Texas System Liposomal phosphodiester, phophorothioate, and p-ethoxy oligonucleotides
US5858397A (en) * 1995-10-11 1999-01-12 University Of British Columbia Liposomal formulations of mitoxantrone
WO1997013499A1 (fr) * 1995-10-11 1997-04-17 The University Of British Columbia Formulations de liposomes a base de mitoxantrone
US6013278A (en) * 1996-05-14 2000-01-11 Burzynski Research Institute Liposomal antineoplaston therapies with markedly improved antineoplastic activity
US6977244B2 (en) 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7285288B1 (en) 1997-10-03 2007-10-23 Board Of Regents, The University Of Texas System Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7704962B1 (en) 1997-10-03 2010-04-27 Board Of Regents, The University Of Texas System Small oligonucleotides with anti-tumor activity
EP1307220A4 (fr) * 2000-03-31 2004-06-16 Aronex Pharmaceuticals Inc Interferon alfa et acide all-trans retinoique liposomal encapsule combines, preparation et utilisation
EP1307220A1 (fr) * 2000-03-31 2003-05-07 Aronex Pharmaceuticals, Inc. Interferon alfa et acide all-trans retinoique liposomal encapsule combines, preparation et utilisation
US8709379B2 (en) 2006-03-29 2014-04-29 Scitech Development, Llc Liposomal nanoparticles and other formulations of fenretinide for use in therapy and drug delivery
WO2017176916A1 (fr) * 2016-04-05 2017-10-12 The Research Foundation For The State University Of New York Compositions contenant de la phosphosérine pour l'induction d'une tolérance immunitaire
US11083782B2 (en) 2016-04-05 2021-08-10 The Research Foundation For The State University Of New York Phosphoserine containing compositions for immune tolerance induction
CN115089506A (zh) * 2022-05-16 2022-09-23 北京化工大学 应用超重力技术制备视黄醇棕榈酸酯脂质体的方法及所得视黄醇棕榈酸酯脂质体
WO2024174011A1 (fr) * 2023-02-24 2024-08-29 Rossi Bergmann Bartira Liposome, procédé de préparation d'un liposome, composition intranasale comprenant un liposome, procédé de préparation d'une composition intranasale, kit et utilisation de la composition

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