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WO1996004891A1 - Preparation a base de carotenoides et leur utilisation pour le traitement du cancer - Google Patents

Preparation a base de carotenoides et leur utilisation pour le traitement du cancer Download PDF

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Publication number
WO1996004891A1
WO1996004891A1 PCT/US1995/010044 US9510044W WO9604891A1 WO 1996004891 A1 WO1996004891 A1 WO 1996004891A1 US 9510044 W US9510044 W US 9510044W WO 9604891 A1 WO9604891 A1 WO 9604891A1
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WIPO (PCT)
Prior art keywords
retinoic acid
composition
lipid
carotenoid
liposomes
Prior art date
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PCT/US1995/010044
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English (en)
Inventor
Kapil Mehta
Roman Perez-Soler
Gabriel Lopez-Berestein
Robert P. Lenk
Alan C. Hayman
Original Assignee
Board Of Regents, The University Of Texas System
Argus Pharmaceuticals, Inc.
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Application filed by Board Of Regents, The University Of Texas System, Argus Pharmaceuticals, Inc. filed Critical Board Of Regents, The University Of Texas System
Priority to AU33615/95A priority Critical patent/AU3361595A/en
Publication of WO1996004891A1 publication Critical patent/WO1996004891A1/fr

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    • 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
    • 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

Definitions

  • the present invention relates to therapeutic compositions of carotenoids encapsulated in liposomes or other lipid carrier particles.
  • retinoids the family of molecules comprising both the natural and synthetic analogues of retinol (vitamin A)
  • vitamin A the analogues of retinol
  • retinoids can suppress the development of malignant phenotype m vitro (for review, see e.g. , Bertram et al.. In: M.S. Arnott et al.. (eds.), Molecular interactions of nutrition and cancer, pp 315- 335. New York, Raven Press, 1982; Lotan et al.. The modulation and mediation of cancer by vitamins, pp 211-223. Basel: S. Karger AG, 1983) thus suggesting a potential use of retinoids in cancer prevention. Also, recently it has been shown that retinoids can exert effects on certain fully transformed, invasive, neoplastic cells leading in certain instances to a suppression of proliferation (Lotan, Biochim.
  • retinoid therapy has been shown to be effective in gram-negative folliculitis, acne fulminans, acne conglobata, hidradenitis suppurativa, 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 drag 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.
  • compositions that are to be administered intravenously typically the composition must provide at least about 100 mg of the active ingredient in a single container; if it contains a lesser amount of the active ingredient, an unpractically large number of vials will be needed for dosing a single patient.
  • a vial having a volume of 120 cc is the largest that can be accommodated in a commercial freeze drier, and 50 cc is the maximum volume of liquid that can be filled in such a vial.
  • 50 cc is the maximum volume of liquid that can be filled in such a vial.
  • the present invention relates to therapeutically useful, reduced toxicity compositions of carotenoids.
  • the compositions comprise a carotenoid, lipid carrier particles, and an intercalation promoter agent.
  • “Carotenoid” is used here to include retinoids, pro-retinoids, carotenes, xanthophylls, and analogs thereof.
  • a preferred example is all-trans retinoic acid.
  • the carotenoid is substantially uniformly distributed with the lipid in the lipid carrier particles. More particularly, the carotenoid is substantially uniformly distributed in an intercalated position throughout a hydrophobic portion of the lipid carrier particles, as opposed to the aqueous phase.
  • substantially uniformly distributed means that at least 50% of the lipid carrier particles will contain carotenoid in a molar ratio between about 5:85 carotenoid: lipid and about 15:70. Preferably at least 75% of all lipid carrier particles will contain such a ratio of the active ingredient.
  • composition is stable in an aqueous environment.
  • stable in an aqueous environment means that the composition (1) will not exhibit any therapeutically significant degradation over a period of at least 24 hours, (2) will not exhibit a substantial degree of fusions of liposomes over that same period, and (3) will not exhibit substantial redistribution of the carotenoid over that same period, including no substantial movement of the drug into the aqueous phase of a liposome, and no substantial state change into a crystalline form.
  • the molar ratio of carotenoid to lipid in the lipid carrier particles is greater than about 1:10, and is most preferably at least about 15:85.
  • the intercalation promoter agent preferably comprises at least about 15% by weight of the composition, and can suitably be, for example, a triglyceride.
  • Lip carrier particles is used here to include liposomes, having a bilayer structure formed of one or more lipids having polar heads and nonpolar tails, as well as micelles, amorphous particulates of lipid, and other lipid emulsion state entities.
  • suitable forms include multilamellar liposomes.
  • the present invention also relates to a pharmaceutical unit dosage formulation of a carotenoid, which comprises a carotenoid, lipid carrier particles, an intercalation promoter agent, and a pharmaceutically acceptable carrier.
  • the carotenoid is substantially uniformly distributed with the lipid in the lipid carrier particles, and the composition is stable in an aqueous environment.
  • the invention in another aspect, relates to a method of inhibiting the growth of cancer cells, in which a therapeutically effective amount of a carotenoid composition is administered to a living subject.
  • the carotenoid composition can be as described above.
  • the composition is preferably administered to the subject in a maintained molar ratio between about 5:85 carotenoid: lipid and about 15:70. "Maintained” in this context means that the stated ratio of drug to lipid lasts for at least 24 hours.
  • the present invention provides the therapeutic benefits of the carotenoid , while substantially reducing the undesirable toxicity of the composition, as compared to the free drag.
  • encapsulation of retinoic acid in liposomes results in a decrease of at least 15 -fold in toxicity as compared to the free drag.
  • the presence of the intercalation promoter agent permits the ratio of active ingredient to lipid to be increased above what has been previously known, and thus makes such formulations useful in a practical sense for lyophilization into a powder, and subsequent reconstitution into solution which can be administered parenterally to a patient.
  • the intercalation promoter agent overcomes steric hindrance that otherwise limits the amount of carotenoid that be incorporated in, for example, a liposome.
  • the encapsulation of carotenoids within, e.g., liposomes permits their direct delivery to intracellular sites and thus circumvents the requirement 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.
  • compositions of the present invention are also substantially improved over prior liposomal retinoid formulations in terms of uniformity of drug distribution.
  • compositions often had substantial percentages of liposomes which contained essentially no drag.
  • 75 % of all liposomes in the composition contain drag with the range specified above.
  • liposome encapsulation of carotenoids and particularly all-trans retinoic acid circumvents the usual hepatic clearance mechanisms. This has resulted in a substantial extension of the efficacy of liposomal carotenoid over free carotenoid or retinoid. It is believed that liposomal all-trans retinoic acid avoids the problems of resistance to non-liposomal all-trans retinoic acid. This resistance is displayed by such parameters as reduced serum concentration upon prolonged treatment typically observed in treatments as extended over 2, 5 or 7 weeks or longer. Here, substantially longer periods of drag administration were unaccompanied by reduced circulating drug levels. Therapeutic i.v.
  • Figure 1 shows a time profile of liposomal retinoic acid (L-RA) stability in the presence (•) and absence (O) of serum.
  • Figure 2 shows human red blood cell (RBC) lysis as a function of time with RA (•) and L-RA (A).
  • FIG. 3 shows RBC lysis as a function of retinoic acid (RA) concentration (•) and L-RA concentration (A).
  • Figure 4 shows the inhibition of THP-1 cell growth as a function of RA concentration (•), L-RA concentration (O) or empty liposome concentration ( ⁇ ).
  • 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 (•), L-RA concentration (O) and empty liposome concentration ( ⁇ ).
  • Figure 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 (O) and large (•) 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 determinations 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 (O) and large (•) 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 +_ SO 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.
  • Cells were cultured in the presence of 5% human AB serum and the absence (O) or presence of 500 nM ROH (A) or RA (•) for varying periods of time. At the end of each time point, the cells were harvested and assayed for enzyme activity. Values shown are the means ⁇ _ SD of six determinations from two independent experiments. Inset, dose-response curve for tissue TGase induction by ROH (A) and RA (•) in HPBM after 72-hr culture.
  • 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 (A), or "empty liposomes" (O) for indicated periods of time. Both the liposomal RA and "empty liposomes" contained 200 ⁇ g/ l 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.
  • A HPBM monolayers were cultured in serum-containing medium alone ( ⁇ ) or medium containing 1 ⁇ M of free- (O) 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.
  • B Western-blot analysis of tissue TGase levels in freshly isolated HPBM (lane 1) and in HPBM cultured for 72 hr in the presence of serum-containing medium alone (lane 2), in medium containing 1 ⁇ M. of free ROH (lane 3), or liposome-encapsulated ROH (lane 4) as described in Materials and Methods. Twenty-five micrograms of cell protein was loaded onto each lane.
  • Fig. 12(A) shows the levels of all-trans retinoic acid in the blood 60 min after oral administration of non-liposomal all-trans retinoic acid or i.v. administration of liposomal all-trans retinoic acid.
  • Fig. 12(B) shows blood clearance of all-trans retinoic acid following administration of the last dose of all-trans retinoic acid.
  • Fig. 13(A) shows the percentage of all-trans retinoic acid metabolized by isolated liver microsomes to animals exposed to 7 weeks of treatment with all-trans retinoic acid, either liposomal i.v. or oral.
  • Fig. 13(B) shows radioactivity (cpm) of all-trans retinoic acid or its polar metabolites (as discussed in association with Fig. 12(A)) as gathered from five animals.
  • Suitable therapeutic carotenoids for encapsulation in accordance with the present invention include various retinoids. Trans-retinoic acid and all-trans-retinol are preferred. Other retinoids that are believed suitable include: retinoic acid methyl ester, retinoic acid ethyl ester, phenyl analog of retinoic acid, etretinate, retinol, retinyl acetate, retinaldehyde, all-trans-retinoic acid, and 13-cis-retinoic acid.
  • Lipid carrier particles such as liposomes
  • Suitable phospholipid compounds include phosphatidyl choline, phosphatidic acid, phosphatidyl serine, sphingolipids, sphingomyelin, cardiolipin, glycolipids, gangliosides, cerebrosides, phosphatides, sterols, and the like.
  • the phospholipids which can be used include dimyristoyl phosphatidyl choline, egg phosphatidyl choline, dilauryloyl phosphatidyl choline, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, l-myristoyl-2- palmitoyl phosphatidyl choline, l-palmitoyl-2-myristoyl phosphatidyl choline, 1- palmitoyl-2-stearoyl phosphatidyl choline, l-stearoyl-2-palmitoylphosphatidylcholine, dioleoyl phosphatidyl choline, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dimyristoyl phosphat
  • Phosphatidyl glycerol more particularly dimyristoyl phosphatidyl glycerol (DMPG) is not preferred for use in the present invention.
  • DMPG dimyristoyl phosphatidyl glycerol
  • the presence of DMPG correlates with the appearance of amorphous structures of anomalous size, which are believed to render the composition much less suitable for intravenous administration.
  • DMPG is omitted, the amorphous structures are not observed.
  • the undesirable effects that are apparently caused by the presence of DMPG may result from the fact that DMPG has a negative charge, which may interact with the carboxylate of the carotenoid.
  • lipids such as steroids and cholesterol may be intermixed with the phospholipid components to confer certain desired and known properties on the resultant liposomes.
  • synthetic phospholipids containing either altered aliphatic portions, such as hydroxyl groups, branched carbon chains, cyclo derivatives, aromatic derivatives, ethers, amides, polyunsaturated derivatives, halogenated derivatives, or altered hydrophilic portions containing carbohydrate, glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate, carboxy, amine, sulfhydryl, imidazole groups and combinations of such groups, can be either substituted or intermixed with the phospholipids, and others known to those skilled in the art.
  • a suitable intercalation promoter agent will permit the high molar ratio of carotenoid to lipid that is desired for the present invention, without substantial crystallization from the liposomes after they are reconstituted in aqueous solution, as can be observed by microscopic analysis, separation techniques based on buoyant density, or other techniques well known to those skilled in the art.
  • Triglycerides are preferred intercalation promoter agents, with soybean oil as one specific example.
  • Other suitable agents include sterols, such as cholesterol, fatty alcohols, fatty acids, fatty acids esterified to a number of moieties, such as polysorbate, propylene glycol, mono- and diglycerides, and polymers such as poly vinyl alcohols.
  • the carotenoid, lipids, and intercalation promoter agent can be dissolved in an organic solvent, such as t-butanol.
  • Lyophilization to form a preliposomal powder can be performed using commercial apparatus which is known to persons skilled in this field. After lyophilization, the powder can be reconstituted as, e.g., liposomes, by adding a pharmaceutically acceptable carrier, such as sterile water, saline solution, or dextrose solution, with agitation, and optionally with the application of heat.
  • a pharmaceutically acceptable carrier such as sterile water, saline solution, or dextrose solution
  • a preferred formulation which can be dissolved in 45 ml of t-butanol, is as follows:
  • a composition of the present invention is preferably administered to a patient parenterally, for example by intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural, or intrathecal injection. Administration could also be by topical application or oral dosage. Preferred dosages are between 40-200 mg/m 2 . The dosage is preferably repeated on a timed schedule until tumor regression or disappearance has been achieved, and may be in conjunction with other forms of tumor therapy such as surgery, radiation, or chemotherapy with other agents.
  • the present invention is useful in the treatment of cancer, including the following specific examples: hematologic malignancies such as leukemia and lymphoma, carcinomas such as breast, lung, and colon, and sarcomas such as Kaposi's sarcoma.
  • hematologic malignancies such as leukemia and lymphoma
  • carcinomas such as breast, lung, and colon
  • sarcomas such as Kaposi's sarcoma.
  • Preparation of lyophilized powder containing all trans-retinoic acid and phospholipids was carried out as follows. A solution of retinoic acid in t-butanol (1-5 mg/ml) was added to a dry lipid film containing dimyristoyl phosphatidyl choline (DMPC) and dimyristoyl phosphatidyl 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.
  • DMPC dimyristoyl phosphatidyl choline
  • DMPG dimyristoyl phosphatidyl glycerol
  • a powder containing dimyristoyl phosphatidyl choline (DMPC), dimyristoyl phosphatidyl glycerol (DMPG), and all- trans retinoic acid was obtained.
  • the lipid: drag 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 drag 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 drag 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 ( ⁇ m), 65% between 2 ⁇ m and 3 ⁇ m, 14% between 3 ⁇ m and 5 ⁇ m, 1 % more than 5 ⁇ m.
  • the method used for encapsulation of retinoids was simple, reproducible and could be used for large scale production, for example, for clinical trials.
  • Liposomal all-trans retinol was prepared by the methods described above for L-RA with DMPC: DMPG, 7:3.
  • Liposomal 3 H-retinoic acid (L- 3 H-RA) was prepared with DMPC: DMPG, 7:3 as described in Example 1. Samples of the L- 3 H-RA were incubated with either phosphate- buffered saline (PBS) or PBS with 20% (by volume) fetal calf serum (FCS). After various periods of incubation at about 37 °C, aliquots were removed and centrifuged to sediment liposomes. The tritium in the supernatant solution was measured to determine 3 H-RA release.
  • Figure 1 shows the release of 3 H-RA over a two day period. The L- 3 H-RA was over about 80% stable over the period of the experiment, even in the presence of 20% FCS.
  • 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: drag 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 drags tested were injected intravenously via tail vein as a single bolus. The injected volumes of free and liposomal-all-trans retinoic acid were the same for each dose.
  • 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 ⁇ (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 LD 50 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
  • 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 ⁇ g 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 concentration of 0.3 ⁇ g 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 ⁇ g/ml or 0.6 ⁇ g/ml RA or L-RA in another experiment, THP-1 cells had increased levels of tissue transglutaminase enzymic activity, a marker for monocytic cell differentiation. As shown in Figure 5, THP-1 cells, at 4 x 10 6 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
  • Liposomal all-trans retinoic acid was shown, therefore, to have antitumor activity at a dose well below the maximum non-toxic dose, against a cell 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). At these sites, peripheral blood monocytes acquire new functional and biochemical characteristics 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 transglutaminase (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 transglutaminase
  • Terminal differentiation 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 phosphatidyl choline
  • DMPG dimyristoyl phosphatidyl glycerol
  • Lipids, culture medium, and serum were screened for endotoxin with the Limulus amebocyte lysate assay (MA Bioproducts, Walkersville, MD), and they were used only when endotoxin contamination was less than 0.25 ng/ml.
  • Limulus amebocyte lysate assay MA Bioproducts, Walkersville, MD
  • HPBM Isolation, Purification, and Culture Pure populations of HPBM were obtained by countercurrent centrifugal elutriation of mononuclear leukocyte-rich fractions obtained from normal donors who were undergoing routine plateletpheresis [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 monocytes was 255 mm 3 , and that of the large monocytes was 280 mm 3 . The small monocytes were 95 %
  • Tissue TGase activity in cell extracts was measured as a Ca + , dependent incorporation of [ 3 H] 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 electroblotted 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] .
  • 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 drag (predissolved in ethanol) in lipid-containing organic solvents before vacuum drying. The dried lipid-drag film was dispersed by agitation in sterile saline solution. Retinoids up to a 1 : 10 drag: 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 radiolabeled retinol and showed that only 5% .+ 2% of the incorporated radioactivity leaked out in the supernatant after 24-hr incubation at 37 °C.
  • HPBM monolayers were washed twice in ice cold medium and resuspended in 0.5 ml of prechilled reaction mixture containing 5.0 microcuries ( ⁇ Ci)/ml [11, 12(n) 3 H] vitamin A (free ROH) in RPMI medium supplemented with 5 % delipidized human AB serum (serum delipidization was done by organic solvent extraction as described earlier [33]. Binding assays were carried out for 1 hr in an ice bath.
  • the monocyte monolayers were washed six times with ice-cold medium and the cells were lysed in 200 ⁇ l of Triton X-100. Fifty- microliter aliquots of cell lysates, in triplicate, were counted for the cell-associated radioactivity. Background counts, obtained by adding the reaction mixture toward the end of the 1-hr incubation before harvesting, were subtracted from the experimental values.
  • Tissue TGase Induction During In Vitro Culture of HPBM 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. After 10 days in 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).
  • 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 activity, whereas in the control cells no significant 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, respectively); after 48 and 72 hr, however, liposomal RA-treated cells accumulated at least 50% more enzyme activity than did free RA-treated cells (Fig. 10A).
  • 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 11 A). After 72 hr of culture, 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 senim-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.
  • Parallel cultures of HPBM maintained under similar conditions were used for assaying enzyme activity 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.
  • Exposure of HPBM to rIFN-g for 72 hr led to enhanced binding of [ 3 H]ROH that was comparable to the binding activity of control HPBM cultured in vitro for 9 days.
  • HPBM maturation induced by in vitro culture or by exposure to rIFN-g was accompanied by similar morphologic and enzymatic changes.
  • the requirement of cell surface receptor for serum retinol-binding protein could be circumvented by direct intracellular delivery of ROH.
  • serum retinoids The factors in serum responsible for induction and accumulation of tissue TGase in cultured HPBM and macrophages 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].
  • RA enzyme-inducing ability of RA was augmented further by encapsulating RA within the liposomes and allowing its intemalization via phagocytosis (Fig. 10).
  • retinoids play an important role in the differentiation process of HPBM support the idea that retinoids are the important regulators of monocyte/macrophage functions.
  • Mehta, K. , and Lopez-Berestein, G Expression of tissue transglutaminase in cultured monocytic leukemia (THP-1) cells during differentiation. Cancer Res. 46, 1388, 1986. 18. Mehta, K. Lopez-Berestein, G., Hersh, E.M., and Juliano, R.L. Uptake of liposomes and liposome-encapsulated muramyl dipeptide by human peripheral blood monocytes. J. Reticuloendothelial Soc. 32, 155, 1982.
  • Liposomes and liposomal all-trans retinoic acid Liposomal all-trans retinoic acid was prepared from lyophilized powder in bottles containing 3 mg of all-trans retinoic acid and 45 mg of a mixture of two phospholipids, dimyristoyl lecithin and dimyristoyl phosphatidylglycerol in a 3:7 ratio (Avanti Polar Lipids, Birmingham, AL). Immediately before use, liposomal all-trans retinoic acid was reconstituted by adding 3 ml of normal saline to each bottle and agitating the suspension on a vortex mixer for 2-3 min. The reconstimted preparation consisted of multilamellar liposomes (average size, 3.1 ⁇ m).
  • CRABP Cellular retinoic acid-binding protein
  • CRABP CRABP I and II
  • cytoplasmic proteins were extracted and 100-200 ⁇ g protein were incubated overnight at 4°C in a 100 ⁇ l solution of 50 nM 3 [H]-all-trans retinoic acid (specific activity 49.3 Ci/mmol; and 2 mM dithiothreitol with or without 200- fold excess of unlabeled all-trans retinoic acid.
  • Reactants were fractionated over vertical slab gel poly aery lamide electrophoresis under native conditions. After electrophoresis the gel was divided into lanes and cut into 5 mm bands; radioactivity was assessed in a liquid scintillation counter. Specific binding was determined from the radioactivity recovered with or without the 200-fold excess of unlabeled retinoid.
  • Liver samples obtained from the animals at the time of death were rinsed in ice- cold saline and homogenized individually in a 3-fold volume of 0.25 M sucrose 0.05 M Tris-HCl (pH 7.4) using a Teflon ® glass homogenizer. Microsomes were isolated by differential centrifugation at (10,000 g for 20 min; 100,000 g, 60 min). The microsomal pellet was suspended in 0.05 M Tris-HCl (pH 7.4), portioned into aliquots and stored at -70°C. Protein content was determined by Biorad Protein Assay using bovine serum albumin as the standard.
  • the assay buffer and conditions used for determining the ability of microsomes to metabolize [carboxyl- 14 C] all-trans retinoic acid (specific activity 13.7 Ci/nmol) were essentially the same as those described by Van Wauwe et al. , J. Pharmac. exp. Ther. , Vol. 245, 718. After 30 min, the reaction was stopped by cooling and the samples were lyophilized to dryness. Dried residues were extracted with methanol containing butylated hydroxyanisole (0.05%, v/v), and the extracts were evaporated and redissolved in small volumes of methanol (25-50 ⁇ l).
  • All-trans retinoic acid and metabolites were then separated by thin layer chromatography by spotting 20,000- 25,000 cpm on 0.25-mm silica-coated plastic sheets, and developing in a solution of benzene, chloroform, and water (4: 1: 1).
  • the radioactive spots were located by spraying, the plates with EN 3 Hance (New England Nuclear) and autoradiography. The radioactive bands were scraped out, extracted with Solvable (New England Nuclear) and counted in a scintillation counter.
  • the extent of all-trans retinoic acid metabolism was determined from the proportions of cpm in appropriate zones and expressed as a percentage of the total amount of radioactivity recovered.
  • the extent of all-trans retinoic acid metabolism by isolated liver microsomes was also determined by HPLC analysis.
  • the reactants were lyophilized and the residues were extracted twice with 2 ml of methanol containing 0.05% butylated hydroxyanisole (Sigma Chemical Co., St. Louis, MO). After centrifugation, the supernatants were aspirated and evaporated. The resulting pellets were re-extracted in a methanol :dichloromethane solution (75:25) and again evaporated in vacuo. More than 80% of the added all-trans retinoic acid was recovered. The final pellet was mixed with 200 ⁇ l of mobile phase for reverse-phase HPLC.
  • the HPLC system included two pumps and a Zorbax-C8 reverse phase column (4 mm x 8 cm; Supelco, PA).
  • the mobile phase consisted of a linear gradient between solvent A (THF and water (25:75) containing 0.04% ammonium acetate. pH 4) and solvent B (100% THF) during a 16 minute run at a flow rate of 1.8 ml/min.
  • the absorbance was monitored at 346 nm. Retention time for all-trans retinoic acid under these conditions was approximately 9.8 minutes.
  • Cytochrome P450 levels in liver microsomes were determined spectrophotometrically.
  • the assay system is based on the carbon monoxide (CO) difference spectra of dithionite-reduced samples, assuming a value of 91 mM/cm for the molar extinction increment between 450 and 490 m ⁇ .
  • the P450 activity was calculated by the following formula: (change in absorbance between dithionite-reduced sample and CO sample alone)/91 x 1000: it was expressed as nm/mg protein.
  • the mean values for the groups were analyzed by using Student's t-test for paired samples.
  • Creatinine (mg%) 0.43 ⁇ 0.05 0.56 ⁇ 0.1 0.51 ⁇ 0.1
  • Bilirubin (mg%) 0.2 ⁇ 00 0.16 ⁇ 0.05 0.13 ⁇ 0.05
  • WBCs white blood cells
  • RBCs red blood cells
  • Hgb hemoglobin
  • Pits platelets
  • Segs segmented neutrophils
  • Lymph lymphocytes
  • BUN blood urea nitrogen
  • SGOT serum glutamic oxaloacetic transaminase
  • SGPT serum glutamic pyruvic transaminase
  • Alk Phos., alkaline phosphatase.
  • spleens from seven of the eight liposomal all-trans retinoic acid-treated subjects showed the presence of numerous small vacuoles throughout the red pulp area. These structures might represent entrapped liposomes that were removed during processing. They were seen throughout the sinusoids and in phagocytes. No such vacuolization was observed in animals that were treated with non-liposomal all-trans retinoic acid or in control animals treated with saline alone.
  • Figure 12(A) shows the levels of all-trans retinoic acid in the blood 60 min after oral administration of non-liposomal all-trans retinoic acid or i.v. administration of liposomal all-trans retinoic acid.
  • these blood levels were higher in rats treated with liposomal all-trans retinoic acid than in those treated with non-liposomal all-trans retinoic acid. This difference became most striking after 7 weeks of continuous drag treatment.
  • the mean level of all-trans retinoic acid in the blood of rats treated with non-liposomal all-trans retinoic acid decreased from 3.01 ⁇ 0.33 ⁇ g/ml on day 1 to 1.97 ⁇ 0.17 ⁇ g/ml (p ⁇ 0.01) after 7 weeks of treatment, whereas the mean blood all-trans retinoic acid levels of rats treated with liposomal all- trans retinoic acid did not change significantly.
  • the mean all-trans retinoic acid concentration on day 1 (4.42 ⁇ 1.2 ⁇ g/ml) was similar to that at the end of treatment (4.41 ⁇ 0.2 ⁇ g/ml). Also studied was blood clearance of all-trans retinoic acid following administration of the last dose of all-trans retinoic acid.
  • FIG. 12 Blood concentrations of all-trans retinoic acid in rats after 7 weeks treatment with non-liposomal all-trans retinoic acid or liposomal all-trans retinoic acid are presented.
  • Fig. 12(A) presents data from groups of eight rats administered (5 mg/kg body weight) either p.o. non-liposomal all-trans retinoic acid (cross-hatched bars) or i.v. liposomal all-trans retinoic acid (solid bars) twice a week for a total of 7 weeks.
  • Blood samples (200 ⁇ l) were collected 60 min after the administration of the first, sixth, and fifteenth doses, and 150 ⁇ l aliquots of the blood were analyzed for all-trans retinoic acid by HPLC.
  • Fig. 12(B) data is presented following administration of the last dose.
  • Blood samples were collected from animals treated with non-liposomal all-trans retinoic acid (open circles) or liposomal all-trans retinoic acid (solid dots) at indicated time intervals and analyzed by HPLC for all- trans retinoic acid content. The results shown are mean plasma drug concentrations in six rats ⁇ S.D.
  • cytochrome P450-dependent accelerated catabolism and induction of CRABP have been implicated in the acquisition of clinical resistance to all-trans retinoic acid
  • the liver microsomes isolated from rats that were treated with non-liposomal all-trans retinoic acid exhibited significant rapid catabolism of all-trans retinoic acid.
  • Microsomes from all liposomal all-trans retinoic acid-treated and control animals induced much slower catabolism of all-trans retinoic acid than those from rats administered non-liposomal all-trans retinoic acid (Fig. 13(B)).
  • Liver microsomes isolated from rats that were treated with "empty liposomes" without all-trans retinoic acid showed rates of conversion of all-trans retinoic acid to its metabolites similar to those of the untreated controls.
  • the reaction products generated by incubating all-trans retinoic acid in the presence of NADPH and liver microsomes were further analyzed by reverse phase HPLC.
  • the metabolite that eluted at 11.1 min from the non-liposomal all- trans retinoic acid microsome reaction mixture was not seen in the liposomal all-trans retinoic acid microsome reaction mixture.
  • the amounts of three other products were much smaller in the reaction mixture incubated with microsomes from liposomal all-trans retinoic acid-treated rats.
  • Fig. 13 The effect of long-term all-trans retinoic acid administration on drug metabolism by liver microsomes is presented.
  • Fig. 13(A) at the end of the 7 week treatment period, animals were killed and their liver microsomes isolated. The ability of microsomes to metabolize in vitro [ 14 C]all-trans retinoic acid was then determined by incubating microsomes in the presence of NADPH and radiolabeled all-trans retinoic acid (50 nM). The reaction products were fractionated by thin layer chromatography and extent of drag metabolism was determined by counting the metabolite fractions.
  • Results are expressed as a percentage of all-trans retinoic acid metabolized to polar products (cross-hatched bars) or percentage of all-trans retinoic acid remaining intact (solid bars).
  • the vales shown are averages from five rats ⁇ S.D.
  • Fig. 13(B) presents radioactivity (cpm) recovered from intact all-trans retinoic acid (lane 1) or its polar metabolites (lane 2), as discussed in Fig. 13(A), were plotted individually for five different rats.
  • Non-liposomal all-trans retinoic acid has been ineffective in permanently maintaining the remission state of acute promyelocytic leukemia ("APL"). Even when all-trans retinoic acid administration is continued after remission has been achieved, many APL patients still experience relapse. Clearly, some mechanism of resistance develops in relapsed patients whereby the ability of all-trans retinoic acid to induce cellular differentiation is diminished substantially.
  • Several in vitro studies have attempted to explain the evolution of this resistance mechanism, which can be induced in culture after continuous exposure to elevated concentrations of retinoid or carotenoid. Interesting recent clinical pharmacological evidence regarding all-trans retinoic acid resistance (Muindi et al. , Blood.
  • liposomal all-trans retinoic acid of this invention bypasses the clearance mechanism that evolves in the livers of patients treated with the oral formulation. Liposomal formulation is thus not be subject to the same relapse rates as have been demonstrated in clinical trials of the non-liposomal formulation.
  • the toxic effects of liposomal all-trans retinoic acid should be less severe than those associated with non-liposomal all-trans retinoic acid because liposome encapsulation of all-trans retinoic acid decreases direct exposure of the drug during circulation to levels below the orally administered toxic dose. The latter allows greater total exposure of the drag on initial dose accompanied by slower clearance of the all-trans retinoic acid from the site of stem cell seeding.
  • All-trans retinoic acid is metabolized by a hydroxylation reaction of the cyclohexenyl ring, to produce 4-hydroxy metabolites which are further oxidized to the 4-oxo metabolites.
  • the hydroxylation of all-trans retinoic acid to the 4-oxo-all- trans retinoic acid metabolite is mediated by cytochrome P450-dependent enzymes.
  • the most favored explanation of the pharmacological mechanism of all-trans retinoic acid resistance is that continuous all-trans retinoic acid treatment acts to induce catabolic enzymes that are responsible for conversion of the drag.
  • CRABP I and II proteins that are believed to mediate the transfer of the retinoid from cytoplasm to the nucleus of the cell.
  • Increased levels of CRABP may cause the pooling of retinoids in tissues resulting in low plasma levels and accelerated clearance of the drag from the circulation.
  • CRABP is thought to increase with continuous exposure to retinoids.
  • An increase in CRABP has been documented in human skin as a result of repeated topical application of all-trans retinoic acid.
  • a similar increase in skin CRABP levels was also observed by Adamson et al. in rhesus monkeys following chronic i.v. administration of all-trans retinoic acid.
  • Example 8 study coupled with the following data obtained in clinical trials discloses that long term oral admimstration of all-trans retinoic acid is associated with the rapid clearance of the drug from plasma that, in turn, contributes to the relapse of the disease in APL patients, strongly supports the rationale of using liposomal all-trans retinoic acid to induce long-term remissions in APL patients.
  • Liposomal all-trans retinoic acid was admimstered i.v. over one-half hour every other day for 28 days to human subjects with hematological malignancies, including
  • a subject with APL in first relapse 10 months after receiving oral all-trans retinoic acid in three weeks of the liposomal treatment of the present invention displayed a rising white count and evidence of increased cellular differentiation in both the blood and the marrow.
  • the C ⁇ (the concentration in blood at the conclusion of i.v. administration, time 0) in ⁇ g/ml was 6.8 on day one and 7.0 on day 15 after the eighth dose.
  • the AUC in ⁇ g/ml x min was 466 on day 1 and 580 on day 15. Converting to ⁇ g hr/ml these values are 7.76 and 9.66 respectively.

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Abstract

Préparation à base de caroténoïdes à toxicité réduite, stable en milieu aqueux, comportant un caroténoïde, des particules d'excipient lipidique, (par exemple des liposomes) et un promoteur d'intercalation (par exemple un triglycéride). Le caroténoïde se trouve ainsi réparti sensiblement uniformément avec le lipide dans les particules d'excipient lipidique. Le rapport molaire caroténoïde/lipide est supérieur à environ 1/10. Est également présentée une méthode d'inhibition de la croissance des cellules cancéreuses consistant à administrer à un sujet vivant une dose thérapeutique efficace de ladite préparation.
PCT/US1995/010044 1994-08-08 1995-08-08 Preparation a base de carotenoides et leur utilisation pour le traitement du cancer WO1996004891A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068081A1 (fr) * 2000-03-13 2001-09-20 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Agent pour le traitement de maladies du tractus tracheobronchique, en particulier de la bronchopneumopathie chronique obstructive
WO2002064110A3 (fr) * 2001-02-13 2003-01-09 Yissum Res Dev Co Liposomes charges de carotenoides
EP1307220A4 (fr) * 2000-03-31 2004-06-16 Aronex Pharmaceuticals Inc Interferon alfa et acide all-trans retinoique liposomal encapsule combines, preparation et utilisation
EP1005565A4 (fr) * 1997-07-29 2005-02-02 Yissum Res Dev Co Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes

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Publication number Priority date Publication date Assignee Title
GB2050287A (en) * 1979-05-02 1981-01-07 Kureha Chemical Ind Co Ltd Liposomes containing physiologically active substances
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
GB2050287A (en) * 1979-05-02 1981-01-07 Kureha Chemical Ind Co Ltd Liposomes containing physiologically active substances
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes

Non-Patent Citations (1)

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Title
THE JOURNAL OF IMMUNOLOGY, Volume 138, No. 11, issued 01 June 1987, MEHTA, "Suppression of Macrophage Cytostatic Activation by Serum Retinoids: a Possible Role for Transglutaminase", pages 3902-3906. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1005565A4 (fr) * 1997-07-29 2005-02-02 Yissum Res Dev Co Nouvelles especes bacteriennes produisant des carotenoides et procede de production de carotenoides au moyen de ces nouvelles especes
WO2001068081A1 (fr) * 2000-03-13 2001-09-20 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH Agent pour le traitement de maladies du tractus tracheobronchique, en particulier de la bronchopneumopathie chronique obstructive
US7074389B2 (en) 2000-03-13 2006-07-11 Gsf-Forschungszentrum Fur Umwelt Und Gesundheit, Gmbh Agent for treating illnesses of the tracheobronchial tract, especially chronic obstructive pulmonary disease (COPD)
EP1307220A4 (fr) * 2000-03-31 2004-06-16 Aronex Pharmaceuticals Inc Interferon alfa et acide all-trans retinoique liposomal encapsule combines, preparation et utilisation
WO2002064110A3 (fr) * 2001-02-13 2003-01-09 Yissum Res Dev Co Liposomes charges de carotenoides

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