WO1998020866A2 - Procede de traitement du sarcome de kaposi par des agonistes des recepteurs de la vitamine-d¿3? - Google Patents

Procede de traitement du sarcome de kaposi par des agonistes des recepteurs de la vitamine-d¿3? Download PDF

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WO1998020866A2
WO1998020866A2 PCT/US1997/020613 US9720613W WO9820866A2 WO 1998020866 A2 WO1998020866 A2 WO 1998020866A2 US 9720613 W US9720613 W US 9720613W WO 9820866 A2 WO9820866 A2 WO 9820866A2
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vdr
interleukin
cells
composition
agonist
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WO1998020866A3 (fr
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Parkash S. Gill
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Gill Parkash S
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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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2053—IL-8
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones

Definitions

  • VDR agonists in the treatment of Kaposi 's sarcoma KS
  • Kaposi 's sarcoma may appear in three different classes of individuals.
  • Classic Kaposi's sarcoma is a rare, indolent, cancer of mainly elderly men of Jewish or
  • Kaposi's sarcoma is a highly vascular tumor, the cells of which display features of activated endothelial cells, including high expression of several phenotypic markers such as CD34, PAL-e, UEA binding, and various endothelial cell-specific tyrosine kinases. Studies have shown that the tumor cells produce and respond to several factors including IL-1, IL-6, IL-8, VEGF, and bFGF.
  • AIDS-KS Kaposi's sarcoma
  • Lymph node involvement is common with KS, however, the precise frequency is not known due to the lack of routine lymph node biopsy in AIDS-KS. Visceral involvement occurs frequently (in nearly 50% of the cases) especially in patients with advanced or cutaneous disease. Advanced gastrointestinal KS can cause enteropathy, diarrhea, bleeding, obstruction and death.
  • Pulmonary involvement is common and significant pulmonary KS occurs in nearly 20% of the cases.
  • the overall time of survival of patients with symptomatic pulmonary KS is less than 6 months.
  • AIDS-KS The treatment of AIDS-KS is at best palliative. Decisions regarding the type of treatment should be based on a number of parameters. These include the tumor burden, local complications such as tumor associated edema, ulceration, pain and visceral involvement. In addition bone marrow function, immunologic status, especially CD4 lymphocyte count, concurrent opportunistic infections and medications predict the ability to deliver certain drugs, and outcome to therapy. Localized KS can be managed with local therapy including radiation therapy. Radiation therapy produces a high response rate with reduction in the tumor nodules and resolution of pain. Radiation of mucosal tissues of HIV infected patients can cause increased risk for local toxicity such as mucositis and thus should be delivered at lower daily dose.
  • KS cutaneous KS
  • doxorubicin doxorubicin, daunorubicin
  • bleomycin etoposide
  • the anti-proliferative effect of VDR agonists is exemplified by their therapeutic effects in psoriasis, a disease characterized by hyperproliferation of keratinocytes and inflammation.
  • the cell differentiation effect of VDR agonists is exemplified by their ability to inhibit proliferation and induce irreversible phenotypic change in various myeloid cell lines of monocyte/macrophage lineage.
  • VDR agonists are increasingly recognized as immunomodulators because of their ability to inhibit proliferation of activated T-lymphocytes and production of IL-2, IL-l ⁇ , IL-6, TNF ⁇ , IL-8 and IFN- ⁇ by mononuclear cells or T-lymphocytes in vitro.
  • IL-2, IL-l ⁇ , IL-6, TNF ⁇ , IL-8 and IFN- ⁇ by mononuclear cells or T-lymphocytes in vitro.
  • l,25 ⁇ dihydroxyvitamin D 3 increased the production of IL-1 ⁇ , TNF ⁇ , and IL-6 in the monocytic/myeloid cell lines U937, HL-60, and THP-1. Therefore, the down-regulation or induction of various cytokines by VDR agonists in monocytic cells appears to be dependent on the lineage of the cell line.
  • KS is not a curable disease despite the best possible therapy available, therefore, the development of other agents with activity in KS and low toxicity profile that allows for prolonged use are needed.
  • This invention proves vitamin D 3 receptor agonists to be one such class of agents.
  • the current invention discloses methods for treating Kaposi's sarcoma with administration of VDR agonists at therapeutic doses. Specifically, this invention demonstrates that KS lesions can be lessened with topical administration of calcipotriene. This invention also details the parenteral administration of VDR agonists encapsulated in liposomes. This invention postulates that greater efficacy in lessening KS lesions can be achieved by combination therapy with VDR agonists and IL-1 , IL-6 and/or IL-8 inhibitors. This combination of immunomodulators can also be encapsulated in liposomes or modified in a similar nature, for example, lipid emulsification for parenteral administration. The mode or route of administration can be parenteral: i.e., intravenous; oral; or topical for cutaneous tumors. The median time found to achieve partial response is 2-10 weeks.
  • this invention also demonstrates that effective doses of VDR agonists can be determined by measuring the inhibitory effect of the agonist on IL-1 , IL-6 and 11-8 in vitro.
  • FIG. 2 shows that IL-1 is an autocrine growth factor for KS cells by demonstrating the inhibition of KS cell growth by IL-1 receptor antagonist (ILRA). Viable cells were counted with a Coulter particle counter. Each point represents the mean of triplicate assays.
  • IL-1 is an autocrine growth factor for KS cells by demonstrating the inhibition of KS cell growth by IL-1 receptor antagonist (ILRA). Viable cells were counted with a Coulter particle counter. Each point represents the mean of triplicate assays.
  • Figure 3 indicates that IL-6 is an autocrine growth factor for KS cells by demonstrating the inhibition of KS cell growth by anti-sense IL-6 oligonucleotides. Inhibition of growth of KS cells was determined after the treatment of cells with increasing concentrations of anti-sense IL-6 oligonucleotides for 3 days. No significant inhibition of KS cell growth was observed with IL-6 sense oligonucleotides, thus demonstrating the specificity of action of IL-6 anti-sense oligonucleotides. Viable cells were counted by Coulter particle counter. Each point represents the mean of triplicate assays, -o- sense IL-6; -•- antisense IL-6.
  • Figure 4 represents the mean of triplicate assays, -o- sense IL-6; -•- antisense IL-6.
  • Figure 4 demonstrates that KS cell lines produce IL-8 in vitro. IL-8 levels were quantitated in KS cell supematants after culturing the cells for three days at equal density by an ELISA (R&D Systems, Minneapolis, MN). KSC-29, KSC-4 and KSC- 54 are Kaposi's sarcoma cell lines. 38-10 is a transformed T-cell line.
  • Figure 5 details the effect of KS cell-derived IL-8 on endothelial cells in vitro. Endothelial cells were cultured in the presence of KS cell supernatant with and without IL-8 neutralizing antibodies. Induction of endothelial cell growth was observed when incubated with KS cell supematants, which was blocked by the addition of IL-8 neutralizing antibody.
  • Figure 6 shows that IL-8 is autocrine growth factor for KS cells by demonstrating the inhibition of KS cell growth by anti-sense IL-8 oligonucleotides. Inhibition of growth of KS cells was determined after the treatment of cells with varying concentrations of anti-sense IL-8 oligonucleotides for 3 days. No significant inhibition of KS cell growth was observed with IL-8 sense oligonucleotides, thus demonstrating the specificity of action of IL-8 anti-sense oligonucleotides. Viable cells were counted by Coulter particle counter. Each point represents the mean of assays performed in triplicate. - o- sense IL-8; -x- antisense IL-8
  • Figures 7 A and 7B demonstrate that inhibition of IL-6 in KS cells by l ⁇ ,25-hydroxyvitamin D 3 is in a dose dependent manner.
  • IL-6 production in KS cells was determined by ELISA in cell supematants after treatment with varying concentrations of l ⁇ ,25 -dihydroxyvitamin D 3 for 24 hours. Each bar represents the mean + SE of assays performed in triplicate.
  • Figure 8 A and 8B demonstrate the inhibition of IL-8 production by KS cells incubated with l,25 ⁇ dihydroxyvitamin D 3 . The inhibition was found to be in a dose dependent manner. IL-8 production in KS cells was determined by ELISA of cell supematants after treatment with varying concentrations of l,25 ⁇ dihydroxyvitamin D 3 for 24 hours. Each point represents the mean ⁇ SE of assays performed in triplicate. DETAILED DESCRIPTION OF THE INVENTION Definitions "Response” means a halt in the progression of KS lesions and/or a decrease in tumor size without accompanying unwanted side effects. "Partial response” means a complete flattening of more than 50% of the raised lesions lasting for four weeks or more.
  • “Pharmacologically acceptable carrier” means any chemical approved for use by the Food and Drug Administration as part of a dmg formulation.
  • “Therapeutically effective dose” of a VDR agonist means an amount calculated to achieve and maintain a therapeutically effective level in the tumor, if applied topically to the tumor, or in the plasma, if administered systemically as to substantially inhibit the proliferation of KS cells. It is preferred that the therapeutic amount be sufficient to inhibit proliferation of more than 50 percent of KS cells in vitro. Of course, the therapeutic dose will vary with the potency of each VDR agonist in inhibiting KS cell growth in vitro, the amount required for the inhibition of IL-1, IL-6 and IL-8 in KS cells, and the rate of elimination or metabolism of the VDR agonist by the body in the tumor tissue and/or in the plasma.
  • Immunomodulators means a protein capable of affecting a either a positive or negative proliferation of cells of the immune system, i.e., T-cells, monocytes, etc.
  • Ant means a compound capable of binding to another compound's cellular receptor and causing cellular effects as if the original compound had bound to the receptor.
  • Antagonist means compounds that bind to another compound's cellular receptor but do not cause the cellular effects the original compound causes by binding to its receptor.
  • antisense includes reference to a single stranded nucleic acid sequence which selectively hybridizes, under selective hybridization conditions, to a single-stranded “sense” nucleic acid.
  • the sense nucleic acid is, or is processed to, messenger RNA. Translation of the mRNA is interfered with by the antisense nucleic acid resulting in a measurable decrease in the level of protein encoded by the mRNA.
  • An antisense nucleic acid can be produced in vivo by expressing the gene (or a gene subsequence) in reversed orientation such that the antisense strand is transcribed instead of the sense strand.
  • Sense oligonucleotide means a sequence of nucleic acids constructed so as to match the mRNA sequence of a certain protein.
  • Vitamin D 3 biologically active forms of Vitamin D 3 , such as l,25 ⁇ dihydroxyvitamin D 3 , (calcitriol) are vitamin D 3 receptor agonists and have potent anti-proliferative apart from their widely acknowledged role in calcium homeostasis (Suda, et al. Ann. Rev. Nutr. 10:195-211(1990)).
  • the anti-proliferative effects of VDR agonists are exemplified by their therapeutic effects in psoriasis which is characterized by hyperproliferation of keratinocytes and infiltration of immune cells in the epidermis and dermis (Gerritsen, et al, Br. J. Dermatol.
  • VDR agonists are further exemplified by their ability to inhibit the growth of breast, ovarian (Saunders, et al, Anti-Cancer Drugs 6:562-9 (1995) and colon cancer cells (Kane, et al, Cancer Res. 56:623-32 (1996) in vitro and breast cancer in vivo in rats (Auzant, et al, Cancer Res. 54:1653-6 (1994).
  • VDR agonists can be either naturally occurring or a synthetic vitamin D 3 analog having high potency in inhibition of KS cells in vitro.
  • An example of a naturally occurring VDR agonists can be either naturally occurring or a synthetic vitamin D 3 analog having high potency in inhibition of KS cells in vitro.
  • vitamin D 3 analog with activity as a VDR agonist is l ,25-dihydroxyvitamin D 3 :
  • VDR agonists are well known in the art.
  • calcipotriene is a VDR agonist used in the present study to practice the invention by treating tumors of KS patients.
  • the primary mechanism by which VDR agonists inhibit KS cell proliferation is by inhibition of the production of IL-1, IL-6 and IL-8; autocrine growth factors for KS cells.
  • VDR agonists and agents that inhibit IL-1, IL-6 and/or IL-8 production in KS cells or combinations of these agents and VDR agonists can be used for the treatment of patients with KS.
  • Means of detecting IL-1, IL-6 and IL-8 are not critical aspects of the present invention.
  • IL-1, IL-6 and IL-8 are detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • Immunological binding assays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case IL-1, IL-6 and IL-8).
  • the capture agent is a moiety that specifically binds to the analyte.
  • the capture agent is an antibody that specifically binds IL-1, IL-6 or IL-8.
  • the antibody (anti-IL-1, IL-6 and IL-8) may be produced by any of a number of means known to those of skill in the art as described herein.
  • Immunoassays often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a labeled IL-1, IL-6 or IL-8 or a labeled anti-IL-1, IL-6 or IL-8.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/IL-1, IL-6 or IL-8 protein complex.
  • the labeling agent is a second IL-1, IL-6 or IL-8 antibody bearing a label.
  • the second IL-1, IL-6 and IL-8 antibody lacks a label, but it is, in turn, bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second may be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • proteins capable of specifically binding immunoglobulin constant regions such as protein A or protein G are used as the label agent.
  • proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al., J. Immunol. 111:1401-1406 (1973), and Akerstrom, et al, J. Immunol. 135:2589-2542 (1985)).
  • incubation and/or washing steps are typically required after each combination of reagents. Incubation steps vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours.
  • the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like.
  • the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40 °C
  • the method of detecting IL-1, IL-6 and IL-8 in a biological or culture sample generally comprises the steps of contacting the sample with an antibody which specifically reacts, under immunologically reactive conditions, to IL-1, IL-6 or IL-8.
  • the antibody is allowed to bind to IL-1, IL-6 or IL-8 protein under immunologically reactive conditions, and the presence of the bound antibody is detected directly or indirectly.
  • IL1, IL6 and IL-8 production by KS cells in contact with VDR agonists is measured by northern blotting.
  • Northern blotting techniques are well known in the art and are described in Sambrook and Ausubel, supra.
  • other methods of detecting IL-1, IL-6 and IL-8 are known and kits are commerically available.
  • Antisense The effectiveness of antisense molecules in blocking target gene functions has been demonstrated in a number of different systems (Friedman et al, Nature 335:452-54
  • a DNA segment comprising an antisense transcript which is complementary to a segment of a gene, including but not limited to, IL-1, IL-6 and IL-8, is introduced into a target KS tumor cell that expresses the gene.
  • the expressed antisense strand interacts with the sense stand and prevents proper processing of the sense stand.
  • complementary with respect to two nucleic acid sequences includes reference to standard purine:pyrimidine (e.g., G:C, A:T) base-pairing between the referenced sequences, i.e., sense and antisense strands of nucleic acids.
  • purine:pyrimidine e.g., G:C, A:T
  • the antisense nucleotides of the present invention are prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences as discussed in Berger & Kimmel, Guide to Molecular Cloning Techniques: METHODS IN ENZYMOLOGY, vol. 152, Academic Press, Inc., San Diego, CA; Sambrook et al, MOLECULAR CLONING - A LABORATORY MANUAL (2nd ed.) Vol. 1-3 (1989); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994
  • an IL-8 antisense oligonucleotide is 5'- CTTGGCGCAGTGTGGTCCACTCTCAATCAC-3' (SEQ ID NO:l) (Koch, A.E., et al, Science 258:1798 (1992)).
  • VDR Agonists In the method of this invention, a therapeutically effective dose of a VDR agonist having potent IL-1, IL-6 and IL-8 inhibitory activities or other agents having IL-1 and/or IL-6 and/or IL-8 inhibitory activity or a combination of these agents is either applied topically or administered systemically to KS patients.
  • VDR agonist compositions of the present invention will also contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • the VDR agonists of this invention are administered topically.
  • Topical delivery of the compounds of the present invention includes delivery by direct application to the skin or mucous membranes,
  • a preferred way to practice the invention is to apply the compound, in a cream or oil-based carrier, directly to the KS lesions.
  • an aerosol can be used topically whenever appropriate, such as to the skin, oral mucosa, and upper and lower respiratory tracts. See infra for aerosol formulations and administration.
  • Topical administration is preferred in treatment of skin lesions, including lesions of the scalp, and lesions of mucous membranes where such direct application is practical. Mouthwash and oral paste formulations can be advantageous for mucous membrane lesions. Oral administration is a preferred alternative for treatment of skin lesions and other lesions where direct topical application is not as practical, and it is a preferred route for other applications.
  • Formulations suitable for oral administration comprise of liquid solutions, such as an effective amount of the VDR agonist suspended in diluents, such as water, saline or PEG 400; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; suspensions in an appropriate liquid; and suitable emulsions.
  • liquid solutions such as an effective amount of the VDR agonist suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; suspensions in an appropriate liquid; and suitable emulsions.
  • Tablet forms include one or more of the following: lactose; sucrose; mannitol; sorbitol calcium phosphates; com starch; potato starch; tragacanth; microcrystalline cellulose; acacia; gelatin; colloidal silicon dioxide; croscarmellose sodium; talc; magnesium stearate; stearic acid; and other excipients; colorants; fillers; binders; diluents; buffering agents; moistening agents; preservatives; flavoring agents; dyes; disintegrating agents; and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • VDR agonists alone or in combination with other suitable components, are made into aerosol formulations (i.e., nebulized) to be administered via inhalation.
  • aerosol formulations are placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which consist of the VDR agonist in a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the VDR agonist with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions are administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • VDR agonists optionally are presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the kind previously described.
  • VDR agonists utilizing a method of slow release; for example using an encapsulation in a microvesicle, such as liposomes, which are also taken up more efficiently by the KS cells.
  • liposomes target the VDR agonists to a particular tissue, such as a
  • Liposomes include emulsions, foams, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the VDR agonist to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, for example, a receptor prevalent among KS tumor cells. These molecules would include monoclonal antibodies, or with other therapeutic compositions.
  • liposomes filled with a desired composition of the present invention can be directed to the site of tumor cells, where the liposomes then deliver the selected VDR agonist compositions.
  • Liposomes for use in the invention are formed from standard vesicle- forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • the selection of lipids is generally guided by consideration of liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes as described in, e.g., Szoka, et al, Ann. Rev. Biophys. Bioeng 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, the text LIPOSOMES, Marc J. Ostro, ed., Chapter 1, Marcel Dekker, Inc., New York (1983), and Hope, et al, Chem. Phys. Lip. 40:89 (1986), all of which are incorporated herein by reference.
  • Micelles containing VDR agonists can be prepared by methods which are well known to one of skill in the art. For example, see U.S. Pat. No. 5,534,499 herein incorporated by reference.
  • the dose administered to a patient in the context of the present invention will be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular VDR agonist and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a VDR agonist.
  • the physician will evaluate circulating plasma levels of the VDR agonist, toxicity of the agonist, progression of the disease, and the production of anti-agonist antibodies.
  • the concentration of a VDR agonist in a cream or oil is 0.025-2%.
  • the preferred effective amount is in the range of 5-25 ⁇ M at the site of the tumor. More preferred is 10-25 ⁇ M and most preferred is 10-20 ⁇ M. The precise concentration depends on what sense nucleotide is targeted and where the tumor is located on the body.
  • Example 1 Inhibition of KS cell growth in vitro by 1.25 ⁇ dihydroxyvitamin D 3
  • KS cells Primary cultures of KS cells were established from the tumors of KS patients. The cultured KS cells were treated with varying concentrations of l,25 ⁇ dihydroxyvitamin D 3 to study its growth inhibitory effects. The method to isolate KS cells from the tumor tissue and maintenance and propagation of the culture in vitro is reported in Cai, et al, Am. J. Pathol 145:74-9 (1994) and Masood, et al, AIDS Res. Hum. Retrov. 10:968-75 (1994), which are incorporated herein by reference.
  • KS-derived spindle cell strains were isolated from primary tumor tissues as described previously (Nakamura, et al, Science 242:426-30 (1988). Cells were cultured in 75 cm 2 flasks coated with 1.5% gelatin, in KS medium consisting of the following: RPMI 1640 (Life Technologies); 100 U/mL penicillin; 100 ⁇ g/mL streptomycin; 2 mM glutamine; essential and nonessential amino acids; 10% charcoal treated fetal bovine serum (FBS, Life Technologies); and 1% Nutridoma-HU (Boehringer Mannheim). The medium was supplemented with 20% v/v HTLV-II transformed T-cell conditioned medium.
  • the phenotypes of the primary isolates were determined by immunofluorescence assay.
  • the markers used to characterize the cells included endothelial cell markers; UEA-1 binding sites, EN-4, PAL-e, smooth muscle cell specific markers, including vascular smooth muscle cell specific ⁇ -actin, and macrophage specific markers, including CD 14.
  • Primary isolates were expanded for 4 to 5 passages and multiple aliquots were frozen in liquid N 2 for future studies.
  • human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells (HASM) and human skin fibroblasts were cultured in the recommended media (Clonetics, San Diego, CA).
  • the immunofluorescence assay was performed as follows. Cells were prepared on slides with a cytospin (Shandon, Astmoor, U.K.) and fixed in 1% paraformaldehyde in PBS for 30 min. The slides were rinsed twice in PBS and incubated in 10% FCS/PBS with 0.2% Triton-XlOO for 20 min. The slides were then preincubated with 50%) FCS/PBS for 30 min and again with a monoclonal antibody to cell surface markers diluted in PBS and 10%> FCS for 45 min. at 37°C. After washing three times, the slides were incubated with an FITC-conjugated goat anti-mouse IgG antibody (Sigma) for 30 min. at
  • KS spindle cell strains KSC-29, KSC-4 and KSC-5
  • KSC-1 AND KS-SLK neoplastic KS cell lines
  • the cells were allowed to attach overnight and treated with varying concentrations of 1,25 dihydroxyvitamin D 3 on day 1 and day 3.
  • the viable cells were counted using a Coulter 18 particulate counter on day 6.
  • Similar proliferation assays were performed on HUVEC, HASM, keratinocytes and human skin fibroblasts. As shown by results summarized in Figure 1, incubation of KS cells for 3 days with l,25 ⁇ dihydroxyvitamin D 3 resulted in a dose-dependent inhibition of KS cell growth.
  • EXAMPLE 3 Effect of VDR agonists on production of autocrine growth factors by KS cells
  • IL-1 receptor antagonist IL-6
  • IL-8 IL-8 anti-sense oligonucleotides
  • KS cells were plated at equal density in 24-well 1.5% gelatin coated plates in triplicate and cultured in medium containing 10%> FCS. Eight to twelve hours later, the medium was replaced with fresh medium containing various concentrations of ILRA. This was repeated on day three. The viable cells were counted using a Coulter particle counter. As shown by the results in Figure 2, incubation of KS cells for three days with ILRA results in a dose dependent inhibition of KS cell growth as measured by cell counts.
  • antisense oligonucleotides block the expression of specific genes within the cells, including those of autocrine growth factors.
  • the specific anti-sense oligonucleotides bind to the complimentary RNA sequences within the cell and prevent translation of the mRNA to protein.
  • anti-sense IL-6 and IL-8 would result in decreased formation of IL-6 and IL-8 protein.
  • KS cells were plated at equal density (10,000 cells/well) in 24-well 1.5% gelatin coated plates in triplicate, and cultured in medium containing 10% FCS. Eight to twelve hours later, the medium was replaced with fresh medium containing various concentrations of either phosphorothioate-modified IL-6 and IL-8 antisense (SEQ ID NO:l) and sense oligonucleotides. The sense oligonucleotides serve as the control. Cells were treated with the sense or anti-sense oligonucleotides on day 1 and 2 and harvested on day 3. The viable cells were counted using Coulter particle counter.
  • IL-8 is also an autocrine growth factor for KS cells. Inhibition of IL-6 and IL-8 production by VDR agonists
  • the IL-6 and IL-8 inhibitory properties of VDR agonist were analyzed by determining its ability to decrease IL-6 and IL-8 protein levels in KS cell culture supematants by an ELISA, which utilizes specific monoclonal antibodies for IL-6 and IL-8 (R&D Systems).
  • KS cells were plated at equal density (10,000 cells/well) in 24-well 1.5% gelatin coated plates in triplicates, and cultured in medium containing 10% FCS. On day 1, the media was replaced with fresh media containing various concentrations of l,25 ⁇ dihydroxyvitamin D 3 . The supematants were collected at various time points, centrifuged to free the supematants of cell debris and stored at -70°C until assays could be performed.
  • Measurements of IL-6 and IL-8 were performed by ELISA method using the protocol recommended by the manufacturer.
  • KS cells were grown to near 80%> confluence in 75 cm 2 culture flasks and treated with 1 ⁇ M l,25 ⁇ dihydroxyvitamin D 3 for 4 to 24 hours.
  • IL-l ⁇ , VEGF, bFGF, TGF ⁇ l and b-actin at 68°C for 2 hours.
  • an autoradiograph was made.
  • the membranes were hybridized with one probe at a time; with the probes being stripped off the membrane after exposure of the autoradiography film.
  • the membrane was then reprobed with another labeled cDNA sequence.
  • the level of b-actin mRNA was used to normalize for the quantity of mRNA.
  • l,25 ⁇ dihydroxyvitamin D 3 at concentrations of 1 ⁇ M did not affect IL-6 or IL-8 mRNA levels as quantitated by northern analysis. Therefore, l,25 ⁇ dihydroxyvitamin
  • D 3 inhibits production of IL-6 and IL-8 by KS cells at a post-transcriptional level.
  • KS cell line KS Y-l
  • KS Y-l KS cell line
  • Tumor growth was measured and the tumor was excised on day 14, measured and examined histologically.
  • Mice treated with l,25 ⁇ dihydroxyvitamin D 3 showed decreased tumor growth while the mice treated with the carrier alone exhibited increased tumor growth.
  • VDR agonists Nine HIV- positive KS patients were treated with calcipotriene, in a 0.05% ointment, applied once or twice a day to selected lesions. The regimen was continued until tumor response at the site of application, tumor progression at the site of application or tumor progression at distant site from the site of application required alternative treatment. In each of the nine patients, the disease was limited to the skin.

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Abstract

L'invention concerne un procédé nouveau et efficace de traitement du sarcome de Kaposi chez des patients, consistant à administrer une dose efficace d'un agoniste des récepteurs de la vitamine-D3. Ces agonistes sont capables d'inhiber la croissance des cellules du sarcome de Kaposi en culture en abaissant le taux de facteurs de croissance autocrines importants, IL-6 et IL-8, dans les cellules du sarcome de Kaposi. Les agonistes des récepteurs de la vitamine-D3 peuvent être administrés par voie locale, orale ou parentérale. Le traitement local, avec une dose efficace d'un agoniste de récepteurs de la vitamine-D3, devrait donner lieu à une amélioration significative des lésions dues au sarcome de Kaposi.
PCT/US1997/020613 1996-11-13 1997-11-12 Procede de traitement du sarcome de kaposi par des agonistes des recepteurs de la vitamine-d¿3? WO1998020866A2 (fr)

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AU52541/98A AU5254198A (en) 1996-11-13 1997-11-12 Method of treating kaposi's sarcoma by vitamin-d3 receptor agonists

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US3078796P 1996-11-13 1996-11-13
US60/030,787 1996-11-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2801305A1 (fr) * 1999-11-24 2001-05-25 Galderma Res & Dev Analogues de la vitamine d

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021380A1 (fr) * 1991-05-31 1992-12-10 The Regents Of The University Of California Procede de traitement du sarcome de kaposi
WO1996034887A2 (fr) * 1995-05-05 1996-11-07 Imperial College Of Science, Technology & Medicine Peptides anti-sens

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992021380A1 (fr) * 1991-05-31 1992-12-10 The Regents Of The University Of California Procede de traitement du sarcome de kaposi
WO1996034887A2 (fr) * 1995-05-05 1996-11-07 Imperial College Of Science, Technology & Medicine Peptides anti-sens

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DELEURAN ET AL.: "Regulation of the chemotactic cytokines IL-8 and MCAF and their induction in different cell types related toi the skin" ACTA DERMATO-VENEREOL. , vol. supp.189, 1994, pages 1-34, XP002063159 *
IWAGUCHI ET AL.: "Evaluation of antiangiogenic agents" BIOTHERAPY, vol. 9, no. 10, 1995, pages 1244-1251, XP002063156 *
KOCH ET AL.: "Interleukin-8 as a macrophage-derived mediator of angiogenesis" SCIENCE, vol. 258, 11 December 1992, pages 1798-1801, XP002063155 *
M]LLER ET AL.: "1alpha,25-dihydroxyvitamin D3 and a novel vitamin D analogue MC 903 are potent inhibitors of human interleukin 1 in vitro" IMMUNOL. LETT., vol. 17, no. 4, April 1988, pages 361-365, XP002063158 *
M]LLER ET AL.: "Inhibition of production and function of interleukin-6 by 1,25-dihydroxyvitamin D3" IMMUNOL. LETT., vol. 28, no. 2, 1991, pages 115-120, XP002063157 *
MASOOD ET AL.: "Antiproliferative effects of 1,25 dihydroxyvitamin D3 on Kaposi's sarcoma cells by inhibiting IL-6 and IL-8 expression" BLOOD, vol. 86, no. 10sup1, December 1995, page 382a XP002063154 *
SIPOS E P ET AL: "INHIBITION OF TUMOR ANGIOGENESIS" ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 732, 1994, pages 263-272, XP000650511 *
THIERRY A R ET AL: "IN VITRO AND IN VIVO INHIBITION OF TUMORIGENICITY OF NEOPLASTIC KAPOSI'S SARCOMA CELL LINE (KS Y-1) BY LIPOSOMAL IL-6, IL-8 AND VEGF ANTISENSE OLIGODEOXYNUCLEOTIDES" JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 36, March 1995, page 413 XP002038488 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2801305A1 (fr) * 1999-11-24 2001-05-25 Galderma Res & Dev Analogues de la vitamine d
WO2001038303A2 (fr) * 1999-11-24 2001-05-31 Galderma Research & Development, S.N.C. Analogues de la vitamine d
WO2001038303A3 (fr) * 1999-11-24 2002-01-17 Galderma Res & Dev Analogues de la vitamine d
US6831106B1 (en) 1999-11-24 2004-12-14 Galderma Research & Development, S.N.C. Vitamin D analogues

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