WO2001028532A2 - Methods and compositions for predicting, diagnosing and treating lipodystrophy - Google Patents

Methods and compositions for predicting, diagnosing and treating lipodystrophy Download PDF

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Publication number
WO2001028532A2
WO2001028532A2 PCT/CA2000/001208 CA0001208W WO0128532A2 WO 2001028532 A2 WO2001028532 A2 WO 2001028532A2 CA 0001208 W CA0001208 W CA 0001208W WO 0128532 A2 WO0128532 A2 WO 0128532A2
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group
members
composition
upodystrophy
levels
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PCT/CA2000/001208
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French (fr)
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WO2001028532A3 (en
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Kevin C. Kain
Lena Serghides
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Kain Kevin C
Lena Serghides
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Priority claimed from CA002289365A external-priority patent/CA2289365A1/en
Application filed by Kain Kevin C, Lena Serghides filed Critical Kain Kevin C
Priority to AU78949/00A priority Critical patent/AU7894900A/en
Priority to EP00969122A priority patent/EP1223983A2/en
Priority to CA002387919A priority patent/CA2387919A1/en
Publication of WO2001028532A2 publication Critical patent/WO2001028532A2/en
Publication of WO2001028532A3 publication Critical patent/WO2001028532A3/en

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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
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    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
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    • A61K31/201—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 having one or two double bonds, e.g. oleic, linoleic acids
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
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    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
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    • 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
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • this invention relates to a method of predicting those individuals at risk of developing Upodystrophy associated with antiretroviral therapy, by measuring a decrease in CD36 levels, as well as compositions and methods for up- regulating CD36 for the treatment of Upodystrophy associated with protease inhibitor therapy for HTV infection.
  • Combination antiretroviral therapy that includes protease inhibitors, represents a significant advance in the therapy of HTV-1 infection, conferring virologic, morbidity and survival benefits to users (1-4).
  • Protease inhibitors (Pis) interfere with post-translational processing of viral proteins by binding to the active site of the HTV aspartyl protease (4-7).
  • Pis in combination with HTV reverse transcription inhibitors, are now in widespread usage as the standard for antiretroviral therapy (8,9).
  • all currently used HTV-1 ARV has been associated with side effects that include peripheral fat wasting, central adiposity, hyperlipidemia, and insulin resistance as part of a syndrome known as Upodystrophy (7, 10-12).
  • Pis may mediate these effects by binding to two proteins, cytoplasmic retinoic-acid binding protein 1 (CRABP-1) and low density lipoprotein-receptor-related protein (LRP), that are involved in lipid metabolism (7). If Pis bind to CRABP-1, as speculated, they might inhibit the synthesis of cis-9-retinoic acid, which is the sole ligand for the retinoid X receptor (RXR).
  • CRABP-1 cytoplasmic retinoic-acid binding protein 1
  • LRP low density lipoprotein-receptor-related protein
  • RXR functions as a heterodimer with the peroxisome-proliferator- activated receptor type gamma (PPAR- ⁇ ), a nuclear receptor which regulates adipocyte differentiation, function and apoptosis (16-19).
  • PPAR- ⁇ has also been demonstrated to promote monocyte/macrophage (m ⁇ ) maturation and uptake of oxidized low density lipoprotein (OxLDL) (20).
  • RXR and PPAR- ⁇ agonists have been shown to improve hyperlipidemia and insulin response in rodent animal models of diabetes and obesity (21,22) but there has been no reported use of these compounds as treatment for HTV -associated Upodystrophy.
  • PPAR- ⁇ has been demonstrated to promote the uptake of OxLDL by transcriptional induction of the scavenger receptor CD36 (20).
  • CD36 has also been demonstrated to function as a free fatty acid receptor and transporter in adipose tissue (18,19).
  • Lipodystrophy may be a significant cardiovascular risk factor and several reports of cardiac deaths have been linked to ARV and PI therapy (7, 10-15). This concern together with ARV-induced changes in body appearance are major obstacles to the successful long term use of combination therapy for HTV-1 infection (7, 10-15). For these reasons, many patients and their physicians are turning to Pi-sparing regimens. However, these Pi-sparing regimens are disadvantageous because they are less effective for treating HTV-1 infection, and not all patients on Pis develop this syndrome. Accordingly, there is a need for a method to predict those at risk of developing Upodystrophy and severe metabolic complications, in order to facilitate the appropriate use of this powerful class of antiretroviral drugs. In addition, there is a need for methods and compositions for the treatment of Upodystrophy.
  • a method of predicting which patients receiving antiretroviral or protease inhibitor therapy are at risk of developing Upodystrophy is provided.
  • the method comprises the steps of:
  • CD36 levels prior to ARV therapy the drop in CD36 levels being predictive of the subsequent development of Upodystrophy.
  • a composition for the prevention or treatment of Upodystrophy associated with antiretroviral therapy comprises, as an active ingredient, a product which up- regulates CD36 in admixture with a pharmaceutically acceptable carrier, excipient, or diluent.
  • the active ingredient comprises one or more of a PPAR ⁇ agonist, a RXR agonist, a PPAR ⁇ -RXR agonist and natural or synthetic ligands thereof.
  • a method for the prevention or treatment of Upodystrophy is provided. The method comprises the steps of:
  • compositions for the treatment of HTV infection are provided.
  • the composition comprises, as an active ingredient, a product which up-regulates CD36 in combination with a pharmaceutically effective amount of a protease inhibitor, or other anti-retroviral agent in admixture with a pharmaceutically acceptable carrier, excipient, or diluent.
  • the product comprises a PPAR ⁇ -RXR agonist, a PPAR ⁇ agonist, a RXR agonist, or natural or synthetic ligands thereof.
  • a method for treatment of HTV infection comprises the steps of:
  • composition by combining a protease inhibitor or other antiretroviral agent, one of a PPAR ⁇ agonist, PPAR ⁇ -RXR agonist, a RXR agonist, and natural or synthetic ligands thereof with an acceptable carrier, excipient or diluent; and (b) administering an effective dose of the composition to a person infected with
  • the invention also includes a method of determining whether a test compound causes Upodystrophy in a mammal comprising:
  • test compound (a) administering the test compound to (i) a mammal, or (ii) a cell expressing CD36 or a mimetic thereof or (iii) an in vitro cell free system capable of expressing CD36 or a mimetic thereof;
  • the cell usefully comprises a mammalian, insect or yeast cell.
  • the mammal is preferably a human.
  • the human will typically have HTV infection and/or AIDS.
  • the level of CD36 is measured in a blood sample or a fraction thereof.
  • the cells can include, but are not limited to, mammalian or human monocyte/macrophage cell lines (for example, THP-1, U937 cell lines etc.), mammalian or human adipocyte cell lines (for example 3T3L1 cell line etc.), other mammalian or human cell lines expressing CD36 (for example C32 melanoma cells etc.), or COS, Chinese Hamster Ovary (CHO) cell lines or other mammalian, insect or yeast cells transfected with or expressing CD36.
  • the decrease in CD36 levels can be in the range of
  • Another embodiment of the invention includes a method of (i) identifying an individual at risk of antiretroviral or protease inhibitor-associated Upodystrophy or (ii) diagnosing an individual having antiretroviral or protease inhibitor-induced Upodystrophy comprising the steps of:
  • the decrease is optionally measured i) relative to the level of CD36 in the individual prior to administration of protease inhibitor or ii) relative to the average CD36 level in a healthy individual.
  • the decrease in CD36 levels can be in the range of 20%- 75% or greater than 75%.
  • the tested compound optionally preferably comprises a HTV protease inhibitor or other antiretroviral agent.
  • Monocytes were fixed in 3% paraformaldehyde and were then stained for CD36 expression by incubation with a 1:100 dilution of the monoclonal anti-CD36 antibody FA6-152 (Immunotech) in RPMI-0 with 1% BSA, on ice for 30 minutes, followed by a 1: 100 dilution of FTTC linked anti-mouse IgG secondary antibody (Serotec). A secondary antibody only and an unstained control were also performed.
  • CD36 expression was quantitated by flow cytometry using the Epics Elite flow cytometer (Beckman-Coulter) on at least 10,000 monocytes when possible. Data was analyzed using Elite software (Beckman-Coulter).
  • CD36 expression was again determined on the isolated monocytes as above. CD36 expression was expressed as fluorescence intensity over secondary only stained controls.
  • THP-1 and C32 cells were treated with THP-1 and C32 cells.
  • monocyte CD36 from the two healthy controls at baseline and after 1 and 2
  • THP-1 cells, a human pre-monocytic cell line, treated with ritonavir expressed -20% less CD36 than control treated cells (P ⁇ 0.05, paired Student's t, N 4).
  • Antiretroviral therapy that included the protease inhibitors, nelfinavir, ABT-378, and ritonavir, induced a marked decrease in CD36 expression in a proportion of healthy and HTV-infected individuals (-70%) taking these drugs.
  • ARVs and Pis may induce CD36 deficiency by inhibiting retinoic acid metabolism by their action on cytochrome P450 3 A and/or CRABP-1 (7), leading to lowered 9-cis-retinoic acid levels, reduced activation of the RXR-PPAR ⁇ heterodimer and decreased CD36 expression (20).
  • Ritonavir has been shown to be the most potent inhibitor of 9-cis-retinoic acid levels and was the most likely to induce Upodystrophy as well (7,10).
  • the present invention also provides a composition and method of up-regulating CD36 for preventing or treating Upodystrophy associated with protease inhibitor or antiretroviral therapy.
  • PPAR ⁇ is a member of the nuclear hormone receptor superfamily, and as a heterodimer with the retinoid X receptor (RXR) activates transcription of target genes by binding to DR-1 (direct repeat with 1 nucleotide spacer) type hormone response elements (27).
  • RXR retinoid X receptor
  • PPAR ⁇ is primarily expressed in adipose tissue, mammary and colonic epithelium and in myelomonocytic cells (28).
  • the CD36 promoter contains a PPAT ⁇ -RXR binding site, and the PPAR ⁇ -RXR complex can modulate CD36 gene expression through direct promoter interaction (28).
  • PPAR ⁇ and RXR ligands are now known including but not limited to prostaglandins A, D, and especially prostanoid 15-deoxy- ⁇ I2 14 -prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs (including ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone etc.), non- steroidal-anti-inflammatory drugs (NSAIDs including indomethacin, flufenamic acid, fenoprofen, ibuprofen), L-tyrosine-based agonists (including GW1929), cytokines (including TL-4, GM-CSF, M-CSF
  • Upodystrophy associated with PI therapy can be reversed by treating individuals with PPAR ⁇ agonists, RXR agonists, PPAR ⁇ -RXR agonists and natural or synthetic ligands thereof.
  • Upodystrophy in susceptable patients can be prevented by combining ARV therapy with with PPAR ⁇ agonists, RXR agonists, PPAR ⁇ -RXR agonists and natural or synthetic ligands thereof .
  • Toward these objectives we have initiated a number of pilot studies. In one prospective cohort study we examine whether different PI regimens and ARV drugs differ in their ability to induce CD36 deficiency and subsequent Upodystrophy.
  • Participants are randomly assigned to different ARV treatment arms and CD36 levels, blood lipids and other metabolic parameters are assessed prior to and after initiating PI treatment. Patients are followed for 24 months to determine the proportion in each treatment arm developing Upodystrophy.
  • individuals identified as at increased risk of developing Pi-induced Upodystrophy as determined by CD36 levels are randomized to a PPAR ⁇ -RXR agonist or placebo and followed for 1 year to determine if PI therapy combined with a PPAR ⁇ -RXR agonist prevents Upodystrophy.
  • a sample of consecutive patients with Pi-induced Upodystrophy are randomized to receive a PPAR-RXR agonist or placebo, to demonstrate that the active agents can reverse CD36 deficiency in vivo.
  • products which upregulate CD36 can be selected and designed in the manufacture of pharmaceutical compositions for the treatment of Upodystrophy.
  • the pharmaceutical compositions can be administered to patients by methods known to those skilled in the art, such as orally, aerosol administration, direct lavage and parenteral injection. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration.
  • the products may be introduced using in vivo delivery vehicles such as liposomes. They may also be introduced into cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes.
  • compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the products are combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA 1990).
  • the pharmaceutical compositions could include an active ingredient, in association with one or more pharmaceutically acceptable vehicles, such as carriers, excipients or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids.
  • suitable products such as carriers, excipients or diluents
  • the methods of combining suitable products with the vehicles is well known to those skilled in the art.
  • the composition could include a targeting agent for the transport of the active compound to specified sites within tissue.
  • such compounds, peptides or vectors may be administered by infection, microinjection, electroporation and by other methods known in the art.
  • oral formulations of the composition according to the present invention are preferred.
  • the oral formulations contain a pharmaceutically inert carrier, including conventional solid carriers, which are conveniently presented in tablet or capsule form.
  • peptides or peptide mimetics are preferred with subdermal or intramuscular administration most preferred.
  • Intravenous administration or use of implanted milliosmol pumps may also be used.
  • compositions of the present invention may be formulated in a variety of ways.
  • Aqueous solutions of composition of the present invention may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. (Examples thereof may be found, for example, in Remington's Pharmaceutical Sciences, 18th Edition, 1990.) Doses are selected to provide effective upregulation of CD36.
  • compositions including a liquid pharmaceutically inert carrier such as water may also be considered for both parenteral and oral administration.
  • a liquid pharmaceutically inert carrier such as water
  • Other pharmaceutically compatible liquids may also be used.
  • the use of such liquids is well known to those of skill in the art. (Examples thereof may be found, for example, in Remington's
  • the dose level and schedule of administration may vary depending on the particular product used, the method of administration, and such factors as the age and condition of the patient.
  • formulations may also be considered for other means of administration such as parenterally, per rectum, and transdermally.
  • the usefulness of these formulations may depend on the particular compound used and the particular patient receiving the compound.
  • compositions containing a pharmaceutically inert carrier including conventional solid carriers, which are conveniently presented in tablet or capsule form.
  • a pharmaceutically inert carrier including conventional solid carriers, which are conveniently presented in tablet or capsule form.
  • Formulations for rectal or transdermal use may contain a liquid carrier that may be oily, aqueous, emulsified or contain certain solvents suitable to the mode of
  • Suitable formulations are known to those of skill in the art. (Examples thereof may be found, for example, in Remington's Pharmaceutical Sciences, 18th Edition, 1990.)
  • the invention also includes methods of determining whether a test compound causes Upodystrophy in a mammal, preferably a human.
  • the method may include (a) administering the test compound to (i) a mammal, or (ii) a cell expressing CD36 or a mimetic thereof or (iii) an in vitro cell free system capable of expressing CD36 or a mimetic thereof; (b) determining whether the test compound changes the level of CD36 in the mammal, the cell or the system.
  • a change in the level of CD36 is predictive that the mammal will develop Upodystrophy.
  • Any suitable cell may be used, such as a mammalian, insect or yeast cell.
  • Any suitable cell free system may also be used, such as a system capable of expressing CD36 or a mimetic thereof.

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Abstract

This invention relates to a method of predicting those individuals at risk of developing lipodystrophy associated with antiretroviral therapy is disclosed. The method includes the steps of: (a) measuring CD36 levels in the patient prior to protease inhibitor or antiretroviral therapy; (b) measuring CD36 levels in the patient after initiating protease inhibitor or antiretroviral therapy; (c) determining whether CD36 levels after antiretroviral or protease inhibitor therapy are changed after therapy, a change in CD36 levels being predictive of the subsequent development of lipodystrophy. The invention also includes a composition for treating or preventing lipodystrophy associated with PI or antiretroviral therapy. The composition includes PPARη-RXR agonist to up-regulate CD36 in admixture with a pharmaceutically acceptable carrier, excipient, or diluent. The composition can be used in a method to prevent or treat lipodystrophy. The invention also includes a method of determining whether a test compound causes lipodystrophy in a mammal.

Description

METHODS AND COMPOSITIONS FOR PREDICTING. DIAGNOSING AND
TREATING UPODYSTROPHY
FIELD OF THE INVENTION This invention relates to methods and compositions for diagnosing and treating
Upodystrophy. In particular, this invention relates to a method of predicting those individuals at risk of developing Upodystrophy associated with antiretroviral therapy, by measuring a decrease in CD36 levels, as well as compositions and methods for up- regulating CD36 for the treatment of Upodystrophy associated with protease inhibitor therapy for HTV infection.
BACKGROUND OF THE INVENTION
Combination antiretroviral therapy (ARV) that includes protease inhibitors, represents a significant advance in the therapy of HTV-1 infection, conferring virologic, morbidity and survival benefits to users (1-4). Protease inhibitors (Pis) interfere with post-translational processing of viral proteins by binding to the active site of the HTV aspartyl protease (4-7). Pis, in combination with HTV reverse transcription inhibitors, are now in widespread usage as the standard for antiretroviral therapy (8,9). However, all currently used HTV-1 ARV has been associated with side effects that include peripheral fat wasting, central adiposity, hyperlipidemia, and insulin resistance as part of a syndrome known as Upodystrophy (7, 10-12).
The mechanism by which ARV therapy and protease inhibitors (Pis) induce these metabolic alterations is unknown. Carr and colleagues have hypothesized that Pis may mediate these effects by binding to two proteins, cytoplasmic retinoic-acid binding protein 1 (CRABP-1) and low density lipoprotein-receptor-related protein (LRP), that are involved in lipid metabolism (7). If Pis bind to CRABP-1, as speculated, they might inhibit the synthesis of cis-9-retinoic acid, which is the sole ligand for the retinoid X receptor (RXR). RXR functions as a heterodimer with the peroxisome-proliferator- activated receptor type gamma (PPAR-γ), a nuclear receptor which regulates adipocyte differentiation, function and apoptosis (16-19). PPAR-γ has also been demonstrated to promote monocyte/macrophage (mφ) maturation and uptake of oxidized low density lipoprotein (OxLDL) (20). In addition, RXR and PPAR-γ agonists have been shown to improve hyperlipidemia and insulin response in rodent animal models of diabetes and obesity (21,22) but there has been no reported use of these compounds as treatment for HTV -associated Upodystrophy. Furthermore, the molecular details of the cellular receptor(s) which are influenced by RXR-PPAR-γ and which actually mediate the metabolic effects remain unknown. PPAR-γ has been demonstrated to promote the uptake of OxLDL by transcriptional induction of the scavenger receptor CD36 (20). CD36 has also been demonstrated to function as a free fatty acid receptor and transporter in adipose tissue (18,19). Using cDNA microarrays and positional data from linkage studies, Aitman and colleagues recently reported that the phenotype observed in spontaneous hypertensive rats (SHR), which includes defects in fatty acid metabolism, hypertriglyceridemia, and insulin resistance, are linked to defective CD36 expression (23). Furthermore, transgenic mice over-expressing CD36 were shown to have lower blood triglycerides and non-esterified fatty acids (23). Finally, Febbraio and colleagues have recently reported that CD36 knockout mice have elevated cholesterol and free fatty acids indicating that CD36 plays a significant role in lipoprotein and fatty acid metabolism (24). However, none of these studies have linked CD36 levels to
Upodystrophy and in particular that associated with HTV or with PI or antiretroviral use.
Lipodystrophy may be a significant cardiovascular risk factor and several reports of cardiac deaths have been linked to ARV and PI therapy (7, 10-15). This concern together with ARV-induced changes in body appearance are major obstacles to the successful long term use of combination therapy for HTV-1 infection (7, 10-15). For these reasons, many patients and their physicians are turning to Pi-sparing regimens. However, these Pi-sparing regimens are disadvantageous because they are less effective for treating HTV-1 infection, and not all patients on Pis develop this syndrome. Accordingly, there is a need for a method to predict those at risk of developing Upodystrophy and severe metabolic complications, in order to facilitate the appropriate use of this powerful class of antiretroviral drugs. In addition, there is a need for methods and compositions for the treatment of Upodystrophy.
SUMMARY OF THE INVENTION
Accordingly it is an object of the invention to provide a method of predicting which patients receiving antiretroviral or protease inhibitor therapy are at risk of developing Upodystrophy. According to a first aspect of the present invention, a method of predicting the development of Upodystrophy in a patient is provided. The method comprises the steps of:
(a) measuring CD36 levels in the patient prior to ARV therapy;
(b) measuring CD36 levels in the patient after initiating ARV therapy; (c) determining whether CD36 levels after initiating ARV therapy are below the
CD36 levels prior to ARV therapy, the drop in CD36 levels being predictive of the subsequent development of Upodystrophy.
According to a second aspect of the present invention, a composition for the prevention or treatment of Upodystrophy associated with antiretroviral therapy is provided. The composition comprises, as an active ingredient, a product which up- regulates CD36 in admixture with a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, the active ingredient comprises one or more of a PPARγ agonist, a RXR agonist, a PPARγ-RXR agonist and natural or synthetic ligands thereof. According to a third aspect of the invention, a method for the prevention or treatment of Upodystrophy is provided. The method comprises the steps of:
(a) forming a composition by combining one or more of a PPARγ agonist, a RXR agonist, a PPARγ-RXR agonist and natural or synthetic ligands thereof with an acceptable carrier, excipient or diluent; and (b) administering an effective dose of the composition to a person at risk of, or having HTV-associated Upodystrophy. According to a fourth aspect of the present invention, a composition for the treatment of HTV infection is provided. The composition comprises, as an active ingredient, a product which up-regulates CD36 in combination with a pharmaceutically effective amount of a protease inhibitor, or other anti-retroviral agent in admixture with a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, the product comprises a PPARγ-RXR agonist, a PPARγ agonist, a RXR agonist, or natural or synthetic ligands thereof.
According to a fifth aspect of the present invention, a method for treatment of HTV infection is provided. The method comprises the steps of:
(a) forming a composition by combining a protease inhibitor or other antiretroviral agent, one of a PPARγ agonist, PPARγ-RXR agonist, a RXR agonist, and natural or synthetic ligands thereof with an acceptable carrier, excipient or diluent; and (b) administering an effective dose of the composition to a person infected with
HTV. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The invention also includes a method of determining whether a test compound causes Upodystrophy in a mammal comprising:
(a) administering the test compound to (i) a mammal, or (ii) a cell expressing CD36 or a mimetic thereof or (iii) an in vitro cell free system capable of expressing CD36 or a mimetic thereof;
(b) determining whether the test compound changes the level of CD36 in the mammal, the cell or the system, wherein a change in the level of CD36 is predictive that the mammal will develop Upodystrophy. The cell usefully comprises a mammalian, insect or yeast cell. The mammal is preferably a human. The human will typically have HTV infection and/or AIDS.
In one variation, the level of CD36 is measured in a blood sample or a fraction thereof. The cells can include, but are not limited to, mammalian or human monocyte/macrophage cell lines (for example, THP-1, U937 cell lines etc.), mammalian or human adipocyte cell lines (for example 3T3L1 cell line etc.), other mammalian or human cell lines expressing CD36 (for example C32 melanoma cells etc.), or COS, Chinese Hamster Ovary (CHO) cell lines or other mammalian, insect or yeast cells transfected with or expressing CD36. The decrease in CD36 levels can be in the range of
20%-75% or greater than 75%. The tested compound optionally comprises a HTV protease inhibitor or other antiretroviral agent.
Another embodiment of the invention includes a method of (i) identifying an individual at risk of antiretroviral or protease inhibitor-associated Upodystrophy or (ii) diagnosing an individual having antiretroviral or protease inhibitor-induced Upodystrophy comprising the steps of:
(a) measuring the level of CD36 in the individual after a protease inhibitor or other antiretroviral is administered to the individual; (b) determining whether the level of CD36 in the individual is decreased, the decrease in CD36 levels being predictive of the subsequent development of
Upodystrophy or the presence of Upodystrophy in the individual.
The decrease is optionally measured i) relative to the level of CD36 in the individual prior to administration of protease inhibitor or ii) relative to the average CD36 level in a healthy individual. The decrease in CD36 levels can be in the range of 20%- 75% or greater than 75%. The tested compound optionally preferably comprises a HTV protease inhibitor or other antiretroviral agent. DETAILED DESCRIPTION OF THE INVENTION Methods: Patients:
Between February 1999 and July 1999, we studied five healthy volunteers and ten treatment-naive HTV-1 infected individuals prior to and during protease inhibitor therapy. The healthy volunteers were consecutive enrollees in a pharmacokinetic study of ritonavir at the Ottawa General Hospital. Volunteers received 200 mg of ritonavir by mouth, twice daily for 7 days. HTV infected individuals studied were consecutive treatment-naive individuals enrolled in a clinical trial of combination antiretroviral therapy at the Toronto General Hospital Immunodeficiency Clinic. All patients received stavudine (40 mg twice daily) plus lamivudine (150 mg twice daily) and were randomized to receive either nelfϊnavir (750 mg three times daily) or ABT-378/ritonavir (300 mg/100 mg, respectively, twice daily) in a blinded, placebo-controlled manner. These patients were consecutive enrollees in an ongoing prospective trial assessing metabolic and fat distribution manifestations of Upodystrophy. All were asymptomatic and had CD4 counts of >500 cells/μL. None of the subjects were receiving concurrent therapy with corticosteroids, anabolic steroids, or drugs known to modulate immune function. This study was approved by the Institutional review Boards of the Ottawa and Toronto General Hospitals and all subjects gave informed consent.
Measurements:
Thirty mLs of blood was collected in heparin anticoagulant, from controls and HTV-infected subjects prior to commencing ritonavir or combination antiretroviral drug therapy, respectively. Their peripheral monocytes were isolated using separation on Ficoll-paque followed by Percoll gradients as previously described (25,26). Monocytes were fixed in 3% paraformaldehyde and were then stained for CD36 expression by incubation with a 1:100 dilution of the monoclonal anti-CD36 antibody FA6-152 (Immunotech) in RPMI-0 with 1% BSA, on ice for 30 minutes, followed by a 1: 100 dilution of FTTC linked anti-mouse IgG secondary antibody (Serotec). A secondary antibody only and an unstained control were also performed. CD36 expression was quantitated by flow cytometry using the Epics Elite flow cytometer (Beckman-Coulter) on at least 10,000 monocytes when possible. Data was analyzed using Elite software (Beckman-Coulter).
Following 7 days of protease inhibitor therapy (steady state), CD36 expression was again determined on the isolated monocytes as above. CD36 expression was expressed as fluorescence intensity over secondary only stained controls.
We also conducted flow cytometric analysis demonstrating the effects of
protease inhibitor treatment on THP-1 and C32 cells. THP-1 and C32 cells were
cultured for 3 days in the presence of lOμM ritonavir or appropriate controls.
Cells were stained with a monoclonal anti-CD36 antibody followed by a secon¬
dary FTTC conjugated antibody. CD36 expression was analysed by flow cytome-
try. Representative flow cytometry showed (1) levels of CD36 in control treated
and ritonavir treated THP-1 cells, (2) levels of CD36 in control treated and ritona¬
vir treated C32 cells. Histograms summarized the effects of ritonavir treatment on
CD36 levels of THP-1 and C32 cells as determined by flow cytometry. CD36 ex¬
pression was compared between control treated and ritonavir treated cells. CD36
levels of ritonavir treated cells were shown as a percent change from CD36 levels
of control treated cells.
Histograms summarized the effects of protease inhibitor therapy on CD36
levels as determined by flow cytometry. CD36 expression was compared between
baseline (pre-treatment) and after 7 days treatment with protease inhibitors. Post-
treatment levels were presented as a percent change over baseline levels. We de¬
termined (1) levels of monocyte CD36 from the four healthy controls before and
after 7 days of ritonavir therapy (hatched bars), (2) levels of monocyte CD36 from ten HTV- 1 -infected subjects before and after 7 and 30 days of combination ARV
that included ritonavir, nelfinavir, or ABT-378/ritonavir therapy, (3) levels of
monocyte CD36 from the two healthy controls at baseline and after 1 and 2
months of follow up.
Results:
Four of the five volunteers and all ten of the HTV infected individuals continued protease inhibitor therapy and provided a 7 day follow up blood sample. Adherence in controls was confirmed with ritonavir levels. One of the five volunteers withdrew from the study secondary to adverse effects of ritonavir. All patients in the clinical trial demonstrated a marked decrease in viral load on treatment (at 1 month), an indirect measure of adherence.
Seven day treatment with ritonavir resulted in a marked decrease in CD36 surface levels in 3 of the 4 volunteers. In the fourth volunteer, there was no significant change in CD36. Similarly, marked decreases in CD36 ( >75 % decrease) were observed in 6 of 10 HTV-infected individuals, moderate decreases in 2 of 10 (30-40% decrease), and in 2 individuals the levels were unchanged or increased. The decrease in monocyte CD36 was more marked in HTV-infected subjects compared to healthy controls taking Pis. No decrease in CD36 was observed in 5 individuals who received reverse transcriptase inhibitors alone or in the level of monocyte CD36 expressed by healthy individuals over time .
Protease inhibitors affect CD36 expression levels in vitro
THP-1 and C32 cells treated with lOmM ritonavir for a period of 3 days expressed significantly lower CD36 levels than appropriate controls as quantitated by flow cytometry. THP-1 cells, a human pre-monocytic cell line, treated with ritonavir expressed -20% less CD36 than control treated cells (P<0.05, paired Student's t, N=4). C32 cells, a human melanoma cell line, treated with ritonavir showed an -40% decline in CD36 surface expression (P<0.05, paired Student's t, N=5).
Discussion: Antiretroviral therapy that included the protease inhibitors, nelfinavir, ABT-378, and ritonavir, induced a marked decrease in CD36 expression in a proportion of healthy and HTV-infected individuals (-70%) taking these drugs. ARVs and Pis may induce CD36 deficiency by inhibiting retinoic acid metabolism by their action on cytochrome P450 3 A and/or CRABP-1 (7), leading to lowered 9-cis-retinoic acid levels, reduced activation of the RXR-PPARγ heterodimer and decreased CD36 expression (20). Ritonavir has been shown to be the most potent inhibitor of 9-cis-retinoic acid levels and was the most likely to induce Upodystrophy as well (7,10).
Not all individuals taking ARV therapy or protease inhibitors in this study showed reduced levels of CD36. Likewise, most, but not all, HTV patients receiving ARV that includes protease inhibitors develop Upodystrophy (10,12). If ARVs induce marked or persistent CD36 deficiency in a susceptible population of patients, then those patients will be at risk of developing this syndrome. By measuring CD36 expression at baseline and again early during ARV therapy, our findings have shown that CD36 levels predict those at risk of Upodystrophy. In addition, our findings indicate that certain classes of protease inhibitors or other ARV agents are more or less likely to induce these metabolic adverse effects.
In summary, we demonstrate that ARV therapy results in a marked decrease in CD36 in a proportion of users of these drugs. Consequently, measurement of CD36 levels early during treatment with ARVs, predicted those at greatest risk of this syndrome.
The present invention also provides a composition and method of up-regulating CD36 for preventing or treating Upodystrophy associated with protease inhibitor or antiretroviral therapy. PPARγ is a member of the nuclear hormone receptor superfamily, and as a heterodimer with the retinoid X receptor (RXR) activates transcription of target genes by binding to DR-1 (direct repeat with 1 nucleotide spacer) type hormone response elements (27). PPARγ is primarily expressed in adipose tissue, mammary and colonic epithelium and in myelomonocytic cells (28). The CD36 promoter contains a PPAT γ -RXR binding site, and the PPARγ -RXR complex can modulate CD36 gene expression through direct promoter interaction (28). Several natural and synthetic PPARγ and RXR ligands are now known including but not limited to prostaglandins A, D, and especially prostanoid 15-deoxy- ΔI2 14-prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs (including ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone etc.), non- steroidal-anti-inflammatory drugs (NSAIDs including indomethacin, flufenamic acid, fenoprofen, ibuprofen), L-tyrosine-based agonists (including GW1929), cytokines (including TL-4, GM-CSF, M-CSF), lipoproteins and their components (oxidized LDL, 13-hydroxyoctadecadienoic acid [13-HODE], 15-hydroxyeicosatetraenoic acid [15- HETE]), polyunsaturated fatty acids, methoprene acid, vitamin A, and vitamin A metabolites especially 9-cis-retinoic acid, BRL49653, LG100268, LG1069, LGD49653, and compounds which are inhibitors of antagonists of PPARγ and or RXR (22,28-31).
Based on the above observations, established Upodystrophy associated with PI therapy can be reversed by treating individuals with PPARγ agonists, RXR agonists, PPARγ-RXR agonists and natural or synthetic ligands thereof. Similarly Upodystrophy in susceptable patients can be prevented by combining ARV therapy with with PPARγ agonists, RXR agonists, PPARγ-RXR agonists and natural or synthetic ligands thereof . Toward these objectives we have initiated a number of pilot studies. In one prospective cohort study we examine whether different PI regimens and ARV drugs differ in their ability to induce CD36 deficiency and subsequent Upodystrophy. Participants are randomly assigned to different ARV treatment arms and CD36 levels, blood lipids and other metabolic parameters are assessed prior to and after initiating PI treatment. Patients are followed for 24 months to determine the proportion in each treatment arm developing Upodystrophy. In a second prospective cohort study, individuals identified as at increased risk of developing Pi-induced Upodystrophy as determined by CD36 levels, are randomized to a PPARγ-RXR agonist or placebo and followed for 1 year to determine if PI therapy combined with a PPARγ-RXR agonist prevents Upodystrophy. Finally in a pilot study, a sample of consecutive patients with Pi-induced Upodystrophy are randomized to receive a PPAR-RXR agonist or placebo, to demonstrate that the active agents can reverse CD36 deficiency in vivo.
In view of results described above, products which upregulate CD36 can be selected and designed in the manufacture of pharmaceutical compositions for the treatment of Upodystrophy. The pharmaceutical compositions can be administered to patients by methods known to those skilled in the art, such as orally, aerosol administration, direct lavage and parenteral injection. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. The products may be introduced using in vivo delivery vehicles such as liposomes. They may also be introduced into cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes.
The pharmaceutical compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the products are combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA 1990).
On this basis, the pharmaceutical compositions could include an active ingredient, in association with one or more pharmaceutically acceptable vehicles, such as carriers, excipients or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids. The methods of combining suitable products with the vehicles is well known to those skilled in the art. The composition could include a targeting agent for the transport of the active compound to specified sites within tissue.
In vitro, such compounds, peptides or vectors may be administered by infection, microinjection, electroporation and by other methods known in the art.
Oral formulations of the composition according to the present invention are preferred. The oral formulations contain a pharmaceutically inert carrier, including conventional solid carriers, which are conveniently presented in tablet or capsule form.
For those unable to take oral medications, in vivo parenteral administration of the compounds, peptides or peptide mimetics is preferred with subdermal or intramuscular administration most preferred. Intravenous administration or use of implanted milliosmol pumps (available from Alza) may also be used.
When used for parenteral administration, the pharmaceutical compositions of the present invention may be formulated in a variety of ways. Aqueous solutions of composition of the present invention may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art. (Examples thereof may be found, for example, in Remington's Pharmaceutical Sciences, 18th Edition, 1990.) Doses are selected to provide effective upregulation of CD36.
Compositions including a liquid pharmaceutically inert carrier such as water may also be considered for both parenteral and oral administration. Other pharmaceutically compatible liquids may also be used. The use of such liquids is well known to those of skill in the art. (Examples thereof may be found, for example, in Remington's
Pharmaceutical Sciences, 18th Edition, 1990.)
The dose level and schedule of administration may vary depending on the particular product used, the method of administration, and such factors as the age and condition of the patient.
As discussed previously, oral administration is preferred, but formulations may also be considered for other means of administration such as parenterally, per rectum, and transdermally. The usefulness of these formulations may depend on the particular compound used and the particular patient receiving the compound.
Oral formulations of products may optionally and conveniently be used in compositions containing a pharmaceutically inert carrier, including conventional solid carriers, which are conveniently presented in tablet or capsule form. Formulations for rectal or transdermal use may contain a liquid carrier that may be oily, aqueous, emulsified or contain certain solvents suitable to the mode of
administration. Suitable formulations are known to those of skill in the art. (Examples thereof may be found, for example, in Remington's Pharmaceutical Sciences, 18th Edition, 1990.)
The invention also includes methods of determining whether a test compound causes Upodystrophy in a mammal, preferably a human. For example, the method may include (a) administering the test compound to (i) a mammal, or (ii) a cell expressing CD36 or a mimetic thereof or (iii) an in vitro cell free system capable of expressing CD36 or a mimetic thereof; (b) determining whether the test compound changes the level of CD36 in the mammal, the cell or the system. A change in the level of CD36 is predictive that the mammal will develop Upodystrophy. Any suitable cell may be used, such as a mammalian, insect or yeast cell. Any suitable cell free system may also be used, such as a system capable of expressing CD36 or a mimetic thereof.
Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. All modifications coming within the scope of the following claims are claimed.
All publications, patents and patent applications referred to in this application are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. References:
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Claims

WE CLAIM:
1. A method of predicting individuals at risk of protease inhibitor or other antiretroviral-associated Upodystrophy comprising the steps of:
(a) measuring CD36 levels in the patient prior to ARV therapy; (b) measuring CD36 levels in the patient after initiating ARV therapy;
(c) determining whether CD36 levels after ARV therapy are below the CD36 levels prior to ARV therapy, the drop in CD36 levels being predictive of the subsequent development of Upodystrophy.
2. The method of claim 1, wherein the drop in CD36 levels is in the range of 20%-75%.
3. The method of claim 1, wherein the drop in CD36 levels is greater than 75%.
4. The method of claims 1-3, wherein the protease inhibitor is selected from one or more members of a group consisting of ritonavir, nelfinavir, indinavir, saquinavir, and ABT-378.
5. A composition for the treatment of Upodystrophy, comprising, as an active ingredient, a product which up-regulates CD36 in admixture with a pharmaceutically acceptable carrier, excipient or diluent.
6. The composition of claim 5, wherein the active ingredient is selected from one or
more members of a group consisting of a PPARγ agonist, a RXR agonist, a PPARγ-
RXR agonist and natural or synthetic ligands thereof.
7. The composition of claim 7, wherein the active ingredient is selected from one or more members of a group consisting of prostaglandin A, prostaglandin D, prostanoid 15- deoxy-Δ12'14-prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs, non-steroidal-anti-inflammatory drugs (NSATDs), L-tyrosine-based agonists, cytokines, lipoproteins and their components, polyunsaturated fatty acids, methoprene acid, vitamin A, vitamin A metabolites, and compounds which are inhibitors of antagonists of PPAR or RXR, a natural or synthetic ligand of any of the foregoing, and a mimetic of any of the foregoing.
8. The composition of claim 7 wherein the thiazolidinedione (TZD) class of antidiabetic drugs are selected from one or more members of a group consisting of ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone, and a mimetic of any of the foregoing.
9. The composition of claim 7 wherein the non-steroidal-anti-inflammatory drugs (NSATDs) are selected from one or more members of a group consisting of indomethacin, flufenamic acid, fenoprofen, ibuprofen.
10. The composition of claim 7, wherein the L-tyrosine-based agonist is GW1929.
11. The composition of claim 7 wherein the cytokines are selected from one or more members of a group consisting of TL-4, GM-CSF, M-CSF.
12. The composition of claim 7 wherein the lipoproteins and their components are selected from one or more members of a group consisting of oxidized LDL, 13- hydroxyoctadecadienoic acid [13-HODE], 15-hydroxyeicosatetraenoic acid [15-HETE].
13. The composition of claim 7, wherein the vitamin A metabolite is selected from one or more members of a group consisting of a 9-cis-retinoic acid, BRL49653, LG100268, LG1069, LGD49653,and a mimetic of any of the foregoing.
14. A method for treating or preventing Upodystrophy comprising administering to a patient in need thereof a composition as claimed in claims 1-13.
15. A composition for the treatment of HTV infection and/or AIDS, comprising, as an active ingredient, a product which up-regulates CD36 in combination with a pharmaceutically effective amount of a protease inhibitor or other anti-retroviral agent, in admixture with a pharmaceutically acceptable carrier, excipient or diluent.
16. The composition of claim 15, wherein the product is selected from one or more
members of a group consisting of a PPARγ agonist, a RXR agonist ,a PPARγ-RXR
agonist and natural or synthetic ligands thereof.
17. The composition of claim 15, wherein the product is selected from one or more members of a group consisting of prostaglandin A, prostaglandin D, prostanoid 15- deoxy-Δ12 14-prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs, non-steroidal-anti-inflammatory drugs (NSATDs), L-tyrosine-based agonists, cytokines, lipoproteins and their components, polyunsaturated fatty acids, methoprene acid, vitamin A, vitamin A metabolites, and compounds which are inhibitors of
antagonists of PPARγ or RXR, a natural or synthetic ligand of any of the foregoing, and a
mimetic of any of the foregoing.
18. The composition of claim 17 wherein the thiazolidinedione (TZD) class of antidiabetic drugs are selected from one or more members of a group consisting of ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone, and a mimetic of any of the foregoing.
19. The composition of claim 17 wherein the non-steroidal-anti-inflammatory drugs (NSATDs) are selected from one or more members of a group consisting of indomethacin, flufenamic acid, fenoprofen, ibuprofen.
20. The composition of claim 17, wherein the L-tyrosine-based agonist is GW1929.
21. The composition of claim 17 wherein the cytokines are selected from one or more members of a group consisting of JL-4, GM-CSF, M-CSF.
22. The composition of claim 17 wherein the lipoproteins and their components are selected from one or more members of a group consisting of oxidized LDL, 13- hydroxyoctadecadienoic acid [13-HODE], 15-hydroxyeicosatetraenoic acid [15-HETE].
23. The composition of claim 17, wherein the vitamin A metabolite is selected from one or more members of a group consisting of a 9-cis-retinoic acid, BRL49653, LG100268, LG1069, LGD49653,and a mimetic of any of the foregoing.
24. The composition of claims 15-25, wherein the protease inhibitor is selected from one or more of the group including but not limited to ritonavir, nelfinavir, indinavir, saquinavir, and ABT-378.
25. A method for treating HTV infection and/or AIDS comprising administering to a patient in need thereof a composition as claimed in claims 15-25.
26. A method for treating Upodystrophy, comprising the steps of:
(a) forming a composition by combining an active ingredient selected from one or more members of the group consisting of a PPARγ agonist, a PPARγ-RXR agonist, a RXR agonist, and natural or synthetic ligands thereof with an acceptable carrier, excipient or diluent; and (b) administering an effective dose of the composition to a person having
HTV-associated Upodystrophy.
27. The method of claim 26, wherein the active ingredient is selected from one or more members of a group consisting of prostaglandin A, prostaglandin D, prostanoid 15- deoxy-Δ12 14-prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs, non-steroidal-anti-inflammatory drugs (NSAIDs), L-tyrosine-based agonists, cytokines, lipoproteins and their components, polyunsaturated fatty acids, methoprene acid, vitamin A, vitamin A metabolites, and compounds which are inhibitors of antagonists of PPAR or RXR, a natural or synthetic ligand of any of the foregoing, and a mimetic of any of the foregoing.
28. The method of claim 27 wherein the thiazolidinedione (TZD) class of antidiabetic drugs are selected from one or more members of a group consisting of ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone, and a mimetic of any of the foregoing.
29. The method of claim 27 wherein the non-steroidal-anti-inflammatory drugs (NSATDs) are selected from one or more members of a group consisting of indomethacin, flufenamic acid, fenoprofen, ibuprofen.
30. The method of claim 27 , wherein the L-tyrosine-based agonist is GW1929.
31. The method of claim 27, wherein the cytokines are selected from one or more members of a group consisting of TL-4, GM-CSF, M-CSF.
32. The method of claim 27, wherein the lipoproteins and their components are selected from one or more members of a group consisting of oxidized LDL, 13- hydroxyoctadecadienoic acid [13-HODE], 15-hydroxyeicosatetraenoic acid [15-HETE].
33. The method of claim 27, wherein the vitamin A metabolite is selected from one or more members of a group consisting of a 9-cis-retinoic acid, BRL49653, LG100268,
LGD49653, and a mimetic of any of the foregoing.
34. A method for treating HTV, comprising the steps of:
(a) forming a composition by combining a protease inhibitor, or other antiretroviral agent, with a compound selected from one or more members of a group consisting of a PPARγ agonist, PPARγ-RXR agonist, a RXR agonist, and natural or synthetic ligands thereof, the composition being combined with an acceptable carrier, excipient or diluent; and
(b) administering an effective dose of the composition to a person infected with HTV.
35. The method of claim 34, wherein the compound is selected from one or more members of a group consisting of prostaglandin A, prostaglandin D, prostanoid 15- deoxy-Δ12 14-prostaglandin J2 (15d-PGJ2), thiazolidinedione (TZD) class of antidiabetic drugs, non-steroidal-anti-inflammatory drugs (NSAIDs), L-tyrosine-based agonists, cytokines, lipoproteins and their components, polyunsaturated fatty acids, methoprene acid, vitamin A, vitamin A metabolites, and compounds which are inhibitors of antagonists of PPAR or RXR, a natural or synthetic ligand of any of the foregoing, and a mimetic of any of the foregoing.
36. The method of claim 34 wherein the thiazolidinedione (TZD) class of antidiabetic drugs are selected from one or more members of a group consisting of ciglitazone, troglitazone, pioglitazone, rosiglitazone, englitazone, and a mimetic of any of the foregoing.
37. The method of claim 34 wherein the non-steroidal-anti-inflammatory drugs (NSAIDs) are selected from one or more members of a group consisting of indomethacin, flufenamic acid, fenoprofen, ibuprofen.
38. The method of claim 34, wherein the L-tyrosine-based agonist is GW1929.
39. The method of claim 34 wherein the cytokines are selected from one or more members of a group consisting of TL-4, GM-CSF, M-CSF.
40. The method of claim 34 wherein the lipoproteins and their components are selected from one or more members of a group consisting of oxidized LDL, 13- hydroxyoctadecadienoic acid [13-HODE], 15-hydroxyeicosatetraenoic acid [15-HETE].
41. The method of claim 34 wherein the vitamin A metabolite is selected from one or more members of a group consisting of a 9-cis-retinoic acid, BRL49653, LG100268, LGD49653,and a mimetic of any of the foregoing.
42. The method of claims 34-41, wherein the protease inhibitor is selected from one or more of the group including but not limited to ritonavir, nelfinavir, indinavir, saquinavir, and ABT-378.
43. A method of determining whether a test compound causes Upodystrophy in a mammal comprising:
(a) administering the test compound to (i) a mammal, or (ii) a cell expressing CD36 or a mimetic thereof or (iii) an in vitro cell free system capable of expressing CD36 or a mimetic thereof; (b) determining whether the test compound changes the level of CD36 in the mammal, the cell or the system, wherein a decrease in the level of CD36 is predictive that the mammal will develop Upodystrophy.
44. The method of claim 42, wherein the cell comprises a mammalian, insect or yeast cell.
45. The method of claim 42, wherein the mammal is a human.
46. The method of claim 42, wherein the human has HTV infection and/or AIDS.
47. The method of claim 42, wherein the level of CD36 is measured in a blood sample or a fraction thereof.
48. The method of claim 42, wherein the cells comprise mammalian or human monocyte/macrophage cell lines, mammalian or human adipocyte cell lines, mammalian or human cell lines expressing CD36, COS, Chinese Hamster Ovary (CHO) cell lines or mammalian, insect or yeast cell transfected with or expressing CD36.
49. The method of claim 42, wherein the decrease in CD36 levels is in the range of 20%- 75%.
50. The method of claim 42 , wherein the decrease in CD36 levels is greater than 75%.
51. The method of claim 42, wherein the compound comprises a HTV protease inhibitor or other antiretroviral agent.
52. A method of (i) identifying an individual at risk of antiretroviral or protease inhibitor-associated Upodystrophy or (ii) diagnosing an individual having antiretroviral or protease inhibitor-induced Upodystrophy comprising the steps of:
(a) measuring the level of CD36 in the individual after protease inhibitor is administered to the individual;
(b) determining whether the level of CD36 in the individual is decreased, the decrease in CD36 levels being predictive of the subsequent development of Upodystrophy or the presence of Upodystrophy in the individual.
53. The method of claim 52, where the decrease is measured i) relative to the level of CD36 in the individual prior to administration of protease inhibitor or other antiretroviral agent or ii) relative to the average CD36 level in a healthy individual.
PCT/CA2000/001208 1999-10-19 2000-10-18 Methods and compositions for predicting, diagnosing and treating lipodystrophy WO2001028532A2 (en)

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