WO2020128033A1 - Métabolisme cellulaire de l'ensemencement d'un réservoir de vih-1 dans des lymphocytes t cd4+ - Google Patents

Métabolisme cellulaire de l'ensemencement d'un réservoir de vih-1 dans des lymphocytes t cd4+ Download PDF

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WO2020128033A1
WO2020128033A1 PCT/EP2019/086782 EP2019086782W WO2020128033A1 WO 2020128033 A1 WO2020128033 A1 WO 2020128033A1 EP 2019086782 W EP2019086782 W EP 2019086782W WO 2020128033 A1 WO2020128033 A1 WO 2020128033A1
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cells
hiv
metabolic
infection
infected
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Asier Saez-Cirion
Jose Carlos VALLE-CASUSO
Michaela Muller-Trutwin
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Institut Pasteur
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • the present invention relates to compounds and methods for partially inhibiting of cellular metabolism, and particularly glycolysis, in cells targeted by pathogens, such as human immunodeficiency viruses.
  • Combination antiretroviral treatment blocks HIV-1 replication but does not eliminate infected cells.
  • Replication competent HIV-1 persists in cellular reservoirs that are the origin of rapid viral rebound when treatment is interrupted (Finzi et al., 1997). Identifying the factors underlying the seeding and survival of HIV-infected cells is a priority in the search for an HIV cure (Deeks et al., 2016).
  • CD4+ T-cells are the major target for HIV-1 infection and are thought to constitute most of the HIV-1 reservoir. However, not all CD4+ T-cells contribute equally to the pool of persistently infected cells during cART.
  • the composition of CD4+ T-cells that remain infected is mainly determined by the susceptibility of CD4+ T-cell subsets to HIV infection, their resistance to HIV-induced apoptosis and their life span and turnover potential (Barton et al., 2016).
  • Naive CD4+ T-cells are highly resistant to HIV-1 infection, while HIV-1 susceptibility increases in more differentiated cell subsets (Roederer et al., 1997; Thomasman et al., 1990). Accordingly, there is a minimal contribution of naive CD4+ T-cells to the HIV reservoir during cART, which is mainly restricted to the memory cell subsets (Chomont et al., 2009).
  • CD4+ T-cells The susceptibility of CD4+ T-cells to HIV-1 infection depends on the relative abundance of cell factors required by the virus to complete its replication cycle and of cellular restriction factors that counteract infection (Lever and Jeang, 2011). T-cell activation sharply increases the expression of HIV dependency factors and thereby cell susceptibility to HIV-1 infection (Pan et al., 2013; Stevenson et al., 1990), despite the concomitant presence of some restriction factors that the virus can most often circumvent. However, responsiveness to TCR activation (Byrne et al., 1988; Roederer et al., 1997) and susceptibility to HIV infection are not homogeneous across or within CD4+ T-cell subsets.
  • the glucose transporter 1 (GLUT) is the main receptor for HTLV-1 (Manel et al., 2003); phosphate transporters PiTl and PiT2 have been reported as surface receptors for koala retrovirus, feline leukemia virus and murine leukemia viruses (Oliveira et al., 2006; Takeuchi et al., 1992; von Laer et al., 1998); and the amino acid transporters ASCT1 and ASCT2 are the receptors for the feline RD-114 endogenous retrovirus (Shimode et al., 2013).
  • HIV-1 does not use metabolite transporters as its main receptors
  • GLUT1 expression is necessary for the post entry steps of HIV-1 replication in CD4+ T-cells (Loisel-Meyer et al., 2012).
  • the metabolism of nucleotides is critical for HIV-1 reverse transcription (Amie et al., 2013).
  • the invention encompasses compositions comprising metabolic inhibitors and methods of using these compositions.
  • the invention encompasses a method of reducing HIV production from a T cell comprising contacting a population of cells comprising HIV-infected CD4+ T cells with 2-deoxy-glucose (2-DG) at a concentration that reduces the production of HIV from said HIV-infected CD4+ T cells.
  • the cells are exposed to a concentration of 1-25 mM 2-DG.
  • the cells are exposed to a concentration of 3-10 mM 2-DG.
  • the cells are exposed to a concentration of 5 mM 2-DG.
  • the method further comprises contacting the population of cells with a metabolic inhibitor that blocks fatty acid transport to mitochondria.
  • the metabolic inhibitor is Etomoxir.
  • the cells are contacted with the metabolic inhibitor in vitro. In one embodiment, the cells are contacted with the metabolic inhibitor in vivo. In one embodiment, the cells are contacted with an additional metabolic inhibitor of metabolic function in CD4+ T cells.
  • the population of cells has received cART treatment.
  • the cART can comprise Combivir, Kaletra, Trizivir, Epzicom, Kivexa, Truvada, Atripla, Complera, Eviplera, Stribild, Triumeq, Evotaz, Prezcobix, Dutrebis, Genvoya, or Descovy.
  • the cART can comprise at least 2 or 3 of any of the following compounds: lamivudine; zidovudine; lopinavir; ritonavir; abacavir; tenofovir disoproxil fumarate; emtricitabine; efavirenz; rilpivirine; elvitegravir; cobicistat; dolutegravir; atazanavir; cobicistat; darunavir; and raltegravir.
  • the invention encompasses a method of reducing virus or bacterial pathogen production from a cell comprising contacting a population of cells pathogen-infected cells with at least 2 metabolic inhibitors of different metabolic functions in the cells at a concentration that reduces the production of the pathogen from said pathogen-infected cells.
  • one of the metabolic inhibitors blocks glycolysis. In one embodiment, one of the metabolic inhibitors blocks fatty acid transport to mitochondria. In one embodiment, both of the metabolic inhibitors are selected from Table 1. In one embodiment, two of the metabolic inhibitors are DON and 2DG. In one embodiment, three of the metabolic inhibitors are DON and etomoxir and 2DG. In one embodiment, concentration of the metabolic inhibitor(s) is a suboptimal concentration.
  • the pathogen is HIV-1.
  • the invention encompasses a method for measuring the effect of a metabolic inhibitor of metabolic function in cells in a pathogen-infected human comprising: administering at least one dose of a metabolic inhibitor of metabolic function in cells to the human; and measuring the level of pathogen infection in the pathogen-infected human.
  • measuring the level of pathogen infection in the pathogen-infected human comprises measuring the level of plasma HIV RNA in an HIV-infected human.
  • measuring the level of plasma HIV RNA in the HIV-infected human is performed by a reverse transcription and amplification reaction.
  • the level of HIV infection in the human is measured at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
  • Figure 1A-D depict CD4+ T-cells subsets have different susceptibilities to HIV-1 infection.
  • Figure 1A depicts representative example of the proportion of 5-days activated CD4+ T cells expressing GFP in the absence of infection (top) or 72 h after challenge with HIV-1 G FP-VSV (bottom).
  • Figure IB depicts relative distribution of CD4+ T-cell subsets in non-activated (NA) and activated (aCD3 5d) cells before HIV challenge and in activated cells not expressing GFP (aCD3 5d GFP-) or expressing GFP (aCD3 5d GFP+) 72 h post challenge.
  • the bottom panels represent the fold change in the CD4+ T- cells subset contribution relative to the non-activated condition (NA). *p ⁇ 0.05; ** p ⁇ 0.01.
  • sorted CD4+ T-cell subsets were cultured under NA or activated conditions for 3 (3d) or 5 days (5d) and challenged with HIV-1 G FP-VSV.
  • Figure 1C depicts representative example of infection levels in Tn, Tcm, Ttm and Tern cells from a donor in the different conditions analyzed.
  • Figure ID depicts Medians and IQR values for experiments with cells from 6 donors. Symbols represent the individual data points. Significant differences between experimental conditions are shown for each T-cell subset as horizontal lines. The median infection level in NA Tn cells is displayed as a reference dashed line to facilitate comparison between T-cell subsets.
  • Figure 2A-B depict HIV-1 infection levels in CD4+ T-cell subsets correlate with the expression levels of genes related to cell metabolism.
  • Figure 2B shows Spearman's correlation between the levels of gene expression at the time of HIV-1 challenge and HIV-1 infection levels 72 h after challenge. Only significant correlations (p ⁇ 0.05) are represented in the graphs (green bars). Genes highlighted in red show the group of genes that correlated with infection levels in all conditions.
  • Figure 3A-C depict CD4+ T-cell subsets have different metabolic profiles that coincide with their susceptibility to HIV-1 infection OCR and ECAR in non-activated (NA), 3-day activation (3d) and 5-day activation (5d) CD4+ T-cell subsets.
  • Figure 3A depicts Median values of the metabolic variables obtained for the CD4+ T-cell subsets from the 6 donors in the different conditions analyzed.
  • Figure 3B depicts Median and IQR basal OCR (left panel) and ECAR (right panel).
  • Figure 3B depicts basal ECAR/OCR ratio for CD4+ T-cell subsets in different activation states (Od, 3d, 5d). Median values in NA Tn cells are indicated by dashed lines as a reference.
  • Figure 3C shows a Summary of correlations between metabolic parameters at the time of infection in NA, 3d and 5d activated CD4+ T-cell subsets and the % of infected cells 72 h post infection.
  • the green color indicates p ⁇ 0.05.
  • the size of the circle represents Spearman's coefficients.
  • Figure 4A-C depict HIV-l-infected CD4+ T-cells are characterized by higher metabolic activity levels.
  • OCR and ECAR Metabolic activity
  • XF bioenergetic phenotypes of GFP+ and GFP- cells
  • Figure 4C depicts Representative analyses of OCR and ECAR (measured as above) for each cell fraction (left) and the median and IQR basal OCR and ECAR for 6 (high activation) and 4 (low activation) donors (right).
  • Figure 5A-B depicts rate of glucose uptake by CD4+ T-cell subsets is associated with their susceptibility to HIV-1 infection CD4+ T cells were sorted based on their differentiation status (Tn or Tcm) and their rate of 2NBDG uptake. Sorted cells were then challenged with HIV-l GFp - VSV.
  • Figure 5A depicts Representative example of 2NBDG content after sorting (top panels) and the levels of GFP expression 72 h after challenge in CD4+ T-cell fractions exposed (HIV-1) or not (control) to the virus.
  • Figure 6A-F depict inhibition of cell metabolic pathways blocks HIV-1 infection of CD4+ T- cells.
  • Figure 6B depicts Infection and cell death in CD4+ T-cells exposed to HIV-1 in glucose-containing medium in the absence or presence of 2-DG (5 mM) or in culture medium without glucose (starvation) (left) or in the absence or presence of UK5099 (25 mM) (right).
  • Figure 6C depicts Relative number of U5-Gag copies in CD4+ T-cells at 6h, 15h or 72h after infection with H IV-1 G FP- VSV in the absence or presence of 2-DG. Individual values (symbols), medians and IQRs (horizontal lines) for five different donors are shown.
  • Figure 6F depicts Percentage of HIV-1 productively (left panel) or latently (right panel) infected cells 72h after the infection of CD4+ T-cells with HIV-lDuoFluo VSVG particles in the presence of 2-DG or etomoxir. Median and IQR values from experiments with 6 donors are shown.
  • Figure 6G depicts p24 production in supernatants from CD4+T-cell cultures 3 and 7 days after infection with HIV-1 BaL in the absence (blue bars) or presence of 2- DG (5 mM) (orange bars). Means and standard deviations for three replicates are shown at each time point for experiments done with cells from three different donors.
  • Figure 7A-D depict suboptimal inhibition of glucose metabolism selectively eliminates preinfected CD4+ T-cells and inhibits HIV-1 amplification from reservoirs.
  • Figure 7A depicts cell viability in sorted pre-infected GFP+ (green) or noninfected GFP- (red) CD4+ T cells cultured for 48 h in the absence or presence of 2-DG. One representative example is shown.
  • Figure 7B depicts relative survival of 2-DG treated cells (circles) was compared to that of nontreated cells (squares) at 24 h and 48 h.
  • Figure 7C depicts changes in the CD4+ T-cell subset distribution 48h after the treatment of infected bulk CD4+ T-cells with 2-DG when compared with the distribution in the control condition.
  • Figure 7D depicts HIV-1 reactivation from CD4+ T-cells from six individuals on cART upon PHA/IL-2 stimulation in the absence (blue line/symbols) or presence of 2-DG (5 mM) (orange line/symbols) (mean and SD, 3 replicates). Mean p24 values in the absence or presence of 2-DG on day 14 post stimulation are shown for all six experiments (right panel).
  • Figure 8 depicts treatment of CD4+ T cells from the same donor with various metabolic inhibitors at the indicated concentrations, alone or in combination, and shows the synergistic effect of suboptimal amounts of 2DG, DON and ETOMOXIR on the extent of HIV-1 infection.
  • the levels of 2-NDBG captured by the different CD4+ T cell subsets are shown in the overlapping histogram (middle panels).
  • the relative contributions of CD4+ T cell subsets to the low, medium and high 2-NBDG cell fractions are displayed in the cumulative bar chart (lower panels).
  • A) Flow cytometry gating strategy used to sort CD4+ Tn and Tcm cells according to their glucose (2- N BDG ) u pta ke levels a nd the 2-N BDG content of the CD4+ T cel l fractions after sorti ng. Representations are displayed as standard pseudocolor dot plots. Sorted cells were used for HIV-1 susceptibility assays.
  • B) Representative example (left) and summary (median and IQR, n 3 donors) (right) of the glycolytic activity of sorted Tcm cells with high glucose uptake (TcmHG Iu) and low glucose uptake (TcmLGIu).
  • FIG 15 A representative example of GFP and mCherry expression in noninfected CD4+ T cells or in CD4+ T cells 72 h post infection with the HIV Duo- Fluo I virus.
  • I productively infected
  • L latently infected
  • Nl noninfected
  • B) Relative infection levels (with respect to naive cells) in 5 days-activated CD4+ T cells from donors (n 9) 72h after chal lenge with H IV Bal or VSVG pseudotyped N L4.3AenvGFP particles.
  • I nfection levels were determined by flow cytometry quantification of intracellular p24 and G FP respectively (top panels). Changes in the distribution of cell subsets in HIV+ cells in relation to non- infected cells (bottom panels). The asterisks represent statistically significant differences (*p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001). C) I nfection levels in 5 days
  • Transcript profiling at the time of infection showed that among the CD4+ T-cell subsets, there were positive correlations between the frequencies of HIV-infected cells and the expression levels of multiple genes related to cell metabolism. Negative correlations were found between the susceptibility of CD4+ T-cells to HIV-1 infection and the expression of SAMHD1, an efficient HIV-1 restriction factor that also plays an important role in the regulation of cell metabolism (Descours et al., 2012; Mathews, 2015). Surprisingly, strong positive correlations were found between the levels of HIV-infected cells and the expression of a cluster of genes related to the interferon response.
  • T-cell activation and cellular metabolism Although there are well-established links between T-cell activation and cellular metabolism, it is increasingly clear that T-cell functions, including proliferation, the secretion of cytokines and cell survival, are supported through different engagements of the various metabolic pathways (Jones and Bianchi, 2015). This may explain the partial dichotomy between T-cell activation and cell metabolism in HIV infection that we observed in our experiments. Additionally, we found Tn cells expressing high levels of activation markers upon anti-CD3 stimulation, but these cells remained mostly resistant to HIV-1 infection. In contrast, the frequency of infected Tn cells sharply increased when we challenged highly glycolytic Tn cells.
  • HIV infection has been shown to induce increased expression of several glucose transporters in in vitro experiments (Kavanagh Williamson et al., 2018; Sorbara et al., 1996). Overall, viruses appear to possess different mechanisms to enhance cell metabolism to favor viral replication (Goodwin et al., 2015; Sanchez and Lagunoff, 2015), and this deserves additional exploration in the context of HIV infection.
  • CD4+ T-cells expressing PD-1 and other immune checkpoints are enriched in HIV in HIV- infected individuals receiving cART (Banga et al., 2016; Chomont et al., 2009; Fromentin et al., 2016).
  • these immune checkpoints appear to mediate their inhibitory activities through the metabolic reprogramming of the cells (Lim et al., 2017; Patsoukis et al., 2015). This suggests that the metabolic requirements of HIV-1 replication might enduringly imprint the infected cells.
  • the invention provides compositions, methods and uses of a compound for inhibiting and treating viral and bacterial pathogen infections (e.g. HIV infections) and methods for assessing the effects of these compounds on pathogen infections.
  • Compounds that inhibit the metabolic function cells can be used for inhibiting and treating pathogen infections in vitro and in vivo.
  • compounds that inhibit the metabolic function of CD4+ cells can be used for inhibiting and treating HIV infections in vitro and in vivo.
  • the invention encompasses various screening methods for determining the effect of a compound that inhibits the metabolic function of cells in a pathogen-infected human and in vitro.
  • metabolism refers to chemical transformations within living cells that serve three main purposes: the conversion of food or fuel to energy to run cellular processes, the conversion of food or fuel to building blocks for proteins, lipids, nucleic acids and carbohydrates, and the elimination of waste.
  • metabolic function means a defined metabolism pathway, not including upstream or downstream effects.
  • the metabolic function(s) is(are) selected from those listed in Table 1 and/or Table 2.
  • metabolic enzyme means an enzyme within a defined metabolism pathway. More preferably, the targeted metabolic enzyme(s) is(are) selected from those listed in Table 1.
  • metabolic inhibitor means an inhibitor of metabolic process or enzyme within a defined metabolism pathway. Most preferably, the metabolic inhibitor(s) is(are) selected from those listed in Table 1.
  • the metabolic inhibitor (metabolic pathway/enzyme) is selected from l-DON (Glutaminolysis), 2DG (Glycolysis), etomoxir (fatty acid transport/ Carnitine palmitoyltransferase-1), UK5099 (mitochondrial pyruvate carrier (MPC)), BPTES (Glutaminolysis), Orlistat (fatty acid synthesis), Metformin (fatty acid oxydation), and Rapamycin (mTOR).
  • the metabolic inhibitor is active in human CD4+ cells and/or macrophages.
  • l-DON or DON refers to 6-Diazo-5-oxo-L-norleucine, also named as 6-diazo-5-oxo-l-norleucine or (L)-DON.
  • the invention encompasses various screening methods for determining the effect of a metabolic inhibitor that inhibits the metabolic function of cells, preferably CD4+ T cells, in a human, preferably a Human Immunodeficiency Virus-infected human, and in vitro.
  • the method comprises administering at least one dose of the metabolic inhibitor to a human or to cells in vitro; and measuring the level of pathogen (e.g., HIV) infection in the human or cells.
  • pathogen e.g., HIV
  • the metabolic inhibitor is active in human CD4+ cells.
  • the method comprises administering at least one dose of the metabolic inhibitor to the human or cells; and measuring the level of plasma HIV RNA in the human or in the cell supernatant. In one embodiment, the method comprises administering at least one dose of the metabolic inhibitor to the human; and measuring the level of HIV-infected reservoir cells in the human.
  • the metabolic inhibitor reduces glycolysis or blocks fatty acid transport to mitochondria.
  • the metabolic inhibitor is administered a concentration that reduces the production of HIV from HIV-infected CD4+ T cells.
  • the metabolic inhibitor is 2-deoxy-glucose (2-DG).
  • the cells are exposed to a concentration of 1-25 mM 2-DG, 3-10 mM 2-DG, or 5 mM 2-DG.
  • the metabolic inhibitor is Etomoxir.
  • the metabolic inhibitor can be used by itself, or preferably, in combination with additional metabolic inhibitor, preferably one that affects a different metabolic function.
  • the metabolic inhibitor targets one or more of the metabolic functions or enzymes listed in Table 1 and/or Table 2.
  • the metabolic inhibitors listed in Table 1 and Table 2, and modified versions thereof, are particularly preferred.
  • the measurement can provide for a comparison to another infected individual that does not receive the metabolic inhibitor or to a prior measurement from that same infected individual, preferably before treatment with the metabolic inhibitor.
  • the measurement of the level of HIV infection in the human is performed at least twice.
  • the measurement is taken 3, 4, 5, 6, 7, 8, 9, or 10 times. In this way, the measurements can provide for a comparison over time within that infected individual, most preferably with a measurement taken before treatment with the metabolic inhibitor
  • the level of HIV infection can be assessed by different techniques known to the skilled artisan.
  • the level of HIV infection in the human can be determined by measuring the level of plasma HIV RNA in the human.
  • the level of plasma HIV RNA in the human can be measured by a reverse transcription and amplification reaction.
  • reverse transcription of the RNA of an HIV can be performed with a "reverse primer” specific for HIV.
  • a "reverse primer” is one that, based on its 5'-3' orientation, can bind to a single-stranded RNA and serve to initiate generation of a complementary DNA (cDNA) copy of the RNA.
  • the reverse transcription can be accomplished using well known and routine methods.
  • the reaction mix for reverse transcription contains the reagents for the reaction, for example, a reverse primer, dNTPs (dATP, dCTP, dGTP and dTTP), a buffer, and a reverse transcriptase. Exemplary reaction conditions are set forth in the examples.
  • Amplification of the cDNA copy of an HIV generated by reverse transcription can be performed with a "forward primer” specific for HIV.
  • a "forward primer” is one that, based on its 5'-3' orientation, can bind to a single-stranded antisense cDNA copy of an RNA generated by reverse transcription and serve to initiate generation of a double-stranded DNA copy of the RNA.
  • the amplification can be accomplished using well known and routine methods.
  • the reagent mix for amplification contains the reagents for the reaction, for example a forward primer, a reverse primer, dNTPs, a buffer, and a DNA polymerase.
  • the method of the invention is performed using a single RT-PCR reagent mix containing the reagents for the reverse transcription and amplification reactions.
  • the reverse primer used for the reverse transcription reaction is also used for the amplification reaction.
  • the reverse transcription and amplification reactions are performed in a plastic or glass container, most preferably in the same container.
  • Amplification methods known in the art include RCA, MDA, NASBA, TMA, SDA, LCR, b- DNA, PCR (all forms including RT-PCR), RAM, LAMP, ICAN, SPIA, QB-replicase, or Invader.
  • a preferred amplification method is the polymerase chain reaction (PCR) amplification. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. linis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res.
  • PCR polymerase chain reaction
  • PCR-HRM High-Resolution DNA Melting
  • Amplification techniques include in particular isothermal methods and PCR-based techniques.
  • Isothermal techniques include such methods as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA), exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others (see e.g. Asiello and Baeumner, Lab Chip; 11(8): 1420-1430, 2011).
  • NASBA nucleic acid sequence-based amplification
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependent amplification
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • EXPAR exponential amplification reaction
  • ICANs isothermal and chimeric primer-init
  • the PCR technique quantitatively measures starting amounts of DNA, cDNA, or RNA.
  • PCR-based techniques include techniques such as, but not limited to, quantitative PCR (Q-PCR), reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative reverse-transcriptase PCR (QRT-PCR), or digital PCR. These techniques are well known and easily available technologies for those skilled in the art.
  • the method is a one-step real-time RT-PCR assay, for example, as described in the Examples.
  • the method is a one-step real-time RT-PCR assay based on TAQMAN probe technology capable of detecting the recently described African E and F genogroups and including a competitive RNA internal control (1C), for example, as described in the Examples.
  • a probe is used to detect the amplified product.
  • the probe can be labeled with a fluorescent, radioactive, or enzymatic label.
  • the amplified product can be detected with a specific detection chemistry such as fluorescence resonance energy transfer (FRET) probes, TAQMAN probes, molecular beacons, scorpion probes, fluorescently labeled (or other labeled) primers, lightup probes or a dye-based chemistry, DNA, PNA, LNA, or RNA including modified bases that bind to the amplified product to detect the sequence of interest.
  • FRET fluorescence resonance energy transfer
  • Detection of the amplified products can be real-time (during the amplification process) or endpoint (after the amplification process).
  • the invention allows for detection of the amplification products in the same vessel as amplification occurs.
  • a DNA internal control is used to monitor the amplification reaction.
  • RNA internal control is used to monitor the reverse transcription and amplification reactions.
  • the metabolic inhibitor e.g., 2-DG
  • the metabolic inhibitor is administered in at least one administration of 1-200 mg/kg/day 5-160 mg/kg/day, 10-80 mg/kg/day, 20-70 mg/kg/day, 30-60 mg/kg/day, or 20-40 mg/kg/day.
  • the administration is at least 1-5, 5-10, 10-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, or 140-160 mg/kg/day.
  • the administration is at least 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, or 160 mg/kg/day of the metabolic inhibitor.
  • the administration is at least 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, or 160 mg/kg/day of 2-DG.
  • the administration of the metabolic inhibitor can be by many methods known in the art, most preferably intravenous, intra-arterial, subcutaneous, intramuscular, sublingual, transmucosal, or oral.
  • multiple administrations are given.
  • at least 1-100, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-20, 20-50, or 50-100, administrations are given.
  • the administration is at least twice/ day, twice/week, once/day, once/week, three times/week, or once/every 2 days.
  • the administration can be given continuously (e.g., with a pump).
  • At least .01, .05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, 120, 160 mg/kg/day of a metabolic inhibitor is administered for at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, or 1, 2, 3, 4, 5, 6, etc. months.
  • At least .01, .05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, 120, 160 mg/kg/day is administered every 2 days or 3 times/week for at least 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, or 1, 2, 3, 4, 5, 6, etc. months.
  • Pathogens within the scope of the invention include:
  • Acinetobacter baumannii Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans
  • the invention encompasses methods of treatment using the metabolic inhibitors described above and the use of compositions comprising these metabolic inhibitors in the treatment of a pathogen infection in a human patient.
  • the method comprises administering at least one metabolic inhibitor to a pathogen-infected human at a concentration that reduces the production of the pathogen from said pathogen-infected cells. In one embodiment, the method comprises administering at least 2 metabolic inhibitors of different metabolic functions in the cells at concentrations that reduce the production of the pathogen from said pathogen-infected cells.
  • a suboptimal dosage of the metabolic inhibitor is administered.
  • the term “suboptimal” refers to a dose at which the metabolic inhibitor does not have on its own maximum effects either on the metabolic pathways, but also do not cause more than 10% cell death of the corresponding uninfected cells (e.g. uninfected CD4+ T cells).
  • the method comprises administering an effective amount of at least one metabolic inhibitor to an HIV-infected human.
  • An effective amount is an amount of the metabolic inhibitor(s) that reduces the level of detectable pathogen (e.g., plasma HIV RNA in an HIV-infected patient) at least 2-fold.
  • the administration of metabolic inhibitor(s) reduces the level of detectable pathogen (e.g., plasma HIV RNA in an HIV-infected patient) at least 2-, 4-, 10-, 30-, 50-, or 100-fold.
  • the administration of the metabolic inhibitor reduces the pathogen (e.g., viral or bacterial) load in the patient at least 2-, 4-, 10-, 30-, 50-, or 100-fold. In some embodiments, the administration of the metabolic inhibitor reduces the number of HIV-1 infected reservoir cells at least 2-, 4-, 10-, 30-, 50-, or 100-fold. In some embodiments, the administration of the metabolic inhibitor reduces active viral or bacterial replication at least 2-, 4-, 10-, 30-, 50-, or 100-fold. .
  • pathogen e.g., viral or bacterial
  • the above reductions can be determined by routine techniques in the art, such as by comparing the levels in the patient before and after administration of the metabolic inhibitor, for example by standard PCR amplification methods with patient plasma samples.
  • the reduction can be assessed at various times after administration, for example at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 40, or 52 weeks after administration of the metabolic inhibitor.
  • the metabolic inhibitor is administered in at least one administration of 1-200 mg, 5-160 mg, 10-80 mg, or 20-40 mg.
  • the administration is at least .01-0.1, 0.1-1, 1-5, 5-10, 10-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, or 140- 160 mg.
  • the administration is at least .01, .05, 0.1, 0.5, 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, or 160 mg of the metabolic inhibitor.
  • the administration of the metabolic inhibitor can be by many methods known in the art, most preferably intravenous, intra-arterial, subcutaneous, intramuscular, sublingual, transmucosal, or oral.
  • multiple administrations are given.
  • at least 1-100, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-20, 20-50, or 50-100, administrations are given.
  • the administration is at least twice/ day, twice/week, once/day, once/week, three times/week, or once/every 2 days.
  • At least .01, .05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, 120, 160 mg/kg/day is administered for at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, or 1, 2, 3, 4, 5, 6, etc. months.
  • At least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 100, 120, 160 mg is administered every 2 days or 3 times/week for at least 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, or 1, 2, 3, 4, 5, 6, etc. months.
  • the methods, uses, and compositions of this invention can be used with HIV-infected patients.
  • the patient is infected with Human Immunodeficiency Virus type
  • the patient is infected with Human Immunodeficiency Virus type
  • HIV-1 infected patient is acutely infected with HIV. In one embodiment, the HIV-1 infected patient is chronically infected with HIV.
  • the HIV-1 infected patient is undergoing cART. In one embodiment, the HIV-1 infected patient has never initiated cART. In various embodiments, the HIV-1 infected patient has previously undergone cART, and either ceases or continues cART.
  • the invention encompasses pharmaceutical compositions comprising one or more metabolic inhibitors.
  • the compositions are preferably for the treatment of a pathogen infection in a human, particularly, preferably comprising a combination of at least two, three, or four metabolic inhibitors.
  • the invention further encompasses the use of these compositions in the manufacture of a medicament for the treatment of a pathogen infection and the use of these compositions in the treatment of a pathogen infection.
  • the composition contains 1-200 mg, 5-160 mg, 10-80 mg, or 20- 40 mg of one or more metabolic inhibitor.
  • the composition contains at least 1-5, 5- 10, 10-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, or 140-160 mg of one or more metabolic inhibitor.
  • all values and subranges within the above ranges are specifically included as if explicitly written out.
  • the metabolic inhibitors may also be advantageously administered for therapeutic purposes together with other metabolic inhibitors, such as HIV inhibitors, particularly cART, known in the general art to be of value in treating HIV infection.
  • Particularly preferred combinations contain at least one, two, three, or four of the HIV inhibitors listed below.
  • Most preferably, the combination contains at least one of the combination antiretroviral therapies listed below.
  • Effective concentrations or amounts of a metabolic inhibitor can be mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration to form pharmaceutical compositions.
  • the metabolic inhibitor is included in an amount effective for treating the pathogen (e.g., HIV) infection.
  • the concentration of active agent in the composition will depend on absorption, inactivation, excretion rates of the active agent, the dosage schedule, amount administered, particular formulation as well as other factors known to those of skill in the art.
  • compositions are intended to be administered by a suitable route, including by way of example and without limitation orally, parenterally, rectally, topically and locally.
  • a suitable route including by way of example and without limitation orally, parenterally, rectally, topically and locally.
  • capsules and tablets can be used for oral administration.
  • the compositions are in liquid, semi-liquid or solid foul and are formulated in a manner suitable for each route of administration.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components, in any combination: a sterile diluent, including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent including by way of example without limitation, water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
  • antimicrobial agents such as benzyl alcohol and methyl parab
  • solubilizing agents may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using co-solvents, such as dimethylsulfoxide (DMSO), using surfactants, such as TWEEN ® , or dissolution in aqueous sodium bicarbonate.
  • co-solvents such as dimethylsulfoxide (DMSO)
  • surfactants such as TWEEN ®
  • dissolution in aqueous sodium bicarbonate such as sodium bicarbonate.
  • Pharmaceutically acceptable derivatives of the agents may also be used in formulating effective pharmaceutical compositions.
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier or vehicle.
  • the effective concentration is sufficient for treating one or more symptoms of at least one disease state.
  • the pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable quantities of the agents or pharmaceutically acceptable derivatives thereof.
  • the pharmaceutically therapeutically active agents and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms.
  • Unit-dose foams as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active agent sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof.
  • a multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.
  • the composition can contain along with the active agent, for example and without limitation: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.
  • a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose
  • a lubricant such as magnesium stearate, calcium stearate and talc
  • a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active agent as defined above and optional pharmaceutical adjuvants in a carrier, such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, by way of example and without limitation, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, such as, by way of example and without limitation, acetate, sodium citrate, cyclodextr
  • compositions containing active agent in the range of 0.005% to 100% with the balance made up from non-toxic carrier may be prepared.
  • a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example and without limitation, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose, magnesium carbonate or sodium saccharin.
  • compositions include solutions, suspensions, tablets, capsules, powders and sustained release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparation of these compositions are known to those skilled in the art.
  • the contemplated compositions may contain 0.001%-100% active agent, such as 0.1-85%, or such as 75-95%.
  • the active agents or pharmaceutically acceptable derivatives may be prepared with carriers that protect the agent against rapid elimination from the body, such as time release formulations or coatings.
  • the compositions may include other active agents to obtain desired combinations of properties.
  • Oral pharmaceutical dosage forms include, by way of example and without limitation, solid, gel and liquid.
  • Solid dosage forms include tablets, capsules, granules, and bulk powders.
  • Oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated.
  • Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent forms with the combination of other ingredients known to those skilled in the art.
  • the formulations are solid dosage forms, such as capsules or tablets.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or agents of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.
  • binders include, by way of example and without limitation, microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose, and starch paste.
  • Lubricants include, by way of example and without limitation, talc, starch, magnesium or calcium stearate, lycopodium and stearic acid.
  • Diluents include, by way of example and without limitation, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate.
  • Glidants include, by way of example and without limitation, colloidal silicon dioxide.
  • Disintegrating agents include, by way of example and without limitation, crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.
  • Coloring agents include, by way of example and without limitation, any of the approved certified water soluble FI) and C dyes, mixtures thereof; and water insoluble ID and C dyes suspended on alumina hydrate.
  • Sweetening agents include, by way of example and without limitation, sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
  • Flavoring agents include, by way of example and without limitation, natural flavors extracted from plants such as fruits and synthetic blends of agents which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.
  • Wetting agents include, by way of example and without limitation, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene laural ether.
  • Emetic-coatings include, by way of example and without limitation, fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.
  • Film coatings include, by way of example and without limitation, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
  • the agent could be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active agent in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the agents can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like.
  • a syrup may contain, in addition to the active agents, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics.
  • Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents.
  • Enteric-coated tablets because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines.
  • Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied.
  • Film-coated tablets are compressed tablets which have been coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned.
  • Coloring agents may also be used in the above dosage forms.
  • Flavoring and sweetening agents are used in compressed tablets, sugar-coated, multiple compressed and chewable tablets. Flavoring and sweetening agents are useful in the formation of chewable tablets and lozenges.
  • Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Aqueous solutions include, for example, elixirs and syrups.
  • Emulsions are either oil-in-water or water-in-oil.
  • Elixirs are clear, sweetened, hydroalcoholic preparations.
  • Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.
  • An emulsion is a two-phase system in which one liquid is dispersed in the form of small globules throughout another liquid.
  • Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.
  • Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form include diluents, sweeteners and wetting agents.
  • Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents may be used in any of the above dosage forms.
  • Solvents include by way of example and without limitation, glycerin, sorbitol, ethyl alcohol and syrup.
  • preservatives include without limitation glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol.
  • Non-aqueous liquids utilized in emulsions include by way of example and without limitation, mineral oil and cottonseed oil.
  • Emulsifying agents include by way of example and without limitation, gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.
  • Suspending agents include, by way of example and without limitation, sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia.
  • Diluents include, by way of example and without limitation, lactose and sucrose.
  • Sweetening agents include, by way of example and without limitation, sucrose, syrups, glycerin and artificial sweetening agents such as saccharin.
  • Wetting agents include by way of example and without limitation, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.
  • Organic acids include, by way of example and without limitation, citric and tartaric acid.
  • Sources of carbon dioxide include, by way of example and without limitation, sodium bicarbonate and sodium carbonate.
  • Coloring agents include, by way of example and without limitation, any of the approved certified water soluble FD and C dyes, and mixtures thereof.
  • Flavoring agents include, by way of example and without limitation, natural flavors extracted from plants such fruits, and synthetic blends of agents which produce a pleasant taste sensation.
  • the solution or suspension in for example propylene carbonate, vegetable oils or triglycerides, is encapsulated in a gelatin capsule.
  • a gelatin capsule Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the solution for example in a polyethylene glycol, may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.
  • a pharmaceutically acceptable liquid carrier e.g., water
  • liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active agent or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells.
  • Other useful formulations include those set forth in U.S. Patent Nos. Re 28,819 and 4,358,603.
  • such formulations include, but are not limited to, those containing an agent provided herein, a dial kylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.
  • BHT butylated
  • formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal.
  • Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol.
  • Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.
  • Tablets and capsules formulations may be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.
  • Parenteral administration generally characterized by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients include by way of example and without limitation, water, saline, dextrose, glycerol or ethanol.
  • compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • the metabolic inhibitor can be dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropy
  • a solid inner matrix e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate,
  • Parenteral administration includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions.
  • the solutions may be either aqueous or nonaqueous.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • thickening and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • Aqueous vehicles include, by way of example and without limitation, Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection.
  • Nonaqueous parenteral vehicles include, by way of example and without limitation, fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Isotonic agents include, by way of example and without limitation, sodium chloride and dextrose. Buffers include phosphate and citrate.
  • Antioxidants include sodium bisulfate.
  • Local anesthetics include procaine hydrochloride.
  • Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.
  • Emulsifying agents include Polysorbate 80 (TWEEN ® 80).
  • a sequestering or chelating agent of metal ions include EDTA.
  • Pharmaceutical carriers also include, by way of example and without limitation, ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • the concentration of the pharmaceutically active agent is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect.
  • the exact dose depends on the age, weight and condition of the patient or animal as is known in the art.
  • the unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle.
  • Preparations for parenteral administration should be sterile, as is known and practiced in the art.
  • intravenous or intra-arterial infusion of a sterile aqueous solution containing an active agent is an effective mode of administration.
  • Another embodiment is a sterile aqueous or oily solution or suspension containing an active agent injected as necessary to produce the desired pharmacological effect.
  • Injectables are designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, such as more than 1% w/w of the active agent to the treated tissue(s).
  • the active agent may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the tissue being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated.
  • the agent may be suspended in micronized or other suitable form or may be derivatized, e.g., to produce a more soluble active product or to produce a prodrug or other pharmaceutically acceptable derivative.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.
  • Lyophilized powders can be reconstituted for administration as solutions, emulsions, and other mixtures or formulated as solids or gels.
  • the sterile, lyophilized powder is prepared by dissolving an agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent.
  • the solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
  • the solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH.
  • each vial will contain, by way of example and without limitation, a single dosage (10-1000 mg, such as 100-500 mg) or multiple dosages of the agent.
  • the lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.
  • Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration.
  • about 1-50 mg, such as about 5-35 mg, for example, about 9-30 mg of lyophilized powder is added per mL of sterile water or other suitable carrier.
  • the precise amount depends upon the selected agent. Such amount can be empirically determined.
  • Topical mixtures are prepared as described for the local and systemic administration.
  • the resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.
  • the agents or pharmaceutically acceptable derivatives thereof may be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma).
  • These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose.
  • the particles of the formulation will, by way of example and without limitation, have diameters of less than about 50 microns, such as less than about 10 microns.
  • the agents may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application.
  • Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active agent alone or in combination with other pharmaceutically acceptable excipients can also be administered.
  • solutions particularly those intended for ophthalmic use, may be formulated, by way of example and without limitation, as about 0.01% to about 10% isotonic solutions, pH about 5-7, with appropriate salts.
  • Transdermal patches including iotophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Patent Nos.
  • Rectal suppositories are used herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients.
  • Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used.
  • Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is, by way of example and without limitation, about 2 to 3 gm.
  • Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
  • Kit of Parts The invention includes a kit of parts for simultaneous, separate, sequential administration to a pathogen-infected patient.
  • the kit can comprise any combination of the metabolic inhibitors of the invention in pharmaceutical compositions together.
  • the kit of parts can contain at least 1-200 mg, 5-160 mg, 10-80 mg, or 20-40 mg of two or more metabolic inhibitors.
  • the kit of parts contains at least 1-5, 5-10, 10-20, 20- 40, 40-60, 60-80, 80-100, 100-120, 120-140, or 140-160 mg of two or more metabolic inhibitors.
  • the kit of parts contains at least 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, or 160 mg of 2-DG.
  • the kit of parts contains additionally contains at least one, two, three, or four of the HIV inhibitors listed below. Most preferably, the kit of parts contains at least one of the combination antiretroviral therapies listed below.
  • Entry inhibitors interfere with binding, fusion and entry of HIV-1 to the host cell by blocking one of several targets (Wikipedia).
  • Maraviroc works by targeting CCR5, a co-receptor located on human helper T-cells.
  • Enfuvirtide is a peptide drug that must be injected and acts by interacting with the N-terminal heptad repeat of gp41 of HIV to form an inactive hetero six-helix bundle, therefore preventing infection of host cells.
  • Nucleoside reverse transcriptase inhibitors and nucleotide reverse transcriptase inhibitors (NtRTI) are nucleoside and nucleotide analogues which inhibit reverse transcription.
  • NRTIs include zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir.
  • Non-Nucleoside reverse transcriptase inhibitors inhibit reverse transcriptase by binding to an allosteric site of the enzyme.
  • 1st generation NNRTIs include nevirapine and efavirenz.
  • 2nd generation NNRTIs include etravirine and rilpivirine.
  • Integrase inhibitors (also known as integrase nuclear strand transfer inhibitors or INSTIs) inhibit the viral enzyme integrase. Integrase inhibitors include raltegravir, elvitegravir, and dolutegravir. Protease inhibitors block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane. Examples of HIV protease inhibitors are lopinavir, indinavir, nelfinavir, amprenavir, ritonavir, darunavir, and atazanavir.
  • Maturation inhibitors have a similar effect by binding to gag, and include bevirimat and becon.
  • Combination antiretroviral therapy is a mixture of at least two, and preferably three or more different classes of antiretroviral therapy. All different combinations of the antiretroviral therapies specified herein are specifically contemplated. Examples of cART include:
  • Trizivir abacavir + lamivudine + zidovudine
  • Epzicom in USA
  • Kivexa in Europe and Russia
  • lamivudine Epzicom
  • Truvada tenofovir disoproxil fumarate + emtricitabine.
  • Complera in USA
  • Eviplera in Europe and Russia
  • Stribild elvitegravir + cobicistat + emtricitabine + tenofovir disoproxil fumarate.
  • Triumeq abacavir + dolutegravir + lamivudine.
  • Dutrebis lamivudine + raltegravir.
  • Genvoya elvitegravir + cobicistat + emtricitabine + tenofovir alafenamide fumarate.
  • Rev inhibitors interfere with the biogenesis of viral RNA required for the replication of HIV.
  • Rev inhibitor can function through binding to the Cap Binding Complex at the 5' end of the mRNA coding for 3 structural proteins of the virus. By promoting HIV RNA splicing, these inhibitors can reduce the level of genomic RNA and inhibit HIV replication.
  • Preferred compounds can be found in U.S. Patents 9,145,367 and 9,061,999, which are hereby incorporated by reference.
  • Particularly preferred compounds are 10-chloro-2,6- dimethyl-2H-pyrido [3',4':4,5]pyrrolo[2,3-g]isoquinoline (IDC16), 8-chloro-N-(4- (trifluoromethoxy)phenyl)quinolin-2-amine (ABX464) and 8-chloro-N-glucuronide-N-(4- (trifluoromethoxy)phenyl)quinolin-2-amine) (ABX464-N-glucuronide) compounds, as set forth in Campos et al. Retrovirology (2015) 12:30, which is hereby incorporated by reference.
  • a particularly preferred compound has the formula:
  • CD4+ T-cells were purified (>90%) from freshly isolated PBMCs by negative selection with antibody-coated magnetic beads (EasySepTM Human CD4+ T-cell Enrichment Kit Ref.19052) in a Robosep instrument (Stem Cell Technology).
  • CD4+ cells (10 6 cell/mL) were cultured in RPMI 1640 containing GlutaMAX, 10% FCS, penicillin (10 lU/mL) and streptomycin (10 pg/mL) in the presence of IL-2 (Miltenyi) at 50 lU/mL (Culture media).
  • IL-2 Miltenyi
  • cells were left unstimulated or were stimulated for 3 or 5 days with 0.5 pg/mL soluble antiCD3 (BioLegend, Ref.300414, Clone UCHT1) in the absence of CD28 co-stimulation as previously described (Saez-Cirion et al., 2011).
  • a glucose-free culture media was used in some infection experiments and is described in the results section [RPMI non-glucose, GlutaMAX, containing 10% FCS, penicillin (10 lU/mL) and streptomycin (10 pg/mL) in the presence of IL-2 (Miltenyi) at 50 lU/mL (culture media)].
  • RPMI non-glucose, GlutaMAX containing 10% FCS, penicillin (10 lU/mL) and streptomycin (10 pg/mL) in the presence of IL-2 (Miltenyi) at 50 lU/mL (culture media)
  • IL-2 Miltenyi
  • HIV-1 NL4.3AenvAnef/GFP Amara et al., 2003
  • HIV-l-DuoFluoAenv(R7GEmC) provided by Professor Eric Verdin and Dr. Calvanese, NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Cat# 12595 DuoFluo (R7GEmC)
  • Both viruses were pseudotyped with the VSV-G envelope protein by transiently cotransfecting (SuperFect; Qiagen) 293T cells with the proviral vectors and the VSV-G expression vector pMD2.G.
  • Nonactivated or activated CD4 + T-cells were infected in triplicate (5xl0 4 cells/well, 200 m
  • Active HIV-1 infection was estimated by flow cytometry (BD LSRII, BD bioscience) as the percentage of GFP-expressing CD4+ T-cells 72 h after infection.
  • Latent HIV infection was estimated by flow cytometry as the percentage of mCherry+GFP-CD4+ T-cells 72 h after infection with HIV-l-DuoFluo(R7GEmC) particles.
  • HIV-1 reverse transcripts (U5-Gag) were quantified by real-time PCR with an Applied Biosystems 7500 Real-Time PCR System 6, 16 and 72 h after infection of CD4+ T-cells with VSV- G-pseudotyped HIV-1 particles as described in (David et al., 2006). Briefly, total DNA was extracted with the NucleoSpin 8/96 Tissue Core kit (Macherey-Nagel, Ref. 740453.4) and 100 ng of template DNA were used per reaction. DNA loading was controlled by concurrently amplifying the albumin gene by real-time PCR and quantifying with reference to a control human genomic DNA (Roche).
  • the reaction mixture contained lx TaqMan Universal PCR master mix, 300 nM of primers and 200 nM of the fluorogenic probe, in a final volume of 30 pi.
  • PCR cycle conditions were: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.
  • Copy numbers of U5-Gag were determined with reference to a standard curve prepared by concurrent amplification of serial dilutions of 8E5 cells containing one integrated copy of HIV-1 per cell.
  • Cells were first selected based on size and structure to eliminate cellular debris. Then cell singlets and living cells (not stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Thermofisher) are gated before proceeding with further selection based on phenotypical or functional markers ( Figures 9, 12, 13).
  • CD4+ T-cell subsets [naive (Tn; CD3+, CD4+, CD45RA+, CCR7+, CD27+, CD95-), central memory (Tcm; CD3+, CD4+, CD45RA-, CCR7+, CD27+), transitional memory (Ttm; CD3+, CD4+, CD45RA-, CCR7-, CD27+) or effector memory (Tem; CD3+, CD4+, CD45RA-, CCR7-, CD27-)] were sorted on a FACS ARIA III cell sorter (BD) using the following antibody panel: CD3-eFLuor450 (eBioscience), CD4-alexaFluor700 (BD), CD45RA-ECD (BC), CCR7-PE_Cy7 (BioLegend), CD27-APC (Miltenyi), CD95-PE (Miltenyi), CD25-FITC (BD
  • GFP+ and GFP- CD4+ T-cells were sorted 72 h after infection with VSV-G pseudotyped NL4.3AenvAnef/GFP particles ( Figure 12A).
  • GFP+ and GFP- cells were also sorted into the following categories based on their expression of activation markers (CD25- ECD, HLA-DR_PerCyP5.5) ( Figure 4 and Figure 12B): high activation GFP+ [H/+ (GFP+, CD25+,HLA-DR+)]; high activation GFP- [H/- (GFP-, CD25+, HLA-DR+)]; low activation GFP+ [L/+, (GFP+, CD25-,HLA-DR-)]; and low activation GFP- [L/- (GFP-, CD25-,HLA-DR-)].
  • CD4+ Tn and Tcm cells were sorted based on their level of glucose uptake after 5 days of stimulation with anti-CD3 (Figure 13A).
  • the cells were washed and incubated with 2-NBDG (2-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino)-2-deoxyglucose) (Thermo Fisher, Ref. N13195) at 75 mM in PBS for 30 min at 37°C.
  • 2-NBDG 2-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino)-2-deoxyglucose
  • Tn HGIu CD3+, CD4+, CD45RA+, CCR7+, CD27+, 2NBDG+
  • Tn LGIu CD3+, CD4+, CD45RA+, CCR7+, CD27+, 2NBDG-
  • Tcm HGIu CD3+, CD4+, CD45RA-, CCR7+, CD27+, 2NBDG+
  • Tcm LGIu CD3+, CD4+, CD45RA-, CCR7+, CD27+, 2NBDG-
  • CD4+ T-cells were stained with HRBD-rFc, a recombinant fusion protein that specifically binds GLUT1 (Metafora-biosystems, Paris, France), and a secondary goat-anti-Mouse Alexa Fluor 647 antibody (Thermofisher).
  • HRBD is derived from the receptor-binding domain of the human T-cell leukemia virus envelope glycoprotein that binds the extracellular domain of GLUT1 (Manel et al., 2005).
  • CD4+T cell subsets CD3- eFLuor450 (eBioscience), CD4-alexaFluor700 (BD), CD45RA-ECD (BC), CCR7-PE_Cy7 (BioLegend), CD27-PE (BD bioscience).
  • RNA trace kit Macherey-Nagel, Ref. 740731.4
  • DNase DNase-free DNA sequence
  • Twenty microliters of RNA was reverse transcribed with Reverse Transcription Master Mix (Fluidigm, 100-6298) (5 minutes at 25°C, 30 minutes at 42°C, and 5 minutes at 85°C).
  • STA specific target preamplification
  • Sample premix (SsoFast EvaGreen Supermix with Low ROX (Biorad), DNA Binding Dye (Fluidigm), preamplified Exo 1-treated sample) and assay mix (assay loading reagent (Fluidigm), Delta Gene primers (Fluidigm)) were then loaded on primed 96.96 Dynamic Array chips (Fluidigm). The chips were transferred into a Biomark HQ device (Fluidigm) for thermocycling, and fluorescence was acquired with the GE 96x96 PCR+Melt v2 program. Linear derivative mode baseline correction was applied.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • Drug Panel A (1) XFmedia 2) oligomycin (2.5 pM), 3) FCCP (0.9 pM) and 4) rotenone (1 pM) and antimycin A (1 pM)) was injected through ports A, B, C and D, respectively, for the mitochondrial stress test.
  • Drug Panel B ( 1) XFmedia 2) glucose (10 mM) 3) oligomycin (2.65 pM), and 4) 2-DG (100 mM)) was used for the glycolysis stress test.
  • sorted GFP+/- CD4+ T-cells subset or CD4+ T bulk cells previously infected with NL4.3Anef/GFP/VSV-G with or without 2-DG or Etomoxir were incubated with CD3-eFLuor450 (eBioscience), CD4-alexaFluor700 (BD Biosciences), CD45RA-ECD (BC), CCR7- PE_Cy7 (BioLegend) and CD27-APC (Miltenyi) to determine the CD4+ T-cell subset distribution.
  • CD3-eFLuor450 eBioscience
  • CD4-alexaFluor700 BD Biosciences
  • CD45RA-ECD BC
  • CCR7- PE_Cy7 BioLegend
  • CD27-APC Miltenyi
  • EXAMPLE 8 HIV-1 reactivation in CD4+ T-cells from HIV-l-infected individuals.
  • Freshly isolated CD4+T-cells (negative selection kit, Stem Cell) from HIV-individuals undergoing successful cART were seeded in 48-well plates (lxlO 6 cells/well, in triplicate) and stimulated with phytohemagglutinin-L (PHA-L, Roche, 1 pg/mL) and IL-2 (Miltenyi) 100UI with or without 2-DG (5 mM).
  • the culture supernatants were collected every 3 to 4 days, and fresh medium +/- 2-DG was added to the cultures.
  • Supernatants were stored at -80°C, and HIV-1 p24 was analyzed later by ultrasensitive digital ELISA (Simoa, Quanterix) (Passaes et al., 2017).
  • CD4+ T-cell subsets have heterogeneous susceptibility to HIV-1 infection
  • Nonactivated CD4+ T-cell subsets showed distinct transcriptional profiles that were further enhanced after activation (e.g., 34 genes and 49 genes differently expressed between CD4+ T- cell subsets without activation and after 3 days of anti-CD3 treatment, respectively, Figure 2A). These genes were mostly related to signal transduction and the response to stimulus, which could be related to the previously described different susceptibility to CD3 activation of the CD4+ T-cell subsets (Croft et al., 1994; Kumar et al., 2011). The level of HIV-infected cells correlated with the expression of several genes at the time of infection in the different conditions studied (Figure 2B and Figure 10). SAMHD1 showed a negative association with infection.
  • Tn cells showed a modest increase only in mitochondrial function and not in glycolysis and only after 5 days of activation, when their metabolism was heavily relying on OXPHOS (Figure 3C). Accordingly, important differences were also found between CD4+ T-cell subsets regarding their capacity to uptake glucose and their levels of the surface expression of the GLUT1 receptor, which were lowest in Tn cells and highest in Tem cells ( Figures 11A, B). The relative metabolic activity levels of the different cell subsets matched their relative susceptibility to HIV-1 infection (Figure 1C), and we found positive correlations between HIV infection levels and multiple metabolic functions in cells that had been activated (Figure 3D, Figure 11C). These results further point to an influence of the metabolic activity of CD4+ T-cells on their susceptibility to HIV-1.
  • HIV-infected CD4+ T-cells are characterized by higher levels of metabolic activity independent of cell activation levels
  • CD4+ T-cells first based on their expression of either high or low levels of both HLA-DR and CD25 and then based on whether they were GFP+ or GFP- ( Figures 4B and S4). After 5 days of stimulation, the CD4+ T-cell subsets expressed different levels of activation markers ( Figure 12B), which were highest in Tem cells and lowest in Tn cells. This was translated to different contributions of CD4+ T-cell subpopulations in the high- and low-activation sorted cell fractions ( Figure 4B).
  • HIV-1 infection is preferentially established in CD4+ T-cells with high metabolic activity levels
  • CD4+ T cells from a same donor were treated with various metabolic inhibitors, alone or in combination.
  • the results are shown in Figure 8, which shows the synergistic effect of suboptimal amounts of 2DG, DON and ETOMOXIR.
  • HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nature medicine 15, 893-900.
  • HIV-1 uncoating is facilitated by dynein and kinesin 1. J Virol 88, 13613-13625.
  • the transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells. Nature Immunology 14, 1155-1165.
  • the ubiquitous glucose transporter GLUT-1 is a receptor for HTLV. Cell 115, 449-459.
  • Tumor lactic acidosis suppresses CTL function by inhibition of p38 and JNK/c-Jun activation. Int J Cancer 131, 633-640.
  • Patsoukis N., Bardhan, K., Chatterjee, P., Sari, D., Liu, B., Bell, L.N., Karoly, E.D., Freeman, G.J., Petkova, V, Seth, P., et al. (2015).
  • PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. Nature Communications 6, 6692.
  • Pearce E.L., Poffenberger, M.C., Chang, C.H., and Jones, R.G. (2013). Fueling immunity: insights into metabolism and lymphocyte function. Science 342, 1242454.
  • Roederer M., Raju, P.A., Mitra, D.K., Herzenberg, L.A., and Herzenberg, L.A. (1997). HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. J Clin Invest 99, 1555-1564.
  • Hyperthermia stimulates HIV-1 replication.
  • HIV-1 replication is controlled at the level of T cell activation and proviral integration.
  • CD4+ memory stem cells are infected by HIV-1 in a manner regulated in part by SAMHD1 expression.
  • J Virol 88, 4976-4986. Takeuchi, Y., Vile, R.G., Simpson, G., O'Hara, B., Collins, M.K., and Weiss, R.A. (1992).
  • Feline leukemia virus subgroup B uses the same cell surface receptor as gibbon ape leukemia virus. J Virol 66, 1219-1222.
  • CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability. Proc Natl Acad Sci U S A 110, 14336- 14341.
  • BECN1 was the most stable gene present in our panel and was used to normalize our RT- qPCR data

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Abstract

Le VIH persiste dans des cellules infectées à longue durée de vie qui ne sont pas affectées par un traitement antirétroviral. Ces réservoirs de VIH sont principalement situés sans des cellules T CD4+ mais leur distribution est variable dans les différents sous-ensembles. La susceptibilité au VIH-1 augmente avec la différenciation des lymphocytes T CD4+. Nous avons évalué si la programmation métabolique qui prend en charge la différenciation et la fonction de lymphocytes T CD4+ affectait leur susceptibilité au VIH-1. Nous avons découvert que des différences de susceptibilité au VIH entre des sous-ensembles naïfs et plus différenciés étaient associées à l'activité métabolique des cellules. En effet, le VIH-1 a sélectivement infecté les lymphocytes T CD4+ avec une phosphorylation oxydative et une glycolyse élevées, indépendamment de leur phénotype d'activation. De plus, l'inhibition partielle de la glycolyse (i) a altéré l'infection par le VIH-1 in vitro dans tous les sous-ensembles de lymphocytes T CD4+, (ii) a diminué la viabilité de cellules pré-infectées, et (iii) a empêché la réactivation du VIH -1 dans des cellules provenant d'individus infectés par le VIH. Nos résultats permettent d'élucider la liaison entre le métabolisme cellulaire et l'infection par le VIH et d'identifier une vulnérabilité permettant de neutraliser les réservoirs de VIH et des infections par d'autres pathogènes.
PCT/EP2019/086782 2018-12-20 2019-12-20 Métabolisme cellulaire de l'ensemencement d'un réservoir de vih-1 dans des lymphocytes t cd4+ Ceased WO2020128033A1 (fr)

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