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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
• • 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
  • A61K31/12—Ketones
  • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
• • 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
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
  • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
  • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • 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
  • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G8/00—Condensation polymers of aldehydes or ketones with phenols only
  • C08G8/28—Chemically modified polycondensates
  • C08G8/30—Chemically modified polycondensates by unsaturated compounds, e.g. terpenes

Definitions

• the present invention broadly relates to the use of certain ingenol derivatives as latent HIV virus reactivators in viral reservoirs.
• the present invention relates to combinations and compositions comprising said ingenol derivatives and substantially active antiretroviral agents active on replicating viruses.
• the human immunodeficiency virus is known to be the etiological agent responsible for AIDS - acquired immunodeficiency syndrome, a fatal disease characterized by the destruction of the immune system, incapacitating the body to respond properly to life-threatening opportunistic infections.
• HAART Highly active antiretroviral therapy
• HAART Highly active antiretroviral therapy
• It is a treatment commonly called an anti-AIDS "cocktail" with at least three active antiretroviral medications containing reverse transcriptase, integrase, entry, and protease inhibitors.
• latently infected memory CD4 + T lymphocyte cell reservoir virus i.e. containing residual latent proviral DNA integrated into the host cell genome
• HIV remains a chronic viral infection when such persistent latent infection is not combated.
• activation of the latent virus contained in such reservoirs in the presence of antiretrovirals, seeks to make it detectable by the body's immune system and accessible to active virus medication to cause destruction to occur.
• cells expressing viral proteins by reaction of the host immune system and / or such that the cells are driven to apoptosis, inhibiting the replication of viruses leaving the reservoirs by the action of antiretrovirals, thus depleting the reservoir of persistent HIV infection, making the complete eradication of the infection possible.
• selective induction of latent infection enables antiretroviral drugs and the antiviral immune response to access and eradicate residual HIV infection - that is, not only temporarily stabilizing the immune system without future use of antiretrovirals, but definitely suppressing antiretroviral drugs. HIV infection in the human body.
• active antiretroviral agents in actively replicating viruses refers to agents that do not act substantially or act only limitedly on HIV viral reservoirs in the human body.
• the present invention relates, within a first aspect, to the use of one or more ingenol derivatives of formula I below:
• x and y being integers, x ranging from 2 to 10, and y ranging from 2 to 7.
• q being an integer ranging from 1 to 10, preferably from 2 to 6, and w being an integer ranging from 1 to 10, preferably from 8 to 10, provided that:
• formula I of the ingenol derivatives of the invention has the following conformation:
• adjuvant is an active principle or agent acting in parallel with or assisting the action of another active principle or agent in the treatment of a particular disease or condition.
• the typical individual effect of "adjuvant” is not that which alone effectively treats the disease or disorder, achieving cure.
• the invention relates to the use of ingenol derivatives of formula I above, characterized in that it is in reactivation of latent HIV virus in viral reservoirs of the human body. It is a use in medical therapy.
• the invention relates to an association useful in the treatment or prevention of HIV virus infection characterized in that it comprises one or more ingenol derivatives of formula I, and at least one active replicating virus active antiretroviral agent, particularly selected from nucleoside or non-nucleoside reverse transcriptase inhibitors, protease inhibitors, co-receptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, cyclin dependent kinase inhibitors, and combinations thereof.
• active replicating virus active antiretroviral agent particularly selected from nucleoside or non-nucleoside reverse transcriptase inhibitors, protease inhibitors, co-receptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, cyclin dependent kinase inhibitors, and combinations thereof.
• “combination” means any suitable dosage form in combination, admixture, formulation, preparation or equivalent administered to a person containing at least one ingenol derivative of formula I (component a). ) and at least one substantially active antiretroviral agent in actively replicating viruses (component b).
• “combination” means that one or more ingenol derivatives of formula I (component a) and one or more antiretroviral agents (component b) may be available in a single dosage unit (eg tablet, capsule, ampoule, pouch, etc.) or in separate dosage units, wherein components (a) and (b) are available to a patient. for joint or separate administration, either simultaneously or sequentially.
• a single dosage unit eg tablet, capsule, ampoule, pouch, etc.
• components (a) and (b) are available to a patient. for joint or separate administration, either simultaneously or sequentially.
• the invention relates to pharmaceutical compositions containing the aforementioned association and pharmaceutically acceptable excipients.
• composition of the invention may additionally contain other active ingredient (s) distinct from both ingenol (s) of formula I and antiretroviral agents.
• composition of the invention further comprises one or more compounds capable of reactivating the latent HIV virus in viral reservoirs of the human body, distinct from the ingenol derivatives of formula I.
• the invention relates to an adjuvant useful in reactivating latent HIV viruses in viral reservoirs of the human body characterized by comprising one or more ingenol derivatives of formula I, and pharmaceutically acceptable excipients.
• the invention in another aspect, relates to a method of treating or preventing HIV infection characterized in that it comprises administering to a patient in need of such treatment an association as mentioned above. Said treatment comprises administering the combination components concomitantly or sequentially.
• the invention relates to a method for reactivating the latent HIV virus in human body viral reservoirs characterized by administering to one patient one or more ingenol derivatives of formula I.
• nucleoside reverse transcriptase inhibitors suitable for the invention without excluding any other, can be mentioned the compounds ⁇ (zidovudine), 3TC (lamivudine), d4T (stavudine), abacavir, ddl (didanosine), ddC (zalcitabine), FTC ( emtricitabine), PMPA ((R) -9- (2-phosphonylmethoxypropyl) adenine), tenofovir, adefovir, amdoxovir, elvucitabine, alovudine, racivir, apricitibine, phosphatide and fozivudine.
• non-nucleoside reverse transcriptase inhibitors suitable for the invention without excluding any other, nevirapine, efavirenz, delavirdine, loviride, etravirine,
• (+) calanolide, rilpivirine and lersivirine (+) calanolide, rilpivirine and lersivirine.
• protease inhibitors suitable for the invention without excluding any other, the compounds ritonavir, lopinavir, nelfinavir, saquinavir, indinavir, atazanavir, amprenavir, darunavir, fosamprenavir and tipranavir may be cited.
• the compounds raltegravir, elvitegravir and dolutegravir may be cited.
• fusion inhibitors suitable for the invention without excluding any other, the enfuvirtide and tifuvirtide compounds may be cited.
• co-receptor inhibitors suitable for the invention without excluding any other, CCR5 vicriviroc and maraviroc co-receptor inhibitors may be cited.
• the ingenol derivatives of the invention, the active replicating virus-active antiretrovials or the combination of the invention containing them, may be administered to a patient by any suitable route, for example, orally, parenterally, intraperitoneally, intravenously, intrauterine. arterial, transdermal, sublingual, intramuscular, rectal, transbucal, intra-nasal, liposomal, inhalation, vaginal, intraocular, subcutaneous, intra-adipose, intraarticular, intrathecal, by catheter or stent administration, etc.
• the dosage form may be immediate, slow or controlled release.
• Figure 1B represents a latency induction graph in clone Jlat 6.3 with different concentrations of the above mentioned KyoII derivative. 20 ng TNF- ⁇ was used as positive control and results are shown as% induced cells.
• Figure 1C represents a histogram showing the induction of latency in clone Jlat 6.3 with 4 ⁇ M of the above mentioned KyoII derivative.
• 20 ng TNF-Î ⁇ was used as a positive control and the results are also shown as% induced cells.
• Figure 2 is a histogram showing the activation of apoptosis in human peripheral blood mononuclear cells (PBMC) cells cultured for 72 hours with different concentrations of the above mentioned ingenol KyoII derivative. Drug concentration is placed next to each graph and% apoptotic cells are marked in the left quadrant of each graph.
• PBMC peripheral blood mononuclear cells
• Figures 7 and 8 are PCR (polymerase chain reaction) graphs for efavirenz-treated patient blood cells only (Fig. 7), and with a mixture of efavirenz and ingenol B derivative of the invention (Fig. 8). .
• J-lat a cell line derived from Jurkat strains, which functions as an in vitro model of latent HIV-1. Similar to resting CD4 + T cells infected with latent HIV-1 virus, J-Lat cells carry a complete HIV-1 genome, integrated into regions of the cell genome that can be activated; however, the transcription of these regions is momentarily inhibited. Additionally, the latent pro-virus integrated into J-Lat cell lines encodes a green fluorescent protein (GFP) gene, thus providing a fluorescent reporter of HIV-1 transcriptional activity. These cells were treated with TNF-a (20 ng / ml) for viral re-activation as a positive control and the effect compared with the mixture of Z1 derivatives of ingenol. HIV viral gene expression was monitored by the GFP reporter gene for 48-72 hours after treatment with TNF- ⁇ through flow cytometry.
• GFP green fluorescent protein
• the cells were washed with RPMI medium and resuspended in RPMI medium containing 10% fetal bovine serum and cultured for a further 24 hours to induce latent viruses.
• 30,000 cells were read on a BD-Excalibur flow cytometer (from Beckton Dickinson and Co., USA) for reading cells expressing the GFP marker protein.
• a cell sample was not induced and was cultured for 48 hours as a mock control (thus referred to as "mock") thus marking the spontaneous induction of the provirus (background).
• FIG. 1A and 1B show that the 24-hour induction KyoII sample was able to activate the latent viruses present in clones Jlat 6.3 and 8.4 in a dose-dependent manner, and even at very low doses (0.4 ⁇ ) was able to induce up to 8% of Jlat clone 6.3 and 8.4 cells in culture, and at the concentration of 40 ⁇ was able to induce almost 30% of the cells, overlapping the potency of TNF-a.
• Figure 1C shows histograms showing raw data from Cellquest software (from Becton Dickinson and Company, USA) showing latency induction in Jlat 6.3 clone following the same protocol as above with 4 ⁇ M KyoII. In this case 20 ng TNF- ⁇ was used as a positive control and the results are also shown as% induced cells.
• PBS Phosphate Buffered Saline
• Example 3 Cytotoxicity Check vs. reactivation
• we sought to correlate the cytotoxicity and reactivation effects of HIV, both for the KyoII mixture of ingenol derivatives where Z — Zl of examples 1 and 2 above, and for 3 ingenol derivatives of formula I where Z Z2. , indicated above as A, B and C.
• a concentration of 10 6 cells / ml JLat 6.3 were induced with different concentrations of KyoII, A, B and C for 24 hours.
• the cells were washed with RPMI medium and resuspended in RPMI medium containing 10% fetal bovine serum and cultured for a further 24 hours to induce latent viruses.
• 30,000 cells were read on a BD-Excalibur flow cytometer for reading cells expressing the GFP marker protein.
• a cell sample was not induced and was cultured for 48 hours as a mock control (thus referred to as "mock") thus marking the spontaneous induction of the provirus (background).
• the propidium iodide staining technique was used. Thus the cells were centrifuged at 1000G for 3 minutes and washed at the same volume in 1 X PBS (without Ca2 + and Mg2 +, "Cat # 9240" from Irvine Scientific, USA) containing 2% fetal bovine serum.
• PBMC cells were isolated and placed in RPMI culture medium with 10% fetal bovine serum, with 50 IU / ml IL2 (interleukin 2) and grown in 2 bottles with 5ml identical cells (106 / ml) with different selective compositions.
• EDTA ethylene diamine tetraacetic acid
• IL2 interleukin 2
• 5ml identical cells 106 / ml
• antiviral efavirenz ⁇
• both efavirenz (10uM) and derivative B of the invention (luM) were included.
• RNA from PBMCs was extracted with an RNEasy kit (from QiaGen, USA) and the amount of HIV-1 genomic RNA was assayed by real time PCR reaction.
• DNase I was inactivated in an incubation at 70 ° C for 10 min in the presence of 1mM EDTA and 50mM DT (dithiothreitol).
• Reverse RNA transcription was performed with random hexamer primers and SuperScript III (cDNA synthesis enzyme label from Invitrogen, USA) at 42 ° C for 60 min.
• the cDNA was then subjected to PCR reactions.
• the primer pair used in this 1st round of PCR was GAG1 and SK431, which amplifies the region within the HIV-1 gag.
• PCR was run on a conventional machine in a volume of 25ul with 5 ⁇ 1 cDNA, 20 mM tris (hydroxymethyl) aminomethane (pH 8.3), 50 mM KC1, 2 mM MgCl2, 0.4 mM dNTPs ( deoxynucleotide triphosphates) and 1U of Ampli-Taq (DNA polymerase from Applied Biosystems, USA), and 50 ng of each primer.
• PCR conditions were: 94 ° C for 3 min, followed by 15 cycles of 94 ° C for 30 s, 55 ° c for 30 s, and 72 ° C for 1 min.
• the product of this I PCR was subjected to a 2nd real-time PCR Semi-nested, this a machine ABI PRISM 7000 real-time PCR (company Applied Biosystems, USA) using TaqMan reaction mix in a total volume of 25 ⁇ 1, 2 ⁇ do I the round diluted 50 times with 0.2 ⁇ M of GAG1 and GAG2 primers, and 0.2 ⁇ of FAM GAG3 probe.
• Real-time PCR conditions were: 50 ° C for 2 min, and 95 ° C for 10 min, followed by 50 cycles of 95 ° C for 15s and 60 ° C for 1 min
• the size of the amplicons were 221 bp for the round PCR I and 83 bp for (real-time) PCR.
• CD4 + T lymphocytes and human macrophages and pigtail monkeys (Macaca nemestrina).
• human and monkey PBMC cells at a concentration of 10 6 cells / ml were cultured in RPMI medium with 10% fetal bovine serum for 24hs and the adhered cells (macrophage differentiating monocytes) were separated from the cell supernatant containing the total lymphocytes.
• the cells were exposed to different concentrations of derivatives A, B and C of the invention, and left to culture for 72 hours. Following exposure, cells were stained with lymphocyte-specific monoclonal (anti-CD3) and monocyte / macrophage (anti-CD14) simultaneously with an anti-CD4.
• ingenol Z2 derivatives of the invention were able to down-modulate the expression of the HIV-1 CD4 receptor on the surface of human lymphocytes ( Figure 11) and Macaca nemestrine ( Figure 9). Similarly these compounds also down modulated CD4 from the surface of human monocytes / macrophages ( Figure 12) and Macaca nemestrina ( Figure 10). Notable was the greater potency of ingenol Z2 derivatives in down-modulating CD4 from monkey cells when compared to comparator molecules (prostratin, bryostatin and PMA, see Figures 9 and 10).
• Ingenol A, B and C derivatives were able to down modulate the major HIV receptor (CD4) on the surface of the target cells of the infection.
• CD4 major HIV receptor

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