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Research Area
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Description
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Immunology, Infection |
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Biological Activity
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Description
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Raltegravir (MK-0518, Isentress) is a potent integrase (IN) inhibitor for WT and S217Q PFV IN with IC50 of 90 nM and 40 nM, respectivly. |
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Targets
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IN (WT PFV) |
IN (S217Q PFV) |
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IC50 |
90 nM |
40 nM [1] |
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In Vitro
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PFV IN carrying the S217H substitution is 10-fold less susceptible to Raltegravir with IC50 of 900 nM. PFV IN displays 10% of WT activity and is inhibited by Raltegravir with an IC50 of 200 nM, indicating a ~twofold decrease in susceptibility to the IN strand transfer inhibitor (INSTI) compared with WT IN. S217Q PFV IN is as sensitive to Raltegravir as the WT enzyme. [1] Raltegravir is metabolized by glucuronidation, not hepatically. Raltegravir has potent in vitro activity against HIV-1, with a 95% inhibitory concentration of 31±20 nM, in human T lymphoid cell cultures. Raltegravir is also active against HIV-2 when Raltegravir is tested in CEMx174 cells, with an IC95 of 6 nM. Raltegravir metabolism occurs primarily through glucuronidation. Drugs that are strong inducers of the glucuronidation enzyme, UGT1A1, significantly reduce Raltegravir concentrations and should not be used. Raltegravir exhibits weak inhibitory effects on hepatic cytochrome P450 activity. Raltegravir does not induce CYP3A4 RNA expression or CYP3A4-dependent testosterone 6-β-hydroxylase activity. [2] Raltegravir cellular permeativity is reduced in the presence of magnesium and calcium. [3] Raltegravir and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs efficiently block viral replication. [4] In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication is efficiently inhibited by Raltegravir, which shows an EC90 in the low nanomolar range. [5] |
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In Vivo
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Raltegravir induces viro-immunological improvement of nonhuman primates with progressing SIVmac251 infection. One non-human primate shows an undetectable viral load following Raltegravir monotherapy. [5] |
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Clinical Trials
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Raltegravir has entered in a phase II clinical trial in the treatment of HIV infections. |
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Features
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Raltegravir is the first approved human immunodeficiency virus type 1 (HIV-1) integrase inhibitor. |
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Combination Therapy
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Description
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Additive to synergistic activity is observed when human T lymphoid cell culture samples infected with the H9IIIB variant of HIV-1 are incubated with Raltegravir and a panel of available nucleoside analogue reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors, and protease inhibitors. Raltegravir-containing regimens have potent antiretroviral activity and are well tolerated in HIV-1-infected individuals. [2] Antiretroviral therapy (ART) regimens consisting of two NRTIs/NtRTIs plus Raltegravir maintains stably suppressed SIVmac251 viral load, but not the proviral DNA, in non-human primates. [5] Raltegravir plus Truvada has entered in a phase III clinical trial in the treatmento of HIV infection. |
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Protocol
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Kinase Assay
[1]
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| PFV integration assay |
For quantitative strand transfer assays, donor DNA substrate is formed by annealing HPLC grade oligonucleotides 5′-GACTCACTATAGGGCACGCGTCAAAATTCCATGACA and 5′-ATTGTCATG GAATTTTGACGCGTGCCCTATAGTGAGTC. Reactions (40 μL) contains 0.75 μM PFV IN, 0.75 μM donor DNA, 4 nM (300 ng) supercoiled pGEM9-Zf(?) target DNA, 125 mM NaCl, 5 mM MgSO4, 4 μM ZnCl2, 10 mM DTT, 0.8% (vol/vol) DMSO, and 25 mM BisTris propane–HCl, pH 7.45. Raltegravir is added at indicated concentrations. Reactions are initiated by addition of 2 μL PFV IN diluted in 150 mM NaCl, 2 mM DTT, and 10 mM Tris-HCl, pH 7.4, and stopped after 1 hour at 37 °C by addition of 25 mM EDTA and 0.5% (wt/vol) SDS. Reaction products, deproteinized by digestion with 20 μg proteinase K for 30 minutes at 37 °C followed by ethanol precipitation, are separated in 1.5% agarose gels and visualized by staining with ethidium bromide. Integration products are quantified by quantitative real-time PCR, using Platinum SYBR Green qPCR SuperMix and three primers: 5′-CTACTTACTCTAGCTTCCCGGCAAC, 5′-TTCGCCAGTTAATAGTTTGCGCAAC, and 5′-GACTCACTATAGGGCACGCGT. PCR reactions (20 μL) contained 0.5 μM of each primer and 1 μL diluted integration reaction product. Following a 5-min denaturation step at 95 °C, 35 cycles are carried out in a CFX96 PCR instrument, using 10 seconds denaturation at 95 °C, 30 seconds annealing at 56 °C and 1 minutes extension at 68 °C. Standard curves are generated using serial dilutions of WT PFV IN reaction in the absence of INSTI. |
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Cell Assay
[5]
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| Cell Lines |
Human MT-4 cells |
| Concentrations |
0.0001-1 μM |
| Incubation Time |
5 days |
| Methods |
Human MT-4 cells are infected for 2 hours with the SIVmac251, HIV-1 (IIIB) and HIV-2 (CDC 77618) stocks at a multiplicity of infection of, approximately, 0.1. Cells are then washed three times in phosphate buffered saline, and suspended at 5 × 105/mL in fresh culture medium (to primary cells 50 units/mL of IL-2 are added) in 96-well plates, in the presence or absence of a range of triplicate raltegravir concentrations (0.0001 μM -1 μM). Untreated infected and mock-infected controls are prepared too, in order to allow comparison of the data derived from the different treatments. Viral cytopathogeniciy in MT-4 cells is quantitated by the methyl tetrazolium (MTT) method (MT-4/MTT assay) when extensive cell death in control virus-infected cell cultures is detectable microscopically as lack of capacity to re-cluster. The capability of MT-4 cells to form clusters after infection. Briefly, clusters are disrupted by pipetting; and, after 2 hours of incubation at 37 °C, the formation of new clusters is assessed by light microscopy (100 × magnification). Cell culture supernatants are collected for HIV-1 p24 and HIV-2/SIVmac251 p27 core antigen measurement by ELISA. In CEMx174-infected cell cultures, which show a propensity to form syncytia induced by the virus envelope glycoproteins, syncytia are counted, in blinded fashion, by light microscopy for each well at 5 days following infection. |
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Animal Study
[5]
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| Animal Models |
Indian Rhesus macaques |
| Formulation |
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| Doses |
50 mg/kg or 100 mg/kg |
| Administration |
Oral administration |
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References |
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[1] Hare S, et al. Proc Natl Acad Sci U S A. 2010, 107(46), 20057-20062.
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[2] Hicks C, et al. Clin Infect Dis. 2009, 48(7), 931-939.
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[3] Moss DM, et al. Antimicrob Agents Chemother. 2012, 56(6), 3020-3026.
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[4] Hare S, et al. Mol Pharmacol. 2011, 80(4), 565-572.
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[5] Lewis MG, et al. Retrovirology. 2010, 7, 21.
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