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Raltegravir potassium (formerly also known as MK-0518 potassium; trade name: Isentress) is a novel, potent integrase (IN) inhibitor for WT and S217Q PFV IN with IC50 of 90 nM and 40 nM in cell-free assays, respectively. Raltegravir is an antiretroviral drug used to treat HIV infection. Raltegravir binds to and inhibits integrase, an HIV enzyme that inserts viral genetic material into the genetic material of the infected human cell. Inhibition of integrase prevents insertion of HIV DNA into the human DNA genome, thus blocking HIV replication. It is the first integrase inhibitors that received FDA approval for HIV treatment.
Physicochemical Properties
| Molecular Formula | C20H20FKN6O5 |
| Molecular Weight | 482.51 |
| Exact Mass | 482.111 |
| CAS # | 871038-72-1 |
| Related CAS # | Raltegravir;518048-05-0;Raltegravir-d3 potassium;1246816-98-7;Raltegravir sodium;1292804-07-9 |
| PubChem CID | 23668479 |
| Appearance | White to off-white solid powder |
| Density | 1.46 g/cm3 |
| Melting Point | 282ºC |
| LogP | 2.131 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 9 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 33 |
| Complexity | 843 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | IFUKBHBISRAZTF-UHFFFAOYSA-M |
| InChi Code | InChI=1S/C20H21FN6O5.K/c1-10-25-26-17(32-10)16(30)24-20(2,3)19-23-13(14(28)18(31)27(19)4)15(29)22-9-11-5-7-12(21)8-6-11;/h5-8,28H,9H2,1-4H3,(H,22,29)(H,24,30);/q;+1/p-1 |
| Chemical Name | potassium;4-[(4-fluorophenyl)methylcarbamoyl]-1-methyl-2-[2-[(5-methyl-1,3,4-oxadiazole-2-carbonyl)amino]propan-2-yl]-6-oxopyrimidin-5-olate |
| Synonyms | MK-0518; MK0518; MK 0518; MK-0518 potassium; Raltegravir; trade name: Isentress |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | PFV IN with the S217H alteration has an IC50 of 900 nM, making it ten times less sensitive to raltegravir. PFV IN exhibited 10% of WT's activity and was inhibited by Raltegravir at an IC50 of 200 nM, suggesting that PFV IN is less sensitive to IN strand transfer inhibitors (INSTIs) than WT IN is. Similar to the WT enzyme, S217Q PFV IN is also susceptible to raltegravir [1]. Glucuronidation, not the liver, is the mechanism of raltegravir metabolism. With a 95% inhibitory concentration of 31±20 nM in human T cell cultures, raltegravir demonstrates strong anti-HIV-1 action in vitro. Raltegravir exhibited anti-HIV-2 activity in CEMx174 cells as well, with an IC95 of 6 nM. Glucuronidation is the main mechanism of raltegravir metabolism. Strong glucuronidase UGT1A1 inducers should not be utilized since they drastically lower raltegravir concentrations. Hepatic cytochrome P450 activity is only slightly inhibited by raltegravir. Neither CYP3A4-dependent testosterone 6-beta-hydroxylase activity nor CYP3A4 RNA expression are induced by raltegravir [2]. Magnesium and calcium have been shown to decrease raltegravir's cellular permeability [3]. Effectively preventing viral replication is possible with raltegravir and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) [4]. Latisavue successfully suppressed SIVmac251 replication in the acutely infected human lymphoid CD4+ T cell lines MT-4 and CEMx174, suggesting an EC90 in the low nanomolar range [5]. |
| ln Vitro |
PFV IN with the S217H alteration has an IC50 of 900 nM, making it ten times less sensitive to raltegravir. PFV IN exhibited 10% of WT's activity and was inhibited by Raltegravir at an IC50 of 200 nM, suggesting that PFV IN is less sensitive to IN strand transfer inhibitors (INSTIs) than WT IN is. Similar to the WT enzyme, S217Q PFV IN is also susceptible to raltegravir [1]. Glucuronidation, not the liver, is the mechanism of raltegravir metabolism. With a 95% inhibitory concentration of 31±20 nM in human T cell cultures, raltegravir demonstrates strong anti-HIV-1 action in vitro. Raltegravir exhibited anti-HIV-2 activity in CEMx174 cells as well, with an IC95 of 6 nM. Glucuronidation is the main mechanism of raltegravir metabolism. Strong glucuronidase UGT1A1 inducers should not be utilized since they drastically lower raltegravir concentrations. Hepatic cytochrome P450 activity is only slightly inhibited by raltegravir. Neither CYP3A4-dependent testosterone 6-beta-hydroxylase activity nor CYP3A4 RNA expression are induced by raltegravir [2]. Magnesium and calcium have been shown to decrease raltegravir's cellular permeability [3]. Effectively preventing viral replication is possible with raltegravir and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) [4]. Latisavue successfully suppressed SIVmac251 replication in the acutely infected human lymphoid CD4+ T cell lines MT-4 and CEMx174, suggesting an EC90 in the low nanomolar range [5]. Raltegravir potently inhibited wild-type (WT) prototype foamy virus (PFV) integrase strand transfer activity in an in vitro assay, with an IC50 of 90 nM. [1] PFV integrase carrying the S217H substitution (equivalent to HIV-1 Q148H) displayed reduced susceptibility to Raltegravir, with an IC50 of approximately 900 nM, indicating a 10-fold decrease in potency compared to the WT enzyme. [1] PFV integrase carrying the S217Q substitution remained sensitive to Raltegravir, with an IC50 of 40 nM. [1] PFV integrase carrying the N224H substitution (equivalent to HIV-1 N155H) also showed reduced susceptibility to Raltegravir, with an IC50 of 200 nM, representing an approximately 2-fold decrease in potency. [1] |
| ln Vivo |
Rateltelevir improves the viro-immunological status of nonhuman primates infected with SIVmac251 as it progresses. Raltegravir monotherapy results in an undetectable viral load in one non-human primate[5]. In SIVmac251-infected rhesus macaques (Group 1), ten-day monotherapy with raltegravir (50 or 100 mg twice daily orally with food) resulted in a significant decrease in viral load (P = 0.031) and a significant increase in CD4+ T cell counts (P = 0.017) in all animals. One animal in the 100 mg group achieved undetectable viral load (<40 copies/mL). [5] After adding two reverse transcriptase inhibitors, tenofovir (PMPA, 20 mg/kg/day subcutaneously) and emtricitabine (FTC, 50 mg/kg/day subcutaneously), to the raltegravir regimen (all animals switched to 100 mg twice daily) starting at day 11, viral load continued to decrease, becoming undetectable in all animals within two weeks and remaining undetectable until the end of follow-up (day 52). CD4 counts continued to increase, restoring to pre-inoculation levels. [5] In a second group of SIVmac251-infected macaques (Group 2), raltegravir monotherapy (100 mg twice daily) for seven days also resulted in a significant decrease in viral load compared to pre-treatment historical values, confirming the reproducible antiviral effect. One animal with undetectable viral load after monotherapy showed a rebound upon treatment suspension. [5] Despite suppression of viral load to undetectable levels with the combination therapy, proviral DNA levels in PBMCs remained stable and did not change significantly after 52 days of treatment, indicating persistence of the lentiviral reservoir. [5] |
| Enzyme Assay |
A quantitative PFV integrase strand transfer assay was performed. The reaction mixture (40 µL) contained 0.75 µM purified PFV integrase, 0.75 µM donor DNA (a double-stranded oligonucleotide mimicking the preprocessed viral U5 DNA end), 4 nM (300 ng) supercoiled plasmid target DNA, 5 mM MgSO4 (as the divalent metal cofactor), 4 µM ZnCl2, 125 mM NaCl, 10 mM DTT, 25 mM Bis-Tris propane-HCl buffer (pH 7.4), and 0.8% DMSO. Raltegravir was added to the reaction at the indicated concentrations. Reactions were initiated by adding PFV integrase, incubated at 37°C for 1 hour, and stopped by adding EDTA and SDS. Reaction products were deproteinized, ethanol precipitated, and separated on a 1.5% agarose gel stained with ethidium bromide for visualization. For quantification, integration products were measured by quantitative real-time PCR using specific primers and a fluorescent DNA-binding dye. A standard curve generated from serial dilutions of a WT PFV integrase reaction (without inhibitor) was used to quantify the integration efficiency in inhibitor-treated samples. [1] |
| Cell Assay |
Caco-2 Monolayer Permeability Assay: Caco-2 cells were seeded on polycarbonate membrane Transwells and cultured for 21 days to form monolayers. Monolayer integrity was confirmed by transepithelial electrical resistance (TEER >600 Ω) and low permeability to [14C]mannitol. For permeability studies, the pH in the apical compartment was varied (using buffers like MES for lower pH and HEPES/Tricine for higher pH), while the basolateral compartment was maintained at pH 7.4. Raltegravir (50 µM for pH effect studies; 1 µM for metal/omeprazole studies) was added to either the apical (for apical-to-basolateral transport) or basolateral (for basolateral-to-apical transport) compartment. Plates were incubated at 37°C with 5% CO2. Samples were taken from the receiver compartment at various time points (e.g., 0, 30, 60, 90, 120 min) and replaced with fresh buffer. Raltegravir concentrations were analyzed by LC-MS/MS or scintillation counting. Apparent permeability (Papp) and efflux ratios were calculated. Cellular Accumulation Assay: Caco-2 cells were seeded in 6-well plates and grown for 5 days. Cells were washed and equilibrated with pH-buffered incubation solutions (pH 5-8). Raltegravir (1 µM) was added with or without pre- and co-incubation with the ABCB1 inhibitor tariquidar (300 nM). After a 10-minute incubation at 37°C with 5% CO2, extracellular samples were taken, cells were washed with ice-cold buffer, and lysed with water. Cell lysates were processed with acetonitrile, centrifuged, and the supernatant was analyzed for raltegravir content by LC-MS/MS. [3] |
| Animal Protocol |
Indian rhesus macaques were mucosally inoculated (intrarectally or intravaginally) with 300 MID50 of pathogenic SIVmac251. Viral loads stabilized by week 12 post-infection. For drug treatment, animals were randomized to receive raltegravir monotherapy orally at 50 mg or 100 mg twice daily with food for 10 days. At day 11, animals originally on 50 mg were switched to 100 mg twice daily, and all animals received subcutaneous injections of tenofovir (PMPA, 20 mg/kg/day) and emtricitabine (FTC, 50 mg/kg/day) in addition to raltegravir. Treatment continued until day 52. Plasma samples were collected regularly for viral load measurement by quantitative RT-PCR. PBMCs were collected for proviral DNA quantification by PCR and for flow cytometric analysis of CD4+ T cell counts. Clinical chemistry and hematology were monitored. [5] |
| ADME/Pharmacokinetics |
Raltegravir solubility is pH-dependent. At 10 mM, it was partly insoluble at pH 6.6 and below, but fully soluble at pH 6.8 to 8. The lipophilicity (log P) of raltegravir, determined using octanol-water partition, decreased from ~1.06 to -1.29 as pH increased from 5 to 9. The pKa of raltegravir was determined to be 6.7 using UV spectroscopy. In vitro cellular permeability (apical-to-basolateral) of raltegravir across Caco-2 monolayers is significantly reduced with increasing extracellular pH (from 27.3 x 10^-6 cm/s at pH 5 to 2.9 x 10^-6 cm/s at pH 8.5). Divalent cations (Mg2+, Ca2+) reduce the in vitro cellular permeability of raltegravir. Clinical studies referenced show that co-administration with omeprazole (increasing gastric pH) increases raltegravir AUC and Cmax in healthy volunteers, and that antacids containing magnesium/aluminum reduce raltegravir C12. The primary metabolic route of raltegravir is glucuronidation via UGT1A1. It is a weak substrate for drug transporters ABCB1, SLC22A6, and SLC15A1. [3] |
| Toxicity/Toxicokinetics |
Raltegravir is not a substrate or inhibitor of major cytochrome P450 enzymes. Drug-drug interactions exist: Atazanavir (UGT1A1 inhibitor) increases raltegravir exposure, while rifampin (UGT1A1 inducer) decreases it. Co-administration with antacids containing magnesium/aluminum resulted in 75% of subjects having a raltegravir C12 lower than the IC95 (15 ng/mL in 50% human serum). [3] |
| References |
[1]. Molecular mechanisms of retroviral integrase inhibition and the evolution of viral resistance. Proc Natl Acad Sci U S A, 2010. 107(46): p. 20057-62. [2]. Raltegravir: the first HIV type 1 integrase inhibitor. Clin Infect Dis. 2009 Apr 1;48(7):931-9. [3]. Divalent metals and pH alter raltegravir disposition in vitro. Antimicrob Agents Chemother. 2012 Jun;56(6):3020-6. [4]. Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572). Mol Pharmacol. 2011 Oct;80(4):565-72. [5]. Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy. Retrovirology, 2010. 7: p. 21. |
| Additional Infomation |
Raltegravir (brand names: Isentress and Isentress HD) is a prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection in adults and children. One form of raltegravir, Isentress, is approved for adults and children weighing at least 4 lb and 4 oz (2 kg). Another form of raltegravir, Isentress HD, is approved for adults and children weighing at least 88 lb (40 kg). Raltegravir is always used in combination with other HIV medicines. Raltegravir Potassium is the orally bioavailable potassium salt of a human immunodeficiency virus (HIV) integrase strand transfer inhibitor (HIV-1 INSTI) with HIV-1 antiviral activity. Raltegravir binds to and inhibits integrase, an HIV enzyme that inserts viral genetic material into the genetic material of the infected human cell. Inhibition of integrase prevents insertion of HIV DNA into the human DNA genome, thus blocking HIV replication. A pyrrolidinone derivative and HIV INTEGRASE INHIBITOR that is used in combination with other ANTI-HIV AGENTS for the treatment of HIV INFECTION. See also: Raltegravir (has active moiety); Lamivudine; raltegravir potassium (component of). Drug Indication Isentress is indicated in combination with other anti-retroviral medicinal products for the treatment of human immunodeficiency virus (HIV 1) infection. Raltegravir is the first integrase strand transfer inhibitor (INSTI) approved for clinical use against HIV-1 infection. [1] Crystal structures of the prototype foamy virus (PFV) intasome (integrase-DNA complex) bound to Raltegravir revealed its binding mode. Raltegravir binds to the active site of integrase, chelating the two catalytic divalent metal ions (Mg²⁺ or Mn²⁺) via a triad of heteroatoms in its pharmacophore. This binding displaces the 3’-terminal adenosine nucleotide of the viral DNA from the active site, thereby inactivating the intasome and blocking the strand transfer reaction. [1] A key interaction involves a face-to-face π-π stacking between the oxadiazole ring of Raltegravir and the side chain of Tyr212 in PFV integrase (equivalent to Tyr143 in HIV-1 integrase). Substitutions at this position (e.g., Y143H/R/C in HIV-1) are known to confer resistance, which is explained by the loss of this stabilizing interaction. [1] The study used PFV integrase as a model to understand resistance mutations in HIV-1 integrase. The S217H and N224H mutations in PFV integrase (analogous to Q148H and N155H in HIV-1) confer reduced susceptibility to Raltegravir. Crystal structures of mutant intasomes showed that these substitutions necessitate unfavorable conformational rearrangements in the active site to accommodate the inhibitor, explaining the mechanism of resistance. Specifically, the S217H (Q148H) mutation requires a significant backbone shift for inhibitor binding, while the N224H (N155H) mutation disrupts a stabilizing interaction between the asparagine and the DNA backbone that must be broken upon inhibitor binding. [1] The presence of a serine at position 209 in PFV integrase (equivalent to Gly140 in HIV-1) likely explains why the single S217H mutant in PFV shows a high level of resistance similar to the HIV-1 Q148H/G140S double mutant. In the S217H mutant structure, Ser209 forms a hydrogen bond with His217, illustrating the potential interaction and co-evolution of residues at positions 140 and 148 in HIV-1 integrase under drug selection pressure. [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL Solubility in Formulation 5: 25 mg/mL (51.81 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0725 mL | 10.3625 mL | 20.7250 mL | |
| 5 mM | 0.4145 mL | 2.0725 mL | 4.1450 mL | |
| 10 mM | 0.2072 mL | 1.0362 mL | 2.0725 mL |