HIV integrase strand transfer (IC50 = 2.7 nM)

Dolutegravir (S/GSK1349572) has an EC50 of 0.51 nM against HIV-1 in PBMCs, 0.71 nM in MT-4 cells, and 2.2 nM in the pseudotyped self-inactivating virus (PHIV) assay. Dolutegravir's 50% cytotoxic concentrations (CC50) in proliferating IM-9, U-937, MT-4, and Molt-4 cells are, in order, 4.8, 7.0, 14, and 15 μM. The CC50 values in unstimulated and stimulated PBMCs are 189 μM and 52 μM, in that order. Dolutegravir's 0.51 nM EC50 against HIV-1 in PBMCs indicates that a cell-based therapeutic index of at least 9,400 is required[1].

In rats (0.23 mL/min/kg) and monkeys (2.12 mL/min/kg), the plasma clearance after a single intravenous (IV) dose of dolutegravir is low. Both the rat and monkey have half-lives of roughly six hours, and their steady-state volume of distribution (VSS) is small. When given orally as a solution to one male monkey and five fast-fasting rats, dolutegravir is highly bioavailable and quickly absorbed (75.6 and 87.0%, respectively). After oral administration of a suspension to non-fasted rats up to 250 mg/kg and non-fasted monkeys up to 50 mg/kg, dolutegravir exposure (Cmax and AUC) increased with increasing dose, however the rise is less than proportional[3].

In vitro strand transfer assay. The inhibitory potencies of S/GSK1349572 and other INIs were measured in a strand transfer assay using recombinant HIV integrase as previously described (5). A complex of integrase and biotinylated preprocessed donor DNA-streptavidin-coated Acintillation proximity assay (SPA) beads was formed by incubating 2 μM purified recombinant integrase with 0.66 μM biotinylated donor DNA-4 mg/ml streptavidin-coated SPA beads in 25 mM sodium morpholinepropanesulfonic acid (MOPS) (pH 7.2), 23 mM NaCl, and 10 mM MgCl2 for 5 min at 37°C. These beads were spun down and preincubated with diluted INIs for 60 min at 37°C. Then a 3H-labeled target DNA substrate was added to give a final concentration of 7 nM substrate, and the strand transfer reaction mixture was incubated at 37°C for 25 to 45 min, which allowed for a linear increase in the strand transfer of donor DNA to radiolabeled target DNA. The signal was read using a Wallac MicroBeta scintillation plate reader [1].

Antiviral assay in MT-4 cells.[1]
MT-4 cells growing exponentially at a density of 5 × 105 or 6 × 105/ml were infected with HIV-1 strain IIIB at a viral multiplicity of infection of 0.001 or a 50% tissue culture infective dose of 4 to 10. The cells were then aliquoted to 96-well plates in the presence of varying concentrations of compounds. After incubation for 4 or 5 days, antiviral activity was determined by a cell viability assay that either measured bioluminescence with a CellTiter-Glo luminescent reagent or measured absorbance at 560 and 690 nm using the yellow tetrazolium MTT reagent [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide].

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Antiviral assay in PBMCs.[1]
In one 96-well culture plate, PHA- and IL-2-stimulated PBMCs (4 × 105/well) were preincubated with a compound for 1 h, while HIV-1 strain Ba-L was mixed with the same compound in a second plate. An aliquot of the Ba-L-compound mixture was then transferred to the PBMC-compound mixture and was incubated for 7 days. After this incubation, supernatants were assayed for reverse transcriptase (RT) activity by incorporation of [methyl-3H]dTTP to measure viral replication as previously described.

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Cytotoxicity assays.[1]
In vitro growth inhibition (cytotoxicity) studies were conducted with S/GSK1349572 in proliferating human leukemic and lymphomic cell lines (IM-9, U-937, MT-4, and Molt-4) as well as in stimulated and unstimulated human PBMCs. ATP levels were quantified by using the CellTiter-Glo luciferase reagent to measure the ability of a compound to inhibit cell growth as an indicator of the compound's potential for cytotoxicity.

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Mechanistic cellular studies.[1]
To determine if S/GSK1349572 was inhibiting HIV replication in cellular assays through an integrase inhibition mechanism, the effects on the synthesis of HIV NL432 DNA species in MT-4 cells were measured in a single-round infection assay using quantitative PCR methods. Quantitative PCR analysis was performed to measure the synthesis of HIV DNA species in MT-4 cells in the presence of an INI or NNRTI as described previously, with minor modifications. Briefly, 293T cells were transfected with the NL432 plasmid to generate infectious virus, and the supernatant was filtered through 0.45-μm-pore-size filters and was treated with DNase I. MT-4 cells were infected with HIV-1 NL432 for 1 h, incubated with dilutions of a compound, and collected after 6 or 18 h of incubation. All cells were incubated with 0.5 μM ritonavir in order to limit HIV replication to a single cycle. Total-DNA PCR to detect late RT products was performed by incubating the samples for 6 h. Nested Alu-PCR to detect integrated provirus and 2-LTR PCR to detect 2-LTR circles were performed by incubating the samples for 18 h. Reaction products were analyzed using the ABI Prism 7900HT-3 sequence detection system

For rat and monkey PK studies, Dolutegravir is administered as the free acid or the sodium salt. All doses are presented in terms of the free acid. Dolutegravir is administered by intravenous (IV) short-term (within 2 min) bolus (1 mg/kg) to three male rats and two male monkeys. For single oral administration, Dolutegravir as a solution (5 mg/kg) is administered to three fasted male rats and two fasted male monkeys. Dolutegravir is administered as single oral doses of 5, 50, 100, and 250 mg/kg to non-fasted male rats (n=2/dose level) and 3, 10, and 50 mg/kg to non-fasted female monkeys. For intravenous administration, blood samples are collected from rats (0.2 mL via jugular vein cannula) and monkeys (approximately 0.2 or 0.5 mL via saphenous vein in a hindlimb) into Na2EDTA-treated syringes at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h. For oral administration, samples are collected at 0.25 (rats only), 0.5, 1, 2, 4, 6 [rats (solution and suspension) and monkey (solution only)], 8, and 24 h. Following collection, the blood is immediately put on wet ice and then centrifuged within an hour at 1740 g for 10 min at 4°C to obtain plasma. All samples are stored at approximately -20°C or colder prior to analysis by using a method based on protein precipitation and LC-MS/MS analysis.[1]

2.7 mg/kg/day; administrated orally for two weeks.

C57BL/6 mice

[1]. Kobayashi M, et al. In Vitro antiretroviral properties of S/GSK1349572, a next-generation HIV integrase inhibitor. Antimicrob Agents Chemother. 2011 Feb;55(2):813-21.
[2]. Hare S, et al. Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572). Mol Pharmacol. 2011 Oct;80(4):565-72.
[3]. Moss L, et al. The comparative disposition and metabolism of dolutegravir, a potent HIV-1 integrase inhibitor, in mice, rats, and monkeys. Xenobiotica. 2015 Jan;45(1):60-70

C20H19F2N3O5

419.38

419.12927

C, 57.28; H, 4.57; F, 9.06; N, 10.02; O, 19.08

1051375-16-6

Dolutegravir sodium;1051375-19-9;Cabotegravir;1051375-10-0;Dolutegravir-d3;Dolutegravir-d5;2249814-82-0

White to off-white solid

-1.32

104.36

O=C(C1=CN(C2=C(O)C1=O)C[C@]3([H])OCC[C@@H](C)N3C2=O)NCC4=CC=C(F)C=C4F

RHWKPHLQXYSBKR-BMIGLBTASA-N

InChI=1S/C20H19F2N3O5/c1-10-4-5-30-15-9-24-8-13(17(26)18(27)16(24)20(29)25(10)15)19(28)23-7-11-2-3-12(21)6-14(11)22/h2-3,6,8,10,15,27H,4-5,7,9H2,1H3,(H,23,28)/t10-,15+/m1/s1

(4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.62 mg/mL (6.25 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.96 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.96 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 25.0 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.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.96 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)