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

Absorption, Distribution and Excretion
When 50 mg of dolutegravir once daily was orally administered to HIV-1 infected adults, the AUC, Cmax, and Cmin is 53.6 mcg h/mL, 3.67 mcg/mL, and 1.11 mcg/mL, respectively. The peak plasma concentration was observed 2 to 3 hours post-dose. Steady state is achieved within approximately 5 days with average accumulation ratios for AUC, Cmax, and C24h ranging from 1.2 to 1.5. When 50 mg once daily is given to pediatric patients (12 to < 18 years and weighing ≥40 kg) the Cmax, AUC, and C24 is 3.49 mcg/mL, 46 mcg.h/mL, and 0.90 mcg/mL respectively.
When a single oral dose of dolutegravir is given, nearly all complete dose is recovered in a proportion of 53% excreted unchanged in the feces and 31% excreted in urine. The renal eliminated recovered dose consists of ether glucuronide of dolutegravir (18.9%), a metabolite formed by oxidation at the benzylic carbon (3.0%), a hydrolytic N-dealkylation product (3.6%) and unchanged drug (< 1%).
The administration of a dose of 50 mg of dolutegravir presents an apparent volume of distribution of 17.4 L. The median dolutegravir concentration in CSF was 18 ng/mL after 2 weeks of treatment.
The apparent clearance rate of dultegravir is 1.0 L/h.
... After a single oral dose of [14C] dolutegravir, 53% of the total oral dose was excreted unchanged in feces. Thirty-one percent of the total oral dose was excreted in urine, represented by an ether glucuronide of dolutegravir (18.9% of total dose), a metabolite formed by oxidation at the benzylic carbon (3.0% of total dose), and its hydrolytic N-dealkylation product (3.6% of total dose). Renal elimination of unchanged drug was low (<1% of the dose).
Dolutegravir is highly bound (=98.9%) to human plasma proteins based on in vivo data and binding is independent of plasma concentration of dolutegravir. The apparent volume of distribution (Vd/F) following 50-mg once-daily administration is estimated at 17.4 L based on a population pharmacokinetic analysis.
Food increased the extent of absorption and slowed the rate of absorption of dolutegravir. Low-, moderate-, and high-fat meals increased dolutegravir AUC(0-8) by 33%, 41%, and 66%; increased Cmax by 46%, 52%, and 67%; and prolonged Tmax to 3, 4, and 5 hours from 2 hours under fasted conditions, respectively.
Following oral administration of dolutegravir, peak plasma concentrations were observed 2 to 3 hours postdose. With once-daily dosing, pharmacokinetic steady state is achieved within approximately 5 days with average accumulation ratios for AUC, Cmax, and C24 h ranging from 1.2 to 1.5. Dolutegravir plasma concentrations increased in a less than dose-proportional manner above 50 mg. Dolutegravir is a P-glycoprotein substrate in vitro. The absolute bioavailability of dolutegravir has not been established.

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Metabolism / Metabolites
Dolutegravir is highly metabolized through three main pathways and it forms no long-lived metabolites. The first pathway is defined by the glucuronidation by UGT1A1, the second pathway by carbon oxidation by CYP3A4 and the third pathway is what appears to be a sequential oxidative defluorination and glutathione conjugation. The main metabolite found in blood plasma is the ether glucuronide form (M2) and its chemical properties disrupt its ability to bind metal ions, therefore, it is inactive.
Dolutegravir is primarily metabolized via UGT1A1 with some contribution from CYP3A. ... ether glucuronide of dolutegravir (18.9% of total dose), a metabolite formed by oxidation at the benzylic carbon (3.0% of total dose), and its hydrolytic N-dealkylation product (3.6% of total dose). ...

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Biological Half-Life
The half-life of dolutegravir is 14 hours.
Dolutegravir has a terminal half-life of approximately 14 hours and an apparent clearance (CL/F) of 1.0 L/h based on population pharmacokinetic analyses.

Hepatotoxicity
In large clinical trials, therapy with dolutegravir was associated with alanine aminotransferase (ALT) elevations of greater than 3 times the upper limit of normal (ULN) in 2% to 5% of patients, but these rates were similar to those in comparator groups receiving matched background optimized antiretroviral therapy without dolutegravir. These elevations were not associated with clinical symptoms and generally did not require dose modification. A few instances of acute liver injury with jaundice were described in the registration trials for dolutegravir which occurred in association with hypersensitivity reactions and resolved with drug discontinuation. The clinical features of these cases were not provided and their association with dolutegravir as opposed to the concurrent antiretroviral agents was not fully established. Since its approval and more wide spread use, however, several case reports of acute hepatitis attributable to dolutegravir have appeared. The latency to onset varried from 1 to 8 months and the pattern of serum enzyme elevations was hepatocellular. Immunoallergic and autoimmune features were not present. At least one published case resulted in acute liver failure and need for liver transplanation. The product label for dolutegravir mentions hepatitis and hepatic failure as potential adverse reactions and states that patients with hepatitis B or C coinfection are susceptible to worsening or flares of hepatitis with initiation of dolutegravir therapy, perhaps as a consequence of immune reconstitution syndrome. Monitoring of liver tests is recommended in patients starting regimens that include dolutegravir.
Likelihood score: D (possible cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Dolutegravir is detectable in maternal milk in low amounts. It appears that elimination by newborn infants is prolonged and the drug has been detected in infant plasma during breastfeeding. Dolutegravir has been used safely in HIV-positive mothers during breastfeeding and is recommended as a first-line drug during breastfeeding. Achieving and maintaining viral suppression with antiretroviral therapy decreases breastfeeding transmission risk to less than 1%, but not zero. Individuals with HIV who are on antiretroviral therapy with a sustained undetectable viral load and who choose to breastfeed should be supported in this decision. If a viral load is not suppressed, banked pasteurized donor milk or formula is recommended.
◉ Effects in Breastfed Infants
An HIV-positive mother took a combination tablet containing dolutegravir 50 mg, abacavir sulfate 600 mg and lamivudine 300 mg (Triumeq) once daily. Her infant was exclusively breastfed for about 30 weeks and partially breastfed for about 20 weeks more. No obvious side effects were noted.
In a study of African women with HIV infection received a dolutegravir-based regimen of 50 mg dolutegravir, 300 mg tenofovir disoproxil fumarate, and either 200 mg emtricitabine in South Africa or 300 mg lamivudine in Uganda during pregnancy and breastfeeding. This regimen was as safe for breastfed as an efavirenz-based regimen with the same other drugs.
An open-label, controlled, multicenter phase 3 trial women who were confirmed HIV-positive were randomized to receive one of 3 regimens: dolutegravir, emtricitabine, and tenofovir alafenamide (n = 208); dolutegravir, emtricitabine, and tenofovir disoproxil fumarate (n = 202); or efavirenz, emtricitabine, and tenofovir disoproxil fumarate (n = 207). The regimens were started at 14 to 28 weeks of pregnancy and continued postpartum. Of the 617 liveborn infants, 99% were breastfeeding at time of last infant HIV test, which was as late as 50 weeks of age. The mean infant duration on the study was 47.6 weeks of age. Infants who had any clinical or laboratory adverse event of grade 3 or higher ranged from 25 to 31%, but was not statistically significant between groups. Dolutegravir-containing regimens resulted in lower rates of virological failure, HIV drug resistance, and infant mortality up to 50 weeks postpartum compared with efavirenz, emtricitabine, and tenofovir disoproxil fumarate.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Dolutegravir is highly protein bound to human plasma proteins reaching a percentage 98.9% of the administered dose.

[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

Dolutegravir is a monocarboxylic acid amide obtained by formal condensation of the carboxy group of (4R,12aS)-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-carboxylic acid with the amino group of 2,4-difluorobenzylamine. Used (as its sodium salt) for treatment of HIV-1. It has a role as a HIV-1 integrase inhibitor. It is a monocarboxylic acid amide, an organic heterotricyclic compound, a secondary carboxamide and a difluorobenzene. It is a conjugate acid of a dolutegravir(1-).
Dolutegravir (brand names: Tivicay and Tivicay PD) is a prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection in combination with:
Other HIV medicines in adults;
Other HIV medicines in children at least 4 weeks of age and older who weigh at least 6.6 lb (3 kg) and who meet specific requirements, as determined by a health care provider; or
Rilpivirine (brand name: Edurant) in adults to replace their current HIV medicines when their health care provider determines that they meet certain requirements.
Dolutegravir is always used in combination with other HIV medicines.
Dolutegravir is an HIV-1 integrase inhibitor that blocks the strand transfer step of the integration of the viral genome into the host cell (INSTI). The effect of this drug has no homology in human host cells, which gives it excellent tolerability and minimal toxicity. Dolutegravir was developed by ViiV Healthcare and FDA-approved on August 12, 2013. On November 21, 2017, dolutegravir, in combination with rilpivirine, was approved as part of the first complete treatment regimen with only two drugs for the treatment of adults with HIV-1 named Juluca.
Dolutegravir is a Human Immunodeficiency Virus Integrase Strand Transfer Inhibitor. The mechanism of action of dolutegravir is as a HIV Integrase Inhibitor, and Multidrug and Toxin Extrusion Transporter 1 Inhibitor, and Organic Cation Transporter 2 Inhibitor.
Dolutegravir is a human immunodeficiency virus (HIV) integrase inhibitor, the third in this class of agents that target the viral integrase. Dolutegravir is used only in combination with other antiretroviral agents in the treatment of HIV infection, and it has had limited use. Dolutegravir is associated with a low rate of serum aminotransferase elevations during therapy, but has not been linked to instances of acute, clinically apparent liver injury.
Dolutegravir is an orally bioavailable integrase strand-transfer inhibitor (INSTI), with activity against human immunodeficiency virus type 1 (HIV-1) infection. Upon oral administration, dolutegravir binds to the active site of integrase, an HIV enzyme that catalyzes the transfer of viral genetic material into human chromosomes. This prevents integrase from binding to retroviral deoxyribonucleic acid (DNA), and blocks the strand transfer step, which is essential for the HIV replication cycle. This prevents HIV-1 replication.
See also: Dolutegravir Sodium (has salt form); Dolutegravir sodium monohydrate (is active moiety of); Dolutegravir; rilpivirine (component of) ... View More ...

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Drug Indication
Dolutegravir is indicated in combination with other antiretroviral agents for the treatment of patients with HIV-1 infection that comply with the characteristics of being adults or children aged 12 years and older and present at least a weight of 40 kg. The FDA combination therapy approval of dolutegravir and [rilpivirine] is indicated for adults with HIV-1 infections whose virus is currently suppressed (< 50 copies/ml) on a stable regimen for at least six months, without history of treatment failure and no known substitutions associated to resistance to any of the two components of the therapy. Dolutegravir is also available in combination with [lamivudine] and [abacavir] for the treatment of adult and pediatric patients with HIV-1 who weigh ≥10kg.
FDA Label
Tivicay is indicated in combination with other anti-retroviral medicinal products for the treatment of Human Immunodeficiency Virus (HIV) infected adults, adolescents and children of at least 6 years of age or older and weighing at least 14 kg. Tivicay is indicated in combination with other anti-retroviral medicinal products for the treatment of Human Immunodeficiency Virus (HIV) infected adults, adolescents and children of at least 4 weeks of age or older and weighing at least 3 kg.

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Mechanism of Action
Dolutegravir is an HIV-1 antiviral agent. It inhibits HIV integrase by binding to the active site and blocking the strand transfer step of retroviral DNA integration in the host cell. The strand transfer step is essential in the HIV replication cycle and results in the inhibition of viral activity. Dolutegravir has a mean EC50 value of 0.5 nM (0.21 ng/mL) to 2.1 nM (0.85 ng/mL) in peripheral blood mononuclear cells (PBMCs) and MT-4 cells.
Dolutegravir inhibits HIV integrase by binding to the integrase active site and blocking the strand transfer step of retroviral deoxyribonucleic acid (DNA) integration which is essential for the HIV replication cycle. Strand transfer biochemical assays using purified HIV-1 integrase and pre-processed substrate DNA resulted in IC50 values of 2.7 nM and 12.6 nM.

C20H19F2N3O5

419.38

419.129

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

54726191

White to off-white solid

1.53 g/cm3

669.0±55.0 °C at 760 mmHg

190-193ºC

358.4±31.5 °C

0.0±2.1 mmHg at 25°C

1.650

-1.32

2

8

3

30

829

2

FC1=CC(F)=C(C=C1)CNC(C2=CN(C3=C(C2=O)O)C[C@@]([H])(N4C3=O)OCC[C@H]4C)=O

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.)