RdRp (RNA-dependent RNA-polymerases)

After being continuously incubated with 1 µM remdesivir (GS-5734) for 72 hours, the levels of GS-443902 (GS-441524 triphosphate; remdesivir metabolite; compound 4tp) were determined at 2, 24, 48, and 72 hours. , and in macrophages, HMVEC, and HeLa cell lines, respectively, obtained Cmax values of 300, 110, and 90 pmol/million cells [1]. Compound 8a, GS-443902, is a derivative of triphosphate (TP) [2]. GS-443902 (NTP; 0.01, 0.1, 1, 10, 100 μM) integrates into immature viral RNA transcripts and causes their premature termination, hence inhibiting RSV RdRp-catalyzed RNA production. After being effectively converted to GS-443902 in cells, GS-5734 specifically inhibits EBOV replication by targeting its RdRp and suppressing viral RNA synthesis [3].

Remdesivir (GS-5734; 10 mg kg; intravenous injection) enters peripheral blood mononuclear cells (PBMC) quickly, and after two hours of administration, it is significantly and efficiently transformed to GS-443902 (GS-441524 III). NTP; phosphate; metabolite of remdesivir. Rhesus monkey. With a t1/2 length of 14 hours and levels necessary for >50% viral suppression at 24 hours, GS-443902 is the primary metabolite in PBMC [3].

Transcription reactions contained 25 µg of crude RSV RNP complexes in 30 µL of reaction buffer (50 mM TRIS-acetate [pH 8.0], 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl2, 3 mM DTT, 2 mM EGTA, 50 µg/mL BSA, 2.5 U RNasin, 20 µM ATP, 100 µM GTP, 100 µM UTP, 100 µM CTP, and 1.5 µCi [α-32P]ATP [3,000 Ci/mmol]). The radiolabeled nucleotide used in the transcription assay was selected to match the nucleotide analog being evaluated for inhibition of RSV RNP transcription. To determine whether nucleotide analogs inhibited RSV RNP transcription, compounds were added using a 6-step serial dilution in 5-fold increments. After a 90 min incubation at 30 °C, the RNP, reactions were stopped with 350 µL of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager[1].

EBOV HMVEC Antiviral Assay[1]
Antiviral assays were conducted in biosafety level-4 containment (BSL-4) at the CDC. HMVEC-TERT cells were seeded in 96 well plates. Eight to ten concentrations of compound were diluted in 3-fold serial dilution increments in media and 100 uL/well of each dilution was transferred in triplicate onto 96 well plates containing pre-seeded HMVEC-TERT monolayers. The plates were transferred to BSL-4 containment and the appropriate dilution of EBOV-GFP virus stock, previously determined by titration and prepared in cell culture media, was added to test plates containing cells and serially diluted compounds. Each plate included three wells of infected untreated cells and three wells of uninfected cells that served as 0% and 100% virus inhibition control, respectively. Following the infection, test plates were incubated for 3 to 4 days in a tissue culture incubator. After the incubation, virus replication was measured in an Envision plate reader by direct fluorescence to measure GFP expression from the reporter virus. The percentage inhibition was calculated for each tested concentration relative to the 0% and 100% inhibition controls and the EC50 value for each compound was determined by non-linear regression as the effective concentration of compound that inhibited virus replication by 50%.

In vivo efficacy[3]
Rhesus monkeys (Macaca mulatta) were challenged on day 0 by intramuscular injection with a target dose of 1,000 PFU of EBOV Kikwit (Ebola virus H. sapiens-tc/COD/1995/Kikwit), which was derived from a clinical specimen obtained during an outbreak occurring in the Democratic Republic of the Congo (formerly Zaire) in 1995. Challenge virus was propagated from the clinical specimen using cultured cells (Vero or Vero E6) for a total of four passages. Animals (3–6 years old) were randomly assigned to experimental treatment groups, stratified by sex (with equal number of males and females per group) and balanced by body weight, using SAS statistical software. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals. The primary endpoint for efficacy studies was survival to day 28 following virus challenge. GS-5734 was formulated at Gilead Sciences in water with 12% sulfobutylether-β-cyclodextrin (SBE- β-CD), pH adjusted to 3.0 using HCl. Formulations were administered to anaesthetized animals by bolus intravenous injection at a rate of approximately 1 min per dose in the right or left saphenous vein. The volume of all vehicle or GS-5734 injections was 2.0 ml kg−1 body weight. Animals were anaesthetized using intramuscular injection of a solution containing ketamine (100 mg ml−1) and acepromazine (10 mg ml−1) at 0.1 ml kg−1 body weight.[3]

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Animals were observed at least twice daily to monitor for disease signs, and animals that survived to day 28 were deemed to be protected. Study personnel alleviated unnecessary suffering of infected animals by euthanizing clinically moribund animals. The criteria used as the basis for euthanasia of moribund animals were defined before study initiation and included magnitude of responsiveness, reduced body temperature, and/or specified alterations to serum chemistry parameters35. Serum chemistry was analysed using a Vitros 350 Chemistry System, and coagulation parameters were evaluated using a Sysmex CA-1500 coagulation analyser. Haematology analysis was conducted using a Siemens Advia 120 Hematology System with multispecies software. On days in which GS-5734 or vehicle dosing were scheduled with blood sample collection for clinical pathology or viraemia analysis, blood samples were collected immediately before dose administration.[3]

[1]. Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. Med Chem. 2017 Mar 9;60(5):1648-1661.

[2]. Synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine C-nucleosides. Bioorg Med Chem Lett. 2012 Apr 15;22(8):2705-7.

[3]. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016 Mar 17;531(7594):381-5.

GS-443902 is an organic triphosphate that is GS-441524 in which the 5'-hydroxy group has been replaced by a triphosphate group. It is the active metabolite of remdesivir. It has a role as a drug metabolite, an antiviral drug and an anticoronaviral agent. It is a C-nucleoside, an aromatic amine, a nitrile, a pyrrolotriazine and an organic triphosphate. It is functionally related to a GS-441524.

531.202425003052

530.995

1355149-45-9

GS-441524;1191237-69-0;Remdesivir;1809249-37-3;GS-443902 trisodium;1355050-21-3

56832906

Typically exists as White to light yellow solids at room temperature

2.4±0.1 g/cm3

1.841

-5.92

7

17

8

33

941

4

P(=O)(O)(OP(=O)(O)OP(=O)(O)O)OC[C@@H]1[C@H]([C@H]([C@](C#N)(C2=CC=C3C(N)=NC=NN23)O1)O)O

DFVPCNAMNAPBCX-LTGWCKQJSA-N

InChI=1S/C12H16N5O13P3/c13-4-12(8-2-1-6-11(14)15-5-16-17(6)8)10(19)9(18)7(28-12)3-27-32(23,24)30-33(25,26)29-31(20,21)22/h1-2,5,7,9-10,18-19H,3H2,(H,23,24)(H,25,26)(H2,14,15,16)(H2,20,21,22)/t7-,9-,10-,12+/m1/s1

((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methyl tetrahydrogen triphosphate

Remdesivir triphosphate metabolite; GS-441524 triphosphate; GS 441524 triphosphate; GS441524 triphosphate; GS443902; GS-443902; GS-441524 Triphosphate; RDV-TP; AEL0YED4SU; UNII-AEL0YED4SU; [[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate; GS443902

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

H2O : ~100 mg/mL (~188.25 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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