HCMV
Human cytomegalovirus (HCMV) UL97 protein kinase (IC50 for wild-type UL97 autophosphorylation: ~35 nM; Ki for ATP-competitive inhibition: 10 nM).[1]

Maribavir, with a mean IC50 of 35 nM, is a strong inhibitor of autophosporylation of both the wild type and all of the major Ganciclovir (GCV) resistant UL97 mutants that have been studied. With an IC50 of 4.8 nM, the M460I mutation causes hypersensitivity to maribavir. L397R, a UL97 mutant resistant to maribavir, exhibits reduced function as a Ganciclovir kinase and protein kinase (~10% of wild type levels). Maribavir is a competitive inhibitor of ATP with a Ki of 10 nM, as shown by enzyme kinetic studies[1]. Using a multicycle DNA hybridization assay, maribavir (1263W94) inhibits viral replication in a dose-dependent manner with an IC50 of 0.12±0.01 μM. Maribavir significantly inhibits the pUL97 protein kinase, with 50% inhibition happening at 3 nM[2].

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Maribavir potently inhibited the autophosphorylation of wild-type HCMV UL97 protein kinase with a mean IC50 of 35 nM [1].
Maribavir also potently inhibited the autophosphorylation of major ganciclovir (GCV)-resistant UL97 mutants (M460I, H520Q, A594V, L595F) with a similar mean IC50 (~35 nM). The M460I mutant was hypersensitive to maribavir with an IC50 of 4.8 nM [1].
Maribavir acted as a competitive inhibitor of ATP binding to UL97, with a calculated Ki of 10 nM [1].
The maribavir-resistant UL97 mutant (L397R) showed severely impaired GCV kinase activity (~10% of wild-type levels) and autophosphorylation activity (~8% of wild-type levels), and its autophosphorylation was not inhibited by maribavir even at high concentrations (up to 100 µM) [1].

Using increasing concentrations of ATP (2 μM to 20 μM), enzyme kinetic analysis is carried out on the purified wild type and mutant UL97 protein species. To calculate the Km for ATP for each UL97 species, the amount of incorporated radiolabelled phosphate is plotted against the concentration of ATP in a Lineweaver Burke plot. Protein kinase assays are used to measure the impact of Maribavir on the rate of radiolabelled phosphate incorporation by either wild type or mutant UL97.The assays can be conducted at a fixed concentration of 0.55 μM of Maribavir, as previously mentioned, or with increasing concentrations of 0.01 μM to 5.0 μM of Maribavir to determine the IC50 of Maribavir for each species of UL97.Plots of 1/v vs 1/ATP with increasing Maribavir concentrations are created to ascertain the type of inhibition mediated by Maribavir. If the family of lines converged at 1/Vmax on the y-axis, then there was clear evidence of competitive inhibition. The Ki is computed using the variation in slope brought about by the addition of Maribavir[1].

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The autophosphorylation activity of purified wild-type and mutant UL97 proteins was assessed in a standard protein kinase assay. Purified UL97 protein was incubated in a reaction buffer containing Tris-HCl (pH 9.0), MgCl2, DTT, NaCl, ATP (with tracer [γ-32P]ATP) at 37°C. Reactions were terminated by adding SDS sample buffer and boiling. The amount of radiolabeled phosphate incorporated via autophosphorylation was analyzed by SDS-PAGE and quantified using a phosphoimager or densitometry [1].
To determine the IC50 of maribavir, the protein kinase assay was performed with increasing concentrations of maribavir (0.01 µM to 5.0 µM). The percentage of inhibition was plotted against drug concentration, and an exponential decay curve was fitted to calculate the IC50 [1].
To determine the mechanism of inhibition and the Ki value, the protein kinase assay was performed with increasing concentrations of ATP (2 µM to 20 µM) in the presence of different, fixed concentrations of maribavir. Data were analyzed using Lineweaver-Burk plots. The convergence of lines on the y-axis at 1/Vmax indicated competitive inhibition, and the Ki was calculated using the standard equation for competitive inhibition [1].

MRC-5 cells are cultivated for three days in MEM 8-1-1 until confluence (~1.1×105 cells/well), after being seeded in 24-well plates at a density of approximately 5×104 cells/well. In MEM 2-1-1, the cells are infected with AD169 at a multiplicity of infection (MOI) of 1 to 3, and the infection is allowed to adsorb for 90 minutes at 37°C. One milliliter of MEM 2-1-1 is added in place of the unadsorbed virus. Maribavir, BDCRB, or GCV are added to the medium at the concentrations specified for each experiment to examine the impact of the compounds on viral DNA synthesis or maturation[2].

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The ability of wild-type and mutant UL97 proteins to phosphorylate ganciclovir (GCV) was assessed in insect cells. Sf21 insect cells were infected with recombinant baculoviruses expressing UL97 variants at a high multiplicity of infection (MOI of 10). At 48 hours post-infection, the culture medium was replaced with fresh medium containing 1.0 mM GCV and tritiated [³H]GCV. After a further 24 hours of incubation, cells were harvested, and nucleotides were extracted by acid hydrolysis and neutralization. Aliquots of the supernatant were spotted onto DEAE-cellulose filter discs to bind monophosphorylated GCV. The discs were washed, dried, and the retained radioactivity was measured by liquid scintillation counting to quantify GCV phosphorylation [1].

Absorption, Distribution and Excretion
Pharmacokinetic models of patients receiving 400 mg maribavir twice daily showed AUC0-tau and Cmax of 128 µg·h/mL and 17.2 µg/mL, respectively. The median Tmax ranged from 1 to 3 hours. Maribavir is primarily eliminated via hepatic metabolism. After oral administration of radiolabeled maribavir, 61% of the dose is excreted in the urine (<2% unchanged drug) and 14% in the feces (5.7% unchanged drug). The mean apparent steady-state volume of distribution of maribavir is 27.3 L. In post-transplant patients, the mean oral clearance of maribavir is 2.85 L/h. Metabolism/Metabolites After oral administration, maribavir is primarily metabolized by CYP3A4, followed by CYP1A2. Its major circulating metabolite is VP 44469, an inactive N-dealkylated metabolite.

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Biological half-life>
In post-transplant patients, the mean elimination half-life was 4.32 hours.

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Metabolic studies in human fibroblasts (MRC-5) showed that maribavir was not phosphorylated to monophosphate or triphosphate in either uninfected or HCMV-infected cells. Cell extracts were analyzed by anion exchange and reversed-phase high-performance liquid chromatography, and no phosphorylated metabolites were detected. This method is sensitive enough to detect intracellular concentrations as low as 10 nM [2].

Hepatotoxicity
In large pre-registration clinical trials, the incidence of ALT elevation in patients with refractory cytomegalovirus infection following hematopoietic stem cell transplantation was 3.5% in the maribavir group, compared to less than 1% in the standard treatment group. All ALT elevations were transient, mild, and asymptomatic. No clinically significant liver injury with jaundice was reported in pre-marketing studies. Since maribavir's approval, no cases of clinically significant liver injury with jaundice associated with maribavir use have been reported. However, current clinical experience with maribavir treatment remains limited. Probability score: E (unlikely to be the cause of clinically significant liver injury). Protein Binding Across all tested concentrations, maribavir is extensively bound to proteins in plasma (approximately 98%), with serum albumin and α1-acid glycoprotein likely being the primary binders. Compared to ganciclovir, maribavir exhibits lower cytotoxicity against primary human bone marrow progenitor cells. The mean IC50 values for inhibition of granulocyte-macrophage colony-forming unit (CFU-GM) were 90 ± 4 μM for maribavir and 15 ± 3 μM for ganciclovir. The mean IC50 values for inhibition of erythroid burst-forming unit (BFU-E) were 88 ± 4 μM for maribavir and 39 ± 10 μM for ganciclovir [2]. Growth inhibition studies in human leukemia cell lines (Molt-4, CEM, CEM-CD4+, IM9) yielded IC50 values ranging from 35 μM to 72 μM [2].

[1]. The effects of Maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein. Herpesviridae. 2010 Dec 7;1(1):4.

[2]. Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action. Antimicrob Agents Chemother. 2002 Aug;46(8):2365-72.

Maribavir is a cytomegalovirus (CMV; HHV5) pUL97 kinase inhibitor used to treat CMV infection in post-transplant patients. Most standard CMV therapies, such as ganciclovir or foscarnet, target the CMV DNA polymerase—while these drugs are generally effective, they tend to promote CMV resistance to DNA polymerase-based therapies, and their use is often limited by toxicities such as myelosuppression and kidney damage. Maribavir's innovation lies in its targeting of the CMV pUL97 kinase, thus providing an effective alternative treatment for drug-resistant infections. Maribavir was approved by the FDA in November 2021 under the brand name Livtencity (Takeda Pharmaceutical) for the treatment of drug-resistant CMV infection in post-transplant patients. The drug was approved by Health Canada in September 2022 and by the European Commission in November 2022. Maribavir's mechanism of action is as an inhibitor of cytomegalovirus pUL97 kinase, cytochrome P450 3A4, P-glycoprotein, and breast cancer resistance protein. Maribavir is an oral antiviral drug that inhibits the pUL97 kinase of cytomegalovirus (CMV) and is used to treat refractory CMV infection after transplantation. Mild to moderate elevations in serum transaminases are rare during maribavir treatment, but have not been found to be associated with clinically significant acute liver injury. Maribavir is an oral benzimidazole nucleoside compound with anti-cytomegalovirus (CMV) activity. Maribavir is a selective ATP competitor that inhibits viral UL97 kinase, which is involved in viral nuclear maturation processes such as viral DNA assembly and viral capsid removal from the infected cell nucleus. Maribavir is effective against CMV strains resistant to standard anti-cytomegalovirus (CMV) drugs.

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Drug Indications
Maribavir is indicated for the treatment of post-transplant cytomegalovirus (CMV) infection (after hematopoietic stem cell transplantation or solid organ transplantation) that is unresponsive to standard treatments such as ganciclovir, valganciclovir, cidofovir, or foscarnet. In the United States, patients receiving this treatment should weigh more than 35 kg and be at least 12 years old. In Canada and Europe, maribavir is approved for use in adults only.
LIVTENCITY is indicated for the treatment of cytomegalovirus (CMV) infection and/or disease unresponsive to one or more prior therapies (including ganciclovir, valganciclovir, cidofovir, or foscarnet), with or without resistance, in adult patients who have undergone hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Official guidelines on the rational use of antiviral drugs should be consulted. Treatment of Cytomegalovirus (CMV) Infection
Cytomegalovirus Disease

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Mechanism of Action
Human cytomegalovirus (CMV) is a herpesvirus that commonly causes infection in patients after stem cell or organ transplantation. Like other herpesviruses, CMV tends to persist in the host and reactivate under immunosuppression—therefore, patients requiring multiple immunosuppressive drugs to combat transplant rejection are at much higher risk of severe CMV infection. Maribavir belongs to a class of anti-CMV drugs called benzimidazole nucleosides. It competitively inhibits the human CMV pUL97 viral protein kinase, resulting in the virus surviving replication with severe defects, although the exact mechanism is not fully understood. Furthermore, maribavir can also inhibit viral release from the nucleus to the cytoplasm by inhibiting pUL97-dependent phosphorylation of laminin A/C, a component of nuclear lamina, but the extent to which this activity contributes to its antiviral efficacy is unclear.

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Pharmacodynamics
Compared to traditional CMV antiviral drugs, maribavir exerts its antiviral effect through a different target, and therefore can be used to treat CMV infections that have developed resistance to standard therapy. Maribavir should not be used concurrently with ganciclovir or valganciclovir, as both molecules require activation via CMV pUL97 to exert their antiviral effect. Therefore, concurrent administration with maribavir (an inhibitor of the same kinase) will significantly reduce their antiviral activity.

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Maribavir (also known as 1263W94 and benzimidavir) is a benzimidazole nucleoside compound, a novel HCMV replication inhibitor that targets the UL97 protein kinase [1].
It has potent antiviral activity against HCMV and Epstein-Barr virus (EBV). It has been reported that its in vitro IC50 value against HCMV is 4 to 10 times lower than that of ganciclovir, and it is effective against HCMV strains resistant to ganciclovir, foscarnet, and cidofovir [1]. Long-term culture of HCMV cells in the presence of maribavir leads to resistance, which is mainly related to a single amino acid alteration in UL97: L397R. Other resistance mutations in UL97 (e.g., mutations at amino acid sites 411, 409, and 353) have also been reported [1]. This study concludes that maribavir is a potent ATP-competitive inhibitor of the UL97 kinase. GCV resistance mutations in UL97 (except M460I) do not confer cross-resistance to maribavir. The M460I mutation confers hypersensitivity to maribavir. Conversely, the maribavir resistance mutation L397R severely impairs the GCV kinase and protein kinase activity of UL97 [1].

C15H19CL2N3O4

376.23506

375.075

C, 47.89; H, 5.09; Cl, 18.84; N, 11.17; O, 17.01

176161-24-3

471161

Solid powder

1.7±0.1 g/cm3

611.0±65.0 °C at 760 mmHg

323.3±34.3 °C

0.0±1.8 mmHg at 25°C

1.703

2.71

4

6

4

24

447

4

ClC1=C(Cl)C=C2C(N=C(NC(C)C)N2[C@@H]3[C@@H](O)[C@@H](O)[C@H](CO)O3)=C1

KJFBVJALEQWJBS-XUXIUFHCSA-N

InChI=1S/C15H19Cl2N3O4/c1-6(2)18-15-19-9-3-7(16)8(17)4-10(9)20(15)14-13(23)12(22)11(5-21)24-14/h3-4,6,11-14,21-23H,5H2,1-2H3,(H,18,19)/t11-,12-,13-,14-/m0/s1

5,6-Dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole

1263-W94; GW-1263; SHP-620; 1263 W94; 1263W-94; BW1263W 94; GW1263; SHP620; BW-1263W94; VP41263; Livtencity; BW 1263W94; VP-41263; GW-257406X; GW257406-X; GW 257406X; GW 257406 X; GW257406X

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)

DMSO : ~200 mg/mL (~531.58 mM)

Solubility in Formulation 1: ≥ 2.87 mg/mL (7.63 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 2: ≥ 2.87 mg/mL (7.63 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 3: ≥ 2.5 mg/mL (6.64 mM) 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 4: ≥ 2.5 mg/mL (6.64 mM) 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 5: ≥ 2.5 mg/mL (6.64 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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Solubility in Formulation 6: 5% DMSO+40% PEG300+5% Tween-80+50% Saline: ≥ 2.87 mg/mL (7.63 mM)

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