Letermovir (formerly known as MK-8828 and AIC-246; trade name: Prevymis) is a new potent anticytomegalovirus drug in clinical development. On 11/8/2017, Letermovir was approved by FDA to prevent infection after bone marrow transplant. Despite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance.

Letermovir (formerly known as MK-8828 and AIC-246; trade name: Prevymis) is a new potent anticytomegalovirus drug in clinical development. On 11/8/2017, Letermovir was approved by FDA to prevent infection after bone marrow transplant. Despite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance.

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Letermovir exhibited potent antiviral activity against a panel of 17 different clinical HCMV isolates (WT1-WT17) in plaque reduction assays. The 50% effective concentrations (EC50) were consistently in the low nanomolar range (0.0008 µM to 0.0031 µM), approximately 1,000-fold more potent than ganciclovir (GCV).
Letermovir remained highly active against HCMV laboratory strains (AD169, EC50 = 0.0051 µM) and a series of AD169-derived drug-resistant variants. These variants carried confirmed resistance mutations to GCV and/or cidofovir (CDV) in the UL97 (viral kinase) and UL54 (viral DNA polymerase) genes. The EC50 values for letermovir against these resistant viruses (0.0016 µM to 0.0039 µM) were comparable to or lower than that for the parental AD169 strain, indicating no cross-resistance.
Letermovir demonstrated remarkable selectivity for HCMV. In cell culture-based replication assays, it showed no significant activity (EC50 >10 µM) against other human herpesviruses, including varicella-zoster virus (VZV), herpes simplex virus types 1 and 2 (HSV-1, HSV-2), human herpesvirus 6 (HHV-6), and Epstein-Barr virus (EBV). Activity against murine CMV (MCMV) was very low (EC50 = 4.5 µM), and no activity was detected against rat CMV (RCMV, EC50 >10 µM).
Letermovir showed no inhibitory activity (EC50 >10 µM to >32 µM) against a panel of important human pathogenic viruses from other families, including human adenovirus type 2 (HAdV-2), hepatitis B virus (HBV), human immunodeficiency virus type 1 (HIV-1), influenza A virus (H1N1), and hepatitis C virus (HCV) replicon.
Cytotoxicity studies performed in parallel (using alamarBlue and/or microscopic evaluation) indicated no toxicity at the highest drug concentrations used in the antiviral assays (up to 32 µM).[1]

Letermovir (10-100 mg/kg/day, p.o.) leads to a dose-dependent reduction of the HCMV titer in transplanted cells compared to that of the placebo-treated control group using the mouse xenograft model

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Letermovir (AIC246) exerts potent in vivo efficacy in a mouse xenograft model of HCMV infection. [2]
Phase I trials demonstrated that Letermovir was generally well tolerated and showed high and long-lasting exposure in human subjects, allowing once-daily dosing. [2]
A proof of concept was shown in a phase IIa trial and in a patient infected with a multidrug-resistant HCMV strain causing multiorgan disease. [2]

AIC246 has consistent antiviral efficacy, and there is remarkable selectivity of AIC246 for human cytomegaloviruses. AD169 mutant strains and designated rAIC246-1 and rAIC246-2 are highly resistant to Letermovir (AIC246), with EC50s of 5.6 nM, 1.24 μM, 0.37 μM, respectively. Letermovir inhibits HCMV replication through a specific antiviral mechanism that involves the viral gene product UL56. Letermovir inhibits HCMV replication in cell culture by interfering with the proper cleavage/packaging of HCMV progeny DNA[2]. Letermovir inhibits the current gold standard GCV by more than 400-fold with respect to EC50s (mean, 4.5 nM versus 2 μM) and by more than 2,000-fold with respect to EC90 values (mean, 6.1 nM versus 14.5 μM)[3]. Letermovir in conbination with anti-HCMV drugs causes additive antiviral effects, but there is no interaction between letermovir and anti-HIV drugs.

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The paper references the mode of action of letermovir, stating it interferes with DNA concatemer maturation by targeting the viral terminase complex. [1]
A functional viral DNA cleavage assay was performed to assess the impact of Letermovir on terminase activity. Cells were infected with HCMV and treated with the drug. After incubation, total DNA was extracted and digested with a restriction enzyme (KpnI). The digested DNA was size-fractionated by gel electrophoresis, transferred to a membrane, and hybridized with a digoxigenin-labeled probe specific for the terminal region of the HCMV genome. The presence of a ~4 kb fragment indicates proper terminase cleavage and maturation of unit-length genomes, while its absence indicates inhibition of cleavage/packaging. Letermovir inhibited the formation of the ~4 kb fragment in a concentration-dependent manner, confirming it interferes with terminase-mediated DNA processing. [2]

Briefly, 5×103 AD169-infected NHDF cells/well are seeded into the wells of 30 96-well microtiter plates. The infection is allowed to proceed under the exposure of 50 nM AIC246 (10×EC50) until a CPE developed in one or more of the compound-treated wells (indicative of resistant virus breakthrough). Noninfected and nontreated cells serve as controls on each plate. Mutant virus amplification is accomplwashed after cultures achieved maximum CPE by the passage of cell-free supernatant virus in the presence of 50 nM AIC246. The resultant AIC246-resistant progeny virus mutants are plaque purified three times by limiting dilutions in the presence of AIC246. The stability of resistance is tested by serially passaging plaque-purified viruses without selective pressure (8 to 10 times).

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The primary antiviral activity of letermovir was assessed using standard plaque reduction assays for HCMV. Briefly, virus isolates were used to infect permissive cell monolayers in the presence of serial dilutions of the compound. After an incubation period, plaques were visualized (e.g., by staining) and counted to determine the drug concentration that reduced plaque formation by 50% (EC50).
For some HCMV variants and other viruses, cytopathic effect (CPE)-based assays or other specific cell culture-based replication assays were employed. These methods measured the compound's ability to inhibit virus-induced cell death or other virus-specific replication markers (e.g., GFP expression from recombinant viruses, viral antigen production).
Cytotoxicity was assessed in parallel using cell viability assays (e.g., alamarBlue) and microscopic evaluation to ensure the observed antiviral effects were not due to general cellular toxicity.[1]

10 mL/kg.; oral
Mice

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The in vivo antiviral activity was assessed using a mouse xenograft model. Immunodeficient mice were used. Gelfoam sponges were seeded with HCMV (strain Davis)-infected human fibroblasts and implanted subcutaneously in the dorsoscapular area.
Letermovir (AIC246) and the control drug valganciclovir (VGCV) were formulated in 2% dimethyl sulfoxide in 0.5% methylcellulose–99.5% phosphate-buffered saline.
Starting 4 hours after transplantation, mice were treated once daily via oral gavage for nine consecutive days. The administration volume was 10 ml/kg. Doses of Letermovir tested were 1, 3, 10, 30, and 100 mg/kg/day.
After 9 days of treatment, mice were sacrificed, implants were removed and digested with collagenase to recover human cells. Virus titers in the cell suspensions were determined by plaque assay. [3]

Absorption, Distribution and Excretion
In healthy subjects, the bioavailability of letermovir was 94% when not used in combination with cyclosporine; in hematopoietic stem cell transplantation (HSCT) recipients, the bioavailability was 35% when not used in combination with cyclosporine; and in HSCT recipients, the bioavailability was 85% when used in combination with cyclosporine. The time to peak concentration (Tmax) of letermovir ranged from 45 minutes to 2.25 hours. The time required to reach steady-state plasma concentrations was 9–10 days. Co-administration with food increased Cmax by a mean of 129.82% (range 104.35%–161.50%). No significant effect on AUC was observed. Letermovir is absorbed in the liver via the OATP1B1/3 transporter. 93% of the drug is excreted in the feces, of which 70% is the unchanged drug. <2% is excreted in the urine. The mean steady-state volume of distribution is 45.5 L.
The mean clearance rate in healthy subjects was 11.25 L/h.

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Metabolism/Metabolites
Letermovir is primarily metabolized in small amounts via UGT1A1/1A3.

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Biological Half-Life
After a once-daily intravenous injection of 480 mg of letermovir, a mean terminal half-life of 12 hours was observed.

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The paper mentions that letermovir showed favorable pharmacokinetic characteristics in a phase I clinical trial, but this in vitro study did not provide specific ADME/PK parameters (e.g., half-life, Cmax, AUC, bioavailability). [1]

Hepatotoxicity
In large pre-registration clinical trials, following hematopoietic stem cell transplantation, elevated ALT levels occurred in 18.5% of patients in the letermovir group and 21.9% in the placebo group. Of these, 3.5% had ALT levels exceeding five times the upper limit of normal, compared to only 1.6% in the placebo group. ALT elevations are typically transient, mild, and asymptomatic. There have been reports of recurrent elevated serum ALT levels after letermovir re-administration. In premarketing studies, jaundice and liver injury occurred in 0.5% of subjects; however, in the case of hematopoietic stem cell transplantation, all cases had other more likely causes of liver injury that could not be convincingly attributed to letermovir treatment. Since letermovir's approval, no clinically manifested cases of jaundice-related liver injury have been reported; however, overall clinical experience with letermovir treatment is limited. Probability Score: E (Unlikely to cause clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the use of letermovir during lactation. Because letermovir binds to plasma proteins at a rate of up to 99%, its concentration in breast milk may be very low. However, especially in breastfed newborns or preterm infants, other medications may be preferred.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
In vitro observations showed that letermovir binds to plasma proteins at a rate of up to 99% at concentrations of 0.2–50 mg/L.

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The paper states that letermovir showed good safety in a Phase I clinical trial. In the described in vitro experiments, cytotoxicity assessments showed no toxicity observed at the tested concentrations. [1]

[1]. Antimicrob Agents Chemother. 2012 Feb;56(2):1135-7.

[2]. J Virol. 2011 Oct;85(20):10884-93.

[3]. Antimicrob Agents Chemother. 2010 Mar;54(3):1290-7.

[4]. Antimicrob Agents Chemother. 2015;59(6):3140-8.

On November 8, 2017, letermovir was approved by the U.S. Food and Drug Administration (FDA) for the prevention of cytomegalovirus (CMV) infection in patients who have undergone allogeneic hematopoietic stem cell transplantation. It is the first novel CMV anti-infective drug classified as a DNA terminal transferase complex inhibitor. Letermovir has received FDA priority and orphan drug designation. Currently, it is marketed under the brand name Prevymis. Letermovir is a cytomegalovirus DNA terminal transferase complex inhibitor. Its mechanism of action is as an inhibitor of DNA terminal transferase complexes, cytochrome P450 3A, organic anion transport peptide 1B1, organic anion transport peptide 1B3, cytochrome P450 2C8, cytochrome P450 2C9, and cytochrome P450 2C19. Letermovir is an antiviral drug that targets the DNA terminal transferase complex of cytomegalovirus (CMV) to prevent CMV reactivation in immunocompromised patients. Mild to moderate elevations in serum transaminases may occur during literovir treatment, but no clinically significant cases of acute liver injury have been observed. Litermovir is a highly bioavailable, oral non-nucleoside analogue belonging to the 3,4-dihydroquinazoline acetate class. It is an inhibitor of the pUL56 subunit of the cytomegalovirus (CMV) viral terminal enzyme complex and possesses potential CMV-specific antiviral activity. After oral administration, literovir binds to the pUL56 subunit of the CMV viral terminal enzyme complex, preventing the tandem DNA from cleaving into monoclonal DNA of genome length. Because this drug interferes with viral DNA processing and subsequent viral DNA packaging into the procapsid, it blocks CMV replication, thereby preventing CMV infection. Drug Indications: Litermovir is indicated for the prevention of CMV infection and disease in CMV-seropositive adult allogeneic hematopoietic stem cell transplantation (HSCT) recipients. It is also indicated for the prevention of CMV disease in at-risk adult kidney transplant recipients (i.e., donor CMV seropositive/recipient CMV seronegative).
FDA Label
Previmix is indicated for the prevention of cytomegalovirus (CMV) reactivation and disease in CMV seropositive [R+] adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. Antiviral medications should be used correctly according to official guidelines.
Prevention of Cytomegalovirus Infection

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Mechanism of Action
Cytomegalovirus (CMV) relies on a DNA terminal enzyme complex composed of multiple subunits (pUL51, pUL56, and pUL89) to process viral DNA. Viral DNA is produced as single-stranded repeat sequences, which are then cleaved by the DNA terminal enzyme complex into individual viral genomes, which can subsequently be packaged into mature viral particles. Letemovir inhibits the activity of this complex, thereby preventing the production of mature viral genomes and the formation of active viral particles. The exact mechanism by which letemovir binds to this complex is currently unknown. Initially, resistance mutations observed in pUL56 suggested that this subunit was the binding site for letemovir. However, resistance mutations have now been observed in pUL51, pUL56, and pUL89. A change in the amino acid sequence of one subunit could lead to a conformational change in the interacting subunit, thus affecting letemovir binding; or letemovir might interact with multiple subunits of the complex, but no evidence has yet been found to support either possibility. pUL89 is known to contain endonuclease activity of the complex, but because all members of the complex are essential for targeting and preventing proteasome degradation, it is difficult to determine whether letemovir directly inhibits the activity of pUL89.

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Pharmacodynamics
Letemovir inhibits the activity of the cytomegalovirus (CMV) DNA terminal enzyme complex, thereby preventing the viral DNA from being cleaved into a mature-length genome, thus preventing its packaging into viral particles. The EC50 value of letemovir for the DNA terminal enzyme complex is 2.1 nM. Human cytomegalovirus (HCMV) is a clinically significant herpesvirus that can cause complications in immunocompromised individuals. Current treatments (ganciclovir, valganciclovir, foscarnet, cidofovir) target viral DNA polymerases, but have limitations such as toxicity, low oral bioavailability, and drug resistance. Letermovir (AIC246) is a novel 3,4-dihydroquinazoline compound currently in clinical development with a unique mechanism of action that targets the viral terminal enzyme complex, for which there is no corresponding human counterpart. This unique mechanism enables it to combat HCMV strains resistant to existing standard treatments, as demonstrated in this study and in reported clinical cases of multidrug-resistant HCMV disease. The high selectivity of letermovir against HCMV relative to other herpesviruses and unrelated human pathogens suggests good safety and tolerability, and may address the limitations of existing therapies. [1]

C29H28F4N4O4

572.56

572.204

C, 60.84; H, 4.93; F, 13.27; N, 9.79; O, 11.18

917389-32-3

45138674

Solid powder

1.4±0.1 g/cm3

706.5±70.0 °C at 760 mmHg

381.1±35.7 °C

0.0±2.4 mmHg at 25°C

1.601

3.47

1

10

7

41

931

1

C([C@H]1C2C=CC=C(C=2N=C(N2CCN(C3C=CC=C(OC)C=3)CC2)N1C1C=C(C(F)(F)F)C=CC=1OC)F)C(=O)O

FWYSMLBETOMXAG-QHCPKHFHSA-N

InChI=1S/C29H28F4N4O4/c1-40-20-6-3-5-19(16-20)35-11-13-36(14-12-35)28-34-27-21(7-4-8-22(27)30)23(17-26(38)39)37(28)24-15-18(29(31,32)33)9-10-25(24)41-2/h3-10,15-16,23H,11-14,17H2,1-2H3,(H,38,39)/t23-/m0/s1

(S)-2-(8-fluoro-3-(2-methoxy-5-(trifluoromethyl)phenyl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)-3,4-dihydroquinazolin-4-yl)acetic acid

MK-8828; MK 8828; MK8828; AIC-246; AIC 246; AIC246; Letermovir; Prevymis

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 : ~100 mg/mL ( ~174.65 mM )
Ethanol : ~100 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.37 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 2: ≥ 2.5 mg/mL (4.37 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 3: ≥ 2.5 mg/mL (4.37 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 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.37 mM)

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