Broad-spectrum antiviral

The RNA-dependent RNA polymerase of the severe acute respiratory syndrome coronavirus 2 is an important target in current drug development efforts for the treatment of coronavirus disease 2019. Molnupiravir is a broad-spectrum antiviral that is an orally bioavailable prodrug of the nucleoside analogue β-D-N4-hydroxycytidine (NHC). Molnupiravir or NHC can increase G to A and C to U transition mutations in replicating coronaviruses. These increases in mutation frequencies can be linked to increases in antiviral effects; however, biochemical data of molnupiravir-induced mutagenesis have not been reported. Here we studied the effects of the active compound NHC 5’-triphosphate (NHC-TP) against the purified severe acute respiratory syndrome coronavirus 2 RNA-dependent RNA polymerase complex. The efficiency of incorporation of natural nucleotides over the efficiency of incorporation of NHC-TP into model RNA substrates followed the order GTP (12,841) > ATP (424) > UTP (171) > CTP (30), indicating that NHC-TP competes predominantly with CTP for incorporation. No significant inhibition of RNA synthesis was noted as a result of the incorporated monophosphate in the RNA primer strand. When embedded in the template strand, NHC-monophosphate supported the formation of both NHC:G and NHC:A base pairs with similar efficiencies. The extension of the NHC:G product was modestly inhibited, but higher nucleotide concentrations could overcome this blockage. In contrast, the NHC:A base pair led to the observed G to A (G:NHC:A) or C to U (C:G:NHC:A:U) mutations. Together, these biochemical data support a mechanism of action of molnupiravir that is primarily based on RNA mutagenesis mediated via the template strand [3].

Molnupiravir has strong antiviral properties and can stop SARS-CoV multiplication and illness [1]. It is administered orally every 12 hours for three days at a dose of 50–500 mg/kg. The duration of fever and viral load are dramatically reduced by molnupiravir (7 mg/kg), when administered orally twice daily for 3.5 days [2].

NTP incorporation and the effect of primer- or template-embedded NHC-MP on viral RNA synthesis[3] NTP incorporation by SARS-CoV-2 RdRp and data acquisition and quantification were done as reported by us. Enzyme concentration was 100 or 200 nM for single and multiple nucleotide incorporation assays, respectively. RNA synthesis incubation time was 10 min. Data from single nucleotide incorporation assays were used to determine the preference for the natural nucleotide over NHC-TP. The selectivity value is calculated as a ratio of the incorporation efficiencies of the natural nucleotide over the nucleotide analogue. The efficiency of nucleotide incorporation is determined by the ratio of Michaelis–Menten constants Vmax over Km. The substrate for nucleotide incorporation is a 5-nt primer generated by incorporation of [α-32P]NTP into a 4-nt primer. Formation of the 5-nt primer is maximal at a given time point; however, its precise concentration is unknown. Hence, the product generated in the reaction is measured by quantifying the signal corresponding to the 6-nt primer product and dividing it to the total signal in the reaction (5-nt primer and 6-nt primer). This defines the product fraction. The product fraction is commonly multiplied by the total substrate concentration in order to determine the molar units of the Vmax, which is here not possible as explained above. Therefore, the unit of Vmax is reported as product fraction over time. The selectivity value is unitless as it is the ratio of two Vmax/Km measurements with the same units. RNA templates with embedded NHC-MP were produced as described by us. NHC-related protocol modifications are explained in Fig. S1.

Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% fetal bovine serum (FBS). Normal primary human bronchial tracheal epithelial cells (HBTECs) from a 30-year old healthy female donor were grown in BronchiaLife cell culture medium. These cells were obtained by the vendor under informed consent and adheres to the Declaration of Helsinki, The Human Tissue Act (UK), CFR Title 21, and HIPAA regulations. All regulatory approval lies with the vendor. Immortalized cell lines used in this study were routinely checked for microbial contamination (in approximately 6-month intervals). HBTECs were tested for microbial contamination on July 25, 2017 by LifeLine Cell Technology. Only HBTECs with a passage number 1-4 were used for this study [2].

Animal/Disease Models: C57BL/6 mice (intranasal infection with SARS-CoV)[1]
Doses: 50, 150, 500 mg/kg
Route of Administration: Oral; every 12 hrs (hours) for 3 days
Experimental Results: Body weight loss is Dramatically diminished or prevented.

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Animal/Disease Models: Ca/09-infected female ferrets[1]
Doses: 7 mg/kg
Route of Administration: Oral; twice (two times) daily for 3.5 days
Experimental Results: Shed virus load and duration of fever were Dramatically diminished.

Absorption, Distribution and Excretion
Following oral administration of 800 mg monopilavir every 12 hours, the peak plasma concentration (Cmax) of the active compound (N4-hydroxycytidine) was 2970 ng/mL, the time to peak concentration (Tmax) was 1.5 hours, and the AUC0-12h was 8360 hng/mL. ≤3% of the oral monopilavir dose is excreted in the urine as the active metabolite N4-hydroxycytidine. Metabolism/Metabolites Monopilavir is hydrolyzed to [N4-hydroxycytidine], which is distributed in tissues. After entering cells, N4-hydroxycytidine is phosphorylated to its 5'-triphosphate form. Biological Half-Life The half-life of the active metabolite N4-hydroxycytidine is 3.3 hours.

Hepatotoxicity
In pre-registration clinical trials, elevated serum transaminases were uncommon and mild, with an incidence rate no higher in the monopelavir group than in the placebo group. Furthermore, in premarketing studies, over 900 patients received monopelavir (800 mg twice daily) for 5 days without any clinically significant liver injury events reported. However, it is puzzling that elevated serum transaminases are common during symptomatic SARS-CoV-2 infection, occurring in up to 70% of patients, and are more common in severely ill patients and those with known risk factors for severe COVID-19 (e.g., male sex, advanced age, high body mass index, and diabetes). Therefore, monopelavir has not been confirmed to cause liver injury, but its overall clinical experience is limited. Probability Score: E (Unlikely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of monopelavir during lactation. Breastfeeding is not recommended during treatment and for 4 days after the last dose of molnupiravir.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.
◈ What is molnupiravir?
Monupiravir is an antiviral drug that has received Emergency Use Authorization from the U.S. Food and Drug Administration (FDA) for the treatment of mild to moderate COVID-19 in certain patients. Molnupiravir must be started within 5 days of the onset of COVID-19 symptoms to be effective. Molnupiravir is marketed as Lagevrio®. The FDA's Emergency Use Guidance for molnupiravir recommends that pregnant women should not use this drug unless there is no other treatment option and treatment is deemed necessary. This is because there is currently insufficient information regarding the use of molnupiravir to determine whether and how it affects pregnancy. However, the benefits of using molnupiravir may outweigh the potential risks. Your healthcare provider can discuss the use of monopelavir with you and the best treatment option for you. For more information on COVID-19, see MotherToBaby's case sheet: https://mothertobaby.org/fact-sheets/covid-19/.
◈ I am taking monopelavir, but I want to get pregnant after taking it. How long will this drug stay in my body?
Everyone clears the drug at a different rate. For non-pregnant adults, on average, most of monopelavir is cleared from the body within 1 day. The U.S. Food and Drug Administration (FDA) Emergency Use Guidance recommends that women avoid trying to conceive while taking monopelavir and for 4 days after the last dose.
◈ I am taking monopelavir. Will it affect my ability to get pregnant?
It is currently unclear whether monopelavir affects pregnancy. The FDA Emergency Use Guidance recommends that women of childbearing potential use effective contraception correctly and continuously while taking monopelavir and for 4 days after the last dose.
◈ Does taking Monupiravir increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for a variety of reasons. There are currently no human studies confirming that Monupiravir increases the risk of miscarriage.
◈ Does taking Monupiravir increase the risk of birth defects?
There is a 3-5% risk of birth defects in every pregnancy. This is called background risk. No human studies have been conducted to determine whether Monupiravir increases the risk of birth defects (above background risk).
◈ Does taking Monupiravir during pregnancy increase the risk of other pregnancy-related problems?
No human studies have been conducted to determine whether Monupiravir increases the risk of pregnancy-related problems such as preterm birth (delivery before 37 weeks of gestation) or low birth weight (birth weight less than 5 pounds 8 ounces [2500 grams]). COVID-19 infection during pregnancy increases the risk of preterm birth, stillbirth, and other pregnancy complications.
◈ Will taking Monupiravir during pregnancy affect the child's future behavior or learning abilities?
Currently, no studies have confirmed whether monopelavir causes behavioral or learning problems in children.
◈ Breastfeeding while taking monopelavir:
The U.S. Food and Drug Administration (FDA) Emergency Use Guidance for Monopelavir recommends that breastfeeding women should not use this medication unless there are no other treatment options and treatment is truly necessary. However, the benefits of taking monopelavir and the benefits of breastfeeding may outweigh the potential risks. Breastfeeding women may consider expressing and discarding breast milk while taking monopelavir and for 4 days after the last dose. Your healthcare provider can discuss the use of monopelavir and the best treatment option for you. Be sure to consult your healthcare provider about all questions regarding breastfeeding.
◈ Will taking monopelavir affect fertility or increase the risk of birth defects in men?
Currently, no studies have assessed whether monopelavir affects male fertility (the ability to impregnate a partner) or increases the risk of birth defects (above background risk). The U.S. Food and Drug Administration (FDA) Emergency Use Guidance recommends that men use a reliable method of contraception correctly and consistently during treatment and for at least three months after their last dose of monopilavir. Generally, exposure to the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please refer to MotherToBaby's "Paternal Exposure" information sheet at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Monopiravir and its active metabolite N4-hydroxycytidine are not bound to proteins in plasma.

[1]. Sheahan TP, et al. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med. 2020 Apr 6. pii: eabb5883.
[2]. Toots M, et al. Characterization of orally efficacious influenza drug with high resistance barrier in ferrets and human airway epithelia. Sci Transl Med. 2019 Oct 23;11(515). pii: eaax5866.
[3]. Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template. Biol Chem. 2021 Jul; 297(1): 100770.

Monupiravir is a nucleoside analog, a derivative of N(4)-hydroxycytidine, in which the 5'-hydroxyl group is replaced by (2-methylpropionyl)oxy. It is a prodrug of the active antiviral ribonucleoside analog N(4)-hydroxycytidine (EIDD-1931), which is active against a variety of RNA viruses, including SARS-CoV-2, MERS-CoV, and seasonal and pandemic influenza viruses. Currently, it is in Phase III clinical trials for the treatment of COVID-19 patients. It can be used as a prodrug, as well as an anticoronavirus drug and an antiviral drug. It is a nucleoside analog, isopropyl ester, and ketooxime. Its function is related to N(4)-hydroxycytidine. Monupiravir (EIDD-2801, MK-4482) is an isopropyl ester prodrug of [N4-hydroxycytidine]. In non-human primates, the oral bioavailability of monoupiravir is improved, and after hydrolysis in vivo, it is distributed to tissues and converted into the active 5'-triphosphate form. This active drug can integrate into the genome of RNA viruses, leading to an accumulation of viral mutations, a phenomenon known as the viral error catastrophe. Recent studies have shown that monopilavirvir can inhibit the replication of human coronaviruses and bat coronaviruses (including SARS-CoV-2) in mouse and human respiratory epithelial cells. Furthermore, a murine hepatitis virus mutant strain resistant to remdesivir has also shown increased sensitivity to N4-hydroxycytidine. On November 4, 2021, the UK Medicines and Healthcare products Regulatory Agency (MHRA) approved monopilavirvir for the prevention of serious consequences of COVID-19 in adults, such as hospitalization and death. On December 23, 2021, the US Food and Drug Administration (FDA) also granted monopilavirvir Emergency Use Authorization. However, it has not yet received full approval. Monopilavirvir is a ribonucleoside analog and antiviral drug used to treat severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) infection, the pathogen of 2019 novel coronavirus disease (COVID-19). Monupiravir therapy, administered orally in the early stages of SARS-CoV-2 infection for 5 days, has not been found to be associated with elevated serum transaminases or clinically significant liver damage. Monupiravir is a highly bioavailable oral prodrug of EIDD-1931, a synthetic ribonucleoside derivative N4-hydroxycytidine and ribonucleoside analog with potential antiviral activity against various RNA viruses. After oral administration, the prodrug monoupiravir is metabolized to its active form, EIDD-1931, and converted to its triphosphate (TP) form. The TP form of EIDD-1931 can be incorporated into RNA, inhibiting the activity of viral RNA-dependent RNA polymerase. This leads to termination of RNA transcription, thereby reducing viral RNA production and replication.

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Drug Indications
[N4-hydroxycytidine] and its prodrug monopelavir are being investigated for their activity against a variety of viral infections, including influenza, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2. Monopelavir has been approved in the UK for reducing the risk of hospitalization and death in patients with mild to moderate COVID-19, particularly for patients at higher risk of severe illness (e.g., obese, diabetic, heart disease, or over 60 years of age). In the US, monopelavir has been approved for emergency use in the treatment of high-risk adult patients with mild to moderate COVID-19.
Prevention of Coronavirus Disease 2019 (COVID-19)
Treatment of Coronavirus Disease 2019 (COVID-19)

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Mechanism of Action
Monupiravir is hydrolyzed in vivo to N4-hydroxycytidine, which is phosphorylated in tissues to its active 5'-triphosphate form and integrated into the genome of new viral particles, leading to the accumulation of inactivating mutations, i.e., viral error catastrophe.
A mutant mouse hepatitis virus resistant to remdesivir also showed increased sensitivity to N4-hydroxycytidine.

C13H19N3O7

329.3059

329.12

C, 47.42; H, 5.82; N, 12.76; O, 34.01

2492423-29-5

Molnupiravir-d7

145996610

White to off-white solid powder

-0.8

4

7

6

23

534

4

O1[C@]([H])([C@@]([H])([C@@]([H])([C@@]1([H])C([H])([H])OC(C([H])(C([H])([H])[H])C([H])([H])[H])=O)O[H])O[H])N1C(N=C(C([H])=C1[H])N([H])O[H])=O

O[C@@H]([C@H]([C@H](N1C(N/C(C=C1)=N/O)=O)O2)O)[C@H]2COC(C(C)C)=O

HTNPEHXGEKVIHG-QCNRFFRDSA-N

InChI=1S/C13H19N3O7/c1-6(2)12(19)22-5-7-9(17)10(18)11(23-7)16-4-3-8(15-21)14-13(16)20/h3-4,6-7,9-11,17-18,21H,5H2,1-2H3,(H,14,15,20)/t7-,9-,10-,11-/m1/s1

MK 4482; EIDD-2801; EIDD 2801; Molnupiravir; MK-4482; MK4482; EIDD2801; prodrug-EIDD-1931; prodrug-EIDD 1931; prodrug-EIDD1931.

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 : 50 mg/mL (151.83 mM)

Solubility in Formulation 1: 12.05 mg/mL (36.59 mM) in 10% PEG400 2.5% Ethoxylated hydrogenated castor oil 87.5% water (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.59 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 (7.59 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 (7.59 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 corn oil and mix evenly.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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