3CLPRO (SARS-CoV 3C-like protease)

3CLPRO cleaves SARS-CoV-2.1's polyproteins 1a and 1ab. Non-structural proteins, including proteins, cannot be released to carry out their roles without the assistance of SARS-CoV-2 3CLPRO, which inhibits viral replication [1].

Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.[2]

Protein binding [2]
Plasma protein binding was measured by the rapid equilibrium dialysis (RED) method to determine the free fraction and the unbound percentage of PF-332 for various species. An equilibrium dialysis was conducted in duplicate for each sample. 200 μl of plasma spiked with PF-332 were added in the plasma chamber and 350 μl of PBS pH = 7.4 were added in the buffer chamber. The dialysis block was then incubated at 37 °C for 6 h with constant shaking at 400 rpm. After 6 h, aliquots of the plasma and the buffer chambers were collected, spiked to obtain a matching homogeneous matrix, and quantified by LC–MS/MS. [2]
Microsomal metabolic stability [2]
Mouse liver microsomes (CD-1 male strain) were purchased from GIBCO. Hamster (Syrian female strain) and human liver microsomes were purchased from Xenotech. 1 ml of liver microsomal (LM) suspension at 20 mg/ml was mixed with 19 ml of 100 mM phosphate buffer. The latter is a titer solution containing 1 (M) KH2PO4 and 1 (M) K2HPO4 diluted in 10-fold distilled water (30 ml buffer + 270 ml of water) to obtain 100 mM phosphate buffer with an adjusted pH at 7.40 ± 0.02. A solution of NADPH Regeneration System (NRS) was prepared using 13 mM NADP, 33 mM Glucose-6-phosphate, 33 mM MgCl2, and 4 U/ml buffer solution of glucose-6-phosphate dehydrogenase.
All plastic materials including tips are incubated at 37 °C overnight. The LM suspension and the NRS solution were incubated at 37 °C for ~15 min before use. 48 μl of buffer was added to the wells of the blank plate. 40 μl of the compound at 1 uM was added to the working plates, 8 μl of NRS solution was added in the 0, 5, 10, 20, 30, and 60 min plates. The reaction is then initiated by adding 32 μl of 1 mg/ml of LM suspension to each plate. The reaction is terminated by adding 240 μl ice-cold acetonitrile at the designated time points. At T = 0, the acetonitrile is added before the LM solution.
The plates are centrifuged (3500 rpm, 20 min, and 15 °C); 110 μl of distilled water are then added to 110 μl of the supernatant and analyzed using an LC–MS/MS.

SARS-CoV-2 in vitro antiviral assays[2]
The assay using Vero E6 cells was derived from a previously established SARS-CoV assay. In this assay, fluorescence of Vero E6-eGFP cells declines after infection with SARS-CoV-2 due to virus-induced cytopathogenic effect. In the presence of an antiviral compound, the cytopathogenicity is inhibited and the fluorescent signal maintained. Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with heat-inactivated 10% v/v fetal calf serum (FCS) and 500 μg/ml Geneticin and kept under 5% CO2 at 37 °C.[2]
The test compounds were serially diluted in assay medium (DMEM supplemented with 2% v/v FCS). Diluted compounds were then mixed with Vero E6-eGFP cells corresponding to a final density of 25,000 cells/well in 96-well blackview plates. The next day, cells were infected with the SARS-CoV-2 at a final MOI of approximately 0.05 TCID50/cell. Final dilution of the different strains was adapted in order to obtain a similar MOI between all variants of interest. The plates were incubated in a humidified incubator at 37 °C and 5% CO2. At 4 days post-infection (pi), the wells were examined for eGFP expression using an argon laser-scanning microscope. The microscope settings were excitation at 488 nm and emission at 510 nm and the fluorescence images of the wells were converted into signal values. Toxicity of compounds in the absence of virus was evaluated in a standard MTS assay as described previously.[2]
A549-Dual™ hACE2-TMPRSS2 cells were cultured in DMEM 10% FCS supplemented with 10 µg/ml blasticidin, 100 µg/ml hygromycin, 0.5 µg/ml puromycin and 100 µg/ml zeocin. For the antiviral assay, cells were seeded in assay medium (DMEM 2%) at a density of 15,000 cells/well. One day after, the compound was serially diluted in assay medium (DMEM supplemented with 2% v/v FCS) and cells were infected with their respective SARS-CoV-2 strain at a MOI of approximately 0.05. The MOI was kept comparable for the variant strains in the different experiments. On day 4 pi., differences in cell viability caused by virus-induced CPE or by compound-specific side effects were analyzed using MTS as described previously.[2]
The results of in vitro antiviral experiments were expressed as EC50 values defined as the concentration of compound achieving 50% inhibition of the virus-reduced eGFP signals as compared to the untreated virus-infected control cells.

SARS-CoV-2 infection model in hamsters[2]
The hamster infection model of SARS-CoV-2 has been described before16,20. Female Syrian hamsters were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages at 21 °C, 55% humidity and 12:12 day/night cycles. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P065-2020). For infection, female hamsters of 6–8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µL containing 104 TCID50 of SARS-CoV-2 Beta variant B.1.351 (day 0). On day 4 pi, animals were euthanized for the sampling of the lungs and further analysis by i.p. injection of 500 μl Dolethal (200 mg/ml sodium pentobarbital). All caretakers and technicians were blinded to group allocation in the animal facility.[2]
Treatment regimen (beta variant study) Hamsters were treated by oral gavage with either the vehicle (n = 12) or PF-332 at 125 (n = 10) or 250 (n = 12) mg/kg/dose twice daily starting from D0, just before the infection with the Beta variant. All the treatments continued until day 3 pi. Hamsters were monitored for appearance, behavior, and weight. At day 4 pi, hamsters were euthanized by i.p. injection of 500 μl Dolethal (200 mg/ml sodium pentobarbital). Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration, respectively as described before17.[2]
Efficacy-transmission study (delta variant study) Two groups of index hamsters were infected intranasally with 50 µl containing 104 TCID50 of SARS-CoV-2 Delta variant and treated with either vehicle or PF-332 at 250 mg/kg/dose twice daily starting from D0. On day 1 pi (just after the morning dose), each index hamster was co-housed with a contact hamster (non-infected, non-treated hamsters) in one cage and the co-housing continued until day 3 pi The treatment of index hamsters was continued until day 2 pi. At day 3 pi, all the index hamsters were euthanized whereas all the contact hamsters were euthanized the day after (i.e., day 4 pi of index) as mentioned before and lungs were collected to assess viral loads.

Absorption, Distribution and Excretion
The median Tmax of nirmatrelvir, when given with ritonavir, is 3 hours. After a single oral dose of 300mg nirmatrelvir and 100mg ritonavir in healthy subjects, the Cmax and AUCinf of nirmatrelvir were 2.21 µg/mL and 23.01 µg*hr/mL, respectively.
The major route of nirmaltrevir elimination is via renal elimination, due in part to its coadministration with ritonavir which inhibits its metabolism. Following oral administration alongside ritonavir, approximately 49.6% of drug-related material was recovered in the feces and 35.3% was recovered in the urine.
The mean volume of distribution of nirmatrelvir, when given with ritonavir, is 104.7 liters.
The mean oral clearance of nirmatrelvir, administered with ritonavir, is 8.99 L/h.

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Metabolism / Metabolites
Nirmatrelvir is a substrate of CYP3A4, but undergoes minimal metabolism when administered alongside ritonavir.

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Biological Half-Life
The mean half-life of nirmatrelvir, administered alongside ritonavir, is 6.05 hours.

Hepatotoxicity
In preregistration clinical trials, serum aminotransferase elevations were uncommon and mild, and were no more frequent with Paxlovid than with placebo. Furthermore, among more than 1000 patients treated with Paxlovid (nirmatrelvir 300 mg with ritonavir 100 mg twice daily) for 5 days in prelicensure studies, there were no reported episodes of clinically apparent liver injury. Confounding the issue is that serum aminotransferase elevations are common during symptomatic SARS-CoV-2 infection, present in up to 70% of patients and are more frequent in patients with severe disease and in those with the known risk factors for COVID-19 severity such as male sex, older age, higher body mass index and diabetes. Thus, Paxlovid has not been shown to cause liver injury, but the total clinical experience with its use is limited.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Nirmatrelvir is given in combination with ritonavir, which enhances its bioavailability. The concentration of nirmatrelvir in breastmilk is low. Ritonavir is excreted into milk in measurable concentrations and low levels can be found in the blood of some breastfed infants. No adverse reactions in breastfed infants have been reported. For more information, refer to the LactMed record on ritonavir. Because of the poor oral bioavailability of nirmatrelvir and small amounts of both drugs in milk, this combination is unlikely to adversely affect the nursing infant.
◉ Effects in Breastfed Infants
In a cross-sectional study of women who had COVID-19 and received nirmatrelvir in combination ritonavir, two women breastfed their infants. No adverse effects were reported in the infants.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Nirmatrelvir, when given with ritonavir, is 69% protein-bound in plasma.

[1]. Considerations for the Discovery and Development of 3-Chymotrypsin-Like Cysteine Protease Inhibitors Targeting SARS-CoV-2 Infection. Current Opinion in Virology Available online 27 April 2021.

Pharmacodynamics
Nirmatrelvir is administered alongside ritonavir, a potent inhibitor of CYP3A enzymes, in order to inhibit its metabolism and increase plasma nirmatrelvir concentrations. While therapeutically beneficial, the use of ritonavir poses a significant risk of drug interaction due to its potent inhibition profile - patients and clinicians should consult the prescribing information for Paxlovid (nirmatrelvir and ritonavir) to evaluate any potential for drug interaction with existing medications prior to the initiation of Paxlovid.

C23H32F3N5O4

499.5265

499.24

C, 55.30; H, 6.46; F, 11.41; N, 14.02; O, 12.81

2628280-40-8

Nirmatrelvir-d9;2861202-76-6

155903259

White to off-white solid powder

2.2

3

8

7

35

964

6

CC1([C@@H]2[C@H]1[C@H](N(C2)C(=O)[C@H](C(C)(C)C)NC(=O)C(F)(F)F)C(=O)N[C@@H](C[C@@H]3CCNC3=O)C#N)C

LIENCHBZNNMNKG-OJFNHCPVSA-N

InChI=1S/C23H32F3N5O4/c1-21(2,3)16(30-20(35)23(24,25)26)19(34)31-10-13-14(22(13,4)5)15(31)18(33)29-12(9-27)8-11-6-7-28-17(11)32/h11-16H,6-8,10H2,1-5H3,(H,28,32)(H,29,33)(H,30,35)/t11-,12-,13-,14-,15-,16+/m0/s1

(1R,2S,5S)-N-((S)-1-cyano-2-((S)-2-oxopyrrolidin-3-yl)ethyl)-3-((S)-3,3-dimethyl-2-(2,2,2-trifluoroacetamido)butanoyl)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Nirmatrelvir; PF-07321332; PF 07321332; P7R9A5P7H32; Nirmatrelvir; Paxlovid; PF-07321332; PF07321332; Nirmatrelvir [USAN]; UNII-7R9A5P7H32; F07321332; brand name Paxlovid;

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 : 100~140 mg/mL ( 200.18~280.26 mM )
Ethanol : 50 ~100 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.16 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 20.8 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: ≥ 2.08 mg/mL (4.16 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 20.8 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: ≥ 2.08 mg/mL (4.16 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 20.8 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+90% Corn Oil: ≥ 2.08 mg/mL (4.16 mM)

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