Rhinovirus
Picornavirus capsid protein [1]
- Picornavirus capsid protein [2]

Broadly acting picornavirus inhibitor pirodavir is a powerful drug. At a dose of 64 ng/mL, pirodavir inhibits 80 out of the 100 human rhinovirus (HRV) strains that were tested. Pirodavir also demonstrated efficacy against 16 enteroviruses in that study, with a mean 80% inhibitory concentration (IC80) of 1,300 ng/mL. Pirodavir has an IC50 of 5,420 nM and an IC90 of >13,350 nM, which inhibits the replication of enterovirus 71. Three of the tested clinical isolates and 56 rhinovirus laboratory strains are inhibited by pirodavir. 59% of the serotypes and isolates with IC50s of less than 100 nM are inhibited by pirodavir[1].Cell growth is reduced by 66% (s.e.m. 0.75) and 28% (s.e.m. 0.25) at 16 and 4μg/mL, respectively, when pirodavir concentrations are used. Pirodavir does not inhibit cell growth at lower concentrations (1μg/mL). For logarithmic cell growth at 37°C, 7μg/mL is the 50% cytotoxic concentration of pirodavir. 50% cytotoxic concentration is greater than 50μg/mL in the antiviral assay with confluent HeLa cells at 33°C[2].

---

1. Broad-spectrum antipicornaviral activity: Pirodavir exhibited potent inhibitory effects against multiple picornavirus species and serotypes. For rhinoviruses (RV), it inhibited 32 tested RV serotypes (e.g., RV1A, RV2, RV3, RV4, RV5, RV6, RV7, RV8, RV9, RV14, RV16, RV18, RV29, RV30, RV31, RV33, RV41, RV43, RV47, RV50, RV53, RV56, RV62, RV63, RV67, RV71, RV73, RV75, RV77, RV85, RV89, RV90, RV95) with EC50 values ranging from 0.003 to 0.08 μM [2]
2. Inhibition of enteroviruses and hepatoviruses: Pirodavir effectively inhibited enteroviruses including coxsackievirus A9, B3, B4; echovirus 6, 11, 19; and poliovirus type 1 (Sabin strain) with EC50 values between 0.01 and 0.5 μM. It also suppressed hepatitis A virus (HAV) replication with an EC50 of approximately 0.1 μM [2]
3. Positive control activity in compound screening: In [1], Pirodavir was used as a positive control, showing an EC50 of 0.01 μM against RV16 and 0.05 μM against coxsackievirus B3 [1]
4. Mechanism of action: Pirodavir binds to the picornavirus capsid protein, preventing the uncoating process that occurs after the virus binds to host cell receptors. This binding stabilizes the viral capsid structure, blocking the release of viral RNA into the host cell cytoplasm and thereby inhibiting viral replication [2]

Picornavirus capsid protein binding assay: Recombinant picornavirus (e.g., rhinovirus) capsid protein was prepared and purified. Purified capsid protein was incubated with various concentrations of Pirodavir in a buffer solution at 37°C for 1 hour. The binding between Pirodavir and the capsid protein was detected using gel filtration chromatography or radioligand binding assay. Binding affinity was analyzed through binding curves, though no specific numerical values were provided [2]

HeLa cells are seeded in six-well plates with 4 mL of growth medium at a density of roughly 180,000 cells per dish. Eagle's basal medium is the growth medium, to which 5% fetal calf serum, 2% sodium bicarbonate, and 1% glutamine are added. The growth medium is removed and replaced with the test solutions (fresh growth medium with or without different concentrations of the antiviral compounds) after a 24-hour incubation period at 37°C in a humidified CO2 atmosphere. The amount of living cells present in triplicate cultures at the time of Pirodavir addition and every 24 hours for three days is counted in order to evaluate the cytotoxicity of the antiviral compounds (e.g., Pirodavir). A Coulter Counter is used to count the number of viable cells in triplicate for each drug concentration after trypsinization[2].

---

1. Plaque reduction assay :
- Host cells (e.g., HeLa cells, WI-38 cells) were seeded into 6-well culture plates at a density of 2×10⁵ cells per well and incubated overnight at 37°C with 5% CO₂ until the cell confluency reached 80%-90% [2]
- Pirodavir was serially diluted and mixed with 100 plaque-forming units (PFU) of picornavirus (e.g., rhinovirus, coxsackievirus), followed by incubation at room temperature for 1 hour [2]
- The virus-drug mixture was added to the cell culture plates and incubated at 37°C for 1 hour, with gentle shaking every 15 minutes to ensure uniform infection [2]

- The virus-drug mixture was aspirated, and the cells were overlaid with maintenance medium containing 2% agarose. Incubation was continued for 48-72 hours [2]
- After plaques formed, the cells were stained with crystal violet, and the number of plaques was counted to calculate the 50% plaque reduction concentration (EC50) [2]
2. Cytopathic effect (CPE) inhibition assay:
- Host cells (e.g., HEp-2 cells) were seeded into 96-well culture plates at a density of 5×10⁴ cells per well and incubated overnight [2]
- Different concentrations of Pirodavir were added to the cells for 1 hour of pretreatment, followed by the addition of picornavirus inoculum that could induce 80%-90% CPE [2]
- After incubation at 37°C for 72 hours, CPE was observed under a microscope, or cell viability was detected using the MTT assay to calculate the 50% CPE inhibition concentration (EC50) [2]
3. Cytopathic effect (CPE) inhibition assay :
- HeLa cells were seeded into 96-well plates at a density of 1×10⁴ cells per well and incubated for 24 hours [1]
- Pirodavir was serially diluted and mixed with rhinovirus type 16, followed by incubation at 37°C for 30 minutes before being added to the cell culture plates [1]
- After 48 hours of incubation, CPE was observed to calculate the EC50 [1]

Pyrodavir exhibits low cytotoxicity to host cells (e.g., HeLa, WI-38, HEp-2 cells), with a 50% cytotoxic concentration (CC50) greater than 10 μM. The therapeutic index (TI = CC50/EC50) ranges from 20 to 1000 [2]

[1]. In vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity. Antimicrob Agents Chemother. 2004 May;48(5):1766-72.

[2]. In vitro activity of pirodavir (R 77975), a substituted phenoxy-pyridazinamine with broad-spectrum antipicornaviralactivity. Antimicrob Agents Chemother. 1992 Jan;36(1):100-7.

Pyrodavir is an anti-microRNA virus drug.

---

1. Pyrodavir (code: R 77975) is a substituted phenoxypyridazinamide compound with broad-spectrum anti-microRNA virus activity[2]
2. MicroRNA viruses include rhinoviruses, enteroviruses, hepatitis A viruses, etc., which can cause respiratory infections, gastrointestinal infections, neurological diseases and other diseases. The development of pyrodavir provides a potential treatment option for these viral infections[2]
3. In [1], pyrodavir was used as a positive control to evaluate the anti-microRNA virus activity of a novel pyridazinoxime ether derivative, and its activity was used as a reference standard[1]
4. Pyrodavir specifically targets the capsid protein of microRNA viruses and has no cross-reactivity with host cell proteins, thus its cytotoxicity is low[2]

C21H27N3O3

369.46

369.205

C, 68.27; H, 7.37; N, 11.37; O, 12.99

124436-59-5

71345

Light yellow to yellow solid powder

1.1±0.1 g/cm3

572.0±40.0 °C at 760 mmHg

124-125℃

299.8±27.3 °C

0.0±1.6 mmHg at 25°C

1.554

3.94

0

6

8

27

446

0

O=C(OCC)C1=CC=C(OCCC2CCN(C3=NN=C(C)C=C3)CC2)C=C1

KCHIOGFOPPOUJC-UHFFFAOYSA-N

InChI=1S/C21H27N3O3/c1-3-26-21(25)18-5-7-19(8-6-18)27-15-12-17-10-13-24(14-11-17)20-9-4-16(2)22-23-20/h4-9,17H,3,10-15H2,1-2H3

Ethyl p-(2-(1-(6-methyl-3-pyridazinyl)-4-piperidyl)ethoxy)benzoate

R 77975; R-77975; R77975; R77,975; R 77,975; R-77,975;

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 : ~10 mg/mL (~27.07 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.71 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

---

Solubility in Formulation 2: ≥ 1 mg/mL (2.71 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 10.0 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.

---

View More

Solubility in Formulation 3: ≥ 1 mg/mL (2.71 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 1 mg/mL (2.71 mM)

---

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