Pyronaridine inhibits Echinococcus granulosus sensu stricto topoisomerase I (Egtopo I) with an IC50 of 209.7 ± 1.1 mM. [2]

Pyronaridine (24 hours) shown P resistance. The IC50 value for Plasmodium falciparum activity is 1.53-3.94 nM [1].

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Pyronaridine exhibited protoscolicidal activity against E. granulosus ss protoscolices (PSCs) in a dose-dependent manner, with an LC50 value of 49.0 ± 0.2 μM. [2]
Pyronaridine induced apoptosis in E. granulosus ss PSCs. This was evidenced by the observation of abundant TdT dUTP nick-end labeling (TUNEL)-positive cells, indicating DNA fragmentation. A clear pattern of DNA fragmentation (DNA ladder) was observed on agarose gel electrophoresis of DNA extracted from treated PSCs. Furthermore, treatment resulted in a 20-fold increase in caspase-3 activity compared to vehicle controls. [2]
Ultrastructural analysis by transmission electron microscopy (TEM) of cysts from treated mice showed devastating damage, including collapsed cysts, germinal layers torn from laminated layers, condensed cytoplasm in parenchyma cells, formation of apoptotic bodies, and complete shedding of microtriches. [2]

In mice infected with Echinococcus granulosus, pyronaridine (57 mg/kg, orally, once day for 30 days) decreases the parasite load [2]. Pyronaridine (57 mg/kg, intraperitoneally, once day for three days) decreases the load of parasites in mice with secondary infection (cyst infection) [2]. Pyranaridine exposure in the liver of male ICR mice (57 mg/kg, i.p., single dose) was greater than exposure in the plasma [2].

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In a murine model of secondary CE infection, intraperitoneal administration of Pyronaridine at 57 mg/kg once daily for three days resulted in a significant reduction in cyst wet weight (38.7% reduction, p < 0.05) and achieved 100% cyst mortality (p < 0.001), compared to vehicle controls. [2]
Oral administration of Pyronaridine at 57 mg/kg once daily for 30 days significantly reduced the parasite burden, with a 42.4% reduction in cyst wet weight (p < 0.05) and a 90.7% cyst mortality rate (p < 0.001 compared to the albendazole group). [2]
Using a microinjection procedure that mimics the clinical PAIR technique, intracystic injection of Pyronaridine (200 mM final concentration in cysts) resulted in a 52.3% reduction in parasite wet weight (p < 0.05) and a significant reduction in cyst counts (6.4 ± 1.2 vs. 14.4 ± 2.1 in saline group, p < 0.05). [2]

The inhibitory activity of Pyronaridine against E. granulosus sensu stricto topoisomerase I (Egtopo I) was analyzed using a DNA topoisomerase I assay kit. The reaction mixture contained DNA (pBR322, 200 ng), Egtopo I (300 ng), 0.1% bovine serum albumin, and various concentrations of Pyronaridine (final concentrations: 50, 100, 150, 200, 300, 400, 500, and 600 μM) in an assay buffer. The mixture was incubated for 30 minutes at 37°C. The reaction was terminated by adding 10% SDS solution and DNA loading buffer. Samples were then electrophoresed on 1% agarose gels, stained with ethidium bromide (5 μg/ml), and photographed at 254 nm. The ability of Pyronaridine to inhibit Egtopo I-mediated relaxation of supercoiled DNA was assessed, and the curve was fitted using a nonlinear regression model to calculate the IC50 value. [2]

For the in vitro drug activity assay, E. granulosus ss protoscolices (PSCs) were cultured in 96-well plates. Pyronaridine was prepared as a 10× stock solution in deionized water, with concentrations ranging from 340 μM to 1140 μM. 200 PSCs per well were incubated with the drug (final concentrations 34-114 μM) in a 5% CO2 atmosphere at 37°C for 24 hours. The viability of PSCs was assessed using a methylene blue staining test. 0.1% methylene blue solution was added to each well, and after 5 minutes, PSCs were observed under an inverted microscope. Dead PSCs stained blue, while living ones remained colorless. Experiments were carried out in triplicate. [2]
To assess apoptosis, TUNEL assay was performed on Pyronaridine-treated PSCs to detect DNA fragmentation. Apoptotic cells were observed by green fluorescence (TUNEL) and nuclei were stained with DAPI (blue). Additionally, DNA extracted from treated PSCs was subjected to agarose gel electrophoresis to visualize DNA fragmentation patterns (DNA ladder). Caspase-3 activity was measured in PSCs treated with or without Pyronaridine using a fluorometric assay. [2]

Animal/Disease Models: Mouse model of Echinococcus granulosus infection[2]
Doses: 57 mg/kg
Route of Administration: Orally, one time/day for 30 days.
Experimental Results: The wet weight of the parasite was diminished by 42.4%, and Echinococcus granulosus was killed. Secondary tapeworm infection (cyst) 90.7% SS.

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For the in vivo efficacy study, female BALB/c mice (6-8 weeks old) were infected intraperitoneally with microcysts. Three months post-infection, mice were randomly allocated into groups. For intraperitoneal (i.p.) administration, Pyronaridine tetraphosphate was dissolved in saline and administered i.p. at dosages of 14, 28, or 57 mg/kg in a volume of 1.0 ml once daily for three days. The control group received saline. [2]
For oral administration, Pyronaridine tetraphosphate was dissolved in deionized water and administered by gavage at 57 mg/kg in a volume of 0.2 ml once daily for 30 days. Albendazole (50 mg/kg) formulated in 0.5% carboxymethyl cellulose was used as a comparator. The unmedicated control group received deionized water. At two weeks post-treatment, all mice were euthanized for necropsy and cyst analysis. [2]
For the microinjection procedure, microcysts (300 μm in diameter) were cultured. Microneedles were used to inject approximately 100 pl of Pyronaridine solution (stock at 14 or 28 mM) into the cyst cavity, resulting in final concentrations of 100 μM or 200 μM. An identical amount of saline was injected into control cysts. After washing, 35 microinjected cysts were intraperitoneally injected into mice (n=5 per group). At 30 days post-treatment, mice were euthanized and therapeutic effects were examined. [2]
For the in vivo pharmacokinetics study, male ICR mice (20-22 g) were given a single intraperitoneal injection of Pyronaridine (57 mg/kg). Plasma, liver, and lung samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 30, and 48 hours post-administration (n=3 per time point). The samples were processed and analyzed by LC-MS/MS. [2]

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For the in vivo efficacy study, female BALB/c mice (6-8 weeks old) were infected intraperitoneally with microcysts. Three months post-infection, mice were randomly allocated into groups. For intraperitoneal (i.p.) administration, Pyronaridine tetraphosphate was dissolved in saline and administered i.p. at dosages of 14, 28, or 57 mg/kg in a volume of 1.0 ml once daily for three days. The control group received saline. [2]
For oral administration, Pyronaridine tetraphosphate was dissolved in deionized water and administered by gavage at 57 mg/kg in a volume of 0.2 ml once daily for 30 days. Albendazole (50 mg/kg) formulated in 0.5% carboxymethyl cellulose was used as a comparator. The unmedicated control group received deionized water. At two weeks post-treatment, all mice were euthanized for necropsy and cyst analysis. [2]
For the microinjection procedure, microcysts (300 μm in diameter) were cultured. Microneedles were used to inject approximately 100 pl of Pyronaridine solution (stock at 14 or 28 mM) into the cyst cavity, resulting in final concentrations of 100 μM or 200 μM. An identical amount of saline was injected into control cysts. After washing, 35 microinjected cysts were intraperitoneally injected into mice (n=5 per group). At 30 days post-treatment, mice were euthanized and therapeutic effects were examined. [2]
For the in vivo pharmacokinetics study, male ICR mice (20-22 g) were given a single intraperitoneal injection of Pyronaridine (57 mg/kg). Plasma, liver, and lung samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 10, 24, 30, and 48 hours post-administration (n=3 per time point). The samples were processed and analyzed by LC-MS/MS. [2]

Following a single intraperitoneal dose (57 mg/kg) in mice, Pyronaridine was highly distributed in the liver and lungs. The average concentrations in the liver and lungs from 0 to 48 hours were 435.7 μmol/kg and 122.5 μmol/kg, respectively, whereas the average plasma concentration was 1.5 μM, resulting in a liver/plasma ratio of 290.5 and a lung/plasma ratio of 81.7. [2]
Pyronaridine sustained high concentrations in the liver without an obvious decline over 48 hours after administration. [2]
In human hepatocytes, Pyronaridine showed great metabolic stability with a half-life (T1/2) of 7.8 hours and a slow clearance rate (Clint) of 3.8 ml/min/kg. [2]
Pyronaridine did not significantly inhibit CYP1A2, CYP2C9, CYP2C19, and CYP3A4 isoforms (IC50 > 10 μM), but showed moderate inhibition of CYP2D6 (IC50 = 1.53 μM). [2]

In the three-dose intraperitoneal regimen in mice (14, 28, 57 mg/kg, q.d. for 3 days), no significant pathological changes in the liver, kidneys, thymus, or body weight were observed compared to the control group, except for a minor increase in liver weight in the high-dosage group. Hematological and biochemical indexes were almost all within normal limits. All mice exhibited normal behavioral activities without obvious adverse effects. [2]
In the 30-day oral administration study in mice (57 mg/kg/day), a minor loss of body weight was observed in the Pyronaridine-treated group, which was deemed acceptable. Hematological and biochemical indexes did not show any statistical difference among the groups. [2]
Based on human equivalent dose (HED) calculations, the oral dosage used in the study (57 mg/kg/day in mice) is equivalent to 4.6 mg/kg in humans. Previous sub-acute toxicity studies (not conducted within this paper) have shown that oral administration of PND at 24 mg/kg/day (HED = 13.0 mg/kg/day) for 30 days was well tolerated by dogs. [2]

[1]. Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo. Acta Trop. 2008 Mar;105(3):222-8.

[2]. Old drug repurposing for neglected disease: Pyronaridine as a promising candidate for the treatment of Echinococcus granulosus infections. EBioMedicine. 2020 Apr;54:102711.

Pyronaridine is an aminoquinoline compound. It has been studied for the treatment of malaria. Pyronaridine has been reported to exist in Acronychia pubescens, and relevant data are available. Pyronaridine is a benzonaphthyl derivative with antimalarial and potential antiviral activity. After administration, Pyronaridine inhibits the production of β-heme, leading to the accumulation of toxic heme in the parasite. Furthermore, Pyronaridine inhibits glutathione-dependent heme degradation. This promotes heme-induced erythrocyte lysis, ultimately leading to parasite death. Pyronaridine also exhibits antiviral activity against certain viruses in vitro, including Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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Pyronaridine is an approved antimalarial drug (China Food and Drug Administration). This study repurposes it for the treatment of cystic echinococcosis (CE), a neglected disease caused by Echinococcus granulosus sensu stricto. [2]
The study proposes that Pyronaridine could be a rapidly translatable option for CE treatment, including its potential use in the PAIR (puncture, aspiration, injection, re-aspiration) clinical procedure. [2]
The mechanism of action against E. granulosus ss is suggested to involve the inhibition of topoisomerase I and the induction of apoptosis. [2]

C29H32CLN5O2

518.058

517.224

74847-35-1

Pyronaridine tetraphosphate;76748-86-2

107771

Typically exists as solid at room temperature

1.4±0.1 g/cm3

621.3±55.0 °C at 760 mmHg

329.6±31.5 °C

0.0±1.9 mmHg at 25°C

1.720

4.8

2

7

7

37

707

0

COC1=NC2=C(C=C1)NC3=C(C=CC(=C3)Cl)C2=NC4=CC(=C(C(=C4)CN5CCCC5)O)CN6CCCC6

DJUFPMUQJKWIJB-UHFFFAOYSA-N

InChI=1S/C29H32ClN5O2/c1-37-26-9-8-24-28(33-26)27(23-7-6-21(30)16-25(23)32-24)31-22-14-19(17-34-10-2-3-11-34)29(36)20(15-22)18-35-12-4-5-13-35/h6-9,14-16,36H,2-5,10-13,17-18H2,1H3,(H,31,32)

4-[(7-chloro-2-methoxybenzo[b][1,5]naphthyridin-10-yl)amino]-2,6-bis(pyrrolidin-1-ylmethyl)phenol

Pyronaridine Benzonaphthyridine 7351Malaridine

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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