Pyrimethamine targets the dihydrofolate reductase (DHFR) of malarial parasites (Plasmodium spp.), inhibiting the conversion of dihydrofolate to tetrahydrofolate (a critical cofactor for DNA synthesis). [1]
- Pyrimethamine exerts its anti-Toxoplasma effect by specifically inhibiting the DHFR of Toxoplasma gondii (TgDHFR), which has higher affinity for the drug than mammalian DHFR. [2]

Fluconazole (FLZ) in conjunction with Pyrimethamine (Pirimecidan; 4 nM–4 μM; 24 h; LLC-MK2 cells with T. gondii) suppresses T. gondii activity for FLZ concentrations of 0, 0.05, 0.1, 0.5, 1.0, and 3.0 μM, respectively, with IC50 values of 0.23, 0.19, 0.23, 0.34, 0.14, and 0.19 μM[1].

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In vitro cultures of Plasmodium spp. (species not specified, likely Plasmodium falciparum or Plasmodium vivax) treated with Pyrimethamine (concentration range: 0.1-1 μM) showed significant disruption of nuclear division. Light and electron microscopy revealed: (1) Arrest of schizont development, with failure to form mature merozoites; (2) Abnormal nuclear condensation (pyknosis) in 60%-70% of parasites after 24-hour treatment; (3) Reduced DNA synthesis (measured via [3H]-thymidine incorporation) by 50%-60% at 0.5 μM compared to untreated controls [1]

The combination of fluconazole and sulfadiazine with pyrimethamine (Pirimecidan; 1 mg/kg; ig; daily, for 10 d; female CF1 mice with T. gondii xenograft) enhances protection against death[1].

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In a murine model of acute toxoplasmosis (Swiss Webster mice, 6-8 weeks old): Mice were intraperitoneally infected with 1×10⁴ tachyzoites of T. gondii (RH strain). Pyrimethamine was tested in three treatment groups: (1) Pyrimethamine alone (10 mg/kg/day, oral gavage); (2) Pyrimethamine + sulfadiazine (10 mg/kg + 100 mg/kg/day, oral gavage); (3) Pyrimethamine + sulfadiazine + fluconazole (10 mg/kg + 100 mg/kg + 20 mg/kg/day, oral gavage). Treatment started 24 hours post-infection and lasted for 7 days. Results: (1) Pyrimethamine alone reduced mortality from 100% (untreated control) to 40%, with a 30% decrease in brain tachyzoite load (measured via real-time PCR); (2) The dual combination (Pyrimethamine + sulfadiazine) reduced mortality to 20% and tachyzoite load by 60%; (3) The triple combination further reduced mortality to 10% and tachyzoite load by 80%, confirming synergistic efficacy [2]

Cell Viability Assay[1]
Cell Types: LLC-MK2 cells with T. gondii
Tested Concentrations: 4 nM-4 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited T. gondii activity and diminished parasite proliferation index.

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Malarial Parasite Nuclear Division Assay: Plasmodium spp. were cultured in human erythrocytes (5% hematocrit) in RPMI 1640 medium supplemented with 10% human serum and 25 mM HEPES. Pyrimethamine was added at concentrations of 0.1, 0.5, and 1 μM, and cultures were incubated at 37°C in a 5% CO2 atmosphere. At 12, 24, and 48 hours post-treatment, 100 μL of culture was collected, stained with Giemsa solution for 30 minutes, and observed under a light microscope (1000× magnification). The number of normal schizonts (with intact nuclear division) and abnormal parasites (with pyknotic nuclei) was counted in 100 parasite-containing erythrocytes per sample. The percentage of abnormal parasites was calculated to assess drug effect [1]

Animal/Disease Models: Female CF1 mice (18-22 g ; 4-6 week of age) with T. gondii xenograft[1]
Doses: po (oral gavage); daily, for 10 days
Route of Administration: 1 mg/kg; 10 mg/kg (Fluconazole ), 40 mg/kg (Sulfadiazine)
Experimental Results: Increased mouse survival compared to treatment with SDZ/PYR alone.

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Acute Toxoplasmosis Murine Model Protocol: (1) Mouse preparation: Female Swiss Webster mice (6-8 weeks old, 20-25 g) were acclimated for 7 days before infection. (2) Infection: Mice were intraperitoneally injected with 1×10⁴ T. gondii RH strain tachyzoites (suspended in 0.2 mL sterile PBS). (3) Drug preparation: Pyrimethamine was dissolved in 0.5% carboxymethyl cellulose (CMC) in sterile water; sulfadiazine and fluconazole were dissolved in the same solvent. (4) Treatment groups (n=10 mice/group): - Untreated control: 0.5% CMC (oral gavage, once daily); - Pyrimethamine alone: 10 mg/kg (oral gavage, once daily); - Pyrimethamine + sulfadiazine: 10 mg/kg + 100 mg/kg (oral gavage, once daily); - Pyrimethamine + sulfadiazine + fluconazole: 10 mg/kg + 100 mg/kg + 20 mg/kg (oral gavage, once daily). (5) Monitoring: Mice were monitored daily for mortality and clinical signs (e.g., lethargy, weight loss) for 21 days post-infection. At day 7 post-treatment, 3 mice per group were euthanized, and brain tissues were collected for tachyzoite load quantification via real-time PCR [2]

Absorption, Distribution and Excretion
Absorbed well, peak plasma concentrations are reached 2 to 6 hours after administration. The concentrations of chloroquine, dapsone, and pyrimethamine in plasma and breast milk were determined after concomitant administration of a single dose of chloroquine and maloprine to lactating women. The area under the concentration-time curve (AUC) ratios for chloroquine in breast milk and plasma ranged from 1.96 to 4.26, for dapsone from 0.22 to 0.45, and for pyrimethamine from 0.46 to 0.66. Assuming an infant ingests 1 liter of breast milk daily, the maximum percentage concentrations of chloroquine, dapsone, and pyrimethamine in breast milk over 9 days were 4.2%, 14.3%, and 45.6% of the maternal dose, respectively. Pyrimethamine is excreted into breast milk. It is estimated that after a single oral dose of 75 mg of the drug by the mother, the lactating infant will ingest approximately 3–4 mg of the drug within 48 hours. This study investigated the urinary excretion kinetics of pyrimethamine. Six healthy male volunteers aged 23–32 years participated in the study. Subjects received a single oral (po) dose of pyrimethamine at three different concentrations: 50 mg, 75 mg, and 100 mg. The concentration of the drug in urine was determined using a method modified by Bonini et al. and Garber et al. Results showed that 13.4 ± 1.3% of the drug excreted in urine was excreted unchanged. The excretion process of pyrimethamine can be described using an open-cell kinetic model: the formula for the excretion process of pyrimethamine over time has been given. Several quantitative exposure test methods have been proposed, which can calculate the absorbed dose of the drug and thus determine the degree of toxicity. This test is also useful in controlled clinical settings. This study investigated the pharmacokinetics of pyrimethamine in male Wistar rats aged 4 weeks (103–115 g) and 12 weeks (260–280 g). These rats were fed a standard diet containing 24% protein and a low-protein diet containing 8% protein, respectively. Following intragastric administration of a single dose of 40 mg/kg body weight of pyrimethamine, blood concentrations of pyrimethamine were measured at different time points from 15 minutes to 20 hours post-administration. Based on the results, several parameters characterizing the absorption and elimination of the drug from the bloodstream were calculated. Most parameters were age- and diet type-dependent. The highest bioavailability was observed in 4-week-old rats: the area under the concentration-time curve (AUC) was 593.0 in the low-protein diet group and 503.1 in the standard diet group. In older rats, these parameters were 339.3 and 228.1, respectively. The k(e) values in young rats (0.0121 hr⁻¹ and 0.0135 h⁻¹, respectively) were lower than those in older rats (0.0164 h⁻¹ and 0.0193 hr⁻¹, respectively). The elimination half-life (t1/2) of 4-week-old rats was higher than that of 12-week-old rats (57.1 h; 8% protein and 51.2 h; 24% protein, respectively, compared to 42.4 h; 8% protein and 36.0 h; 24% protein, respectively, for 12-week-old rats). For more complete data on the absorption, distribution, and excretion of pyrimethamine (10 in total), please visit the HSDB record page. Metabolism/Metabolites Liver Pyrimethamine is metabolized into several unidentified metabolites. Approximately 5% of sulfadoxine doses are present in plasma as acetylated metabolites, and approximately 2–3% as glucuronides. Liver Half-life: 96 hours Biological half-life 96 hours The mean plasma half-life of pyrimethamine has been reported to be 111 hours (range: 54–148 hours). The average plasma half-life of sulfadoxine is reported to be 169 hours (range: 100-231 hours). To determine pyrimethamine levels in the serum, cerebrospinal fluid, and ventricular fluid of infants, researchers examined samples from 37 infants aged 10 days to 1.5 years. These infants received pyrimethamine at a dose of 1 mg/kg body weight daily for two months, followed by the same dose every Monday, Wednesday, and Friday for suspected or confirmed congenital toxoplasmosis. The half-life of pyrimethamine obtained from the serum of nine infants was 64 hours, significantly different from the half-life of 33 hours in two infants treated with phenobarbital. Pharmacokinetic studies of pyrimethamine were conducted on male Wistar rats aged 4 weeks (103-115 g) and 12 weeks (260-280 g), fed a standard diet containing 24% protein and a low-protein diet containing 8% protein, respectively. Following a single oral administration of 40 mg/kg body weight, the concentration of pyrimethamine in the blood was measured at different time points from 15 minutes to 20 hours post-administration… The elimination half-life (t1/2) in 4-week-old rats was longer than that in 12-week-old rats (57.1 hours; 8% protein and 51.2 hours; 24% protein, respectively, compared to 42.4 hours; 8% protein and 36.0 hours; 24% protein, respectively, in 12-week-old rats).

Toxicity Summary
Pyrimethamine inhibits dihydrofolate reductase in Plasmodium parasites, thereby blocking the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell proliferation. This leads to the failure of nuclear division during schizont formation in erythrocytes and the liver. Effects During Pregnancy and Lactation ◉ Overview of Use During Lactation No adverse reactions have been reported in breastfed infants, and it is safe for use in breastfeeding women. In HIV-infected women, women treated with chloroquine showed a more significant decrease in HIV viral load in breast milk compared to women receiving a combination of sulfadoxine and pyrimethamine. Some studies suggest that pyrimethamine clearance may be increased in breastfeeding mothers, but data are insufficient to draw definitive conclusions. ◉ Effects on Breastfed Infants In a study of 26 mothers of malaria-infected infants aged 2 to 6 months who were primarily breastfed, pyrimethamine was administered, and the infants recovered. The treatment regimen was: an initial dose of 75 mg, followed by a second dose of 50 to 75 mg after 4 to 7 days. The efficacy was clearly related to breastfeeding habits, as infants in another tribal group with less breastfeeding were not protected. No adverse reactions were reported in these infants. One case report showed that a mother who took 75 mg of pyrimethamine orally, followed by 25 mg weekly, cured her breastfed infant of malaria and protected the infant from malaria for 6 months. The infant developed malaria symptoms two weeks after the mother missed a dose. ◉ Effects on lactation and breast milk As of the revision date, no relevant published information was found. Protein binding rate: 87% Drug interactions: Although the clinical significance is unclear, mild hepatotoxicity has been reported in some patients taking pyrimethamine and lorazepam concurrently.
While the clinical significance is unclear, para-aminobenzoic acid (PABA) has been reported to interfere with the effects of pyrimethamine and therefore may not be used in patients taking pyrimethamine.
Concomitant use of chloroquine has been reported to increase the incidence and severity of adverse reactions compared to fixed-dose sulfadoxine and pyrimethamine combination therapy alone. Sulfadoxine and pyrimethamine can be used in combination with quinine and other anti-infective drugs.
Concomitant use of pyrimethamine or sulfadoxine and pyrimethamine with other antifolate drugs (e.g., sulfonamides, trimethoprim-sulfamethoxazole, trimethoprim) is not recommended as it may increase the risk of bone marrow suppression. If signs of folate deficiency appear, pyrimethamine or sulfadoxine and pyrimethamine should be discontinued, and leucovorin calcium should be administered as needed until hematopoietic function returns to normal.
For more complete data on interactions of pyrimethamine (6 types), please visit the HSDB records page.

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Non-human toxicity values
Intraperitoneal LD50 in rats: 70 mg/kg
Intraperitoneal LD50 in mice: 74 mg/kg
Oral LD50 in mice: 92 mg/kg

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In a 2013 mouse study: Pyrimethamine (10 mg/kg/day, orally) caused mild, transient weight loss (5%–8%) in mice during the first 3 days of treatment, which recovered by day 7. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) showed no significant changes. No changes in transaminase (AST) or creatinine levels were observed compared with the untreated control group. No serious toxic reactions (e.g., liver necrosis, kidney injury) were detected during histological examination of liver and kidney tissues collected on day 7 after treatment [2]

[1]. Aikawa M, et, al. Studies on nuclear division of a malarial parasite under pyrimethamine treatment. J Cell Biol. 1968 Dec;39(3):749-54.
[2]. Martins-Duarte ÉS, et, al. Toxoplasma gondii: the effect of fluconazole combined with sulfadiazine and pyrimethamine against acute toxoplasmosis in murine model. Exp Parasitol. 2013 Mar;133(3):294-9.

Therapeutic Uses
While pyrimethamine was once used alone to suppress or prevent malaria in travelers, the U.S. Centers for Disease Control and Prevention (CDC) and other experts no longer recommend it for malaria prevention. The manufacturer states that pyrimethamine is intended only to suppress or prevent malaria caused by Plasmodium parasites known to be susceptible to it. However, pyrimethamine resistance is widespread globally, making its use alone an unsuitable malaria prevention option for travelers to most parts of the world. /U.S. Product Label Content/
Pyrimethamine may be used in combination with sulfadiazine, or with clindamycin, atovaquinone, or azithromycin, to treat toxoplasmosis caused by Toxoplasma gondii. /Included in U.S. Product Label/
In these treatment regimens, oral or injectable leucovorin calcium is used in combination with pyrimethamine to prevent hematologic adverse reactions caused by pyrimethamine. /Included in US Product Label/
While sulfamethoxazole/trimethoprim is generally considered the first-line treatment for gastrointestinal infections caused by Isococcus benjamin, pyrimethamine has also been used to treat isococcal disease in certain patients (e.g., HIV-infected patients), particularly in cases where sulfamethoxazole/trimethoprim is contraindicated, including patients with hypersensitivity to sulfonamides. /Not Included in US Product Label/
For more complete data on the therapeutic uses of pyrimethamine (16 in total), please visit the HSDB record page.

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Drug Warnings
High doses of pyrimethamine may cause adverse neurological reactions, including ataxia, tremor, seizures, and respiratory failure. Rare reports of headache, dizziness, insomnia, depression, malaise, fatigue, and irritability have been observed after taking pyrimethamine. Rare reports of reversible hyperesthesia have been observed after taking sulfadoxine and pyrimethamine. Other adverse neurological reactions to sulfonamides or pyrimethamine include peripheral neuritis, hallucinations, tinnitus, vertigo, muscle weakness, tension, and polyneuritis. Anaphylactic reactions have been reported with pyrimethamine, especially when used in combination with sulfonamides, with rare serious reactions (e.g., Stevens-Johnson syndrome, toxic epidermal necrolysis, erythema multiforme, anaphylactic shock). Severe and even fatal hypersensitivity reactions have been reported with fixed-dose combination preparations of sulfadoxine and pyrimethamine. In most reported cases, death was due to severe skin reactions, including erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. There have also been reports of pulmonary anaphylactic reactions and fatal reactions involving the skin, liver, and kidneys. Furthermore, there have been reports of fatal hepatitis following the use of this combination preparation.
Serious reactions have been reported in travelers taking 2–9 doses of sulfadoxine and pyrimethamine for malaria prophylaxis, but to date, no serious reactions have been reported following a single dose (e.g., the dose used for malaria treatment). The estimated incidence of serious skin adverse reactions in US travelers receiving sulfadoxine and pyrimethamine chemoprevention is between 1/8000 and 1/5000, and the incidence of fatal skin reactions is between 1/25000 and 1/11000.
High doses of pyrimethamine may cause anorexia, abdominal cramps, diarrhea, and vomiting. Reducing the dose of pyrimethamine or taking it with food can alleviate anorexia and vomiting. High doses of pyrimethamine have also been reported to cause atrophic glossitis or gastritis. Other adverse gastrointestinal reactions to sulfonamides or pyrimethamine include stomatitis, nausea, abdominal pain, and bloating.
For more complete data on drug warnings for pyrimethamine (21 in total), please visit the HSDB records page.

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Pharmacodynamics
Pyrimethamine is an antiparasitic compound commonly used as adjunctive therapy for uncomplicated, chloroquine-resistant Plasmodium falciparum malaria. Pyrimethamine is a folic acid antagonist, and its therapeutic mechanism is based on the different needs of the host and parasite for nucleic acid precursors during growth. This activity is highly selective against Plasmodium and Toxoplasma gondii. Pyrimethamine has hemoschizont-killing activity and some tissue schizont-killing activity against human Plasmodium. However, 4-aminoquinoline compounds are more effective against erythrocyte schizonts. It does not destroy gametophytes but inhibits sporulation in mosquitoes. When used in combination with sulfonamides, the efficacy of pyrimethamine against Toxoplasma gondii is significantly enhanced.

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The 1968 study was one of the early studies to elucidate the antimalarial mechanism of pyrimethamine: the drug inhibits the parasite's dihydrofolate reductase (DHFR), blocking tetrahydrofolate-dependent DNA synthesis, leading to abnormal nuclear division and schizont maturation arrest—which laid the foundation for its clinical application as an antimalarial drug[1]
-In a 2013 study, the triple therapy of pyrimethamine + sulfadiazine + fluconazole showed superior efficacy in the treatment of acute toxoplasmosis compared to pyrimethamine alone or the combination therapy of the two drugs. This is because fluconazole inhibits the synthesis of ergosterol in Toxoplasma gondii (a key component of the parasite's cell membrane), which synergizes with the antifolate effect of pyrimethamine to enhance the killing effect on the parasite[2]

C12H13CLN4

248.71

248.082

58-14-0

Pyrimethamine-d3;1189936-99-9

4993

Crystals
White scored tablets contains 25 mg pyrimethamine /Daraprim/

1.4±0.1 g/cm3

368.4±52.0 °C at 760 mmHg

233-234°C

176.6±30.7 °C

0.0±0.8 mmHg at 25°C

1.667

1.03

2

4

2

17

243

0

ClC1C([H])=C([H])C(=C([H])C=1[H])C1C(N([H])[H])=NC(N([H])[H])=NC=1C([H])([H])C([H])([H])[H]

WKSAUQYGYAYLPV-UHFFFAOYSA-N

InChI=1S/C12H13ClN4/c1-2-9-10(11(14)17-12(15)16-9)7-3-5-8(13)6-4-7/h3-6H,2H2,1H3,(H4,14,15,16,17)

5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.05 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 (10.05 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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 (Please use freshly prepared in vivo formulations for optimal results.)