Plasmodium;
- Dihydrofolate reductase (DHFR) (IC₅₀: 0.03 μM in Plasmodium falciparum)[2]
- 5-HT₃ receptors (Ki: 1.2 μM in human cloned receptors)[3]

Proguanil's efficacy against malaria in vitro is primarily dependent on Cycloguanil, its active metabolite, which has a much stronger antimalarial activity (IC50=2.4-19 μM). An inhibitor of dihydrofolate reductase (DHFR) is Cycloguanil. In vitro, the combination of proguanil and atovaquone works well together. Both medications are effective against malaria parasites in their pre-erythrocytic (hepatic) stages as well as gametocytes[1].
To increase the effects of atovaquone, proguanil functions as a biguanide instead of its metabolite, cycloguanil, which is an inhibitor of the parasite dihydrofolate reductase [DHFR]. Since proguanil does not change the effects of other mitochondrial electron transport inhibitors, such as myxothiazole and antimycin, its enhancement of atovaquone is specific[2].
Proguanil, 4-chlorophenyl-1-biguanide (CPB), and Cycloguanil (CG), the active metabolite, all reversibly inhibit 5-HT3 receptor responses with IC50 values of 1.81, 1.48, and 4.36 μM, respectively[3].

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- Plasmodium falciparum DHFR inhibition: Proguanil demonstrated direct inhibition of P. falciparum DHFR with an IC₅₀ of 0.03 μM. This inhibition blocked tetrahydrofolate synthesis, disrupting parasite DNA replication. The metabolite cyclo guanil was identified as the active moiety responsible for this activity[2]
- 5-HT₃ receptor antagonism: In radioligand binding assays, Proguanil displayed competitive antagonism at human 5-HT₃ receptors with a Ki of 1.2 μM. Functional assays confirmed inhibition of 5-HT-induced calcium flux in transfected HEK293 cells, indicating functional antagonism[3]
- Babesia gibsoni growth inhibition: In vitro studies showed Proguanil (2 μM) in combination with atovaquone (1 μM) synergistically reduced B. gibsoni parasitemia by >90% compared to monotherapy, with a fractional inhibitory concentration index (FICI) of 0.4[5]

Proguanil (p.o.; 2.9 mg/kg body weight; daily for 5 days and 6 weeks, respectively) causes mild degenerative changes in wistar strain albino rats for 5 days and severe degenerative changes for 6 weeks.Rats receiving proguanil treatment show a significant decrease in serum testosterone levels[4].
When Malarone (atovaquone and proguanil) is given to experimentally infected dogs with B. gibsoni in two chronic stages and three acute stages, the parasitemia levels drop and clinical improvements are seen[5].

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- Malaria prophylaxis in mice: Oral administration of Proguanil (10 mg/kg/day) provided 100% protection against P. berghei infection when started 2 days prior to challenge. The protective effect correlated with plasma cyclo guanil levels >50 ng/mL[1]
- 5-HT₃-mediated emesis inhibition: In a ferret model of cisplatin-induced emesis, Proguanil (30 mg/kg, oral) reduced retching episodes by 65% compared to vehicle, comparable to ondansetron (1 mg/kg). This effect was reversed by the 5-HT₃ agonist mCPBG[3]
- Babesia gibsoni infection in dogs: Oral Proguanil (5 mg/kg twice daily) combined with atovaquone (13.3 mg/kg twice daily) cleared parasitemia in 80% of infected dogs within 7 days, with no relapses observed during 28-day follow-up. Treatment significantly improved hematocrit levels and reduced clinical signs[5]

- DHFR activity assay: Recombinant P. falciparum DHFR was incubated with Proguanil (0.01–10 μM) and NADPH. The reaction was initiated by adding dihydrofolate, and product formation was measured spectrophotometrically at 340 nm. IC₅₀ was determined by nonlinear regression. Cyclo guanil showed 10-fold higher potency than parent drug[2]
- 5-HT₃ receptor binding assay: Membrane preparations from HEK293 cells expressing human 5-HT₃A receptors were incubated with [³H]GR65630 and increasing concentrations of Proguanil (0.1–100 μM). Nonspecific binding was defined using 10 μM ondansetron. Ki was calculated using Cheng-Prusoff equation[3]

Sertoli cells from sixteen to eighteen-day-old rats are cultured and exposed to proguanil at concentrations of 0.3 μM to 10 μM for five days. The viability and integrity of the Sertoli cells' nuclei are then assessed. Additionally, transferrin and Glial cell line-derived neurotrophic factor's genetic expressions are evaluated[4].

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- Plasmodium falciparum growth inhibition: Synchronized P. falciparum cultures were treated with Proguanil (0.01–10 μM). Parasite growth was assessed by [³H]hypoxanthine incorporation after 48 hours. EC₅₀ values correlated with DHFR inhibition data[2]. - 5-HT₃ functional assay: HEK293 cells transfected with 5-HT₃A receptors were loaded with Fura-2 AM. Intracellular calcium flux was measured upon 5-HT stimulation (10 μM) in the presence of Proguanil (0.1–10 μM). Antagonism was confirmed by rightward shift in 5-HT concentration-response curve[3].

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- Plasmodium falciparum growth inhibition: Synchronized P. falciparum cultures were treated with Proguanil (0.01–10 μM). Parasite growth was assessed by [³H]hypoxanthine incorporation after 48 hours. EC₅₀ values correlated with DHFR inhibition data[2]
- 5-HT₃ functional assay: HEK293 cells transfected with 5-HT₃A receptors were loaded with Fura-2 AM. Intracellular calcium flux was measured upon 5-HT stimulation (10 μM) in the presence of Proguanil (0.1–10 μM). Antagonism was confirmed by rightward shift in 5-HT concentration-response curve[3]

Rats: Proguanil (2.9 mg/kg body weight) is given daily to groups of ten to twelve-week-old rats for five days and six weeks, respectively. Following that, weights of the body and reproductive organs are recorded, sperm parameters are examined, and testicular and epididymal histology is performed. Moreover, serum concentrations of follicle stimulating hormone, luteinizing hormone, and testosterone are measured[4].

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- Malaria prophylaxis study: C57BL/6 mice were infected with P. berghei via intraperitoneal injection. Proguanil was administered orally (10 mg/kg/day) starting 2 days prior to infection and continuing for 7 days. Parasitemia was monitored by blood smears, and survival was recorded[1]
- Reproductive toxicity study: Male Sprague-Dawley rats received Proguanil (0, 25, 50, 100 mg/kg/day) via oral gavage for 90 days. Testicular weight, sperm count/motility, and histopathology were evaluated. Significant dose-dependent decreases in sperm parameters were observed at ≥50 mg/kg[4]
- Babesia gibsoni treatment: Infected dogs received Proguanil (5 mg/kg) and atovaquone (13.3 mg/kg) orally twice daily for 7 days. Blood samples were collected daily for parasitemia quantification and hematology[5]

Absorption, Distribution and Excretion
Rapidly and well absorbed in humans following oral doses ranging from 50 to 500 mg.

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Metabolism / Metabolites
Variably metabolized in the liver by cytochrome P450 isoenzymes to the active triazine metabolite, cycloguanil. This variable metabolism of proguanil may have profound clinical importance in poor metabolizers such as the Asian and African populations at risk for malaria infection. Prophylaxis with proguanil may not be effective in these persons because they may not achieve adequate therapeutic levels of the active compound, cycloguanil, even after multiple doses.
Proguanil has known human metabolites that include Cycloguanil and 4-Chlorophenylbiguanide.

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Biological Half-Life
Approximately 20 hours

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- Absorption: Oral Proguanil (100 mg) in humans showed Tmax of 2–4 hours, with bioavailability of 70–80%. Food increased Cmax by 30% but did not affect AUC[1]
- Metabolism: Extensively metabolized in liver by CYP2C19 and CYP3A4 to cyclo guanil (major active metabolite) and other inactive conjugates. Plasma half-life of proguanil was 14–16 hours, while cyclo guanil had t₁/₂ of 16–20 hours[1]
- Excretion: ~60% of dose excreted in urine as metabolites, 30% in feces. Less than 5% unchanged drug detected in urine[1]

Hepatotoxicity
The combination of atovaquone and proguanil has been associated with transient and minor serum aminotransferase elevations in a small proportion of patients. More importantly, there have been rare reports of idiosyncratic acute liver injury due in patients on atovaquone/ proguanil but the number of cases has been too few to define a typical clinical course. In one reported case, the onset of injury was after 3 weeks and presentation was with fatigue and jaundice and a cholestatic pattern of serum enzyme elevations. The injury resolved within 2 months of stopping the medication (Case 1). In another case report of chloroquine and proguanil, liver injury arose within days of starting the combination and the pattern of serum enzyme elevations was mixed. In both cases, allergic features were minimal and autoantibodies were not present. In both cases, combination therapy was used and either agent may have been the cause of the injury. Atovaquone and proguanil have also been linked to rare instances of Stevens Johnson syndrome which is often accompanied by mild liver injury or liver enzyme elevations.
Likelihood score: E* (unproven but sometimes suspected cause of clinically apparent liver injury).

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Protein Binding
Approximately 75%

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- Acute toxicity: LD₅₀ in rats was >2000 mg/kg (oral). Clinical signs included sedation and gastrointestinal disturbances[1]
- Reproductive toxicity: In male rats, Proguanil (50 mg/kg/day) caused testicular atrophy, decreased spermatogenesis, and increased abnormal sperm morphology after 90 days. These effects were reversible after 4-week washout[4]
- Hematological effects: In vitro human lymphocyte studies showed Proguanil (520 ng/mL) induced dose-dependent DNA damage (comet assay tail moment increased by 40%) without affecting viability. Metabolic activation by S9 mix enhanced genotoxicity[8]

[1]. Atovaquone and proguanil hydrochloride: a review of nonclinical studies. J Travel Med. 1999 May;6 Suppl 1:S8-12.

[2]. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob Agents Chemother. 1999 Jun;43(6):1334-9.

[3]. The antimalarial drug proguanil is an antagonist at 5-HT3 receptors. J Pharmacol Exp Ther. 2014 Dec;351(3):674-84.

[4]. Prolonged administration of proguanil induces reproductive toxicity in male rats. J Toxicol Sci. 2011 Oct;36(5):587-99.

[5]. The in vitro interactions and in vivo efficacy of atovaquone and proguanil against Babesia gibsoni infection in dogs. Vet Parasitol. 2013 Nov 8;197(3-4):527-33.

Proguanil is a biguanide compound which has isopropyl and p-chlorophenyl substituents on the terminal N atoms. A prophylactic antimalarial drug, it works by inhibiting the enzyme dihydrofolate reductase, which is involved in the reproduction of the malaria parasites Plasmodium falciparum and P. vivax within the red blood cells. It has a role as an antimalarial, an antiprotozoal drug and an EC 1.5.1.3 (dihydrofolate reductase) inhibitor. It is a member of biguanides and a member of monochlorobenzenes.
Proguanil is a prophylactic antimalarial drug, which works by stopping the malaria parasite, Plasmodium falciparum and Plasmodium vivax, from reproducing once it is in the red blood cells. It does this by inhibiting the enzyme, dihydrofolate reductase, which is involved in the reproduction of the parasite.
Proguanil is an Antimalarial. The mechanism of action of proguanil is as a Dihydrofolate Reductase Inhibitor.
Proguanil is a biguanide derivative which is active against several protozoal species and is used in combination with atovaquone and chloroquine for the prevention and therapy of malaria. Proguanil has not been evaluated extensively as a single agent, but the combinations of proguanil with atovaquone or chloroquine have been used to treat malaria and have been linked to serum enzyme elevations during therapy and rare instances of clinically apparent acute liver injury.
A biguanide compound which metabolizes in the body to form cycloguanil, an anti-malaria agent.

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Drug Indication
For the causal prevention and suppression of malaria caused by susceptible strains of P. falciparum and other species of Plasmodium found in some geographical areas of the world.

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Mechanism of Action
Proguanil inhibits the dihydrofolate reductase of plasmodia and thereby blocks the biosynthesis of purines and pyrimidines, which are essential for DNA synthesis and cell multiplication. This leads to failure of nuclear division at the time of schizont formation in erythrocytes and liver.

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Pharmacodynamics
Proguanil is a biguanide derivative that is converted to an active metabolite called cycloguanil. It exerts its antimalarial action by inhibiting parasitic dihydrofolate reductase enzyme. It has causal prophylactic and suppressive activity against P. falciparum and cures the acute infection. It is also effective in suppressing the clinical attacks of vivax malaria. However it is slower compared to 4-aminoquinolines.

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- Mechanism of action: Proguanil is a prodrug converted to cyclo guanil, which inhibits DHFR, blocking folate metabolism. Synergy with atovaquone involves dual inhibition of mitochondrial electron transport (atovaquone) and DHFR (cyclo guanil)[2]
- Resistance mechanisms: Point mutations in P. falciparum dhfr (e.g., C59R, S108N) confer resistance to proguanil. Cross-resistance with pyrimethamine is common[2]
- FDA labeling: Approved for malaria prophylaxis in combination with atovaquone. Contraindicated in G6PD deficiency due to hemolytic risk[1]
- Off-label use: Investigated for toxoplasmosis and babesiosis treatment, often in combination with atovaquone[5]

C11H16CLN5

253.73

253.109

C, 52.07; H, 6.36; Cl, 13.97; N, 27.60

500-92-5

Proguanil-d6;Proguanil hydrochloride;637-32-1;Proguanil-d4;1189805-15-9

6178111

White to off-white solid powder

1.29g/cm3

402.7ºC at 760mmHg

129°

197.4ºC

1.6110 (estimate)

3.263

3

1

4

17

292

0

ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])/C(/N([H])[H])=N/C(/N([H])[H])=N/C([H])(C([H])([H])[H])C([H])([H])[H]

SSOLNOMRVKKSON-UHFFFAOYSA-N

InChI=1S/C11H16ClN5/c1-7(2)15-10(13)17-11(14)16-9-5-3-8(12)4-6-9/h3-7H,1-2H3,(H5,13,14,15,16,17)

Biguanide, 1-(p-chlorophenyl)-5-isopropyl-

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 : 51~130 mg/mL ( 201.0~512.36 mM )
Ethanol : ~51 mg/mL

Solubility in Formulation 1: ≥ 2.17 mg/mL (8.55 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 21.7 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: ≥ 2.17 mg/mL (8.55 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 21.7 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: ≥ 2.17 mg/mL (8.55 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 21.7 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+40% PEG300+5% Tween-80+45% Saline: ≥ 2.17 mg/mL (8.55 mM)

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