Anti-malarial

When tafenoquine is administered at the 3 mg/kg ED100 values that were established in WT mice, it shows no anti-malarial activity in CYP 2D knock-out mice. When tested at twice its ED100 (6 mg/kg), tafenoquine's anti-malarial activity is partially restored in humanized/CYP 2D6 knock-in mice[1].

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In wild-type (WT) C57BL/6 mice infected with luciferase-expressing Plasmodium berghei sporozoites, oral administration of Tafenoquine at its established 100% efficacious dose (ED₁₀₀) of 3 mg/kg (given on days -1, 0, and +1 relative to infection) completely prevented infection, as evidenced by no detectable luminescence signal at 48 and 72 hours post-inoculation and no blood-stage parasitemia up to 31 days (5/5 mice cured).
In CYP 2D knock-out (KO) mice (lacking the mouse CYP 2D gene cluster), Tafenoquine administered at the same ED₁₀₀ dose (3 mg/kg) showed no anti-malarial activity. All mice (5/5) exhibited robust luminescence signals and developed blood-stage infections, indicating the drug was inactive in the absence of CYP 2D enzymes.
In humanized/CYP 2D6 knock-in (KI) mice (mouse CYP 2D cluster replaced with human CYP2D6), Tafenoquine at its ED₁₀₀ dose (3 mg/kg) also failed to show activity (0/5 mice cured). However, when the dose was doubled to 6 mg/kg (2 x ED₁₀₀), anti-malarial activity was partially restored, with 4 out of 5 mice showing no detectable parasite burden at 31 days.
These results demonstrate that Tafenoquine's in vivo anti-malarial efficacy is critically dependent on metabolism by the CYP 2D enzyme family.[1]

Male, 8- to 12-week-old C57BL/6 wild-type (WT) mice, CYP 2D knock-out (KO) mice, and humanized/CYP 2D6 knock-in (KI) mice on a C57BL/6 background were used.
Mice were inoculated intravenously in the tail vein with approximately 10,000 luciferase-expressing Plasmodium berghei sporozoites on day 0.
Tafenoquine was administered orally (via intragastric gavage) as a suspension. The suspension was prepared by homogenizing the drug powder in a vehicle containing 0.5% (w/v) hydroxyethyl cellulose and 0.2% (v/v) Tween-80 in distilled water. Homogenization was performed on ice using a homogenizer at 20,000–22,000 rpm for 5 minutes.
The dosing regimen was once daily for three consecutive days: day -1 (one day before sporozoite inoculation), day 0 (day of inoculation), and day +1.
Two dose levels of Tafenoquine were tested: its established ED₁₀₀ of 3 mg/kg/day and a double dose of 6 mg/kg/day (2 x ED₁₀₀).
Efficacy was assessed by in vivo imaging system (IVIS) to measure luminescence (parasite burden) in the liver (48 hours post-inoculation) and systemically (72 hours post-inoculation), and by monitoring blood-stage parasitemia via flow cytometry up to 31 days post-inoculation to determine cure rates.[1]

Absorption, Distribution and Excretion
The first human pharmacokinetic study showed a time to peak concentration (tmax) of 13.8 hours, suggesting that prolonged intestinal absorption may be due to slow absorption and clearance from the distal gastrointestinal tract. AUC and Cmax show individual differences. A high-fat diet can increase the bioavailability of tafenoxane by altering the amount absorbed rather than the absorption rate. After absorption, tafenoxane reaches twice the plasma concentration in the system and appears to be widely distributed in the liver, with an AUC approximately 80 times that of plasma concentration. Tafenoxane is degraded via various metabolic pathways and is primarily excreted slowly in feces; very little of the unchanged drug is cleared by the kidneys. Tafenoxane has a large volume of distribution, approximately 2560 liters. Tafenoxane has a low clearance rate, approximately 6 liters/hour. Metabolism/Metabolites Activation of tafenoxane requires the activity of the hepatic microsomal enzyme CYP2D6. This activation step produces the metabolite 5,6-o-quinone tafenoxane. This metabolite is internalized by the parasite and reduced to free radicals by ferredoxin-NADP+ reductase and biflavin reductase. In the human body, tafenoxane is metabolized through multiple metabolic pathways, including O-demethylation, N-dealkylation, N-oxidation and oxidative deamination, and C-hydroxylation of the 8-aminoalkylamino side chain.
Biological Half-Life
Tafenoxane has a relatively long half-life, approximately 14 days.
This study concludes that tafenoxane requires metabolic activation by CYP2D enzymes to exert its antimalarial activity. This is a key ADME characteristic determining its efficacy.

Hepatotoxicity
In premarket clinical trials, tafenoxane was associated with a low incidence of transient and mild elevations in serum transaminases during treatment, but not with elevations in serum enzymes accompanied by jaundice or clinically significant acute liver injury. Despite its limited use, the risk of hepatotoxicity appears to be low, and its safety profile is similar to primaquine. Tafenoxane can cause hemolysis in patients with G6PD deficiency, leading to mild indirect bilirubinemia and jaundice, but without significant evidence of liver injury. Likelihood score: E (unlikely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of tafenoxane during lactation. Tafenoxane can cause hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. If the mother needs to take tafenoxane, both the mother and infant must be tested for G6PD deficiency before administration. Because tafenoxane has a mean half-life of 15 days, the manufacturer recommends against breastfeeding for 3 months after administration if the infant has G6PD deficiency.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding
Tafenoxane has a very high plasma protein binding rate in humans, approximately 99.5%.

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Hemolytic anemia due to G6PD deficiency: Tafenoxane, like other 8-aminoquinoline drugs, can cause hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. In a trial in heterozygous women with G6PD deficiency (Asian mahilon variant, with 40-60% of normal activity), a single dose of 300 mg tafenoxane resulted in a decrease of approximately 23% (lowest value) in hemoglobin, slightly higher than the approximately 16% decrease observed with 15 mg primaquine daily for 14 days. Prescribing tafenoxane requires exclusion of G6PD deficiency (activity below 70% of normal), but may exclude heterozygous women with activity above 70%. [2] - Ocular adverse reactions: Clinical trials observed a high incidence (93%) of mild reversible vortex keratopathy and retinal abnormalities in subjects, with an incidence of 39%. Follow-up studies reported no associated functional visual impairment. [2] - Psychiatric effects: The FDA label contains a warning that serious psychotic adverse reactions may occur in patients with a history of psychosis at the specified dose (for cure) or higher doses (for prophylaxis). [2] - Other adverse reactions: A comprehensive safety analysis reported a higher incidence (≥1%) of diarrhea, nausea, vomiting, sinusitis, gastroenteritis, and back/neck pain compared to placebo, with back/neck pain occurring in more than 5% of cases. Warnings also include methemoglobinemia and hypersensitivity reactions (e.g., angioedema). [2] - Testing requirements: Safe use requires quantitative (not just qualitative) testing for G6PD deficiency to exclude patients with enzyme activity below 70% of normal, as qualitative testing may miss women with intermediate enzyme activity (30-70%). [2]

[1]. Malar J. 2014 Jan 3;13:2.

[2]. J Travel Med.2018 Jan 1;25(1).

[3]. J Travel Med.2018 Dec 11.

N(4)-{2,6-dimethoxy-4-methyl-5-[3-(trifluoromethyl)phenoxy]quinoline-8-yl}pentane-1,4-diamine is an aminoquinoline, a derivative of 8-aminoquinoline, with methoxy groups at positions 2 and 6, a methyl group at position 4, a m-(trifluoromethyl)phenoxy group at position 5, and the amino group at position 8 being replaced by 5-aminopentane-2-yl. It belongs to the (trifluoromethyl)benzene class of compounds, aminoquinoline class of compounds, aromatic ether class of compounds, primary amino compounds, and secondary amino compounds. Tafenoxane is an 8-aminoquinoline analog of primaquine, the only difference being the presence of a phenoxy group at position 5. In 1978, scientists at the Walter Reed Army Institute of Research discovered tafenoxane, believing it could more effectively replace primaquine for the treatment of recurrent vivax malaria. GlaxoSmithKline (GSK) and the Medicines for Malaria Venture further developed the drug, which received FDA approval on July 20, 2018. Tafenoxane is an aminoquinoline drug that can be used in combination with other antimalarial drugs to prevent relapse of Plasmodium vivax malaria, or alone to prevent all types of malaria. A low percentage of transient, asymptomatic elevations of serum enzymes may occur during tafenoxane treatment, but no clinically manifested cases of acute liver injury have been reported. Tafenoxane is an orally bioavailable 8-aminoquinoline derivative with antimalarial activity. Although its mechanism of action is not fully elucidated, after administration, tafenoxane inhibits heme polymerase in the hematogenous phase of Plasmodium. This inhibits the conversion of toxic heme to non-toxic heme, leading to the accumulation of toxic heme within the parasite. Tafenoxane is effective against all stages of Plasmodium, including the pre-hepatic stage. This prevents the development of the erythrocyte stage of Plasmodium, which is the cause of relapses in vivax malaria.
See also: Tafenoquine succinate (active ingredient).
Drug Indications
Tafenoquine is used to treat and prevent relapses of vivax malaria in patients aged 16 years and older. Tafenoquine is not indicated for the treatment of acute vivax malaria. Malaria remains endemic in many tropical countries. vivax malaria (caused by Plasmodium vivax) is less virulent and rarely causes death. However, it imposes a considerable disease burden in endemic areas and is known to exist in hepatocytes as dormant bodies, called dormants, which can remain dormant for weeks or even months. These dormant bodies can lead to persistent relapses.
FDA Label
Mechanism of Action
The mechanism of action of tafenoquine is not fully understood, but studies suggest it is more durable and potent than primaquine. The active ingredient of tafenoquine, 5,6-o-quinoquinone tafenoquine, appears to be upregulated in the gametophyte and hepatic stages of Plasmodium falciparum and undergoes redox cycles. Upon entering the cell, oxidative metabolites produce hydrogen peroxide and hydroxyl radicals. The generation of these free radicals is thought to lead to parasite death. On the other hand, tafenoxane inhibits the activity of heme polymerase during the hematophyte phase of the parasite, explaining its activity against hematophyte parasites.
Pharmacodynamics
In vitro studies showed that tafenoxane had a mean half-maximal inhibitory concentration (IC50) of 0.436 μg against the hematophyte phase of seven Plasmodium species. In chloroquine-resistant Plasmodium strains, tafenoxane had a higher IC50 value than primaquine, ranging from 0.5 to 33.1 μg. In studies evaluating the activity of tafenoxane in blocking transmission during the spore reproductive phase of Plasmodium vivax, results showed that transmission rates decreased at doses above 25 mg/kg. Clinical trials reported that, with chloroquine pretreatment, tafenoxane achieved a 91.9% relapse prevention rate for Plasmodium vivax. In prophylactic studies, tafenoxane showed an efficacy rate of 84% to 87% against falciparum malaria and 99.1% against vivax malaria. Tafenoxane is an 8-aminoquinoline (8AQ) class antimalarial compound currently in late-stage development as a prophylactic agent. It is active against relapsed malaria (anti-dormant activity). This study provides direct preclinical evidence that its antimalarial activity depends on CYP 2D metabolism. Due to the high polymorphism of human CYP 2D6, these findings have significant pharmacogenomical implications. Patients with poor CYP 2D6 metabolism (PM) or potentially intermediate metabolizer (IM) may experience failure when using tafenoxane for treatment or prophylaxis. The necessity of CYP 2D activation may explain some historical reports of "resistance" to 8AQ class drugs such as primaquine, which may also be due to differences in CYP 2D6 activity within the patient population. Tafenoxane showed poor efficacy at an effective dose (ED₁₀₀) in humanized/CYP 2D6 KI mice, but its activity partially recovered after doubling the dose, suggesting a difference in metabolic efficiency between mouse CYP 2D and human CYP 2D6 enzymes. This may help guide dose adjustment in patients with reduced CYP 2D6 activity.

C24H28F3N3O3

463.50

463.208

C, 62.19; H, 6.09; F, 12.30; N, 9.07; O, 10.36

106635-80-7

Tafenoquine Succinate;106635-81-8

115358

Light yellow solid powder

1.237g/cm3

565.6ºC at 760mmHg

295.9ºC

8.17E-13mmHg at 25°C

1.572

6.684

2

9

9

33

597

0

CC1=CC(OC)=NC2=C1C(OC3=CC=CC(C(F)(F)F)=C3)=C(OC)C=C2NC(CCCN)C

LBHLFPGPEGDCJG-UHFFFAOYSA-N

InChI=1S/C24H28F3N3O3/c1-14-11-20(32-4)30-22-18(29-15(2)7-6-10-28)13-19(31-3)23(21(14)22)33-17-9-5-8-16(12-17)24(25,26)27/h5,8-9,11-13,15,29H,6-7,10,28H2,1-4H3

N4-(2,6-dimethoxy-4-methyl-5-(3-(trifluoromethyl)phenoxy)quinolin-8-yl)pentane-1,4-diamine

WR-238605, WR 238605, WR238605, SB-252263-AAB; SB252263-AAB; SB 252263-AAB; Tafenoquine; Krintafel

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 : ~93 mg/mL ( ~200.65 mM ) Ethanol : ~93 mg/mL

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.)