DNA (active metabolite DB75 binds strongly to the minor groove of DNA at AT-rich sites and weakly by intercalation at GC sites; interferes with pathogen’s DNA-dependent enzymes, possibly topoisomerase II). [1]

Caco-2 human colon carcinoma cell monolayer permeability studies: Furamidine (DB75) is hydrophilic with two positive charges and is inefficiently transported across the monolayer via a paracellular route. In contrast, pafuramidine (with O-methylamidoxime moieties masking the positive charges) is lipophilic and efficiently transported transcellularly at an 85-fold greater rate than furamidine. Efflux pumps do not appear to be involved in pafuramidine transport, whereas furamidine transport may involve an efflux pump that does not attenuate apical-to-basolateral transport. [3]
- Antimalarial activity of DB75 (active metabolite of pafuramidine) against Plasmodium falciparum 3D7: IC₅₀ = 128 ± 51 nM at 42 h; 139 ± 43 nM at 66 h; 3.7 ± 0.76 nM at 96 h (using synchronized ring-stage cultures). [4]
- Combination interactions of DB75 with 11 antimalarial drugs (amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, tetracycline) were assessed by checkerboard microdilution growth assays. All combinations showed indifferent interactions (sum of fractional inhibitory concentrations [ΣFIC] ranging from 0.88 to 1.31), with no antagonism or synergy. Positive control (atovaquone + proguanil) showed synergy (ΣFIC = 0.63). For clindamycin, which exhibited a biphasic dose response at 42 h and 66 h, an indifferent interaction was observed at 96 h (ΣFIC = 1.09 ± 0.16). [4]

For five or ten days, trypanosome-infected monkeys can be cured by administering pafuradine (1–10 mg/kg) orally [1]. In some Trypanosoma brucei-infected mice, furamidine (2.5–100 mg/kg; oral; for 4–5 days) can be curative [5].

---

In vervet monkeys (Chlorocebus aethiops) infected intravenously with 10⁴ Trypanosoma brucei rhodesiense KETRI 2537, oral pafuramidine at 10 mg/kg once daily for 5 consecutive days (starting day 7 post-infection, early stage) completely cured all 3 monkeys. Lower doses: 3 mg/kg for 5 days cured 1 of 3 monkeys; 1 mg/kg for 5 days cured 0 of 3 monkeys. For late-stage infections (treatment starting day 14 or day 28 post-infection, 10 mg/kg for 10 days), cure rates were 1 of 3 (day 14 start) and 0 of 3 (day 28 start), indicating limited ability to cross the blood-brain barrier. [1]
- In a Phase 3 randomized, open-label clinical trial (n=273) in patients with first stage T.b. gambiense sleeping sickness, oral pafuramidine (100 mg twice daily for 10 days) achieved a combined cure/probable cure rate of 89% (118/133) at 12 months post-treatment, compared to 95% (123/129) for intramuscular pentamidine (4 mg/kg once daily for 7 days). Pafuramidine was non-inferior to pentamidine (upper bound of 95% CI did not exceed 15%). At 24 months, cure rates were 84% (113/135) for pafuramidine and 89% (116/130) for pentamidine. [2]

Caco-2 cell monolayer permeability assay: Cells were cultured as a model of human intestinal epithelial barrier. Transport of pafuramidine and furamidine across the monolayer was measured to determine the mechanism of enhanced oral bioavailability. Pafuramidine (lipophilic, with masked charges) showed transcellular transport at an 85-fold higher rate than furamidine (hydrophilic, paracellular). Efflux pump involvement was assessed but not found for pafuramidine. [3]
- Yeast (Saccharomyces cerevisiae) studies: DB75 was shown to localize to mitochondria, collapse mitochondrial membrane potential, and inhibit mitochondrial respiration, likely via electron transfer inhibition. DB75 was a more potent respiratory chain inhibitor than pentamidine. [3]
- Antimalarial drug sensitivity assay (microdilution checkerboard method): P. falciparum 3D7 parasites were cultured in 96-well plates. For 42-h assays, parasites were exposed to serial dilutions of DB75 and partner drugs; growth was measured by [³H]hypoxanthine incorporation (added for the final 18-24 h). For 66-h assays, a 0.4% parasite population was used with [³H]hypoxanthine added for the final 18 h. For 96-h assays, synchronized ring-stage cultures (sorbitol-synchronized) were used with [³H]hypoxanthine added for the final 48 h. Fractional inhibitory concentrations (FIC) were calculated, and the sum of FIC (ΣFIC) determined. Synergy defined as ΣFIC ≤ 0.63, antagonism as ΣFIC > 2.0, indifference as ΣFIC ≈ 1. [4]

Animal/Disease Models: vervet monkey (intravenous (iv) (iv)104 trypanosome infection) [1]
Doses: 1, 3, 10 mg/kg (group 1/2/3); 10 mg/kg (group 4/5)
Route of Administration: Oral administration; Group 1/2/3 for 5 days (starting on the 7th day after infection); Group 4 for 10 days (starting on the 14th day after infection); Group 5 for 10 days (starting on the 28th day after infection) ). Post-treatment monitoring continues for 180 days.
Experimental Results: All 3 monkeys in the 10 mg/kg group were cured and there was no recurrence during the monitoring period. All three monkeys in Group 4 were parasitic on day 5 of dosing, but only two of the three monkeys were free of blood parasites when monitored 180 days after treatment. All three monkeys in Group 5 were parasitic on day 4 of dosing, but only two of the three monkeys remained free of blood trypanosomatids at the end of 180 days of post-treatment monitoring.

---

Animal/Disease Models: Female NMRI mice (intraperitoneally (ip) (ip) infected with 2 × 104 STIB900 bloodstream form) [5]
Doses: 2.5, 5, 25 and 50 mg/kg
Route of Administration: p.o.; for 4 days (started on the 4th day postinfection)
Experimental Results: Cured all four mice at 25 and 50 mg/kg.

---

Animal/Disease Models: Female NMRI mice (infected intraperitoneally with 2 × 104 GVR35 bloodstream forms)[5]
Doses: 25, 50 and 100 mg/kg
Route of Administration: p.o.; for 5 days (started on the 21st day postinfection)
Experimental Results: Showed good CNS activity in the GVR35 CNS model, with 3/5 mice cured at 100 mg/kg.

---

Vervet monkey model of T.b. rhodesiense infection: Monkeys (both sexes, 2.5-4.5 kg) were infected intravenously with 10⁴ trypanosomes. Pafuramidine was prepared as a 1% suspension in distilled water containing 0.5% (w/v) carboxymethylcellulose sodium and 0.1% (w/v) Tween 80, stored at 4°C and used within 3 days. The suspension was administered orally via gavage needle once daily for 5 or 10 consecutive days at doses of 1, 3, or 10 mg/kg body weight. Treatment started on day 7 (early stage, groups 1-3), day 14 (group 4), or day 28 (group 5) post-infection. Monitoring included weekly physical exams, blood and cerebrospinal fluid (CSF) collection for 28 days then every 2 weeks up to 100 days post-treatment, then monthly until 180 days. Parasitemia was determined by wet film, hematocrit centrifugation (HCT), and mouse inoculation test (MIT). CSF was examined for trypanosomes and white blood cells. [1]
- Phase 3 clinical trial: Patients (≥12 years, ≥30 kg, first stage T.b. gambiense) were randomized 1:1 to oral pafuramidine 100 mg twice daily for 10 days or intramuscular pentamidine 4 mg/kg once daily for 7 days. Pregnant and lactating women and adolescents (12-15 years) were included. Follow-up examinations (blood, lymph node aspirate, CSF) occurred at 3, 6, 12, 18, and 24 months post-treatment. Lumbar puncture was performed at baseline, 6, 12, 18 months, and when relapse suspected. Efficacy endpoints defined per WHO criteria (cure, probable cure, probable relapse, relapse, death). [2]

Pafuramidine is an orally administered prodrug that is converted to the active compound DB75 (furanidine) in the liver. The enhanced oral activity is attributed to improved oral absorption of the prodrug where cationic functionalities are masked. Limited ability to cross the blood-brain barrier was observed in monkeys (ineffective against late-stage CNS infection). [1]
- In a Phase 1 study (cited), healthy volunteers took oral pafuramidine 100 mg twice daily for 14 days; detailed PK parameters not provided in the attached literature. The lack of proportional conversion of DB289 to DB75 at therapeutic doses precluded using a higher dose to improve efficacy. [2]

In vervet monkeys, pafuramidine at doses up to 10 mg/kg for 5-10 days was well tolerated (no specific toxicity data provided). [1]
- In the Phase 3 clinical trial (n=273), the overall incidence of adverse events during treatment (Day 1-11) was significantly lower in the pafuramidine group (82%) than in the pentamidine group (99%) (p<0.05). Common adverse events with pafuramidine: pyrexia (30.9%), hypotension (44.1%), headache (14.0%), dizziness (6.6%), hypoglycemia (5.9%). Pafuramidine had lower rates of hepatobiliary investigations (7% vs 77%), renal/urinary tract investigations (2% vs 9%), and glucose metabolism disorders (6% vs 18%) compared to pentamidine. Three patients in the pafuramidine group developed glomerulonephritis or nephropathy approximately 8 weeks post-treatment; two were considered possibly related to pafuramidine. A total of 19/136 (14.0%) pafuramidine patients experienced serious adverse events (vs 24/137 [17.5%] pentamidine). Thirteen deaths occurred during follow-up (6 pafuramidine, 7 pentamidine), all considered not related or probably not related to study drug. The clinical development of pafuramidine was discontinued due to delayed post-treatment renal toxicity observed in a supportive Phase 1 study. [2]

[1]. Efficacy of the novel diamidine compound 2,5-Bis(4-amidinophenyl)- furan-bis-O-Methlylamidoxime (Pafuramidine, DB289) against Trypanosoma brucei rhodesiense infection in vervet monkeys after oral administration. Antimicrob Agents Chemother. 2009 Mar;53(3):953-7.

[2]. Efficacy and Safety of Pafuramidine versus Pentamidine Maleate for Treatment of First Stage Sleeping Sickness in a Randomized, Comparator-Controlled, International Phase 3 Clinical Trial. PLoS Negl Trop Dis. 2016 Feb 16;10(2):e0004363.

[3]. Pafuramidine for Pneumocystis jiroveci pneumonia in HIV-infected individuals. Expert Rev Anti Infect Ther. 2007 Dec;5(6):921-8.

[4]. Interactions of DB75, a novel antimalarial agent, with other antimalarial drugs in vitro. Antimicrob Agents Chemother. 2008 Jun;52(6):2253-5.

Palfuramedin is a prodrug of furamidine and has been granted orphan drug designation for the treatment of Pneumocystis carinii pneumonia.

---

Drug Indications
Studied for the treatment of pneumonia, trypanosomiasis, malaria, HIV infection, and infectious and parasitic diseases (not specified).

---

Pafuramidine (2,5-bis(4-amidinophenyl)-furan-bis-O-methylamidoxime) is a novel orally active prodrug of the diamidine DB75 (furanidine). It was developed for the treatment of human African trypanosomiasis (sleeping sickness) to overcome the need for parenteral administration of existing drugs (pentamidine, suramin). [1][2]
- The prodrug approach masks the cationic amidine functionalities to improve oral absorption; conversion to the active DB75 occurs in the liver. DB75 strongly binds to the minor groove of DNA at AT-rich sites, interfering with DNA-dependent enzymes. [1]
- Despite good efficacy and tolerability in Phase 2 and Phase 3 studies, the development program was halted in early 2008 due to delayed renal toxicity (glomerulonephritis/nephropathy) observed approximately 8 weeks post-treatment, considered an unacceptable risk. [2]
- The Phase 3 study successfully enrolled patients in rural Africa (DRC, Angola, South Sudan) with 97% retention at 24 months, providing a model for future clinical trials in resource-limited settings. [2]

C20H20N4O3

364.3978

364.154

186953-56-0

Pafuramidine maleate;837369-26-3

5480200

Off-white to light yellow solid powder

1.25

192.5-193ºC

4.547

2

5

6

27

477

0

CO/N=C(\N)/C1=CC=C(C=C1)C2=CC=C(O2)C3=CC=C(C=C3)/C(=N/OC)/N

UKOQVLAXCBRRGH-UHFFFAOYSA-N

InChI=1S/C20H20N4O3/c1-25-23-19(21)15-7-3-13(4-8-15)17-11-12-18(27-17)14-5-9-16(10-6-14)20(22)24-26-2/h3-12H,1-2H3,(H2,21,23)(H2,22,24)

N'-methoxy-4-[5-[4-[(Z)-N'-methoxycarbamimidoyl]phenyl]furan-2-yl]benzenecarboximidamide

DB289 DB-289 DB 289

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 : ~33.33 mg/mL (~91.47 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.86 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.71 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.71 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 20.8 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.

---

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