pafuramidine and furamidine

Diamidine-containing compounds have a long history of use in the treatment of African trypanosomiasis and leishmaniasis. The discovery that diamidine prodrugs possess in vivo antimicrobial activity when administered orally has led to a renewed interest in this class of compounds for the treatment of parasitic infections. In this review, the selectivity of diamidines against trypanosomes, Leishmania and Plasmodium is rationalized through mechanism-of-action studies. An overview of the antiprotozoal activities of newer diamidines and diamidine prodrugs is also presented, along with a summary of the progress made toward the clinical development of new diamidines for use against these parasitic diseases.

Topics: Animals; Antimalarials; Antiprotozoal Agents; Benzamidines; Chagas Disease; Clinical Trials as Topic; Drug Evaluation, Preclinical; Humans; Leishmania; Leishmaniasis; Malaria, Falciparum; Pentamidine; Plasmodium falciparum; Prodrugs; Trypanocidal Agents; Trypanosoma brucei gambiense; Trypanosoma cruzi; Trypanosomiasis, African

DB-289, an oral diamidoxime prodrug of DB-75 from the University of North Carolina, Georgia State University, Auburn University and Duke University, is being developed by Immtech International as a potential treatment for Pneumocystis carinii pneumonia (PCP), tuberculosis, trypanosomiasis and malaria.

Topics: Animals; Antimalarials; Antiprotozoal Agents; Antitubercular Agents; Benzamidines; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Humans; Prodrugs; Structure-Activity Relationship; Trypanocidal Agents

We evaluated the causal prophylactic antimalarial activity of a single oral dose of pafuramidine (DB289), an experimental prodrug of active metabolite DB75, in a randomized, double-blind, placebo-controlled, outpatient study. Sixteen healthy volunteers were dosed and challenged in a single cohort. Subjects were randomly assigned to one of three treatment arms: 100 mg pafuramidine eight days before challenge, 100 mg pafuramidine the day before challenge, or placebo. Challenge was by the bites of Plasmodium falciparum-infected Anopheles gambiae. Malaria developed in 15 persons but did not develop in one person in the day -8 pafuramidine treatment arm. Plasma levels of DB75 were lower than expected, and as intended were too low to provide suppressive prophylaxis at the earliest appearance of erythrocytic parasites. We conclude that a single dose of 100 mg pafuramidine does not adequately protect non-immune individuals against P. falciparum and shows no clinically or statistically significant evidence of causal prophylactic activity.

Topics: Adult; Animals; Anopheles; Antimalarials; Area Under Curve; Benzamidines; Cohort Studies; Double-Blind Method; Drug Administration Schedule; Erythrocytes; Female; Half-Life; Humans; Insect Vectors; Malaria, Falciparum; Male; Middle Aged; Molecular Structure; Plasmodium falciparum; Young Adult

Human African trypanosomiasis (HAT), also called African sleeping sickness, is a neglected tropical parasitic disease indigenous to sub-Saharan Africa. Diamidine compounds, including pentamidine and CPD-0801, are potent anti-trypanosomal molecules. The latter is a potential drug in the development at the UNC based Consortium for Parasitic Drug Development. An orally bioavailable prodrug of CPD-0801, DB868, is metabolized primarily in the liver to the active form. A monoclonal antibody developed against a pentamidine derivative has shown significant reactivity with CPD-0801 (EC(50) 65.1 nM), but not with the prodrug (EC(50)>18,000 nM). An inhibitory enzyme-linked immunosorbent assay (IELISA) has been used to quantitatively monitor prodrug metabolism by detecting the production of the active compound over time in a sandwich culture rat hepatocyte system and in rats. These results were compared with the results of the standard LC/MS/MS assay. Spearman coefficients of 0.96 and 0.933 (in vitro and in vivo, respectively) indicate a high correlation between these two measurement methods. This novel IELISA provides a facile, inexpensive, and accurate method for drug detection that may aide in elucidating the mechanisms of action and toxicity of existing and future diamidine compounds.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Benzamidines; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Hepatocytes; Humans; Immunoglobulin G; Male; Mice; Mice, Inbred BALB C; Pentamidine; Prodrugs; Rats; Rats, Sprague-Dawley; Rats, Wistar; Tandem Mass Spectrometry; Trypanocidal Agents; Trypanosomiasis, African

African sleeping sickness is a fatal parasitic disease, and all drugs currently in use for treatment have strong liabilities. It is essential to find new, effective, and less toxic drugs, ideally with oral application, to control the disease. In this study, the aromatic diamidine DB75 (furamidine) and two aza analogs, DB820 and DB829 (CPD-0801), as well as their methoxyamidine prodrugs and amidoxime metabolites, were evaluated against African trypanosomes. The active parent diamidines showed similar in vitro profiles against different Trypanosoma brucei strains, melarsoprol- and pentamidine-resistant lines, and a P2 transporter knockout strain (AT1KO), with DB75 as the most trypanocidal molecule. In the T. b. rhodesiense strain STIB900 acute mouse model, the aza analogs DB820 and DB829 demonstrated activities superior to that of DB75. The aza prodrugs DB844 and DB868, as well as two metabolites of DB844, were orally more potent in the T. b. brucei strain GVR35 mouse central nervous system (CNS) model than DB289 (pafuramidine maleate). Unexpectedly, the parent diamidine DB829 showed high activity in the mouse CNS model by the intraperitoneal route. In conclusion, DB868 with oral and DB829 with parenteral application are potential candidates for further development of a second-stage African sleeping sickness drug.

Topics: Animals; Benzamidines; Central Nervous System; Female; Furans; Mice; Molecular Structure; Trypanocidal Agents; Trypanosoma brucei brucei; Trypanosoma brucei gambiense; Trypanosoma brucei rhodesiense; Trypanosomiasis, African

The synthesis and evaluation of 2,5-bis[4-(N-ethoxycarbonyl-N'-isopropyl)amidinophenyl]furan, 2,5-bis[4-(N-2,2,2-trichloroethoxycarbonyl-N'-isopropyl)amidinophenyl]furan and 2,5-bis[4-(N-cyclopentyl-N'-2,2,2-trichloroethoxycarbonyl)amidinophenyl]furan as prodrugs of bis-N-alkylamidines are reported. The results show that the bis-2,2,2-trichloroethyl carbamates function effectively in a rat model for Pneumocystis pneumonia.

Topics: Animals; Benzamidines; Carbamates; Cysts; Disease Models, Animal; Furans; Lung; Pneumocystis carinii; Pneumonia, Pneumocystis; Prodrugs; Rats

The choice of drugs for the treatment of sleeping sickness is extremely limited. To redress this situation, the recently synthesised diamidine, 2,5-bis(4-amidinophenyl)-furan (DB75, furamidine) and its methamidoxime prodrug, 2,5-bis(4-amidinophenyl)-furan-bis-O-methylamidoxime (DB289, pafuramidine) were, together with pentamidine, evaluated for efficacy in acute rodent models. The activity was compared in three common mouse models that mimic the first stage of human African trypanosomiasis. The mice were infected with the pleomorphic T .b. rhodesiense strains KETRI2537 and STIB900 or with the monomorphic T. b. brucei strain STIB795. Importantly, DB75 showed activity superior to that of pentamidine at comparable doses in all three mouse models. Complete cures were achieved with oral dosing of the prodrug DB289 in all three models without any overt toxicity. This shows that the prodrug strategy was successful in terms of reducing toxicity and increasing efficacy and oral bioavailability.

Topics: Administration, Oral; Animals; Antiprotozoal Agents; Benzamidines; Female; Humans; Mice; Molecular Structure; Pentamidine; Prodrugs; Trypanosoma brucei gambiense; Trypanosomiasis, African

Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine.

Topics: Animals; Antimalarials; Atovaquone; Benzamidines; Chloroquine; Drug Interactions; Parasitic Sensitivity Tests; Plasmodium falciparum

DB289 (pafuramidine maleate; 2,5-bis[4-(N-methoxyamidino)phenyl]furan monomaleate) is a prodrug of DB75 (furamidine dihydrochloride; 2,5-bis(4-guanylphenyl)furan dihydrochloride), an aromatic dication related to pentamidine that has demonstrated good efficacy against African trypanosomiasis, Pneumocystis carinii pneumonia, and malaria, but lacks adequate oral availability. The pharmacokinetics and metabolism of 14C-DB289 have been investigated in rat and monkey after oral and intravenous administration. Oral doses were well absorbed (approximately 50-70%) and effectively converted to DB75 in both species but subject to first-pass metabolism and hepatic retention, limiting its systemic bioavailability to 10 to 20%. Clearance of DB289 approximated the liver plasma flow and its large volume of distribution was consistent with extensive tissue binding. Plasma protein binding of DB289 was 97 to 99% in four animal species and humans, but that of DB75 was noticeably less and more species- and concentration-dependent. Together, prodrug and active metabolite accounted for less than 20% of the plasma radioactivity after an oral dose, but DB75 was the major radiochemical component in key organs such as brain and liver and was largely responsible for the persistence of 14C in the body. The predominant route of excretion of radioactivity was via the feces, although biliary secretion was not particularly extensive. High-performance liquid chromatography and liquid chromatography-mass spectrometry investigations showed that the formation of DB75 from the prodrug involved the sequential loss of the two N-methoxy groups, either directly or by O-demethylation followed by reduction of the resulting oxime to the amidine. It was estimated that almost half of an oral dose of DB289 to rats and about one-third of that to monkeys was metabolized to DB75.

Topics: Animals; Antifungal Agents; Antiprotozoal Agents; Benzamidines; Bile; Biotransformation; Blood Proteins; Feces; Female; Humans; Macaca fascicularis; Male; Mice; Milk; Pregnancy; Prodrugs; Rats; Rats, Sprague-Dawley; Tissue Distribution

CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminobenzotriazole and the requirement for NADPH. Apparent K(m) and V(max) values ranged from 0.6 to 2.4 microM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid omega-hydroxylation rates (r(2) = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3-18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics.

Topics: Amidines; Antibodies; Antiparasitic Agents; Arachidonic Acid; Benzamidines; Benzoflavones; Butyrophenones; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 4; Enzyme Inhibitors; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Intestines; Kinetics; Methylation; Microsomes; Oxygenases; Piperidines; Prodrugs; Recombinant Proteins; Stereoisomerism

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b5 and NAD cytochrome b5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b5 and b5 reductase and by chemical inhibitors of b5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b5/b5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b5/b5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. K(m) values were similar to those for microsomes, and V(max) values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b5 or b5 reductase alone or with cytochrome P450 reductase alone or with cytochrome b5. These results indicate that cytochrome b5 and b5 reductase play a direct role in metabolic activation of DB289 to furamidine.

Topics: Benzamidines; Cytochrome-B(5) Reductase; Cytochromes b5; Cytosol; Humans; Microsomes, Liver

Five O-alkoxyamidine analogues of the prodrug 2,5-bis[4-methoxyamidinophenyl]furan were synthesized and evaluated against Trypanosoma brucei rhodesiense in the STIB900 mouse model by oral administration. The observed in vivo activity of these prodrugs demonstrates that compounds with an O-methoxyamidine or O-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. Permeability across Caco-2 cell monolayers and microsomal metabolism were used to identify the underlying mechanisms of prodrug efficacy.

Topics: Administration, Oral; Animals; Benzamidines; Biological Transport; Caco-2 Cells; Disease Models, Animal; Humans; In Vitro Techniques; Mice; Microsomes, Liver; Permeability; Prodrugs; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma brucei rhodesiense; Trypanosomiasis, African

The current epidemic of sleeping sickness, also known as human African trypanosomiasis in sub-Saharan Africa places nearly 60 million people at risk for developing this life threatening infection. Although effective treatments for early-stage sleeping sickness exist, these drugs usually require extended dosing schedules and intravenous administration. New treatments are also needed for cerebral (late) stage trypanosomiasis. 2,5-Bis(4-amidinophenyl)furan (DB75), a pentamidine analog, has potent in vitro and in vivo anti-trypanosomal activity. However, DB75 does not exhibit significant oral bioavailability and has proved to be ineffective against mouse models of late-stage sleeping sickness regardless of administration route. To circumvent the limited oral bioavailability of DB75, an N-methoxy prodrug 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) was designed and developed initially as a compound to treat AIDS-related Pneumocystis carinii pneumonia (PCP). Despite excellent oral activity against early-stage sleeping sickness, oral administration of DB289 exhibited limited efficacy in mouse models of late-stage disease. DB289 has recently entered Phase II(b) clinical trials to treat primary-stage sleeping sickness in Central Africa. The current study takes advantage of the innate fluorescence of DB75 and DB289 along with specific and sensitive quantitative analyses to examine plasma and brain distribution of these compounds. Animals were dosed with intravenous DB75, oral DB289, and intravenous DB289. Following intravenous administration, DB75 was readily detectable in whole brain extracts and persisted for long periods. Fluorescence microscopy revealed that DB75 did not penetrate into brain parenchyma, however, but was sequestered within cells lining the blood-brain and blood-cerebrospinal fluid barriers. In contrast, brain tissue of mice treated with oral DB289 exhibited diffuse fluorescence within the brain parenchyma, suggesting that the prodrug was not trapped within blood-brain barrier cells (BBB). However, maximal brain concentrations of the active compound DB75 were very low (13 nmol/mg of tissue at 24 h). Intravenous administration of DB289 resulted in a qualitatively similar fluorescence pattern to oral DB289, indicating again that DB289 and DB75 were present within brain parenchyma, not only in barrier regions. Furthermore, peak DB75 tissue levels were higher (61 nmol/mg of tissue at 24 h) than with oral prodrug. The near five-fold inc

Topics: Administration, Oral; Animals; Benzamidines; Blood-Brain Barrier; Brain; Injections, Intravenous; Male; Mice; Microscopy, Fluorescence; Prodrugs; Tissue Distribution; Trypanocidal Agents; Trypanosoma brucei gambiense; Trypanosomiasis, African

6-[5-(4-Amidinophenyl)furan-2-yl]nicotinamidine (8a) was synthesized from 6-[5-(4-cyanophenyl)furan-2-yl]nicotinonitrile (4a), through the bis-O-acetoxyamidoxime followed by hydrogenation. Compound 4a was prepared via selective bromination of 6-(furan-2-yl)nicotinonitrile (2a) with N-bromosuccinimide, followed by Suzuki coupling with 4-cyanophenylboronic acid. In a similar way, diamidines 8b and 8c were prepared from the dicyano derivatives 4c and 4d, respectively. N-Methoxy-6-[5-[4-(N-methoxyamidino)phenyl]-furan-2-yl]-nicotinamidine (6a) was prepared via methylation of the respective diamidoxime 5a with dimethylsulfate. Prodrugs 6b and 6c were also prepared by methylation of the respective diamidoximes 5b and 5d. The symmetrical diamidines 14a,b were synthesized through the corresponding bis-O-acetoxyamidoxime followed by hydrogenation. The key compounds 11a,b were conveniently obtained by Stille coupling between 2,5-bis(tri-n-butylstannyl)furan and the corresponding heteroaryl halides. These compounds have been evaluated in vitro for activity against Trypanosoma b.rhodesiense (T. b. r.) and P. falciparum (P. f.). The diamidines 8a, 8c, and 14b gave IC(50) values versus T. b. r. of less than 10 nM. Against P. f. 8a, 8b, and 14b exhibited IC(50) values less than 10 nM. In an in vivo mouse model for T. b. r. four compounds 6a, 6c, 6d, and 8a were curative. Compound 6a produced cures at an oral dosage of 5 mg/kg.

Topics: Animals; Antimalarials; Antiprotozoal Agents; Aza Compounds; Benzamidines; Mice; Plasmodium falciparum; Prodrugs; Structure-Activity Relationship; Trypanosoma brucei rhodesiense; Trypanosomiasis, African

Bovine viral diarrhea virus (BVDV) is an economically significant pathogen of cattle and a problematic contaminant in the laboratory. BVDV is often used as an in vitro model for hepatitis C virus during drug discovery efforts. Aromatic dicationic molecules have exhibited inhibitory activity against several RNA viruses. Thus, the purpose of this research was to develop and apply a method for screening the aromatic cationic compounds for in vitro cytotoxicity and activity against a noncytopathic strain of BVDV. The screening method evaluated the concentration of BVDV in medium and cell lysates after 72 h of cell culture in the presence of either a 25 or 5 microM concentration of the test compound. Five of 93 screened compounds were selected for further determination of inhibitory (90 and 50%) and cytotoxic (50 and 10%) concentration endpoints. The screening method identified compounds that exhibited inhibition of BVDV at nanomolar concentrations while exhibiting no cytotoxicity at 25 microM concentrations. The leading compounds require further investigation to determine their mechanism of action, in vivo activity, and specific activity against hepatitis C virus.

Topics: Animals; Antiviral Agents; Benzamidines; Cations; Cattle; Diarrhea Viruses, Bovine Viral; Furans; Guanidines; Imidazoles; RNA, Viral; Structure-Activity Relationship

DB75 [2.5-bis(4-amidinophenyl)furan] is a promising antimicrobial agent although it has poor oral potency. In contrast, its novel prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289). has excellent oral potency. The mechanisms of transport of DB289 and DB75 across intestinal epithelium have been investigated in these studies to understand differences in their oral potency.. Caco-2 cell monolayers were used as an in vitro model to examine the mechanisms of transport of DB289 and DB75. Samples collected from the transport studies were quantified using high-performance liquid chromatography with ultraviolet and fluorescence detection.. A low permeability coefficient (3.8 x 10(-7) cm/s for transport in apical [AP] to basolateral [BL] direction) and high sensitivity to extracellular Ca2+ suggest that AP to BL transport of DB75 across Caco-2 cell monolayers occurs predominantly via a paracellular route. DB289 has an 85-fold higher transport rate (322.0 x 10(-7) cm/s for transport in the AP to BL direction) across Caco-2 monolayers than that of DB75. This, with its insensitivity to extracellular Ca2+ indicates that AP to BL transport of DB289 across Caco-2 cell monolayers occurs predominantly via a transcellular route.. DB75 is transported across Caco-2 cell monolayers predominantly via paracellular pathways, whereas the prodrug DB289 is transported via transcellular pathways. This could account for the much higher oral activity of DB289 over DB75.

Topics: Anti-Infective Agents; Benzamidines; Caco-2 Cells; Humans; Permeability; Prodrugs

A novel prodrug [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289)] of the promising antimicrobial agent, 2,5-bis(4-amidinophenyl)furan (DB75), has excellent oral activity. It is currently undergoing phase II clinical evaluation as an orally administered drug candidate against African trypanosomiasis and Pneumocystis carinii pneumonia. The sequential product ion (MS(n)) fragmentations of DB289 and selected metabolites were characterized using ion trap mass spectrometry with electrospray ionization. An unusual homolytic bond cleavage, formation of an odd-electron ion from an even-electron ion with the loss of a radical, was commonly seen in the fragmentation patterns of DB289 and its metabolites. Both O-ethyl and N-methyl homologues of DB289 were utilized to confirm this fragmentation pathway. The labile hydrogen atoms in DB289 are readily exchanged with deuterium atoms in the solvent containing deuterium oxide (D2O) instead of water. The mass shift patterns displayed in the product ion spectra of DB289 in D2O proved useful in verifying the fragmentation pathway. Octadeuterated DB289 and DB75 (d-labeling on the diphenyl rings) showed unequivocally that the diphenylfuran moiety is not involved in the fragmentation. The fragmentation pathways uncovered in this work will facilitate structural characterization of all the metabolites produced in the metabolic activation of DB289.

Topics: Benzamidines; Molecular Structure; Prodrugs; Spectrometry, Mass, Electrospray Ionization; Trypanocidal Agents