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1.
J Pharm Sci ; 103(1): 337-49, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24186380

RESUMO

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Benzamidinas/metabolismo , Biotransformação/fisiologia , Citocromo P-450 CYP1A1/metabolismo , Furanos/metabolismo , Pró-Fármacos/metabolismo , Animais , Citocromo P-450 CYP1B1 , Haplorrinos/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Microssomos Hepáticos
2.
PLoS Negl Trop Dis ; 7(6): e2230, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23755309

RESUMO

There are no oral drugs for human African trypanosomiasis (HAT, sleeping sickness). A successful oral drug would have the potential to reduce or eliminate the need for patient hospitalization, thus reducing healthcare costs of HAT. The development of oral medications is a key objective of the Consortium for Parasitic Drug Development (CPDD). In this study, we investigated the safety, pharmacokinetics, and efficacy of a new orally administered CPDD diamidine prodrug, 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868; CPD-007-10), in the vervet monkey model of first stage HAT. DB868 was well tolerated at a dose up to 30 mg/kg/day for 10 days, a cumulative dose of 300 mg/kg. Mean plasma levels of biomarkers indicative of liver injury (alanine aminotransferase, aspartate aminotransferase) were not significantly altered by drug administration. In addition, no kidney-mediated alterations in creatinine and urea concentrations were detected. Pharmacokinetic analysis of plasma confirmed that DB868 was orally available and was converted to the active compound DB829 in both uninfected and infected monkeys. Treatment of infected monkeys with DB868 began 7 days post-infection. In the infected monkeys, DB829 attained a median C(max) (dosing regimen) that was 12-fold (3 mg/kg/day for 7 days), 15-fold (10 mg/kg/day for 7 days), and 31-fold (20 mg/kg/day for 5 days) greater than the IC50 (14 nmol/L) against T. b. rhodesiense STIB900. DB868 cured all infected monkeys, even at the lowest dose tested. In conclusion, oral DB868 cured monkeys with first stage HAT at a cumulative dose 14-fold lower than the maximum tolerated dose and should be considered a lead preclinical candidate in efforts to develop a safe, short course (5-7 days), oral regimen for first stage HAT.


Assuntos
Amidinas/farmacologia , Amidinas/farmacocinética , Antiprotozoários/administração & dosagem , Tripanossomíase Africana/tratamento farmacológico , Administração Oral , Amidinas/efeitos adversos , Animais , Antiprotozoários/efeitos adversos , Antiprotozoários/farmacocinética , Antiprotozoários/farmacologia , Chlorocebus aethiops , Modelos Animais de Doenças , Masculino , Resultado do Tratamento
3.
Antimicrob Agents Chemother ; 54(6): 2507-16, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20368397

RESUMO

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC(50)], <1 microM) against both Leishmania donovani axenic amastigotes and intracellular Leishmania, the causative agent for human visceral leishmaniasis (VL). A systematic lead discovery program was employed to characterize in vitro and in vivo antileishmanial activities, pharmacokinetics, mutagenicities, and toxicities of two novel AIAs, DB745 and DB766. They were exceptionally active (IC(50) < or = 0.12 microM) against intracellular L. donovani, Leishmania amazonensis, and Leishmania major and did not exhibit mutagenicity in an Ames screen. DB745 and DB766, given orally, produced a dose-dependent inhibition of liver parasitemia in two efficacy models, L. donovani-infected mice and hamsters. Most notably, DB766 (100 mg/kg of body weight/day for 5 days) reduced liver parasitemia in mice and hamsters by 71% and 89%, respectively. Marked reduction of parasitemia in the spleen (79%) and bone marrow (92%) of hamsters was also observed. Furthermore, these compounds distributed to target tissues (liver and spleen) and had a moderate oral bioavailability (up to 25%), a large volume of distribution, and an elimination half-life ranging from 1 to 2 days in mice. In a repeat-dose toxicity study of mice, there was no indication of liver or kidney toxicity for DB766 from serum chemistries, although mild hepatic cell eosinophilia, hypertrophy, and fatty changes were noted. These results demonstrated that arylimidamides are a promising class of molecules that possess good antileishmanial activity and desirable pharmacokinetics and should be considered for further preclinical development as an oral treatment for VL.


Assuntos
Amidinas/farmacologia , Antiprotozoários/farmacologia , Furanos/farmacologia , Leishmaniose Visceral/tratamento farmacológico , Amidinas/farmacocinética , Amidinas/toxicidade , Animais , Antiprotozoários/farmacocinética , Antiprotozoários/toxicidade , Disponibilidade Biológica , Cricetinae , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Furanos/farmacocinética , Furanos/toxicidade , Humanos , Técnicas In Vitro , Leishmania donovani/efeitos dos fármacos , Leishmania major/efeitos dos fármacos , Leishmania mexicana/efeitos dos fármacos , Leishmaniose Visceral/metabolismo , Leishmaniose Visceral/parasitologia , Fígado/parasitologia , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Testes de Mutagenicidade , Parasitemia/tratamento farmacológico , Testes de Sensibilidade Parasitária , Baço/parasitologia , Distribuição Tecidual
4.
Antimicrob Agents Chemother ; 53(10): 4185-92, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19620327

RESUMO

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.


Assuntos
Benzamidinas/uso terapêutico , Tripanossomicidas/uso terapêutico , Tripanossomíase Africana/tratamento farmacológico , Animais , Benzamidinas/administração & dosagem , Benzamidinas/química , Benzamidinas/farmacologia , Sistema Nervoso Central/parasitologia , Feminino , Furanos/administração & dosagem , Furanos/química , Furanos/farmacologia , Furanos/uso terapêutico , Camundongos , Estrutura Molecular , Tripanossomicidas/administração & dosagem , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/patogenicidade , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/patogenicidade , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Trypanosoma brucei rhodesiense/patogenicidade , Tripanossomíase Africana/parasitologia
5.
Proteomics ; 8(20): 4186-96, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18792928

RESUMO

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.


Assuntos
Sistema Enzimático do Citocromo P-450/análise , Espectrometria de Massas/métodos , Microssomos Hepáticos/química , Proteômica/métodos , Adolescente , Adulto , Idoso , Criança , Citocromo P-450 CYP3A , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Homologia de Sequência de Aminoácidos , Incerteza
6.
Biochimie ; 90(7): 999-1014, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18343228

RESUMO

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.


Assuntos
Antiparasitários/química , DNA de Protozoário/química , Sistemas de Liberação de Medicamentos , Eucariotos/efeitos dos fármacos , Pentamidina/química , Animais , Antiparasitários/farmacologia , DNA de Protozoário/efeitos dos fármacos , Compostos Heterocíclicos/química , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Pentamidina/análogos & derivados , Pentamidina/farmacologia
7.
J Med Chem ; 50(23): 5807-23, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17948982

RESUMO

Forty three cationic bisbenzofurans were synthesized either by interaction of o-hydroxyaldehydes with alpha-halogenated ketones followed by intramolecular ring closure or by a copper- or palladium-mediated heteroannulation of substituted o-iodophenols with terminal acetylenes. In vitro antiprotozoal activities of compounds 1-43 against Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Leishmania donovani and cytotoxicity against mammalian cells were influenced by the position and the type of cationic substituents as well as the length of the carbon linker between aromatic moieties. One bisamidine displayed an antitrypanosomal efficacy comparable to that of pentamidine and melarsoprol. Twenty two compounds were more potent than pentamidine and seven dications were more effective than artemisinin against P. falciparum. Eight bisbenzofurans displayed activity against L. donovani superior to that of pentamidine. Overall, bisamidines connected by two-carbon linkers exhibited the highest efficacies against T. b. rhodesiense, P. falciparum, and L. donovani.


Assuntos
Antimaláricos/síntese química , Benzofuranos/síntese química , Tripanossomicidas/síntese química , Animais , Antimaláricos/farmacologia , Antimaláricos/toxicidade , Benzofuranos/farmacologia , Benzofuranos/toxicidade , Cátions , Leishmania donovani/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade , Tripanossomicidas/farmacologia , Tripanossomicidas/toxicidade , Trypanosoma brucei rhodesiense/efeitos dos fármacos
8.
Drug Metab Dispos ; 35(11): 2067-75, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17709372

RESUMO

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.


Assuntos
Benzamidinas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Intestinos/enzimologia , Microssomos/enzimologia , Pró-Fármacos/metabolismo , Amidinas/farmacologia , Anticorpos/farmacologia , Antiparasitários/química , Antiparasitários/metabolismo , Antiparasitários/farmacocinética , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Benzamidinas/química , Benzamidinas/farmacocinética , Benzoflavonas/farmacologia , Butirofenonas/farmacologia , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2J2 , Citocromo P-450 CYP3A , Citocromo P-450 CYP4A , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/imunologia , Família 4 do Citocromo P450 , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Mucosa Intestinal/metabolismo , Cinética , Metilação/efeitos dos fármacos , Microssomos/metabolismo , Oxigenases/antagonistas & inibidores , Oxigenases/imunologia , Oxigenases/metabolismo , Piperidinas/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Proteínas Recombinantes/metabolismo , Estereoisomerismo
9.
Antimicrob Agents Chemother ; 51(8): 2801-10, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17517831

RESUMO

Human African trypanosomiasis is a devastating disease with only a few treatment options, including pentamidine. Diamidine compounds such as pentamidine, DB75, and DB820 are potent antitrypanosomal compounds. Previous investigations have shown that diamidines accumulate to high concentrations in trypanosomes. However, the mechanism of action of this class of compounds remains unknown. A long-hypothesized mechanism of action has been binding to DNA and interference with DNA-associated enzymes. The fluorescent diamidines, DB75 and DB820, have been shown to localize not only in the DNA-containing nucleus and kinetoplast of trypanosomes but also to the acidocalcisomes. Here we investigate two series of analogs of DB75 and DB820 with various levels of in vitro antitrypanosomal activity to determine whether any correlation exists between trypanosome accumulation, distribution, and in vitro activity. Despite wide ranges of in vitro antitrypanosomal activity, all of the compounds investigated accumulated to millimolar concentrations in trypanosomes over a period of 8 h. Interestingly, some of the less potent compounds accumulated to concentrations much higher than those of more potent compounds. All of the compounds were localized to the DNA-containing nucleus and/or kinetoplast, and many were also found in the acidocalcisomes. Accumulation in the nucleus and kinetoplast should be important to the mechanism of action of these compounds. The acidocalcisomes may also play a role in the mechanism of action of these compounds. This investigation suggests that the extent of accumulation alone is not responsible for killing trypanosomes and that organelle-specific accumulation may not predict in vitro activity.


Assuntos
Benzamidinas , Furanos , Tripanossomicidas , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Animais , Benzamidinas/química , Benzamidinas/metabolismo , Benzamidinas/farmacocinética , Benzamidinas/farmacologia , DNA de Protozoário/metabolismo , Relação Dose-Resposta a Droga , Furanos/química , Furanos/metabolismo , Furanos/farmacocinética , Furanos/farmacologia , Concentração Inibidora 50 , Organelas/metabolismo , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacocinética , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei rhodesiense/metabolismo
10.
J Med Chem ; 50(10): 2468-85, 2007 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-17439202

RESUMO

3,5-bis(4-amidinophenyl)isoxazole (3)-an analogue of 2,5-bis(4-amidinophenyl)furan (furamidine) in which the central furan ring is replaced by isoxazole-and 42 novel analogues were prepared by two general synthetic pathways. The 43 isoxazole derivatives were assayed against Trypanosoma brucei rhodesiense (T. brucei rhodesiense) STIB900, Plasmodium falciparum (P. falciparum) K1, and rat myoblast L6 cells (for cytotoxicity) in vitro. Eleven compounds (3, 13, 16-18, 22, 26, 29, 31, 37, and 41) exhibited antitrypanosomal IC50 values less than 10 nM, five of which displayed cytotoxic indices (ratios of cytotoxic IC50 to antiprotozoal IC50 values) at least 10 times higher than that of furamidine. Eighteen compounds (4-8, 12, 14, 18-22, 25, 26, 28, 29, 32, and 43) were more active against P. falciparum than furamidine, with IC50 values less than 15 nM. Fourteen of these compounds had cytotoxic indices ranging between 10 and 120 times higher than that of furamidine, and five analogues exhibited high selectivity for P. falciparum over T. brucei rhodesiense.


Assuntos
Antimaláricos/síntese química , Isoxazóis/síntese química , Tripanossomicidas/síntese química , Animais , Antimaláricos/química , Antimaláricos/farmacologia , Benzamidinas/farmacologia , Cátions , Linhagem Celular , Técnicas de Química Combinatória , Isoxazóis/química , Isoxazóis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Ratos , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei rhodesiense/efeitos dos fármacos
11.
Drug Metab Dispos ; 34(12): 1985-94, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16997912

RESUMO

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 microM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.


Assuntos
Antiparasitários/metabolismo , Benzamidinas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Pró-Fármacos/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/metabolismo , Humanos , Técnicas In Vitro , Microssomos Hepáticos/metabolismo
12.
Antimicrob Agents Chemother ; 50(6): 2185-91, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16723581

RESUMO

The aromatic diamidine pentamidine has long been used to treat early-stage human African trypanosomiasis (HAT). Two analogs of pentamidine, DB75 and DB820, have been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis. The diphenyl furan diamidine, DB75, is the active metabolite of the prodrug DB289, which is currently in phase III clinical trials as a new orally active candidate drug to treat first-stage HAT. The new aza analog, DB820, is the active diamidine of the prodrug DB844, currently undergoing preclinical evaluation as a new candidate to treat HAT of the central nervous system. The exact mechanisms of antitrypanosomal activity of aromatic dications remain poorly understood, with multiple mechanisms hypothesized. Pentamidine is known to be actively transported into trypanosomes and binds to DNA within the nucleus and kinetoplast. A long-hypothesized mechanism of action has been that DNA binding ultimately leads to interference with DNA-associated enzymes. Both DB75 and DB820 are intensely fluorescent, which provides an important tool for determining the kinetics of accumulation and intracellular distribution in trypanosomes. We show in the current study that DB75 and DB820 rapidly accumulate and strongly concentrate within trypanosomes, with intracellular concentrations over 15,000-fold higher than mouse plasma concentrations. Both compounds initially accumulate in the DNA-containing nucleus and kinetoplast, but at later time points, they concentrate in non-DNA-containing cytoplasmic organelles. Analyses of the kinetics of uptake and intracellular distribution are necessary to begin to define antitrypanosomal mechanisms of action of DB75, DB820, and other aromatic diamidines.


Assuntos
Benzamidinas/farmacocinética , Furanos/farmacocinética , Pentamidina/análogos & derivados , Pentamidina/farmacocinética , Tripanossomicidas/farmacocinética , Trypanosoma brucei gambiense/metabolismo , Animais , Benzamidinas/sangue , Benzamidinas/farmacologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Fluorescência , Furanos/sangue , Furanos/farmacologia , Técnicas In Vitro , Cinética , Masculino , Camundongos , Estrutura Molecular , Organelas/metabolismo , Pentamidina/farmacologia , Distribuição Tecidual , Tripanossomicidas/sangue , Tripanossomicidas/farmacologia , Trypanosoma brucei gambiense/citologia , Trypanosoma brucei gambiense/efeitos dos fármacos
13.
Drug Metab Dispos ; 33(12): 1886-93, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16131524

RESUMO

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.


Assuntos
Benzamidinas/metabolismo , Citocromo-B(5) Redutase/fisiologia , Citocromos b5/fisiologia , Microssomos Hepáticos/metabolismo , Citosol/metabolismo , Humanos
14.
Curr Med Chem Anticancer Agents ; 5(4): 389-408, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16101490

RESUMO

Fluorescence microscopy of trypanosomes from drug treated mice shows that biologically active heterocyclic diamidines that target the DNA minor groove bind rapidly and specifically to parasite kinetoplast DNA (k-DNA). The observation that the kinetoplast is destroyed, generally within 24 hours, after drug treatment is very important for understanding the biological mechanism, and suggests that the diamidines may be inhibiting some critical opening/closing step of circular k-DNA. Given the uncertainties in the biological mechanism, we have taken an empirical approach to generating a variety of synthetic compounds and DNA minor groove interactions for development of improved and new biological activities. Furamidine, DB75, is a diphenyl-diamidine that has the curvature to match the DNA minor groove as expected in the classical groove interaction model. Surprisingly, a linear diamidine with a nitrogen rich linker has significantly stronger binding than furamidine due to favorable linker and water-mediated DNA interactions. The water interaction is very dependant on compound structure since other linear compounds do not have similar interactions. Change of one phenyl of furamidine to a benzimidazole does not significantly enhance DNA binding but additional conversion of the furan to a thiophene (DB818) yields a compound with ten times stronger binding. Structural analysis shows that DB818 has a very favorable curvature for optimizing minor groove interactions. It is clear that there are many ways for compounds to bind to k-DNA and exert specific effects on kinetoplast replication and/or transcription that are required to obtain an active compound.


Assuntos
DNA/química , DNA/metabolismo , Desenho de Fármacos , Preparações Farmacêuticas/metabolismo , Animais , Antiparasitários/síntese química , Antiparasitários/química , Antiparasitários/farmacologia , DNA/genética , Doença , Humanos , Preparações Farmacêuticas/síntese química , Preparações Farmacêuticas/química
15.
J Med Chem ; 47(17): 4335-8, 2004 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-15294005

RESUMO

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.


Assuntos
Benzamidinas/síntese química , Pró-Fármacos/síntese química , Tripanossomicidas/síntese química , Trypanosoma brucei rhodesiense , Tripanossomíase Africana/tratamento farmacológico , Administração Oral , Animais , Benzamidinas/metabolismo , Benzamidinas/farmacocinética , Transporte Biológico , Células CACO-2 , Modelos Animais de Doenças , Humanos , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Permeabilidade , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Relação Estrutura-Atividade , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacocinética
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