The antiprotozoal compound 1,5-di(4-amidinophenoxy)pentane (pentamidine) and 36 of its analogs were screened for in vitro activity against Leishmania mexicana amazonensis clone 669 C4S (MHOM/BR/73/M2269) and Plasmodium falciparum clones W2 (Indochina HI/CDC) and D6 (Sierra Leone I/CDC). Pentamidine and each of the analogs tested exhibited activity in vitro against L. m. amazonensis and P. falciparum. The pentamidine analogs were more effective against the P. falciparum clones than against L. m. amazonensis. P. falciparum was extremely susceptible to these compounds, with 50% inhibitory concentrations as low as 0.03 FpM. While none of the analogs exhibited marked improvement in antileishmanial activity compared with pentamidine, 12 of the pentamidine analogs showed activity approximately equal to or greater than that of the parent compound. From the promising activity exhibited by the pentamidine analogs in this in vitro study and their potential for reduced toxicity relative to the parent drug, pentamidine-related compounds hold promise as new agents for the treatment of protozoal infections.The efficacy of aromatic diamidines in the treatment of protozoal diseases was first recognized in the 1930s by investigators searching for agents with therapeutic activity against African trypanosomiasis (14). Early clinical trials examining the activities of pentamidine, propamidine, and stilbamidine revealed that these and other aromatic diamidines are effective against the early stages of African trypanosomiasis (6,7,13,14) and against leishmaniasis (11,15,22). Although they are not clinically used in the treatment of malaria, the antiplasmodial activity of aromatic diamidines in monkeys infected with Plasmodium knowlesi was demonstrated during the 1940s (1, 4).Aromatic diamidines not only have antiprotozoal activity but also exhibit activity against bacteria (3), fungi (3), viruses (21), and tumors (12). In the past, their use has mainly been confined to the treatment of protozoal diseases, for which they were first developed. Pentamidine continues to be used in the treatment of the Gambian form of African trypanosomiasis and against antimony-resistant leishmaniasis (17). Pentamidine was first shown to be active against the opportunistic pathogen Pneumocystis carinii in 1958 (10), and in the United States, this compound is primarily used to treat P. carinii pneumonia in patients with the acquired immune deficiency syndrome. The toxicity and side effects associated with the use of pentamidine in the treatment of P. carinii pneumonia in acquired immune deficiency syndrome patients have led to extensive investigations to identify a derivative of pentamidine which is more active against P. carinii pneumonia and less toxic than the parent drug.To this end, over 50 analogs of pentamidine have been synthesized in our laboratory and have been examined for in vivo efficacy against P. carinii in the rat model of disease (10a, 18, 19). The design of more-potent analogs of pentamidine against P. carinii pneumonia has been hampered ...

1,5-Di(4-amidinophenoxy)pentane (pentamidine) and 38 analogs of pentamidine were screened for in vitro activity against the enteric protozoan Giardia lamblia WB (ATCC 30957). All compounds were active against G. lamblia as measured by a [methyl-3H]thymidine incorporation assay. Antigiardial activity varied widely, with 50% inhibitory concentrations (IC50s) ranging from 0.51 +/- 0.13 microM (mean +/- standard deviation) for the most active compound to over 100.0 microM for the least active compounds. The IC50 of the most potent antigiardial agent, 1,3-di(4-amidino-2-methoxyphenoxy)propane compared favorably with the IC50s of the compounds currently used to treat giardiasis, i.e., furazolidone (1.0 +/- 0.03 microM), metronidazole (2.1 +/- 0.80 microM), quinacrine HCl (0.03 +/- 0.02 microM), and tinidazole (0.78 +/- 0.48 microM). A mode of antigiardial activity for these compounds was suggested by the correlation observed between antigiardial activity and the binding of the compounds to calf thymus DNA and poly(dA).poly(dT).

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n ‫؍‬ 31) and other non-Bacillus species (n ‫؍‬ 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per l of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

Nine dicationically substituted bis-benzimidazoles were examined for their in vitro activities against Giardia lamblia WB (ATCC 30957). The potential mechanisms of action of these compounds were evaluated by investigating the relationship among in vitro antigiardial activity and the affinity of the molecules for DNA and their ability to inhibit the activity of giardial topoisomerase II. Each compound demonstrated antigiardial activity, as measured by assessing the incorporation of [methyl-3H]thymidine by giardial trophozoites exposed to the test agents. Three compounds exhibited excellent in vitro antigiardial activities, with 50%o inhibitory concentrations which compared very favorably with those of two currently used drugs, quinacrine HCI and metronidazole. Putative mechanisms of action for these compounds were suggested by the strong correlation observed among in vitro antigiardial activity and the affinity of the molecules for natural and synthetic DNA and their ability to inhibit the relaxation activity of giardial topoisomerase II. A strong correlation between the DNA binding affinity of these compounds and their inhibition of giardial topoisomerase II activity was also observed.Giardia lamblia is a common cause of endemic and epidemic diarrheal disease throughout the world. Some individuals harbor asymptomatic infections, while others may exhibit acute or chronic gastrointestinal disease. Four agents are presently used to treat giardiasis: the nitroimidazoles metronidazole and tinidazole, the nitrofuran furazolidone, and quinacrine HCl, an acridine. Many problems are associated with the currently used chemotherapeutic agents, including treatment failures, unpleasant side effects, activity against normal intestinal flora, and possible carcinogenicity. While treatment of symptomatic individuals is recommended, there is controversy as to whether asymptomatic cyst passers should be treated, especially in light of the problems associated with the antigiardial agents presently available. More-effective and less-toxic agents are therefore needed for the treatment of giardiasis.The search for new antigiardial agents has been aided by improvements in axenic culturing of the organism (13) and drug susceptibility testing (3,12). Among the classes of compounds recently examined for antigiardial activity are anthelminthic benzimidazoles (1,8,14,15) Washington, DC 20307. which are used at present to treat the infection (2). In addition, there was a strong correlation between the antigiardial activities of the pentamidine analogs and their affinity for calf thymus DNA and poly(dA) poly(dT). The ability of pentamidine to bind to DNA has been proposed as a mechanism of action for this and related compounds. Pentamidine and related molecules have also been identified as potential inhibitors of the activity of type II topoisomerases (7,17).A number of dicationically substituted bis-benzimidazoles, originally developed as protease inhibitors and DNA binding agents, were available in our laboratory for in vitro antimi...

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By using high-performance liquid chromatography, the in vitro conversion of pentamidine to the corresponding amidoximes (N-hydroxypentamidine and N,N'-dihydroxypentamidine) was studied in supernatants of rat liver homogenate centrifuged at 9,000 x g. The presence of the two amidoxime peaks in chromatograms was confirmed by liquid secondary ion mass spectrometry and by unequivocal synthesis of the suspected metabolites. The metabolic reactions were found to be catalyzed by the cytochrome P-450 system (mixedfunction oxidases). The formation of the monohydroxylated product was found to have a Km of 0.48 mM and a Vmax of 29.50 pmol/min per mg of protein, while the dihydroxylated metabolite had a Km of 0.73 mM and a Vmax of 4.10 pmol/min per mg of protein. N,N'-Dihydroxypentamidine was found to have highly reduced antiprotozoal activity in vitro relative to that of pentamidine, and neither of the hydroxylated metabolites nor pentamidine was found to be significantly mutagenic by the Ames test. Contrary to previous reports, pentamidine is readily metabolized to at least two hydroxylated products, and this conversion may be relevant to the clinical use of the compound and to future drug design.Pentamidine [1,5-di(4-amidinophenoxy)pentane] was syn-

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