Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors.

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

Identification of (+)-erythro-mefloquine as an active enantiomer with greater efficacy than mefloquine against Mycobacterium avium infection in mice.

Infection caused by Mycobacterium avium is common in AIDS patients who do not receive treatment with highly active antiretroviral therapy (HAART) or who develop resistance to anti-HIV therapy. Mefloquine, a racemic mixture used for malaria prophylaxis and treatment, is bactericidal against M. avium in mice. MICs of (+)-erythro-, (-)-erythro-, (+)-threo-, and (-)-threo-mefloquine were 32 µg/ml, 32 µg/ml, 64 µg/ml, and 64 µg/ml, respectively. The postantibiotic effect for (+)-erythro-mefloquine was 36 h (MIC) and 41 h for a concentration of 4× MIC. The mefloquine postantibiotic effect was 25 h (MIC and 4× MIC). After baseline infection was established (7 days), the (+)- and (-)-isomers of the diastereomeric threo- and erythro-α-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol were individually used to orally treat C57BL/6 bg(+)/bg(+) beige mice that were infected intravenously with M. avium. Mice were also treated with commercial mefloquine and diluent as controls. After 4 weeks of treatment, the mice were harvested, and the number of bacteria in spleen and liver was determined. Mice receiving (+)- or (-)-threo-mefloquine or (-)-erythro-mefloquine had numbers of bacterial load in tissues similar to those of untreated control mice at 4 weeks. Commercial mefloquine had a bactericidal effect. However, mice given the (+)-erythro-enantiomer for 4 weeks had a significantly greater reduction of bacterial load than those given mefloquine. Thus, (+)-erythro-mefloquine is the active enantiomer of mefloquine against M. avium and perhaps other mycobacteria.

Antimalarial activity of 9a-N substituted 15-membered azalides with improved in vitro and in vivo activity over azithromycin.

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

FGI-104: a broad-spectrum small molecule inhibitor of viral infection.

The treatment of viral diseases remains an intractable problem facing the medical community. Conventional antivirals focus upon selective targeting of virus-encoded targets. However, the plasticity of viral nucleic acid mutation, coupled with the large number of progeny that can emerge from a single infected cells, often conspire to render conventional antivirals ineffective as resistant variants emerge. Compounding this, new viral pathogens are increasingly recognized and it is highly improbable that conventional approaches could address emerging pathogens in a timely manner. Our laboratories have adopted an orthogonal approach to combat viral disease: Target the host to deny the pathogen the ability to cause disease. The advantages of this novel approach are many-fold, including the potential to identify host pathways that are applicable to a broad-spectrum of pathogens. The acquisition of drug resistance might also be minimized since selective pressure is not directly placed upon the viral pathogen. Herein, we utilized this strategy of host-oriented therapeutics to screen small molecules for their abilities to block infection by multiple, unrelated virus types and identified FGI-104. FGI-104 demonstrates broad-spectrum inhibition of multiple blood-borne pathogens (HCV, HBV, HIV) as well as emerging biothreats (Ebola, VEE, Cowpox, PRRSV infection). We also demonstrate that FGI-104 displays an ability to prevent lethality from Ebola in vivo. Altogether, these findings reinforce the concept of host-oriented therapeutics and present a much-needed opportunity to identify antiviral drugs that are broad-spectrum and durable in their application.

Enhancement of an analytical method for the determination of squalene in anthrax vaccine adsorbed formulations.

Specific lots of anthrax vaccine adsorbed administered to members of the U.S. Armed Forces have been alleged to contain squalene, a chemical purported to be associated with illnesses of Gulf War veterans. A method of enhanced sensitivity for determining squalene in anthrax vaccine adsorbed using high-performance liquid chromatography with photodiode array detection has been developed, validated, and applied to 44 bottles of 38 lots of anthrax vaccine. In 43 bottles of 37 lots, no squalene was detected within a detection limit of 1ng/0.5ml dose (2 parts-per-billion). One lot, FAV008, was found to contain trace amounts of squalene at 7, 9, and 1microgl(-1), levels considerably below normal human plasma levels (290microgl(-1)). The overall results of this investigation provide direct evidence for the absence of squalene in nearly all of anthrax vaccine preparations tested.

Carbon isosteres of the 4-aminopyridine substructure of chloroquine: effects on pK(a), hematin binding, inhibition of hemozoin formation, and parasite growth.

Unlike diprotic chloroquine (CQ), its two 4-aminoquinoline carbon isosteres (1, 2) are monoprotic at physiological pH. Compared to CQ, hematin binding affinity of 1 decreased 6.4-fold, and there was no measurable binding for 2. Although 1 was a weak inhibitor of hemozoin formation, neither isostere inhibited P. falciparum in vitro. Evidently, the CQ-hematin interaction is largely a function of its pyridine substructure, but inhibition of hemozoin formation and parasite growth depends on its 4-aminopyridine substructure.

Development and application of an analytical method for the determination of squalene in formulations of anthrax vaccine adsorbed.

Specific lots of Anthrax Vaccine Adsorbed, administered to members of the US Armed Forces, have been described on various Internet sites and in news articles as a source of squalene, a chemical purported by these media to be associated with the Gulf War Syndrome. We have developed and validated a method using high-performance liquid chromatography with ultraviolet detection for the determination of squalene in anthrax vaccine preparations. The method has a limit of detection of 140 parts per billion and has been successfully applied to a commercial vaccine known to contain squalene. We have applied this method to 17 lots of Anthrax Vaccine Adsorbed administered to members of the US Armed Forces. No squalene has been detected in any lot. The results of these analyses provide direct evidence for the absence of squalene as an ingredient or a manufacturing contaminant in Anthrax Vaccine Adsorbed.