The use of extracellular vesicles, specifically exosomes, as carriers of biomarkers in extracellular spaces has been well demonstrated. Despite their promising potential, the use of exosomes in the clinical setting is restricted due to the lack of standardization in exosome isolation and analysis methods. The purpose of this review is to not only introduce the different types of extracellular vesicles but also to summarize their differences and similarities, and discuss different methods of exosome isolation and analysis currently used. A thorough understanding of the isolation and analysis methods currently being used could lead to some standardization in the field of exosomal research, allowing the use of exosomes in the clinical setting to become a reality.

Human African trypanosomiasis (HAT) or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. Novel models for funding pharmaceutical development against HAT are beginning to yield results. The Drugs for Neglected Diseases initiative (DNDi) rediscovered a nitroimidazole, fexinidazole, which is currently in Phase I clinical trials. Novel benzoxaboroles, discovered by Anacor, Scynexis and DNDi, have good pharmacokinetic properties in plasma and in the brain and are curative in a murine model of stage two HAT with brain infection. The Consortium for Parasitic Drug Development (CPDD) has identified a series of dicationic compounds that can cure a monkey model of stage two HAT. With other screening programs yielding hits, the pipeline for new HAT drugs might finally begin to fill.

Arylimidamides (AIAs) represent a new class of molecules that exhibit potent antileishmanial activity (50% inhibitory concentration [IC 50 ], <1 M) 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 < 0.12 M) 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.Leishmaniasis, a neglected tropical disease, is caused by parasitic protozoa of the genus Leishmania, including 20 species that are pathogenic for humans (21). Clinical manifestations of leishmaniasis mainly consist of cutaneous, mucocutaneous, visceral, and post-kala-azar dermal leishmaniasis, with symptoms ranging from skin and mucosal ulceration to systemic infection that is fatal if left untreated (6). An estimated 12 million people are currently infected with Leishmania, and up to 350 million people in 88 countries are at risk of infection (35). Approximately 2 million new cases of leishmaniasis are believed to occur annually, with 1.5 million for cutaneous leishmaniasis and 0.5 million for visceral leishmaniasis (VL). In macrophages, Leishmania amastigotes adapt to thrive in an acidic subcellular compartment, the parasitophorous vacuole (PV; pH ϳ5) (2), where they maintain a neutral intracellular pH within the parasite by an energy-dependent process (9). Multiple layers of membrane barriers (i.e., host macrophage plasma membrane, phagolysosomal membrane, and Leishmania amastigote plasma membrane) presumably present a formidable challenge for chemotherapeutic agents to target Leishmania parasites in mammalian hosts.Current chemotherapies for leishmaniasis have many limitations, including resis...

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-demethyl-ation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b 5 /NADH-cytochrome b 5 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 NADPHdependent, with a K m and V max of 0.5 M 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 (Nhydroxy-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.As part of our search for new lead compounds for the treatment of African trypanosomiasis (African sleeping sickness) and other parasitic infections, aromatic dicationic compounds, such as DB75 [2,5-bis(4-amidinophenyl) furan], have been evaluated for efficacy in multiple models of infection. These diamidine-type compounds are effective against a broad range of pathogens in vitro, including Trypanosoma brucei, Leishmania spp., Pneumocystis carinii, and

Hepatic OATPs 1B1, 1B3 and 2B1, as well as P-gp, play important roles in regulating liver uptake and biliary excretion of drugs. The intrinsic ethnic variability in OATP1B1-mediated hepatic uptake of statins has been proposed to underlie the ethnic variability in plasma exposures of statins between Caucasians and Asians. Using a targeted quantitative proteomic approach, we determined hepatic protein concentrations of OATP1B1, OATP1B3, OATP2B1, P-gp, and PMCA4 (a housekeeping protein) in a panel of human livers (n = 141) and compared protein expression across Caucasian, Asian, African-American, and unidentified donors. Using an optimized protocol that included sodium deoxycholate as a membrane protein solubilizer, the hepatic protein expression levels (mean ± S.D.) of these transporters across all livers were determined to be 15.0 ± 6.0, 16.1 ± 8.1, 4.1 ± 1.3, 0.6 ± 0.2, and 2.4 ± 1.0 fmol/μg of total membrane protein, respectively. The scaling factor was 3.5 mg of total membrane protein in 100 mg of wet liver tissue. OATP1B1 protein expression was significantly associated with the c.388A>G (rs2306283, N130D) single nucleotide polymorphism. When compared across ethnicity, the hepatic expression levels of OATP1B1 and OATP1B3 were unexpectedly higher in Asians relative to Caucasians, suggesting that hepatic OATP expression alone does not explain the increased systemic statin levels in Asians compared with Caucasians. These findings may help improve physiologically based pharmacokinetic modeling to predict statin pharmacokinetic profiles and enable extrapolation of pharmacokinetic data of OATP substrates across ethnic groups.