Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

In Vitro and In Vivo Drug Interaction Study of Two Lead Combinations, Oxantel Pamoate plus Albendazole and Albendazole plus Mebendazole, for the Treatment of Soil-Transmitted Helminthiasis.

The current treatments against Trichuris trichiura, albendazole and mebendazole, are only poorly efficacious. Therefore, combination chemotherapy was recommended for treating soil-transmitted helminthiasis. Albendazole-mebendazole and albendazole-oxantel pamoate have shown promising results in clinical trials. However, in vitro and in vivo drug interaction studies should be performed before their simultaneous treatment can be recommended. Inhibition of human recombinant cytochromes P450 (CYPs) CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 was tested by exposure to albendazole, albendazole sulfoxide, mebendazole, and oxantel pamoate, as well as albendazole-mebendazole, albendazole sulfoxide-mebendazole, albendazole-oxantel pamoate, and albendazole sulfoxide-oxantel pamoate. A high-pressure liquid chromatography (HPLC)-UV/visible spectroscopy method was developed and validated for simultaneous quantification of albendazole sulfoxide, albendazole sulfone, mebendazole, and oxantel pamoate in plasma. Albendazole, mebendazole, oxantel pamoate, albendazole-mebendazole, and albendazole-oxantel pamoate were orally applied to rats (100 mg/kg) and pharmacokinetic parameters calculated. CYP1A2 showed a 2.6-fold increased inhibition by albendazole-oxantel pamoate (50% inhibitory concentration [IC50] = 3.1 µM) and a 3.9-fold increased inhibition by albendazole sulfoxide-mebendazole (IC50 = 3.8 µM) compared to the single drugs. In rats, mebendazole's area under the concentration-time curve (AUC) and maximal plasma concentration (Cmax) were augmented 3.5- and 2.8-fold, respectively (P = 0.02 for both) when coadministered with albendazole compared to mebendazole alone. Albendazole sulfone was slightly affected by albendazole-mebendazole, displaying a 1.3-fold-elevated AUC compared to albendazole alone. Oxantel pamoate could not be quantified, translating to a bioavailability below 0.025% in rats. Elevated plasma levels of albendazole sulfoxide, albendazole sulfone, and mebendazole in coadministrations are probably not mediated by CYP-based drug-drug interaction. Even though this study indicates that it is safe to coadminister albendazole-oxantel pamoate and albendazole-mebendazole, human pharmacokinetic studies are recommended.

Bimodal X-ray and Infrared Imaging of an Organometallic Derivative of Praziquantel in Schistosoma mansoni.

An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm.

Approved oncology drugs lack in vivo activity against Trichuris muris despite in vitro activity.

Infections with soil-transmitted helminths (STHs) are considered among the most persistent global health problems. The few available drugs have limitations including low efficacy against Trichuris trichiura infections. As a starting point toward drug repositioning, we studied a set of FDA-approved oncology drugs for activity against Trichuris muris since targets relevant to cancer therapy might have a function in helminth biology. Drugs were tested in vitro on the larval and adult stage of T. muris. Compounds active in vitro were tested in the T. muris mouse model at single oral dosages of 200-400 mg/kg. Of the 114 drugs tested in vitro, 12 showed activity against T. muris larvae (>80 % drug effect at 50 µM). Ten of these drugs were also active on the adult worm stage (>80 % drug effect at 50 µM), of which six revealed IC50 values between 1.8 and 5.0 µM. Except for tamoxifen citrate, all in vitro active drugs were protein kinase inhibitors. None of the drugs tested in vivo showed efficacy, revealing worm burden reductions of 0-24 % and worm expulsion rates of 0-7.9 %. The promising in vitro activities of protein kinases could not be confirmed in vivo. Drug discovery against STH should be strengthened including the definition of compound progression criteria. Follow-up structure-activity relationship studies with modified compounds might be considered.

Activities of N,N'-Diarylurea MMV665852 analogs against Schistosoma mansoni.

There is an unmet need to discover and develop novel antischistosomal drugs. As exemplified by MMV665852, N,N'-diarylureas have recently emerged as a promising antischistosomal chemotype. In this study, we evaluated the structure-activity relationships of 46 commercially available analogs of MMV665852 on newly transformed schistosomula (NTS) and adult Schistosoma mansoni worms in vitro. Active compounds were evaluated with a cytotoxicity assay, in silico calculations, metabolic stability studies, and an in vivo assay with mice harboring adult S. mansoni worms. Of the 46 compounds tested at 33.3 µM, 13 and 14 compounds killed NTS and adult worms, respectively, within 72 h. Nine compounds had 90% inhibitory concentrations (IC90s) of ≤10 µM against adult worms, with selectivity indexes of ≥2.8. Their physicochemical properties and permeation through an artificial membrane indicated good to moderate intestinal absorption. Their metabolic stabilities ranged from low to high. Despite satisfactory in vitro results and in silico predictions, only one compound resulted in a statistically significant worm burden reduction (66%) after administration of a single oral dose of 400 mg/kg of body weight to S. mansoni-infected mice. Worm burden reductions of 0 to 43% were observed for the remaining eight compounds tested. In conclusion, several analogs of the N,N'-diarylurea MMV665852 had high efficacy against S. mansoni in vitro and favorable physicochemical properties for permeation through the intestinal wall. To counteract the low efficacy observed in the mouse model, further investigations should focus on identifying compounds with improved solubility and pharmacokinetic properties.

Elucidation of the in vitro and in vivo activities of bridged 1,2,4-trioxolanes, bridged 1,2,4,5-tetraoxanes, tricyclic monoperoxides, silyl peroxides, and hydroxylamine derivatives against Schistosoma mansoni.

Praziquantel is currently the only drug available to treat schistosomiasis. Since drug resistance would be a major barrier for the increasing global attempts to eliminate schistosomiasis as a public health problem, efforts should go hand in hand with the discovery of novel treatment options. Synthetic peroxides might offer a good direction since their antischistosomal activity has been demonstrated in the laboratory. We studied 19 bridged 1,2,4,5-tetraoxanes, 2 tricyclic monoperoxides, 11 bridged 1,2,4-trioxolanes, 12 silyl peroxides, and 4 hydroxylamine derivatives against newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro. Schistosomicidal compounds were tested for cytotoxicity followed by in vivo studies of the most promising compounds. Tricyclic monoperoxides, trioxolanes, and tetraoxanes revealed the highest in vitro activity against NTS (IC50s 0.4-20.2 µM) and adult schistosomes (IC50s 1.8-22.8 µM). Tetraoxanes showed higher cytotoxicity than antischistosomal activity. Selected trioxolane and tricyclic monoperoxides were tested in mice harboring an adult S. mansoni infection. The highest activity was observed for two trioxolanes, which showed moderate worm burden reductions (WBR) of 44.3% and 42.9% (p>0.05). Complexation of the compounds with ß-cyclodextrin with the aim to improve solubility and gastrointestinal absorption did not increase in vivo antischistosomal efficacy. The high in vitro antischistosomal activity of trioxolanes and tricyclic monoperoxides is a promising basis for future investigations, with the focus on improving in vivo efficacy.

Activity of praziquantel enantiomers and main metabolites against Schistosoma mansoni.

A racemic mixture of R and S enantiomers of praziquantel (PZQ) is currently the treatment of choice for schistosomiasis. Though the S enantiomer and the metabolites are presumed to contribute only a little to the activity of the drug, in-depth side-by-side studies are lacking. The aim of this study was to investigate the in vitro activities of PZQ and its main metabolites, namely, R- and S-cis- and R- and S-trans-4'-hydroxypraziquantel, against adult worms and newly transformed schistosomula (NTS). Additionally, we explored the in vivo activity and hepatic shift (i.e., the migration of the worms to the liver) produced by each PZQ enantiomer in mice. Fifty percent inhibitory concentrations of R-PZQ, S-PZQ, and R-trans- and R-cis-4'-hydroxypraziquantel of 0.02, 5.85, 4.08, and 2.42 µg/ml, respectively, for adult S. mansoni were determined in vitro. S-trans- and S-cis-4'-hydroxypraziquantel were not active at 100 µg/ml. These results are consistent with microcalorimetry data and studies with NTS. In vivo, single 400-mg/kg oral doses of R-PZQ and S-PZQ achieved worm burden reductions of 100 and 19%, respectively. Moreover, worms treated in vivo with S-PZQ displayed an only transient hepatic shift and returned to the mesenteric veins within 24 h. Our data confirm that R-PZQ is the main effector molecule, while S-PZQ and the metabolites do not play a significant role in the antischistosomal properties of PZQ.

Exposure of Heligmosomoides polygyrus and Trichuris muris to albendazole, albendazole sulfoxide, mebendazole and oxantel pamoate in vitro and in vivo to elucidate the pathway of drug entry into these gastrointestinal nematodes.

Millions of people are treated with anthelmintics to control soil-transmitted helminth infections; yet, drug distribution in the plasma and gastrointestinal tract compartments and the pathway of drug uptake into gastrointestinal nematodes responsible for the pharmacological effect are unknown. We assessed the distribution and uptake of albendazole, albendazole sulfoxide, albendazole sulfone in the hookworm Heligmosomoides polygyrus in vitro and in vivo as well as the distribution and uptake of albendazole, mebendazole, and oxantel pamoate in the whipworm Trichuris muris in vitro and in vivo. Oral and intraperitoneal treatments (100 mg/kg) were studied. Drug quantities in helminths and host compartments (stomach, the contents and mucosa of the small and large intestine, and the plasma) were determined using HPLC-UV/vis and anthelmintic activities were recorded using phenotypic readout. The influence of 1-aminobenzotriazole (ABT), an irreversible and unspecific cytochrome P450 inhibitor, on albendazole disposition in mice harboring H. polygyrus was evaluated. In vivo, albendazole was found in quantities up to 10 nmol per ten H. polygyrus and up to 31 nmol per ten T. muris. ABT did not change the levels of albendazole or its metabolites in the plasma of mice harboring H. polygyrus or in H. polygyrus, whereas drug levels in the gastrointestinal tract of host mice doubled. Mebendazole and oxantel pamoate quantities per ten T. muris were as high as 21 nmol and 34 nmol, respectively. Albendazole revealed a very dynamic distribution and high rate of metabolism, hence, H. polygyrus and T. muris are exposed to albendazole and both metabolites via multiple pathways. Diffusion through the cuticle seems to be the crucial pathway of oxantel pamoate uptake into T. muris, and likely also for mebendazole. No relationship between concentrations measured in helminths and concentrations in plasma, intestinal content and mucosa of mice, or drug efficacy was noted for any of the drugs studied.

Treatment of human and livestock helminth infections in a mobile pastoralist setting at Lake Chad: Attitudes to health and analysis of active pharmaceutical ingredients of locally available anthelminthic drugs.

Mobile pastoralists face challenges in accessing quality health care and medication for managing human and animal diseases. We determined livestock disease priorities, health seeking behaviour of people bearing helminthiases and - placing particular emphasis on trematode infections - treatment strategies and outcome satisfaction among mobile pastoralists of four ethnic groups in the Lake Chad area using focus group discussions. People suffering from schistosomiasis were interviewed about symptoms, health seeking behaviour and their satisfaction with respect to the provided treatment. Anthelminthic drugs for human and veterinary use obtained from various health care structures were analysed for active pharmaceutical ingredients (API) and quantity, using high pressure liquid chromatography-UV and liquid chromatography combined with tandem mass spectrometry. Most people suffering from schistosomiasis sought treatment at health care centres. Yet, they also consulted informal providers without medical training. Regarding animal health, self-mediated therapy was common to manage suspected livestock fascioliasis. Self-reported treatment satisfaction for human schistosomiasis and trematodiasis treatment outcome in livestock were low. Mobile pastoralists perceived the purchased drugs to be of low quality. Among 33 products locally sold as anthelminthic drugs for human or veterinary use, 27 contained albendazole or mebendazole, varying between 91% and 159% of the labelled amount. Six products were sold loosely with incomplete information and their API could not be identified. No counterfeit anthelminthic drugs were detected. None of the samples contained praziquantel or triclabendazole, the drugs of choice against schistosomiasis and fascioliasis, respectively. The perceived unsatisfactory treatment outcomes in humans and animals infected with trematodes are most likely due to empiric diagnosis and the resulting use of inadequate therapy for human schistosomiasis and the lack of efficacious drugs against livestock fascioliasis.

Evaluation of an FDA approved library against laboratory models of human intestinal nematode infections.

BACKGROUND: Treatment options for infections with soil-transmitted helminths (STH) - Ascaris lumbricoides, Trichuris trichiura and the two hookworm species, Ancylostoma duodenale and Necator americanus - are limited despite their considerable global health burden. The aim of the present study was to test the activity of an openly available FDA library against laboratory models of human intestinal nematode infections. METHODS: All 1,600 drugs were first screened against Ancylostoma ceylanicum third-stage larvae (L3). Active compounds were scrutinized and toxic compounds, drugs indicated solely for topical use, and already well-studied anthelmintics were excluded. The remaining hit compounds were tested in parallel against Trichuris muris first-stage larvae (L1), Heligmosomoides polygyrus third-stage larvae (L3), and adult stages of the three species in vitro. In vivo studies were performed in the H. polygyrus and T. muris mice models. RESULTS: Fifty-four of the 1,600 compounds tested revealed an activity of > 60 % against A. ceylanicum L3 (hit rate of 3.4 %), following incubation at 200 µM for 72 h. Twelve compounds progressed into further screens. Adult A. ceylanicum were the least affected (1/12 compounds active at 50 µM), while eight of the 12 test compounds revealed activity against T. muris L1 (100 µM) and adults (50 µM), and H. polygyrus L3 (200 µM). Trichlorfon was the only compound active against all stages of A. ceylanicum, H. polygyrus and T. muris. In addition, trichlorfon achieved high worm burden reductions of 80.1 and 98.9 %, following a single oral dose of 200 mg/kg in the T. muris and H. polygyrus mouse model, respectively. CONCLUSION: Drug screening on the larval stages of intestinal parasitic nematodes is feasible using small libraries and important given the empty drug discovery and development pipeline for STH infections. Differences and commonalities in drug activities across the different STH species and stages were confirmed. Hits identified might serve as a starting point for drug discovery for STH.

Repurposing of anticancer drugs: in vitro and in vivo activities against Schistosoma mansoni.

BACKGROUND: Drug discovery for the neglected tropical disease schistosomiasis has a high priority. Anticancer drugs, especially protein kinase inhibitors, might serve as a starting point for drug discovery owing to the importance of protein kinases in helminth growth and development. Furthermore, the Schistosoma mansoni genome encodes several genes for targets of drugs marketed for human use, including several anticancer drugs. METHODS: In this study, we screened the approved oncology drug set of the National Cancer Institute's Developmental Therapeutic Program for antischistosomal activity. Drugs were tested in vitro against the larval and adult stage of S. mansoni. IC50 values and albumin binding were determined for active compounds. Lead compounds were tested in the chronic S. mansoni mouse model. RESULTS: Eleven of the 114 compounds tested revealed IC50 values ≤ 10 µM against both S. mansoni stages. Five of these lost activity against adult S. mansoni in the presence of serum albumin. Of 6 compounds studied in vivo, the highest activity was observed from two kinase inhibitors trametinib, and vandetanib, which reduced worm burden by 63.6 and 48.1% respectively, after a single oral dose of 400 mg/kg body weight. CONCLUSION: Our study has confirmed that oncology drugs possess antischistosomal activity. There is space for further investigation, including elucidation of the mechanisms of action of schistosome-active cancer drugs, application of different treatment courses, and structure-activity relationship studies for improving drug potency.

Orally active antischistosomal early leads identified from the open access malaria box.

BACKGROUND: Worldwide hundreds of millions of schistosomiasis patients rely on treatment with a single drug, praziquantel. Therapeutic limitations and the threat of praziquantel resistance underline the need to discover and develop next generation drugs. METHODOLOGY: We studied the antischistosomal properties of the Medicines for Malaria Venture (MMV) malaria box containing 200 diverse drug-like and 200 probe-like compounds with confirmed in vitro activity against Plasmodium falciparum. Compounds were tested against schistosomula and adult Schistosoma mansoni in vitro. Based on in vitro performance, available pharmacokinetic profiles and toxicity data, selected compounds were investigated in vivo. PRINCIPAL FINDINGS: Promising antischistosomal activity (IC50: 1.4-9.5 µM) was observed for 34 compounds against schistosomula. Three compounds presented IC50 values between 0.8 and 1.3 µM against adult S. mansoni. Two promising early leads were identified, namely a N,N'-diarylurea and a 2,3-dianilinoquinoxaline. Treatment of S. mansoni infected mice with a single oral 400 mg/kg dose of these drugs resulted in significant worm burden reductions of 52.5% and 40.8%, respectively. CONCLUSIONS/SIGNIFICANCE: The two candidates identified by investigating the MMV malaria box are characterized by good pharmacokinetic profiles, low cytotoxic potential and easy chemistry and therefore offer an excellent starting point for antischistosomal drug discovery and development.