Discovery and Preclinical Pharmacology of INE963, a Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cures in Uncomplicated Malaria

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This article references 29 other publications.

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   WHO. Malaria. https://www.who.int/en/news-room/fact-sheets/detail/malaria (accessed 2021–05–01).

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   Balikagala, B.; Fukuda, N.; Ikeda, M.; Katuro, O. T.; Tachibana, S. I.; Yamauchi, M.; Opio, W.; Emoto, S.; Anywar, D. A.; Kimura, E.; Palacpac, N. M. Q.; Odongo-Aginya, E. I.; Ogwang, M.; Horii, T.; Mita, T. Evidence of artemisinin-resistant malaria in Africa. N Engl J. Med. 2021, 385, 1163– 1171,  DOI: 10.1056/NEJMoa2101746

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   Evidence of artemisinin-resistant malaria in Africa

   Balikagala, Betty; Fukuda, Naoyuki; Ikeda, Mie; Katuro, Osbert T.; Tachibana, Shin-Ichiro; Yamauchi, Masato; Opio, Walter; Emoto, Sakurako; Anywar, Denis A.; Kimura, Eisaku; Palacpac, Nirianne M. Q.; Odongo-Aginya, Emmanuel I.; Ogwang, Martin; Horii, Toshihiro; Mita, Toshihiro

   New England Journal of Medicine (2021), 385 (13), 1163-1171CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)

   In the six Southeast Asian countries that make up the Greater Mekong Subregion, Plasmodium falciparum has developed resistance to derivs. of artemisinin, the main component of first-line treatments for malaria. Clin. resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic. methods In this longitudinal study conducted in Northern Uganda, we treated patients who had P. falciparum infection with i.v. artesunate (a water-sol. artemisinin deriv.) and estd. the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes. results From 2017 through 2019, a total of 14 of 240 patients who received i.v. artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 h). Of these 14 patients, 13 were infected with P. falciparum parasites with mutations in the A675V or C469Y allele in the kelch13 gene. Such mutations were assocd. with prolonged parasite clearance half-lives (geometric mean, 3.95 h for A675V and 3.30 h for C469Y, vs. 1.78 h for wild-type allele; P<0.001 and P = 0.05, resp.). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with kelch13 mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, resp.). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia. The independent emergence and local spread of clin. artemisinin-resistant P. falciparum has been identified in Africa. The two kelch13 mutations may be markers for detection of these resistant parasites.
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   Rogers, W. O.; Sem, R.; Tero, T.; Chim, P.; Lim, P.; Muth, S.; Socheat, D.; Ariey, F.; Wongsrichanalai, C. Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia. Malar. J. 2009, 8, 10,  DOI: 10.1186/1475-2875-8-10

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   Failure of artesunate-mefloquine combination therapy for uncomplicated Plasmodium falciparum malaria in southern Cambodia

   Rogers William O; Sem Rithy; Tero Thong; Chim Pheaktra; Lim Pharath; Muth Sinuon; Socheat Duong; Ariey Frederic; Wongsrichanalai Chansuda

   Malaria journal (2009), 8 (), 10 ISSN:.

   BACKGROUND: Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria was assessed in Chumkiri, Kampot Province, Cambodia. METHODS: One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For P. falciparum infected subjects, PCR genotyping of msp1, msp2, and glurp was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the pfmdr1 gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC50 for anti-malarial drugs. RESULTS: Among P. falciparum infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased pfmdr1 copy number, higher initial parasitaemia, higher mefloquine IC50, and longer time to parasite clearance. One P. falciparum isolate, from a treatment failure, had markedly elevated IC50 for both mefloquine (130 nM) and artesunate (6.7 nM). Among P. vivax infected subjects, 42.1% suffered recurrent P. vivax parasitaemia. None acquired new P. falciparum infection. CONCLUSION: The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC50 suggest that artesunate resistance may be emerging on a background of mefloquine resistance.
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   Woodrow, C. J.; White, N. J. The clinical impact of artemisinin resistance in Southeast Asia and the potential for future spread. FEMS Microbiol. Rev. 2017, 41, 34– 48,  DOI: 10.1093/femsre/fuw037

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   The clinical impact of artemisinin resistance in Southeast Asia and the potential for future spread

   Woodrow, Charles J.; White, Nicholas J.

   FEMS Microbiology Reviews (2017), 41 (1), 34-48CODEN: FMREE4; ISSN:1574-6976. (Oxford University Press)

   A review. Artemisinins are the most rapidly acting of currently available antimalarial drugs. Artesunate has become the treatment of choice for severe malaria, and artemisinin-based combination therapies (ACTs) are the foundation of modern falciparum malaria treatment globally. Their safety and tolerability profile is excellent. Unfortunately, Plasmodium falciparum infections with mutations in the 'K13' gene, with reduced ring-stage susceptibility to artemisinins, and slow parasite clearance in patients treated with ACTs, are now widespread in Southeast Asia. We review clin. efficacy data from the region (2000-2015) that provides strong evidence that the loss of first-line ACTs in western Cambodia, first artesunate-mefloquine and then DHA-piperaquine, can be attributed primarily to K13 mutated parasites. The ring-stage activity of artemisinins is therefore crit. for the sustained efficacy of ACTs; once it is lost, rapid selection of partner drug resistance and ACT failure are inevitable consequences. Consensus methods for monitoring artemisinin resistance are now available. Despite increased investment in regional control activities, ACTs are failing across an expanding area of the Greater Mekong subregion. Although multiple K13 mutations have arisen independently, successful multidrug-resistant parasite genotypes are taking over and threaten to spread to India and Africa. Stronger containment efforts and new approaches to sustaining long-term efficacy of antimalarial regimens are needed to prevent a global malaria emergency.
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   van der Pluijm, R. W.; Imwong, M.; Chau, N. H.; Hoa, N. T.; Thuy-Nhien, N. T.; Thanh, N. V.; Jittamala, P.; Hanboonkunupakarn, B.; Chutasmit, K.; Saelow, C.; Runjarern, R.; Kaewmok, W.; Tripura, R.; Peto, T. J.; Yok, S.; Suon, S.; Sreng, S.; Mao, S.; Oun, S.; Yen, S.; Amaratunga, C.; Lek, D.; Huy, R.; Dhorda, M.; Chotivanich, K.; Ashley, E. A.; Mukaka, M.; Waithira, N.; Cheah, P. Y.; Maude, R. J.; Amato, R.; Pearson, R. D.; Goncalves, S.; Jacob, C. G.; Hamilton, W. L.; Fairhurst, R. M.; Tarning, J.; Winterberg, M.; Kwiatkowski, D. P.; Pukrittayakamee, S.; Hien, T. T.; Day, N. P.; Miotto, O.; White, N. J.; Dondorp, A. M. Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study. Lancet Infect. Dis. 2019, 19, 952– 961,  DOI: 10.1016/S1473-3099(19)30391-3

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   Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study

   van der Pluijm, Rob W.; Imwong, Mallika; Chau, Nguyen Hoang; Hoa, Nhu Thi; Thuy-Nhien, Nguyen Thanh; Thanh, Ngo Viet; Jittamala, Podjanee; Hanboonkunupakarn, Borimas; Chutasmit, Kitipumi; Saelow, Chalermpon; Runjarern, Ratchadaporn; Kaewmok, Weerayuth; Tripura, Rupam; Peto, Thomas J.; Yok, Sovann; Suon, Seila; Sreng, Sokunthea; Mao, Sivanna; Oun, Savuth; Yen, Sovannary; Amaratunga, Chanaki; Lek, Dysoley; Huy, Rekol; Dhorda, Mehul; Chotivanich, Kesinee; Ashley, Elizabeth A.; Mukaka, Mavuto; Waithira, Naomi; Cheah, Phaik Yeong; Maude, Richard J.; Amato, Roberto; Pearson, Richard D.; Goncalves, Sonia; Jacob, Christopher G.; Hamilton, William L.; Fairhurst, Rick M.; Tarning, Joel; Winterberg, Markus; Kwiatkowski, Dominic P.; Pukrittayakamee, Sasithon; Hien, Tran Tinh; Day, Nicholas P. J.; Miotto, Olivo; White, Nicholas J.; Dondorp, Arjen M.

   Lancet Infectious Diseases (2019), 19 (9), 952-961CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)

   The current study is part of a multi-country, open-label, randomised clin. trial (TRACII, 2015-18) evaluating the efficacy, safety, and tolerability of triple artemisinin combination therapies. A very high rate of treatment failure after treatment with dihydroartemisinin-piperaquine was obsd. in Thailand, Cambodia, and Vietnam. Patients aged between 2 and 65 years presenting with uncomplicated P falciparum or mixed species malaria at seven sites in Thailand, Cambodia, and Vietnam were randomly assigned to receive dihydroartemisinin-piperaquine with or without mefloquine, as part of the TRACII trial. The primary outcome was the PCR-cor. efficacy at day 42. Next-generation sequencing was used to assess the prevalence of mol. markers assocd. with artemisinin resistance (kelch13 mutations, in particular Cys580Tyr) and piperaquine resistance (plasmepsin-2 and plasmepsin-3 amplifications and crt mutations). Between Sept 28, 2015, and Jan 18, 2018, 539 patients with acute P falciparum malaria were screened for eligibility, 292 were enrolled, and 140 received dihydroartemisinin-piperaquine. The overall Kaplan-Meier est. of PCR-cor. efficacy of dihydroartemisinin-piperaquine at day 42 was 50·0% (95% CI 41·1-58·3). Treatment failure was assocd. independently with plasmepsin2/3 amplification status and four mutations in the crt gene (Thr93Ser, His97Tyr, Phe145Ile, and Ile218Phe).
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   Rosenthal, P. J. Has artemisinin resistance emerged in Africa?. Lancet Infect. Dis. 2021, 21, 1056– 1057,  DOI: 10.1016/S1473-3099(21)00168-7

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   Has artemisinin resistance emerged in Africa?

   Rosenthal Philip J

   The Lancet. Infectious diseases (2021), 21 (8), 1056-1057 ISSN:.

   There is no expanded citation for this reference.
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   Ramharter, M.; Kurth, F. M.; Belard, S.; Bouyou-Akotet, M. K.; Mamfoumbi, M. M.; Agnandji, S. T.; Missinou, M. A.; Adegnika, A. A.; Issifou, S.; Cambon, N.; Heidecker, J. L.; Kombila, M.; Kremsner, P. G. Pharmacokinetics of two paediatric artesunate mefloquine drug formulations in the treatment of uncomplicated falciparum malaria in Gabon. J. Antimicrob. Chemother. 2007, 60, 1091– 1096,  DOI: 10.1093/jac/dkm355

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   Pharmacokinetics of two pediatric artesunate-mefloquine drug formulations in the treatment of uncomplicated falciparum malaria in Gabon

   Ramharter, Michael; Kurth, Florian M.; Belard, Sabine; Bouyou-Akotet, Marielle K.; Mamfoumbi, Modeste Mabika; Agnandji, Selidji T.; Missinou, Michel A.; Adegnika, Ayola A.; Issifou, Saadou; Cambon, Nathalie; Heidecker, Janos L.; Kombila, Maryvonne; Kremsner, Peter G.

   Journal of Antimicrobial Chemotherapy (2007), 60 (5), 1091-1096CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)

   Pediatric drug formulations of artemisinin combination therapies and pharmacokinetic data supporting their use in African children are urgently needed for the effective treatment of young children suffering from falciparum malaria in sub-Saharan Africa. In this study, the pharmacokinetic characteristics of a novel pediatric granule formulation of artesunate-mefloquine therapy were evaluated in comparison to the std. tablet formulation in the treatment of uncomplicated malaria in pediatric patients. Twenty-four patients were assigned to treatment according to body wt. with either a fixed-dose pediatric granule co-formulation (10-20 kg body wt.) or a free-dose co-blister tablet formulation of artesunate-mefloquine (>20-40 kg body wt.). Median values for Cmax (861 and 930 ng/mL), Tmax (1.5 and 1.5 h) and AUC0-t (2050 and 2470 ng/h/mL) were comparable for dihydroartemisinin in the two groups. Exploratory anal. of mefloquine plasma levels revealed a trend towards higher concns. in the younger age group during the absorption phase (2550 and 1815 ng/mL, 54 h after initiation of treatment, resp.). Median mefloquine concns. at day 28 were 197 and 343 ng/mL, resp. The pharmacokinetic characteristics of the two pediatric dosage forms, i.e. the novel fixed-dose co-formulation and the std. co-blister of artesunate-mefloquine show comparable results in the two treatment groups. The novel fixed-dose pediatric formulation is an interesting option for outpatient treatment of uncomplicated malaria in African children.
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   Burrows, J. N.; Duparc, S.; Gutteridge, W. E.; Hooft van Huijsduijnen, R.; Kaszubska, W.; Macintyre, F.; Mazzuri, S.; Mohrle, J. J.; Wells, T. N. C. New developments in anti-malarial target candidate and product profiles. Malar. J. 2017, 16, 26,  DOI: 10.1186/s12936-016-1675-x

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   New developments in anti-malarial target candidate and product profiles

   Burrows, Jeremy N.; Duparc, Stephan; Gutteridge, Winston E.; Hooft van Huijsduijnen, Rob; Kaszubska, Wiweka; MacIntyre, Fiona; Mazzuri, Sebastien; Mohrle, Jorg J.; Wells, Timothy N. C.

   Malaria Journal (2017), 16 (), 26/1-26/29CODEN: MJAOAZ; ISSN:1475-2875. (BioMed Central Ltd.)

   A review. A decade of discovery and development of new anti-malarial medicines has led to a renewed focus on malaria elimination and eradication. Changes in the way new anti-malarial drugs are discovered and developed have led to a dramatic increase in the no. and diversity of new mols. presently in pre-clin. and early clin. development. The twin challenges faced can be summarized by multi-drug resistant malaria from the Greater Mekong Subregion, and the need to provide simplified medicines. This review lists changes in anti-malarial target candidate and target product profiles over the last 4 years. As well as new medicines to treat disease and prevent transmission, there has been increased focus on the longer term goal of finding new medicines for chemoprotection, potentially with long-acting mols., or parenteral formulations. Other gaps in the malaria armamentarium, such as drugs to treat severe malaria and endectocides (that kill mosquitoes which feed on people who have taken the drug), are defined here. Ultimately the elimination of malaria requires medicines that are safe and well-tolerated to be used in vulnerable populations: in pregnancy, esp. the first trimester, and in those suffering from malnutrition or co-infection with other pathogens. These updates reflect the maturing of an understanding of the key challenges in producing the next generation of medicines to control, eliminate and ultimately eradicate malaria.
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   White, N. J.; Pukrittayakamee, S.; Phyo, A. P.; Rueangweerayut, R.; Nosten, F.; Jittamala, P.; Jeeyapant, A.; Jain, J. P.; Lefevre, G.; Li, R.; Magnusson, B.; Diagana, T. T.; Leong, F. J. Spiroindolone KAE609 for falciparum and vivax malaria. N. Engl. J. Med. 2014, 371, 403– 410,  DOI: 10.1056/NEJMoa1315860

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   Spiroindolone KAE609 for falciparum and vivax malaria

   White, Nicholas J.; Pukrittayakamee, Sasithon; Phyo, Aung Pyae; Rueangweerayut, Ronnatrai; Nosten, Francois; Jittamala, Podjanee; Jeeyapant, Atthanee; Jain, Jay Prakash; Lefevre, Gilbert; Li, Ruobing; Magnusson, Baldur; Diagana, Thierry T.; Leong, F. Joel

   New England Journal of Medicine (2014), 371 (5), 403-410, 8 pp.CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)

   Background: KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analog with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum. Methods: We conducted a phase 2, open-label study at three centers in Thailand to assess the antimalarial efficacy, safety, and adverse-event profile of KAE609, at a dose of 30 mg per day for 3 days, in two sequential cohorts of adults with uncomplicated P. vivax malaria (10 patients) or P. falciparum malaria. The primary end point was the parasite clearance time. Results: The median parasite clearance time was 12 h in each cohort (interquartile range, 8 to 16 h in patients with P. vivax malaria and 10 to 16 h in those with P. falciparum malaria). The median half-lives for parasite clearance were 0.95 h (range, 0.68 to 2.01; interquartile range, 0.85 to 1.14) in the patients with P. vivax malaria and 0.90 h (range, 0.68 to 1.64; interquartile range, 0.78 to 1.07) in those with P. falciparum malaria. By comparison, only 19 of 5076 patients with P. falciparum malaria (<1%) who were treated with oral artesunate in Southeast Asia had a parasite clearance half-life of less than 1 h. Adverse events were reported in 14 patients (67%), with nausea being the most common. The adverse events were generally mild and did not lead to any discontinuations of the drug. The mean terminal half-life for the elimination of KAE609 was 20.8 h (range, 11.3 to 37.6), supporting a once-daily oral dosing regimen. Conclusions: KAE609, at dose of 30 mg daily for 3 days, cleared parasitemia rapidly in adults with uncomplicated P. vivax or P. falciparum malaria.
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    White, N. J.; Duong, T. T.; Uthaisin, C.; Nosten, F.; Phyo, A. P.; Hanboonkunupakarn, B.; Pukrittayakamee, S.; Jittamala, P.; Chuthasmit, K.; Cheung, M. S.; Feng, Y.; Li, R.; Magnusson, B.; Sultan, M.; Wieser, D.; Xun, X.; Zhao, R.; Diagana, T. T.; Pertel, P.; Leong, F. J. Antimalarial activity of KAF156 in falciparum and vivax malaria. N. Engl. J. Med. 2016, 375, 1152– 1160,  DOI: 10.1056/NEJMoa1602250

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    Antimalarial activity of KAF156 in falciparum and vivax malaria

    White, Nicholas J.; Duong, Tran T.; Uthaisin, Chirapong; Nosten, Francois; Phyo, Aung P.; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chuthasmit, Kittiphum; Cheung, Ming S.; Feng, Yiyan; Li, Ruobing; Magnusson, Baldur; Sultan, Marc; Wieser, Daniela; Xun, Xiaolei; Zhao, Rong; Diagana, Thierry T.; Pertel, Peter; Leong, F. Joel

    New England Journal of Medicine (2016), 375 (12), 1152-1160CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)

    BACKGROUND: KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites. METHODS: We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a sep. cohort of patients with falciparum malaria who received a single dose (800 mg). RESULTS: Median parasite clearance times were 45 h (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 h (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 h (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 h. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts. CONCLUSIONS: KAF156 showed antimalarial activity without evident safety concerns in a small no. of adults with uncomplicated P. vivax or P. falciparum malaria.
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    Llanos-Cuentas, A.; Casapia, M.; Chuquiyauri, R.; Hinojosa, J. C.; Kerr, N.; Rosario, M.; Toovey, S.; Arch, R. H.; Phillips, M. A.; Rozenberg, F. D.; Bath, J.; Ng, C. L.; Cowell, A. N.; Winzeler, E. A.; Fidock, D. A.; Baker, M.; Mohrle, J. J.; Hooft van Huijsduijnen, R.; Gobeau, N.; Araeipour, N.; Andenmatten, N.; Ruckle, T.; Duparc, S. Antimalarial activity of single-dose DSM265, a novel Plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-of-concept, open-label, phase 2a study. Lancet Infect. Dis. 2018, 18, 874– 883,  DOI: 10.1016/S1473-3099(18)30309-8

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    Antimalarial activity of single-dose DSM265, a novel plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-of-concept, open-label, phase 2a study

    Llanos-Cuentas, Alejandro; Casapia, Martin; Chuquiyauri, Raul; Hinojosa, Juan-Carlos; Kerr, Nicola; Rosario, Maria; Toovey, Stephen; Arch, Robert H.; Phillips, Margaret A.; Rozenberg, Felix D.; Bath, Jade; Ng, Caroline L.; Cowell, Annie N.; Winzeler, Elizabeth A.; Fidock, David A.; Baker, Mark; Mohrle, Jorg J.; Hooft van Huijsduijnen, Rob; Gobeau, Nathalie; Araeipour, Nada; Andenmatten, Nicole; Ruckle, Thomas; Duparc, Stephan

    Lancet Infectious Diseases (2018), 18 (8), 874-883CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)

    DSM265 is a novel, long-duration inhibitor of plasmodium dihydroorotate dehydrogenase (DHODH) with excellent selectivity over human DHODH and activity against blood and liver stages of Plasmodium falciparum. This study aimed to assess the efficacy of DSM265 in patients with P falciparum or Plasmodium vivax malaria infection. This proof-of-concept, open-label, phase 2a study was conducted at the Asociacio´n Civil Selva Amazo´nica in Iquitos, Peru. Patients aged 18-70 years, weighing 45-90 kg, who had clin. malaria (P falciparum or P vivax monoinfection) and fever within the previous 24 h were eligible. Exclusion criteria were clin. or lab. signs of severe malaria, inability to take oral medicine, and use of other antimalarial treatment in the preceding 14 days. Patients were divided into cohorts of those with P falciparum (cohort a) or P vivax (cohort b) infection. Two initial cohorts received single oral doses of 400 mg DSM265. Patients were followed up for efficacy for 28 days and safety for 35 days. Further cohorts received escalated or de-escalated doses of DSM265, after safety and efficacy assessment of the initial dose. The primary endpoints were the proportion of patients achieving PCR-adjusted adequate clin. and parasitol. response (ACPR) by day 14 for patients infected with P falciparum and the proportion of patients achieving a crude cure by day 14 for those infected with P vivax. Cohort success, the criteria for dose escalation, was defined as ACPR (P falciparum) or crude cure (P vivax) in at least 80% of patients in the cohort. The primary anal. was done in the intention-to-treat population (ITT) and the per-protocol population, and safety analyses were done in all patients who received the study drug. This study is registered at ClinicalTrials.gov (NCT02123290). Between Jan 12, 2015, and Dec 2, 2015, 45 Peruvian patients (24 with P falciparum [cohort a] and 21 with P vivax [cohort b] infection) were sequentially enrolled. For patients with P falciparum malaria in the per-protocol population, all 11 (100%) in the 400 mg group and eight (80%) of ten in the 250 mg group achieved ACPR on day 14. In the ITT anal., 11 (85%) of 13 in the 400 mg group and eight (73%) of 11 in the 250 mg group achieved ACPR at day 14. For the patients with P vivax malaria, the primary endpoint was not met. In the per-protocol anal., none of four patients who had 400 mg, three (50%) of six who had 600 mg, and one (25%) of four who had 800 mg DSM265 achieved crude cure at day 14. In the ITT anal., none of five in the 400 mg group, three (33%) of nine in the 600 mg group, and one (14%) of seven in the 800 mg group achieved crude cure at day 14. During the 28-day extended observation of P falciparum patients, a resistance-assocd. mutation in the gene encoding the DSM265 target DHODH was obsd. in two of four recurring patients. DSM265 was well tolerated. The most common adverse events were pyrexia (20 [44%] of 45) and headache (18 [40%] of 45), which are both common symptoms of malaria, and no patients had any treatment-related serious adverse events or adverse events leading to study discontinuation. After a single dose of DSM265, P falciparum parasitemia was rapidly cleared, whereas against P vivax, DSM265 showed less effective clearance kinetics. Its long duration of action provides the potential to prevent recurrence of P falciparum after treatment with a single dose, which should be further assessed in future combination studies. The Global Health Innovative Technol. Fund, the Bill & Melinda Gates Foundation, the National Institutes of Health (R01 AI103058), the Wellcome Trust, and the UK Department of International Development.
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    Sinxadi, P.; Donini, C.; Johnstone, H.; Langdon, G.; Wiesner, L.; Allen, E.; Duparc, S.; Chalon, S.; McCarthy, J. S.; Lorch, U.; Chibale, K.; Mohrle, J.; Barnes, K. I. Safety, tolerability, pharmacokinetics, and antimalarial activity of the novel Plasmodium phosphatidylinositol 4-kinase inhibitor MMV390048 in healthy volunteers. Antimicrob. Agents Chemother. 2020, 64, e01896– 19,  DOI: 10.1128/AAC.01896-19

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    Safety, tolerability, pharmacokinetics, and antimalarial activity of the novel Plasmodium phosphatidylinositol 4-kinase inhibitor MMV390048 in healthy volunteers

    Sinxadi, Phumla; Donini, Cristina; Johnstone, Hilary; Langdon, Grant; Wiesner, Lubbe; Allen, Elizabeth; Duparc, Stephan; Chalon, Stephan; McCarthy, James S.; Lorch, Ulrike; Chibale, Kelly; Mohrle, Jorg; Barnesa, Karen I.

    Antimicrobial Agents and Chemotherapy (2020), 64 (4), e01896CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)

    MMV390048 is a novel antimalarial compd. that inhibits Plasmodium phosphatidylinositol-4-kinase. The safety, tolerability, pharmacokinetic profile, and antimalarial activity of MMV390048 were detd. in healthy volunteers in three sep. studies. A first-in-human, double-blind, randomized, placebo-controlled, single-ascending-dose study was performed. Addnl., a volunteer infection study investigated the antimalarial activity of MMV390048 using the Plasmodium falciparum induced blood-stage malaria (IBSM) model. Due to the high pharmacokinetic variability with the powder-in-bottle formulation used in both of these studies, a third study was undertaken to select a tablet formulation of MMV390048 to take forward into future studies. MMV390048 was generally well tolerated when administered as a single oral dose up to 120 mg, with rapid absorption and a long elimination half-life. Twelve adverse events were considered to be potentially related to MMV390048 in the first-in-human study but with no obvious correlation between these and MMV390048 dose or exposure. Although antimalarial activity was evident in the IBSM study, rapid recrudescence occurred in most subjects after treatment with 20 mg MMV390048, a dose expected to be subtherapeutic. Reformulation of MMV390048 into two tablet formulations (tartaric acid and Syloid) resulted in significantly reduced intersubject pharmacokinetic variability. Overall, the results of this study suggest that MMV390048 is well tolerated in humans, and the pharmacokinetic properties of the compd. indicate that it has the potential to be used for antimalarial prophylaxis or inclusion in a single-dose cure. MMV390048 is currently being tested in a phase 2a study in Ethiopian adults with acute, uncomplicated falciparum or vivax malaria monoinfection.
13. 13

    Gaur, A. H.; McCarthy, J. S.; Panetta, J. C.; Dallas, R. H.; Woodford, J.; Tang, L.; Smith, A. M.; Stewart, T. B.; Branum, K. C.; Freeman, B. B.; Patel, N. D.; John, E.; Chalon, S.; Ost, S.; Heine, R. N.; Richardson, J. L.; Christensen, R.; Flynn, P. M.; Van Gessel, Y.; Mitasev, B.; Möhrle, J. J.; Gusovsky, F.; Bebrevska, L.; Guy, R. K. Safety, tolerability, pharmacokinetics, and antimalarial efficacy of a novel Plasmodium falciparum ATP4 inhibitor SJ733: a first-in-human and induced blood-stage malaria phase 1a/b trial. Lancet Infect. Dis. 2020, 20, 964– 975,  DOI: 10.1016/S1473-3099(19)30611-5

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    Safety, tolerability, pharmacokinetics, and antimalarial efficacy of a novel Plasmodium falciparum ATP4 inhibitor SJ733: a first-in-human and induced blood-stage malaria phase 1a/b trial

    Gaur, Aditya H.; McCarthy, James S.; Panetta, John C.; Dallas, Ronald H.; Woodford, John; Tang, Li; Smith, Amber M.; Stewart, Tracy B.; Branum, Kristen C.; Freeman, Burgess B., III; Patel, Nehali D.; John, Elizabeth; Chalon, Stephan; Ost, Shelley; Heine, Ryan N.; Richardson, Julie L.; Christensen, Robbin; Flynn, Patricia M.; Van Gessel, Yvonne; Mitasev, Branko; Mohrle, Jorg J.; Gusovsky, Fabian; Bebrevska, Lidiya; Guy, R. Kiplin

    Lancet Infectious Diseases (2020), 20 (8), 964-975CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)

    Phase 1b took place at Q-Pharm (Herston, QLD, Australia) and was initiated only after phase 1a showed that exposure exceeding the threshold min. exposure could be safely achieved in humans. Participants were inoculated on day 0 with P falciparum-infected human erythrocytes (around 2800 parasites in the 150 mg dose cohort and around 2300 parasites in the 600 mg dose cohort), and parasitemia was monitored before malaria inoculation, after inoculation, immediately before SJ733 dosing, and then post-dose. Participants were treated with SJ733 within 24 h of reaching 5000 parasites per mL or at a clin. score higher than 6. Phase 1b primary endpoints were calcn. of a parasite redn. ratio (PRR48) and parasite clearance half-life, and safety and tolerability of SJ733 (incidence, severity, and drug-relatedness of adverse events). In both phases of the trial, SJ733 hydrochloride salt was formulated as a powder blend in capsules contg. 75 mg or 300 mg for oral administration. Healthy men and women (of non-childbearing potential) aged 18-55 years were eligible for both studies. Both studies are registered with ClinicalTrials.gov (NCT02661373 for the phase 1a and NCT02867059 for the phase 1b). In the phase 1a, 23 healthy participants were enrolled and received one to three non-consecutive doses of SJ733 between March 14 and Dec 7, 2016.
14. 14

    McCarthy, J.; Yalkinoglu, O.; Odedra, A.; Webster, R.; Oeuvray, C.; Tappert, A.; Bezuidenhout, D.; Giddins, M.; Dhingra, S.; Fidock, D.; Marquart, L.; Webb, L.; Yin, X.; Khandelwal, A.; Bagchus, W. Safety, pharmacokinetics, and antimalarial activity of the novel Plasmodium eukaryotic translation elongation factor 2 inhibitor M5717: a first-in-human, randomised,placebo-controlled, double-blind, single ascending dose study and volunteer infection study. Lancet Infect. Dis. 2021, 21, 1713– 1724,  DOI: 10.1016/S1473-3099(21)00252-8

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    Safety, pharmacokinetics, and antimalarial activity of the novel plasmodium eukaryotic translation elongation factor 2 inhibitor M5717: a first-in-human, randomised, placebo-controlled, double-blind, single ascending dose study and volunteer infection study

    McCarthy, James S.; Yalkinoglu, Ozkan; Odedra, Anand; Webster, Rebecca; Oeuvray, Claude; Tappert, Aliona; Bezuidenhout, Deon; Giddins, Marla J.; Dhingra, Satish K.; Fidock, David A.; Marquart, Louise; Webb, Lachlan; Yin, Xiaoyan; Khandelwal, Akash; Bagchus, Wilhelmina M.

    Lancet Infectious Diseases (2021), 21 (12), 1713-1724CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)

    M5717 is the first plasmodium translation elongation factor 2 inhibitor to reach clin. development as an antimalarial. We aimed to characterize the safety, pharmacokinetics, and antimalarial activity of M5717 in healthy volunteers. This first-in-human study was a two-part, single-center clin. trial done in Brisbane, QLD, Australia. Part one was a double-blind, randomised, placebo-controlled, single ascending dose study in which participants were enrolled into one of nine dose cohorts (50, 100, 200, 400, 600, 1000, 1250, 1800, or 2100 mg) and randomly assigned (3:1) to M5717 or placebo. A sentinel dosing strategy was used for each dose cohort whereby two participants (one assigned to M5717 and one assigned to placebo) were initially randomised and dosed. Randomisation schedules were generated electronically by independent, unblinded statisticians. Part two was an open-label, non-randomised volunteer infection study using the Plasmodium falciparum induced blood-stage malaria model in which participants were enrolled into three dose cohorts. Healthy men and women of non-childbearing potential aged 18-55 years were eligible for inclusion; individuals in the volunteer infection study were required to be malaria naive. Safety and tolerability (primary outcome of the single ascending dose study and secondary outcome of the volunteer infection study) were assessed by frequency and severity of adverse events. The pharmacokinetic profile of M5717 was also characterised (primary outcome of the volunteer infection study and secondary outcome of the single ascending dose study). Parasite clearance kinetics (primary outcome of the volunteer infection study) were assessed by the parasite redn. ratio and the corresponding parasite clearance half-life; the incidence of recrudescence up to day 28 was detd. (secondary outcome of the volunteer infection study). Recrudescent parasites were tested for genetic mutations (exploratory outcome). The trial is registered with ClinicalTrials.gov (NCT03261401). Between Aug 28, 2017, and June 14, 2019, 221 individuals were assessed for eligibility, of whom 66 men were enrolled in the single ascending dose study (eight per cohort for 50-1800 mg cohorts, randomised three M5717 to one placebo, and two in the 2100 mg cohort, randomised one M5717 to one placebo) and 22 men were enrolled in the volunteer infection study (six in the 150 mg cohort and eight each in the 400 mg and 800 mg cohorts). No adverse event was serious; all M5717-related adverse events were mild or moderate in severity and transient, with increased frequency obsd. at doses above 1250 mg. In the single ascending dose study, treatment-related adverse events occurred in three of 17 individuals in the placebo group; no individual in the 50 mg, 100 mg, or 200 mg groups; one of six individuals in each of the 400 mg, 1000 mg, and 1250 mg groups; two of six individuals in the 600 mg group; and in all individuals in the 1800 mg and 2100 mg groups. In the volunteer infection study, M5717-related adverse events occurred in no participants in the 150 mg or 800 mg groups and in one of eight participants in the 400 mg group. Transient oral hypoesthesia (in three participants) and blurred vision (in four participants) were obsd. in the 1800 mg or 2100 mg groups and constituted an unknown risk; thus, further dosing was suspended after dosing of the two sentinel individuals in the 2100 mg cohort. Maximum blood concns. occurred 1-7 h after dosing, and a long half-life was obsd. (146-193 h at doses ≥200 mg). Parasite clearance occurred in all participants and was biphasic, characterised by initial slow clearance lasting 35-55 h (half-life 231·1 h [95% CI 40·9 to not reached] for 150 mg, 60·4 h [38·6 to 138·6] for 400 mg, and 24·7 h [20·4 to 31·3] for 800 mg), followed by rapid clearance (half-life 3·5 h [3·1 to 4·0] for 150 mg, 3·9 h [3·3 to 4·8] for 400 mg, and 5·5 h [4·8 to 6·4] for 800 mg). Recrudescence occurred in three (50%) of six individuals dosed with 150 mg and two (25%) of eight individuals dosed with 400 mg. Genetic mutations assocd. with resistance were detected in four cases of parasite recrudescence (two individuals dosed with 150 mg and two dosed with 400 mg). The safety, pharmacokinetics, and antimalarial activity of M5717 support its development as a component of a single-dose antimalarial combination therapy or for malaria prophylaxis.
15. 15

    Hameed, S. P.; Solapure, S.; Patil, V.; Bharath, B.; Murugan, K.; Viswanath, P.; Puttur, J.; Srivastava, A.; Bellale, E.; Panduga, V.; Shanbag, G.; Awasthy, D.; Landge, S.; Morayya, S.; Koushik, K.; Saralaya, R.; Raichurkar, A.; Rautela, N.; Choudhury, N.; Ambady, A.; Nandishaiah, R.; Reddy, J.; Prabhakar, K. R.; Menasinakai; Suresh Rudrapatna, S.; Chatterji, M.; Bandodkar, B.; Mukherjee, K.; Balasubramanian, V.; Warner, P.; Hosagrahara, V.; Dudley, A.; Iyer, P.; Narayanan, S.; Sambandamurthy, V.; Henrich, P.; Coburn-Flynn, O.; Fidock, D.; Magistrado, P.; Lukens, A.; Wirth, D.; Jiménez-Díaz, M.; Martínez, M.; Sanz, L.; McLaughlin, R.; Waterson, D.; Rosenbrier-Ribeiro, L.; Hickling, K.; Kavanagh, S. Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate. Nat. Commun. 2015, 6, 6715,  DOI: 10.1038/ncomms7715

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    Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate

    Hameed P., Shahul; Solapure, Suresh; Patil, Vikas; Henrich, Philipp P.; Magistrado, Pamela A.; Bharath, Sowmya; Murugan, Kannan; Viswanath, Pavithra; Puttur, Jayashree; Srivastava, Abhishek; Bellale, Eknath; Panduga, Vijender; Shanbag, Gajanan; Awasthy, Disha; Landge, Sudhir; Morayya, Sapna; Koushik, Krishna; Saralaya, Ramanatha; Raichurkar, Anandkumar; Rautela, Nikhil; Roy Choudhury, Nilanjana; Ambady, Anisha; Nandishaiah, Radha; Reddy, Jitendar; Prabhakar, K. R.; Menasinakai, Sreenivasaiah; Rudrapatna, Suresh; Chatterji, Monalisa; Jimenez-Diaz, Maria Belen; Martinez, Maria Santos; Sanz, Laura Maria; Coburn-Flynn, Olivia; Fidock, David A.; Lukens, Amanda K.; Wirth, Dyann F.; Bandodkar, Balachandra; Mukherjee, Kakoli; McLaughlin, Robert E.; Waterson, David; Rosenbrier-Ribeiro, Lyn; Hickling, Kevin; Balasubramanian, V.; Warner, Peter; Hosagrahara, Vinayak; Dudley, Adam; Iyer, Pravin S.; Narayanan, Shridhar; Kavanagh, Stefan; Sambandamurthy, Vasan K.

    Nature Communications (2015), 6 (), 6715CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)

    The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compds., the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clin. candidate (compd. 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg-1 and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concn. for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clin. development.
16. 16

    Avery, V. M.; Bashyam, S.; Burrows, J. N.; Duffy, S.; Papadatos, G.; Puthukkuti, S.; Sambandan, Y.; Singh, S.; Spangenberg, T.; Waterson, D.; Willis, P. Screening and hit evaluation of a chemical library against blood-stage Plasmodium falciparum. Malar. J. 2014, 13, 190,  DOI: 10.1186/1475-2875-13-190

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    Screening and hit evaluation of a chemical library against blood-stage Plasmodium falciparum

    Avery, Vicky M.; Bashyam, Sridevi; Burrows, Jeremy N.; Duffy, Sandra; Papadatos, George; Puthukkuti, Shyni; Sambandan, Yuvaraj; Singh, Shivendra; Spangenberg, Thomas; Waterson, David; Willis, Paul

    Malaria Journal (2014), 13 (), 190/1-190/26, 26CODEN: MJAOAZ; ISSN:1475-2875. (BioMed Central Ltd.)

    Background In view of the need to continuously feed the pipeline with new anti-malarial agents adapted to differentiated and more stringent target product profiles (e g, new modes of action, transmission-blocking activity or long-duration chemo-protection), a chem. library consisting of more than 250,000 compds. has been evaluated in a blood-stage Plasmodium falciparum growth inhibition assay and further assessed for chem. diversity and novelty. Methods The selection cascade used for the triaging of hits from the chem. library started with a robust three-step in vitro assay followed by an in silico anal. of the resulting confirmed hits. Upon reaching the predefined requirements for selectivity and potency, the set of hits was subjected to computational anal. to assess chem. properties and diversity. Furthermore, known marketed anti-malarial drugs were co-clustered acting as 'signposts' in the chem. space defined by the hits. Then, in cerebro evaluation of the chem. structures was performed to identify scaffolds that currently are or have been the focus of anti-malarial medicinal chem. programs. Next, prioritization according to relaxed physicochem. parameters took place, along with the search for structural analogs. Ultimately, synthesis of novel chemotypes with desired properties was performed and the resulting compds. were subsequently retested in a P. falciparum growth inhibition assay. Results This screening campaign led to a 1.25% primary hit rate, which decreased to 0.77% upon confirmatory repeat screening. With the predefined potency (EC50 < 1 μM) and selectivity (SI > 10) criteria, 178 compds. progressed to the next steps where chem. diversity, physicochem. properties and novelty assessment were taken into account. This resulted in the selection of 15 distinct chem. series. Conclusion A selection cascade was applied to prioritize hits resulting from the screening of a medium-sized chem. library against blood-stage P. falciparum. Emphasis was placed on chem. novelty whereby computational clustering, data mining of known anti-malarial chemotypes and the application of relaxed physicochem. filters, were key to the process. This led to the selection of 15 chem. series from which ten confirmed their activity when newly synthesized sample were tested.
17. 17

    Pevarello, P.; García-Collazo, A. M.; García-García, A. B. Substituted imidazo (2,1-b)-1,3,4-thiazole compounds, their pharmaceutical compositions and uses thereof. WO 2009040552 A2, 2009.

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18. 18

    Pastor-Fernández, J.; García-Collazo, A. M.; Noya-Mariño, B.; González Cantalapiedra, E. Amino-imidazolothiadiazoles for use as protein or lipid kinase inhibitors WO 2012020217 A1, 2012.

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    There is no corresponding record for this reference.
19. 19

    Assay performed as previously described:Aguiar, A. C. C.; Pereira, D. B.; Amaral, N. S.; De Marco, L.; Krettli, A. U. Plasmodium vivax and Plasmodium falciparum ex vivo susceptibility to anti-malarials and gene characterization in Rondônia, West Amazon. Brazil Malar. J. 2014, 13, 73,  DOI: 10.1186/1475-2875-13-73

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    There is no corresponding record for this reference.
20. 20

    Assay performed as previously described:Tumwebaze, P. K.; Katairo, T.; Okitwi, M.; Byaruhanga, O.; Orena, S.; Asua, V.; Duvalsaint, M.; Legac, J.; Chelebieva, S.; Ceja, F. G.; Rasmussen, S. A.; Conrad, M. D.; Nsobya, S. L.; Ozkan Aydemir, O.; Bailey, J. A.; Bayles, B. R.; Rosenthal, P. J.; Cooper, R. A. Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study. Lancet Microbe. 2021, 2, e441– e449,  DOI: 10.1016/S2666-5247(21)00085-9

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    Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study

    Tumwebaze, Patrick K.; Katairo, Thomas; Okitwi, Martin; Byaruhanga, Oswald; Orena, Stephen; Asua, Victor; Duvalsaint, Marvin; Legac, Jennifer; Chelebieva, Sevil; Ceja, Frida G.; Rasmussen, Stephanie A.; Conrad, Melissa D.; Nsobya, Samuel L.; Aydemir, Ozkan; Bailey, Jeffrey A.; Bayles, Brett R.; Rosenthal, Philip J.; Cooper, Roland A.

    Lancet Microbe (2021), 2 (9), e441-e449CODEN: LMAIAR; ISSN:2666-5247. (Elsevier Ltd.)

    Background Treatment and control of malaria depends on artemisinin-based combination therapies (ACTs) and is challenged by drug resistance, but thus far resistance to artemisinins and partner drugs has primarily occurred in southeast Asia. The aim of this study was to characterize antimalarial drug susceptibility of Plasmodium falciparum isolates from Tororo and Busia districts in Uganda. Methods In this prospective longitudinal study, P falciparum isolates were collected from patients aged 6 mo or older presenting at the Tororo District Hospital (Tororo district, a site with relatively low malaria incidence) or Masafu General Hospital (Busia district, a high-incidence site) in eastern Uganda with clin. symptoms of malaria, a pos. Giemsa-stained blood film for P falciparum, and no signs of severe disease. Ex-vivo susceptibilities to ten antimalarial drugs were measured using a 72-h microplate growth inhibition assay with SYBR Green detection. Relevant P falciparum genetic polymorphisms were characterised by mol. methods. We compared results with those from earlier studies in this region and searched for assocns. between drug susceptibility and parasite genotypes. Findings From June 10, 2016, to July 29, 2019, 361 P falciparum isolates were collected in the Busia district and 79 in the Tororo district from 440 participants. Of 440 total isolates, 392 (89%) successfully grew in culture and showed excellent drug susceptibility for chloroquine (median half-maximal inhibitory concn. [IC50] 20·0 nM [IQR 12·0-26·0]), monodesethylamodiaquine (7·1 nM [4·3-8·9]), pyronaridine (1·1 nM [0·7-2·3]), piperaquine (5·6 nM [3·3-8·6]), ferroquine (1·8 nM [1·5-3·3]), AQ-13 (24·0 nM [17·0-32·0]), lumefantrine (5·1 nM [3·2-7·7]), mefloquine (9·5 nM [6·6-13·0]), dihydroartemisinin (1·5 nM [1·0-2·0]), and atovaquone (0·3 nM [0·2-0·4]). Compared with results from our study in 2010-13, significant improvements in susceptibility were seen for chloroquine (median IC50 288·0 nM [IQR 122·0-607·0]; p<0·0001), monodesethylamodiaquine (76·0 nM [44·0-137]; p<0·0001), and piperaquine (21·0 nM [7·6-43·0]; p<0·0001), a small but significant decrease in susceptibility was seen for lumefantrine (3·0 nM [1·1-7·6]; p<0·0001), and no change in susceptibility was seen with dihydroartemisinin (1·3 nM [0·8-2·5]; p = 0·64). Chloroquine resistance (IC50>100 nM) was more common in isolates from the Tororo district (11 [15%] of 71), compared with those from the Busia district (12 [4%] of 320; p = 0·0017). We showed significant increases between 2010-12 and 2016-19 in the prevalences of wild-type P falciparum multidrug resistance protein 1 (PfMDR1) Asn86Tyr from 60% (391 of 653) to 99% (418 of 422; p<0·0001), PfMDR1 Asp1246Tyr from 60% (390 of 650) to 90% (371 of 419; p<0·0001), and P falciparum chloroquine resistance transporter (PfCRT) Lys76Thr from 7% (44 of 675) to 87% (364 of 417; p<0·0001). Interpretation Our results show marked changes in P falciparum drug susceptibility phenotypes and genotypes in Uganda during the past decade. These results suggest that addnl. changes will be seen over time and continued surveillance of susceptibility to key ACT components is warranted.
21. 21

    Linares, M.; Viera, S.; Crespo, B.; Franco, V.; Gómez-Lorenzo, M. G.; Jiménez-Díaz, M. B.; Angulo-Barturen, I.; Sanz, L. M.; Gamo, F.-J. Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay. Malar. J. 2015, 14, 441,  DOI: 10.1186/s12936-015-0962-2

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    Identifying rapidly parasiticidal anti-malarial drugs using a simple and reliable in vitro parasite viability fast assay

    Linares, Maria; Viera, Sara; Crespo, Benigno; Franco, Virginia; Gomez-Lorenzo, Maria G.; Jimenez-Diaz, Maria Belen; Angulo-Barturen, Inigo; Sanz, Laura Maria; Gamo, Francisco-Javier

    Malaria Journal (2015), 14 (), 441/1-441/8CODEN: MJAOAZ; ISSN:1475-2875. (BioMed Central Ltd.)

    Background: The emergence of Plasmodium falciparum resistance to artemisinins threatens to undermine the effectiveness of artemisinin-based combination anti-malarial therapy. Developing suitable drugs to replace artemisinins requires the identification of new compds. that display rapid parasite killing kinetics. However, no current methods fully meet the requirements to screen large compd. libraries for candidates with such properties. This study describes the development and validation of an in vitro parasite viability fast assay for identifying rapidly parasiticidal anti-malarial drugs. Methods: Parasite killing kinetics were detd. by first culturing unlabeled erythrocytes with P. falciparum in the presence of anti-malarial drugs for 24 or 48 h. After removing the drug, samples were added to erythrocytes prelabeled with intracellular dye to allow their subsequent identification. The ability of viable parasites to re-establish infection in labeled erythrocytes could then be detected by two-color flow cytometry after tagging of parasite DNA. Thus, double-stained erythrocytes (with the pre-labeled intracellular dye and the parasite DNA dye) result only after establishment of new infections by surviving parasites. The capacity of the test anti-malarial drugs to eliminate viable parasites within 24 or 48 h could, therefore, be detd. Results: The parasite viability fast assay could be completed within 48 h following drug treatment and distinguished between rapidly parasiticidal anti-malarial drugs vs. those acting more slowly. The assay was validated against ten std. anti-malarial agents with known properties and results correlated well with established methods. An abbreviated assay, suitable for adaptation to medium-high throughput screening, was validated and applied against a set of 20 compds. retrieved from the publically available Medicines for Malaria Venture 'Malaria Box'. Conclusion: The quantification of new infections to det. parasite viability offers important advantages over existing methods, and is amenable to medium-high throughput screening. In particular, the parasite viability fast assay allows discrimination of rapidly parasiticidal anti-malarial candidates.
22. 22

    Sanz, L.; Crespo, B.; De-Cozar, C.; Ding, X.; Llergo, J.; Burrows, J.; Garcia-Bustos, J.; Gamo, F. J. P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action. PLoS One 2012, 7, e30949,  DOI: 10.1371/journal.pone.0030949

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    P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action

    Sanz, Laura M.; Crespo, Benigno; De-Cozar, Cristina; Ding, Xavier C.; Llergo, Jose L.; Burrows, Jeremy N.; Garcia-Bustos, Jose F.; Gamo, Francisco-Javier

    PLoS One (2012), 7 (2), e30949CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)

    Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately det. the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artifacts, as viability and metab. are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compds. on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodol. to measure in vitro the direct effect of antimalarial compds. over the parasite viability, which is based on limiting serial diln. of treated parasites and re-growth monitoring. This methodol. allows to precisely det. the killing rate of antimalarial compds., which can be quantified by the parasite redn. ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to det. compd. killing activities that might be otherwise missed by traditional, metab.-based techniques. The anal. of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compds. based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compds., which are crucially needed for the control and possibly the eradication of malaria.
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    Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127– 132,  DOI: 10.1038/nbt1358

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    23

    A quantitative analysis of kinase inhibitor selectivity

    Karaman, Mazen W.; Herrgard, Sanna; Treiber, Daniel K.; Gallant, Paul; Atteridge, Corey E.; Campbell, Brian T.; Chan, Katrina W.; Ciceri, Pietro; Davis, Mindy I.; Edeen, Philip T.; Faraoni, Raffaella; Floyd, Mark; Hunt, Jeremy P.; Lockhart, Daniel J.; Milanov, Zdravko V.; Morrison, Michael J.; Pallares, Gabriel; Patel, Hitesh K.; Pritchard, Stephanie; Wodicka, Lisa M.; Zarrinkar, Patrick P.

    Nature Biotechnology (2008), 26 (1), 127-132CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)

    Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biol. consequences of multikinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. The authors present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global anal. of the results, the authors introduce the concept of a selectivity score as a general tool to quantify and differentiate the obsd. interaction patterns. The authors further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.
24. 24

    Jimenez-Díaz, M. B.; Mulet, V.; Viera, S.; Gomez, V.; Garuti, H.; Ibanez, J.; Alvarez-Doval, A.; Shultz, L. D.; Martínez, A.; Gargallo-Viola, D.; Angulo-Barturen, I. Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgammanull mice engrafted with human erythrocytes. Antimicrob. Agents Chemother. 2009, 53, 4533– 4536,  DOI: 10.1128/AAC.00519-09

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    Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rγnull mice engrafted with human erythrocytes

    Jimenez-Diaz, Maria Belen; Mulet, Teresa; Viera, Sara; Gomez, Vanessa; Garuti, Helen; Ibanez, Javier; Alvarez-Doval, Angela; Shultz, Leonard D.; Martinez, Antonio; Gargallo-Viola, Domingo; Angulo-Barturen, Inigo

    Antimicrobial Agents and Chemotherapy (2009), 53 (10), 4533-4536CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)

    Murine models of Plasmodium falciparum malaria may become crucial tools in drug discovery. Here we show that non-myelodepleted NOD-scid IL2Rγnull mice engrafted with human erythrocytes support an infectious burden up to tenfold higher than that supported by engrafted NOD-scid β2microglobulinnull mice. The new model was validated for drug discovery and was used to assess the therapeutic efficacy of 4-pyridones, selective inhibitors of P. falciparum cytochrome bc1.
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    Hultman, I.; Vedin, C.; Abrahamsson, A.; Winiwarter, S.; Darnell, M. Use of HμREL Human coculture system for prediction of intrinsic clearance and metabolite formation for slowly metabolized compounds. Mol. Pharmaceutics 2016, 13, 2796– 2807,  DOI: 10.1021/acs.molpharmaceut.6b00396

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    Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds

    Hultman, Ia; Vedin, Charlotta; Abrahamsson, Anna; Winiwarter, Susanne; Darnell, Malin

    Molecular Pharmaceutics (2016), 13 (8), 2796-2807CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)

    Design of slowly metabolized compds. is an important goal in many drug discovery projects. Std. hepatocyte suspension intrinsic clearance (CLint) methods can only provide reliable CLint values above 2.5 μL/min/million cells. A method that permits extended incubation time with maintained performance and metabolic activity of the in vitro system is warranted to allow in vivo clearance predictions and metabolite identification of slowly metabolized drugs. The aim of this study was to evaluate the static HμREL coculture of human hepatocytes with stromal cells to be set up inhouse as a std. method for in vivo clearance prediction and metabolite identification of slowly metabolized drugs. Fourteen low CLint compds. were incubated for 3 days, and seven intermediate to high CLint compds. and a cocktail of cytochrome P 450 (P 450) marker substrates were incubated for 3 h. In vivo clearance was predicted for 20 compds. applying the regression line approach, and HμREL coculture predicted the human intrinsic clearance for 45% of the drugs within 2-fold and 70% of the drugs within 3-fold of the clin. values. CLint values as low as 0.3 μL/min/million hepatocytes were robustly produced, giving 8-fold improved sensitivity of robust low CLint detn., over the cutoff in hepatocyte suspension CLint methods. The CLint values of intermediate to high CLint compds. were at similar levels both in HμREL coculture and in freshly thawed hepatocytes. In the HμREL coculture formation rates for five P 450-isoform marker reactions, paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), and 1-OH-midazolam (3A4) were within the range of literature values for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation rate was lower. Further, both phase I and phase II metabolites were detected and an increased no. of metabolites were obsd. in the HμREL coculture compared to hepatocyte suspension. In conclusion, HμREL coculture can be applied to accurately est. intrinsic clearance of slowly metabolized drugs and is now utilized as a std. method for in vivo clearance prediction of such compds. inhouse.
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    Hutzler, J. M.; Ring, B. J.; Anderson, S. R. Low-turnover drug molecules: A current challenge for drug metabolism scientists. Drug Metab. Dispos. 2015, 43, 1917– 1928,  DOI: 10.1124/dmd.115.066431

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    Low-turnover drug molecules: a current challenge for drug metabolism scientists

    Hutzler, J. Matthew; Ring, Barbara J.; Anderson, Shelby R.

    Drug Metabolism & Disposition (2015), 43 (12), 1917-1928CODEN: DMDSAI; ISSN:1521-009X. (American Society for Pharmacology and Experimental Therapeutics)

    In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metab. of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for low-turnover (slowly metabolized) drug mols. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a max. of 1 h with liver microsomes to 4 h with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compds. that are slowly metabolized. Thus, the challenge of accurately estg. low human clearance with confidence has emerged to be among the top challenges that drug metab. scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metab. of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodol. using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addn., more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HμREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metab. scientists with the most up-to-date exptl. options for dealing with the complex issue of low-turnover drug candidates.
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    Trager, W.; Jensen, J. B. Human malaria parasites in continuous culture. Science 1976, 193, 673– 675,  DOI: 10.1126/science.781840

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    Human malaria parasites in continuous culture

    Trager W; Jensen J B

    Science (New York, N.Y.) (1976), 193 (4254), 673-5 ISSN:0036-8075.

    Plasmodium falciparum can now be maintained in continuous culture in human erythrocytes incubated at 38 degrees C in RPMI 1640 medium with human serum under an atmosphere with 7 percent carbon dioxide and low oxygen (1 or 5 percent). The original parasite material, derived from an infected Aotus trivirgatus monkey, was diluted more than 100 million times by the addition of human erythrocytes at 3- or 4-day intervals. The parasites continued to reproduce in their normal asexual cycle of approximately 48 hours but were no longer highly synchronous. The have remained infective to Aotus.
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    Johnson, J. D.; Dennull, R. A.; Gerena, L.; Lopez-Sanchez, M.; Roncal, N. E.; Waters, N. C. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob. Agents Chemother. 2007, 51, 1926– 1933,  DOI: 10.1128/AAC.01607-06

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    Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening

    Johnson, Jacob D.; Dennull, Richard A.; Gerena, Lucia; Lopez-Sanchez, Miriam; Roncal, Norma E.; Waters, Norman C.

    Antimicrobial Agents and Chemotherapy (2007), 51 (6), 1926-1933CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)

    Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examd. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ∼0.5% parasitemia, resp. The MSF assay quality was significantly robust, displaying a Z' range of 0.73 to 0.95. The 50% inhibitory concns. for each drug and culture condition combination were detd. by using the MSF assay. Compared to the std. [3H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r2 ≥ 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.
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    Gubler, H.; Clare, N.; Galafassi, L.; Geissler, U.; Girod, M.; Herr, G. Helios: History and anatomy of a successful in-house enterprise high-throughput screening and profiling data analysis system. SLAS Discovery 2018, 23, 474– 488,  DOI: 10.1177/2472555217752140

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    Helios: History and Anatomy of a Successful In-House Enterprise High-Throughput Screening and Profiling Data Analysis System

    Gubler Hanspeter; Clare Nicholas; Galafassi Laurent; Geissler Uwe; Girod Michel; Herr Guy; Geissler Uwe

    SLAS discovery : advancing life sciences R & D (2018), 23 (5), 474-488 ISSN:.

    We describe the main characteristics of the Novartis Helios data analysis software system (Novartis, Basel, Switzerland) for plate-based screening and profiling assays, which was designed and built about 11 years ago. It has been in productive use for more than 10 years and is one of the important standard software applications running for a large user community at all Novartis Institutes for BioMedical Research sites globally. A high degree of automation is reached by embedding the data analysis capabilities into a software ecosystem that deals with the management of samples, plates, and result data files, including automated data loading. The application provides a series of analytical procedures, ranging from very simple to advanced, which can easily be assembled by users in very flexible ways. This also includes the automatic derivation of a large set of quality control (QC) characteristics at every step. Any of the raw, intermediate, and final results and QC-relevant quantities can be easily explored through linked visualizations. Links to global assay metadata management, data warehouses, and an electronic lab notebook system are in place. Automated transfer of relevant data to data warehouses and electronic lab notebook systems are also implemented.