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Cite This: ACS Infect. Dis. 2020, 6, 3−13 pubs.acs.org/journal/aidcbc
Setting Our Sights on Infectious Diseases
Manu De Rycker,† David Horn,† Bree Aldridge,‡ Richard K. Amewu,§ Clifton E. Barry, III,∥
Frederick S. Buckner,⊥ Sarah Cook,# Michael A. J. Ferguson,† Nathalie Gobeau,◇
Jennifer Herrmann,∇,○ Paul Herrling,◆ William Hope,● Jennifer Keiser,⦶,◓
Maria Jose Lafuente-Monasterio,⌀ Paul D. Leeson,◊ Didier Leroy,◇ Ujjini H. Manjunatha,⧫
James McCarthy,⬡ Timothy J. Miles,⌀ Valerie Mizrahi,⧩ Olena Moshynets,□ Jacquin Niles,■
John P. Overington,⊡ John Pottage,◨ Srinivasa P. S. Rao,⧫ Kevin D. Read,† Isabela Ribeiro,⬒
Lynn L. Silver,⬔ Jen Southern,☆ Thomas Spangenberg,★ Shyam Sundar,◮ Caitlin Taylor,⧩
Wes Van Voorhis,⊥ Nicholas J. White,▶ Susan Wyllie,† Paul G. Wyatt,† and Ian H. Gilbert*,†
†Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, University of Dundee,
Dundee DD1 5EH, United Kingdom
‡Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, United States
§Department of Chemistry, University of Ghana, P.O. Box LG56, Legon, Accra, Ghana
∥National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892,
United States
⊥Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, MS 358061, 750 Republican
Street, Rm E-606, Seattle, Washington 98109-4766, United States
#School of Humanities, University of Glasgow, 1 University Gardens, Glasgow G12 8QQ, United Kingdom
◇Medicines for Malaria Venture (MMV), PO Box 1826, 20 Route de Pre-́Bois, 1215 Geneva 15, Switzerland
∇Helmholtz Institute for Pharmaceutical Research Saarland, Department Microbial Natural Products, Saarland University, Campus
E8.1, 66123 Saarbrücken, Germany
○German Centre for Infection Research, partner site Hannover-Braunschweig, Germany
◆Independent Consultant, Switzerland
●Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, United Kingdom
⦶Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4051
Basel, Switzerland
◓University of Basel, CH-4001 Basel, Switzerland
⌀Tres Cantos Medicines Development Campus, Diseases of the Developing World (DDW), GlaxoSmithKline, Tres Cantos, Spain
◊Paul Leeson Consulting Ltd., United Kingdom
⧫Novartis Institute for Tropical Diseases (NITD), Novartis Institutes for BioMedical Research (NIBR), 5300 Chiron Way,
Emeryville, California 94608, United States
⬡QIMR Berghofer Medical Research Institute, 300 Herston Road, Hertson, Queensland 4006, Australia
□Biofilm Study Group, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, 150 Zabolotnoho
Street, Kiev 03143, Ukraine
■School of Engineering, Massachusetts Institute of Technology, Building 1-206, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139-4307, United States
⊡Medicines Discovery Catapult, Alderley Park, Alderley Edge, Cheshire SK10 4TG, United Kingdom
◨ViiV Healthcare, 980 Great West Road, Brentford, Middlesex TW8 9GS, United Kingdom
⬒Drugs for Neglected Diseases Initiative (DNDi), Chemin Louis-Dunant 15, 1202 Genev̀e, Switzerland
⬔LL Silver Consulting, New Jersey, United States
☆Lancaster Institute for the Contemporary Arts (LICA), The LICA Building, Lancaster University, Lancaster LA1 4YW, United
Kingdom
★Global Health Institute of Merck, Ares Trading S.A., a subsidiary of Merck KGaA Darmstadt Germany, Route de Crassier 1, 1262
Eysins, Switzerland
Received: September 25, 2019
Published: December 6, 2019
© 2019 American Chemical Society 3 DOI: 10.1021/acsinfecdis.9b00371
ACS Infect. Dis. 2020, 6, 3−13
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ACS Infectious Diseases Viewpoint
◮Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
⧩SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Institute of Infectious Disease and Molecular Medicine and
Wellcome Centre for Infectious Disease Research in Africa, University of Cape Town, Observatory, Cape Town 7925, South Africa
▶Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 3/F, 60th Anniversary
Chalermprakiat Building, 420/6 Rajvithi Road, Bangkok 10400, Thailand
ABSTRACT: In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK,
held an international conference with the aim of discussing some key questions around discovering new medicines for infectious
diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs
to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and
between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery,
translational, and clinical development processes. We started with an introductory session on the current situation and then
worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies,
drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
The general introductory session gave the background to thediseases and the problems involved,1 which was started by several University-Academic groups and several pharmaceutical
Nicholas White. There is a very serious inequity in health care companies that have institutes dedicated to global health and
across the world. Child mortality across the world is inversely have drug discovery programs, and other pharmaceutical
correlated to health spending per capita. Infectious diseases are companies are giving significant resources. The Wellcome
still a major cause of mortality and morbidity and, globally, are Trust, the Bill and Melinda Gates Foundation, and the NIH also
the second leading cause of death; respiratory and diarrheal make substantial contributions to Research and Development.
diseases as the major contributors, followed by tuberculosis and However, the combined resources from the public, charitable,
HIV. Treatments for many of these diseases are far from and private sectors, amounting to around $1 billion annually (for
satisfactory, with many drugs currently in use dating from early NTDs, malaria, and TB), pale in comparison with funds
in the 20th century, being essentially poisons; for example, allocated to the development of drugs for the developed world
arsenic used to treat sleeping sickness and antimonials, for conditions (annual BioPharma R&D spending is around $160
leishmaniasis. Successes have been seen in some areas, a primary billion) and do not satisfy the clinical need.
example being HIV-AIDS, which used to be a “death sentence”. With increased investment in drug discovery for neglected
There are now effective treatments that have been made diseases has come a better understanding of the respective drugdiscovery paths. For dengue for example, it has become clear
available to ∼60% of those infected with HIV, greatly extending that target-based approaches are very challenging, while
life expectancy. However, many infected individuals are unaware phenotypic screening has been more successful. In TB (and
of their status, meaning that many infected people continue to many other diseases), the need for pathophysiologically relevant
spread the disease. In other cases, the situation is static in vitro assays to ensure correlation between in vitro and in vivo
(tuberculosis) or worsening (malaria); although distribution (animal models) models and patients has been shown to be
of insecticide treated bed nets and the use of artemisinin-based critical. For instance, routine addition of glycerol to in vitro anti-
antimalarials are thought to have saved 6.8 million lives, case TB screens to increase growth rates yielded several false positive
numbers globally are currently increasing again. For some hits, due to the nonphysiological interplay between glycerol
diseases, such as hepatitis B and hepatitis C, which affect huge metabolism and the inhibitors.2 In the case of malaria,
numbers of people, there are drugs available, but they are outside phenotypic screening against parasites in human red blood
the budget of most patients and health care providers. Bacterial cells appears to be physiologically relevant as several compounds
infections remain a huge problem, which is exacerbated by the discovered in this assay are now in clinical development;
rise of antimicrobial resistance (AMR), which has a massive however, assays looking at different life stages (blood stage
negative effect on global health and the economy. AMR needs to forms, liver schizonts, gametocytes, hypnozoites) are required to
be approached on multiple levels. What used to be considered understand fully the clinical potential of new inhibitors.
“last resort” antibiotics are widely used in pigs and chickens, for Subsequent identification of the molecular targets for some of
example, so better stewardship is required. The economic model these malaria compounds has further increased the oppor-
for antibacterial drug discovery is broken and needs to be tunities to find compounds with suitable properties for clinical
changed to encourage the pharmaceutical industry to invest in development.
the development of new treatments. Newly emerging diseases, In addition to the drug discovery and development process,
such as SARS, “bird flu” (H5N1 with mortality rates of the search for new medicines for neglected diseases should also
approximately 50%), MERS, and Ebola, and these types of take into account the environmental, cultural, and social
outbreaks, potentially with increased transmissibility, remain environment of those affected by these diseases and their access
major threats. to healthcare. Finally, the main goal of this research is new
There are relatively few “players” in the discovery of new medicines not publications. Publications are important but
drugs for neglected diseases, as discussed by Paul Herrling. should not be allowed to drive the agenda.
These include some PDPs (Product Development Partnerships) By way of example of the challenges in neglected disease
and NGOs (Non-Governmental Organizations) such as clinical trials, Shyam Sundar gave an overview of ongoing work
Medicines for Malaria Venture (MMV), Drugs for Neglected on clinical trials for visceral leishmaniasis. This disease, fatal if
Diseases initiative (DNDi), and the TB Alliance. There are left untreated, predominantly affects people living in India, East
4 DOI: 10.1021/acsinfecdis.9b00371
ACS Infect. Dis. 2020, 6, 3−13
ACS Infectious Diseases Viewpoint
Africa, and South America but is also endemic in several there are also slow growing forms (“persisters”) that are less
European countries. A number of drugs are available, including susceptible to drugs. The complexity of human disease means
antimonials, amphotericin B, miltefosine, and paromomycin. that it is challenging to find an animal model that is predictive of
However, antimony is highly toxic, as is amphotericin B, unless human disease. Indeed, several expensive clinical trials have
in the liposomal formulation, which is very expensive, and failed in the past.
miltefosine requires a 28-day treatment course and is Recently, the marmoset (Callithrix jacchus) has been used as a
teratogenic. Trials of combination treatments using existing model, which is naturally infected with TB and more closely
drugs are underway, but there are significant differences in the replicates human disease as well as treatment outcomes. Using
efficacy of these therapies from one region to another. Particular linezolid, for example, TB lesions have been monitored by PET/
challenges in eliminating visceral leishmaniasis include post kala- CT (positron emission tomography/computed tomography)
azar dermal leishmaniasis, which can occur after the initial scanning in this model. This shows that the disease is very
infection has been treated, constituting a transmission reservoir; dynamic. From this, it is possible to see that treatment does not,
these patients often reject the relatively toxic treatment options. and may not need to, give rise to complete resolution of lesions,
In addition, HIV−visceral leishmaniasis coinfection is mutually but it pushes the equilibrium to a level where the immune system
reinforcing, difficult to treat, and common in some areas. The can re-establish control. This model for understanding disease
long-term aim is to develop new drugs and new combinations for progression has now been extended to human clinical studies.
visceral leishmaniasis. The imaging studies give a much clearer understanding of
At the conference, we also wanted to highlight the importance disease progression than measuring the numbers of bacteria in
of public engagement and increasing awareness of neglected sputum, for example. Imaging allows one to study the effects of
tropical diseases. Toward that end, artist Jen Southern described new treatments on individual lesions. This offers the possibility
a public engagement and contemporary art project, Para-Site- of having a major impact on phase 2 clinical studies.
Seeing, created in collaboration with Rod Dillon (entomologist/ James McCarthy presented the many benefits of malaria
microbiologist studying sandfly transmission of Leishmania in Volunteer Infection Studies (VIS). In this human challenge
Brazil3) realized as part of both Scotland’s NEoN Digital Arts model, healthy volunteers are injected with Plasmodium-infected
Festival [www.northeastofnorth.com] and the LifeSpace erythrocytes, resulting in a well standardized course of
Science Art Research gallery program [lifespace.dundee.ac.uk]. parasitaemia with little or no illness. Volunteers are treated
The multispecies relationships of Leishmania can be understood with experimental drugs on day 8 with the collection of both
on both microscopic and global scales, and the project cites pharmacokinetic data and pharmacodynamic data (parasitemia
Leishmania research within geographical, cultural, and social levels derived using PCR). This model allows early pharmaco-
histories. It communicates to audiences, in an engaging and kinetic/pharmacodynamic (PK/PD) characterization of new
accessible way, the journey of Leishmania, all told from the drugs and is ideally suited to investigate new drug combinations.
perspective of the parasite through a series of fictional travel As such, VIS are a game changer for malaria clinical
blogs shared on social media (using Twitter, Instagram, and development.
YouTube). Visitors to both the gallery and the online para-site- In the case of Chagas’ disease, the current burden of infection
seeing.org Web site can follow the story of the parasite in is primarily the chronic, indeterminate stage. This occurs after
photographs, poetry, video, and installations, in the gut of the the initial acute infection. The infected subject experiences little
sandfly, across the world, across the lab, and in its relationship or no clinical symptoms, and the parasites are difficult to detect
with the drugs being developed to eliminate it. in the blood but persist in various tissues. The disease is endemic
in the Americas but is also found in Spain and several countries
■ CLINICAL DEVELOPMENT AND CONNECTIVITY TO in Europe, primarily due to population flows. Isabela Ribeiro
THE PRECLINICAL SITUATION described a number of clinical trials using benznidazole,
Clinical development of candidate drugs takes a long time, costs nifurtimox, and the CYP51 inhibitors posaconazole and
a lot of money, and has a high attrition rate. Advances that can fosravuconazole. On the basis of trial data generated in the
speed up the clinical trials process and provide increased 1990s, benznidazole was recently (2017) approved by the FDA
understanding, such as why drugs work or fail, will be invaluable for use in children aged 2 to 12 years with a 60-day dosing
for infectious diseases, particularly where the patient popula- regimen. A more recent trial with benznidazole (BENDITA:
tions are in areas with limited facilities for conducting clinical NCT03378661) suggests that a shorter regimen of benznidazole
trials. The regulatory agencies (Food and Drug Administration may retain efficacy, while significantly reducing adverse side
[FDA] and European Medicines Agency [EMA]) are viewed as effects that currently lead to frequent treatment discontinuation.
highly amenable and progressive in their thinking but Trials assessing the CYP51 inhibitors all failed to show sustained
consultation should begin very early on in the process. However, parasite clearance.4,5 More encouragingly, a recent trial testing
innovation is sorely needed to improve the economic model and the sleeping sickness drug fexinidazole (DNDi-CH-FEXI-001:
to indicate direct clinical benefits. NCT02498782) showed high sustained parasite clearance rates
Clifton Barry described work on tuberculosis (TB). TB but also unexpected toxicity. A follow-up trial with lower doses is
continues to be a major problem worldwide with: at least 1.5 now ongoing in Chagas’ disease patients (FEXI12:
million deaths per year; a huge proportion of the world NCT03587766). Assessment of parasite clearance in clinical
population latently infected; rapidly rising levels of drug resistant trials typically involves multiple PCR samplings to detect
TB; long and complex treatment regimens. Current treatments parasite DNA in blood; however, parasite densities in blood are
typically require 6 months of therapy. The disease is further typically very low, and negative PCR results do not equate to
complicated by the presence of intracellular and extracellular cure. This presents particular challenges in the chronic stage,
bacteria, some in aerobic conditions and some in anaerobic where reservoirs of parasites may persist in inaccessible tissues
environments. There are also bacteria found in caseous and cardiac involvement may be present prior to therapy. To
granulomas, where many drugs cannot penetrate. Further, increase the sensitivity of the PCRmethod, multiple samples are
5 DOI: 10.1021/acsinfecdis.9b00371
ACS Infect. Dis. 2020, 6, 3−13
ACS Infectious Diseases Viewpoint
tested at multiple time points, but the lack of a simpler, more outputs will be more readily translated to in vivo efficacy. As
robust measure of cure remains a key issue to date. In the case of noted above, however, TB tissue distribution, persisters, and
Chagas’, on one hand, we need shorter trials, but on the other, dosing regimen and time frame are all factors that must be
long-term follow-up may be needed for the qualification of considered during the progression of potential combinations.
markers and the correlation to clinical impact. John Pottage discussed combination treatment in HIV
William Hope described aspects of clinical development of infection. There are currently 1.8 million new cases of HIV
antibacterials. Late-stage clinical trials for antibacterials are infection per year. Since therapy is life-long, toxicity can be
currently too costly, and biomarkers are often poorly predictive cumulative, so a key goal is to reduce dosing and long-term
of clinical benefit. Antibacterial stewardship will be particularly exposure. For HIV, approximately 40 medicines have emerged
important to reduce the widespread and profligate use of during a total of 38 years of the epidemic, but a cure has been
valuable drugs that could otherwise have an extended lifespan. documented for only two individuals following bone marrow
New antibacterial drugs need to be novel and address an unmet transplant; latent reservoirs remain a major challenge.
clinical need. Carefully planned clinical trials to demonstrate Combinations of nucleoside analogues, protease inhibitors,
efficacy and safety are a critical part of the drug discovery and integrase inhibitors are highly effective, but the GEMINI
process. Randomized clinical trials are the typical way to trials (NCT00105079) indicated that two-drug combinations
proceed; however, patient recruitment is very difficult, can be equally effective compared to three-drug combinations.
particularly MDR/XDR patients, at least in high income The question here is whether two-drug combinations will
countries. A noninferiority study relative to the standard of provide sufficient mitigation against the emergence of resistance
care is usually the starting point. As a consequence, relatively over a long period of time. Notably, clinicians favor a daily oral
prevalent complicated urinary tract infections are usually used formulation, whereas many patients favor a monthly parenteral
for the initial studies. Other types of infection can be more administration. This has led to the introduction of several long
challenging, because many of these patients are very sick, already acting parenteral antiretroviral agents into clinical development.
having been exposed to antibiotics and may have very different OlenaMoshynets reported on the combination of a macrolide
PK and PD relative to healthy volunteers. and colistin for treatment of carbapenem-resistant infections
due to Klebsiella pneumoniae. The rationale for this study was
■ COMBINATION THERAPY: CLINICAL AND that azithromycin is good at preventing biofilm formation in
PRECLINICAL SELECTION K. pneumoniae and increases the effectiveness of colistin. Biofilm
For many infectious diseases, combination therapy is used or is formation by bacterial opportunists during an infection process7
being considered. This can be for a variety of reasons: to reduce reduces antibiotic effectiveness. Colistin is a toxic antibiotic,
the risk of resistance; to increase the efficacy through synergy or with relatively low efficacy, which requires very high levels of
additive action; to tackle pathogens in multiple states and/or dosing. It is often dosed with other antibiotics. Some early
locations within the body; to broaden the spectrum of activity; clinical data were presented. Specifically, a combined anti-
to potentially reduce toxicity through reduced dosing. The bacterial therapy including azithromycin, colistin, and tienam
selection of combinations can be very challenging, however, and was applied in two cases of severe sepsis associated with bad
potential drug−drug interactions must also be considered. prognosis caused by carbapenem-resistant K. pneumonia. The
Didier Leroy described how combination therapies are now infection was completely eradicated in both cases. The
standard for malaria treatment, but many currently accessible experimental treatment was performed by Head of the
combinations contain artemisinin, meaning that artemisinin Department of Surgical Treatment of Infective Endocarditis of
failure would have a major detrimental impact on multiple Amosov National Institute of Cardiovascular Surgery, A.
therapies. As noted above, VIS may facilitate the relatively rapid Krykunov (Moshynets and Krykunov, manuscript in prepara-
testing of a number of new drug combinations against malaria. tion).
The humanized mouse model of malaria, involving SCID mice Maria Jose Lafuente reported on combination studies using
transfused with human red blood cells (Plasmodium falciparum Plasmodium falciparum. The tools available include in vitro
does not infect mouse red blood cells), allows for testing of even isobolograms, in vitro PRR (parasite reduction ratio) assay, and
greater numbers of combinations, which may be prioritized for the in vivo humanized mouse model. The time to recrudescence
testing based on in silico predictions. Between March 2017 and is used as the main PD end point, looking for an increase in time
April 2019, 23 combinations have been studied for PK/PD to recrudescence longer than that of each of the individual
relationships by the team of Dr. Inigo Angulo-Barturen (CEO of components.
the Art of Discovery in Bilbao), who pioneered the industrialized
HuSCIDmouse model for malaria. This approach is successfully ■ PK/PD STUDIES AND ANIMAL MODELS
revealing examples of both antagonism and synergy. Notably, Animal models of infection can be highly valuable in optimizing
dosing is typically applied at levels that yield recrudescence PK/PD before progressing into the clinic. However, it is critical
within 60 days such that drug efficacy can be compared in a that the animal models developed should be representative of
quantitative manner. human disease.
Checkerboard or isobologram analyses are typically used to There were two talks on cryptosporidiosis from Wes van
assess drug−drug interactions in vitro and their synergistic or Voorhis and Ujjini Manjunatha. The GEMS study8 highlighted
antagonistic impacts on pathogen viability. Bree Aldridge cryptosporidiosis as the second most common cause of diarrhea
described that there are now new, more efficient sampling and in children and of death in toddlers, and the MAL-ED study9
scoring methods that allow assessment of higher-order highlighted the link to stunting andmalnutrition. The only FDA-
combinations, specifically applied to the TB assay, such as approved drug for cryptosporidiosis is nitazoxanide, but this
DiaMOND (diagonal measurement of n-way drug interac- drug provides only a 34% improvement over a placebo and is
tions).6 These assays are now being applied to TB cultures in unsuitable for HIV-infected individuals or for children under 1
stress conditions rather than in “rich media” in the hope that the year old. The current Target Product Profile indicates the need
6 DOI: 10.1021/acsinfecdis.9b00371
ACS Infect. Dis. 2020, 6, 3−13
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for a therapy that is suitable for babies of less than 6 months, safe the primary site of Cryptosporidium infection is epithelial cells in
for presumptive disease (given the difficulties with current the small intestine, infections outside the GI tract have been
point-of-care diagnosis), inexpensive, and available in a liquid reported in humans,19 mainly in chronic immunocompromised
and stable formulation. A treatment for cattle is also desirable. HIV-positive cryptosporidiosis patients. However, such reports
Mice (Cryptosporidium parvum), calf (C. parvum), and piglet in children are sparse, and it remains to be seen how relevant a
(C. hominis) models of cryptosporidiosis are used preclinically. recently reported Cryptosporidium respiratory infection is to
Two compound series were described: a series of bumped kinase intestinal cryptosporidiosis, diarrhea, and a possible persistence
inhibitors (BKIs) being developed by the University of in the GI.19
Washington10−12 and PI(4)K inhibitors being developed by Formalaria, wheremany compounds are in the drug discovery
Novartis.13 For both of these series, efficacy in mouse models of pipeline, valid PK/PD models are essential to prioritize
infection is not dependent on plasma levels of compounds. In compounds for development. Nathalie Gobeau described how
the case of the BKIs, gastrointestinal (GI) epithelial cell the models are established first on the basis of efficacy and PK
concentrations in the terminal ileum and cecum, where data from the humanized SCIDmouse model, and then together
cryptosporidium predominately replicates, can be used to with the predicted human PK, predicted human doses are
predict in vivo efficacy. BKI GI ileocecal epithelial concen- calculated. Compounds with the lowest predicted dose are
trations can bemodeled byGastroPlus, and these modeled levels prioritized for testing in the above-mentioned human challenge
have been confirmed in mice during a time course after trials, which allows further tuning of the PK/PD model. It is
administering BKIs and harvesting organs for tissue levels post- critically important in malaria therapy to predict appropriate
mortem.14 Interestingly, efflux by Pgps (P-glycoprotein efflux combination partners, and both PK and PD interaction models
pumps) appears to reduce efficacy.15 BKIs that are effective in are used to rank potential combinations. The top combinations
mice have been shown to dramatically reduce diarrhea and are then tested once again in the VIS model (see above).
parasite excretion in both calf16,17 and piglet models18 of In liver stage malaria (pre-erythrocytic development),
established Cryptosporidium infection. Thomas Spangenberg reported that the activity on the
The Cryptosporidium PI(4)K inhibitors were identified from a Plasmodium berghei infected liver cell spheroids of a clinical
phenotypic screen of a focused new chemical library, and compound could be correlated with the corresponding in vivo
CpPI(4)K has been validated as a promising molecular target in efficacy mouse model by comparing IC99 (in vitro) and average
20
both immunocompromised mouse and neonatal calf models.13 systemic concentration (in vivo) values.
The lead candidate displays low clearance and medium to low The cryptosporidiosis case clearly shows that systemic blood
oral bioavailability across a range of species. Mass spectrometry exposure is not always the relevant PK parameter and that drug
imaging (MS-Imaging) is a label-free technique used to visualize exposure for the infectious organism will depend on the location
the spatial distribution of compounds by their molecular masses. of the organism in the human host. Thomas Spangenberg
MS imaging in mice showed that the compound is localized in discussed PK/PD models in schistosomiasis. Schistosoma
the gastrointestinal tract. Intravenous dosing of the compound is mansoni worms live in the mesenteric veins, and systemic
not efficacious, unlike the case of oral delivery, despite similar plasma exposure of praziquantel in the S. mansonimouse model
systemic exposure, indicating that systemic exposure alone is not is not predictive of efficacy. However, estimated portal vein drug
sufficient for efficacy. concentrations do predict the efficacy of praziquantel, indicating
Soft drugs (SD) are commonly used to limit systemic that exposure prior to the first pass metabolism in the liver is
exposure, thereby enhancing the therapeutic margin. An oral critical. This was supported through the use of a cytochrome
soft-drug approach, where systemic exposure was limited P450 metabolism inducer, which resulted in much lower
through fast metabolism after absorption, demonstrated that systemic exposure but no reduction in efficacy of praziquantel.
21
GI exposure is necessary and sufficient for efficacy in the In some pathogens, compound access can be restricted,
immunocompromised mouse model (Manjunatha et al. compounds effluxed or metabolized, affecting compound
unpublished results). As an alternative to the soft-drug exposure within the pathogen. Understanding this is of key
approach, compounds with the right balance of properties importance to understand PK studies. Caitlin Taylor outlined an
(permeability, solubility) can be developed with limited approach usingMycobacterium tuberculosis spheroplasts that lack
systemic exposure, while retaining sufficient permeability to a mycomembrane and peptidoglycan. The concept is to identify
access parasites. potentially promising inhibitory compounds that are not
In patients, watery diarrheal symptoms and shorter GI transit normally taken up. In turn, this knowledge will inform current
time may reduce efficacy; thus, efficacy in the more clinically efforts to understand drug transport and uptake in mycobacteria
relevant calf model and in the controlled human infection model with the aim of facilitating the development of more general
is important. For the soft-drug approach, it will be important to strategies for anti-TB drug delivery. Since small changes in the
consider the therapeutic window in the target patient structure of a compound can have a significant impact on
populations: diarrheal patients under 2 years; children with activity, her work exploits click chemistry using azide-derivatized
severe acute malnutrition (SAM); immunocompromised compounds developed by a collaborator (Dr. Mark Blaskovich,
children due to HIV infection or malnutrition with reduced University of Queensland) to establish intracellular localization
metabolic capacity, who are likely to have reduced metabolic within the (myco)bacterial cell.
activity.
PK/PD for cryptosporidiosis (at least in the mouse, calf, and ■ DRUG DISCOVERY PATHWAYS
piglet models of disease) is driven by gastrointestinal tract In this session, Manu De Rycker discussed drug discovery
distribution rather than systemic distribution, given that the GI pathways for kinetoplastid infections, Jennifer Keiser, helminth
tract may be the sole relevant niche for the parasite for diarrhea infections, Valerie Mizrahi, tuberculosis, and Lynn Silver,
and GI tract dysfunction, such as environmental enteric bacterial infections. There were common challenges among
dysfunction and subsequent malnutrition and stunting. Though these disease areas.
7 DOI: 10.1021/acsinfecdis.9b00371
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ACS Infectious Diseases Viewpoint
For many of these diseases, the preclinical development relevant validated targets, and the availability of appropriate cell-
pathway is poorly understood and in need of further based assays.
development. It is important to define the assays that are Helminth infections are poorly treated and lead to short-term
representative of human disease and can be used to triage issues such as diarrhea, fatigue, and anemia. The long-term
compounds and guide the chemistry optimization. This is consequences include malabsorption of food and growth and
particularly the case where there are no current clinical cognitive retardation. Over 250 million people are infected with
treatments or the only drugs are reactive or form reactive schistosomiasis. The only drug registered to treat this is
intermediates. A particular challenge is heterogeneity in praziquantel, and there is concern over resistance and hence a
infectious organisms. This is very obvious in helminth infections need for a new medicine. The parasite has a complex life cycle,
where different developmental stages as larvae and adult worms and it is not suitable for high throughput screening. A screening
might occur in infected people. Building in vitro assays that can cascade has been established, starting with the schistosomula,
assess all these stages is challenging, in particular for the adult which has the highest throughput. Actives are then screened
22
worms, which typically need to be isolated from animals, against the juvenile and adult worms, which are much lower
severely limiting the throughput of any assays. Usually, more throughput assays. However, there is good interlaboratory
easily accessible stages are used for primary high-throughput confirmation of activity. Several screens have been conducted28
screening, with the risk that compound activity will not translate using the malaria or pathogen box fromMMV, for example. In
to all disease-relevant stages.22 Other pathogens, such as addition, there are several interesting starting points from
Mycobacterium tuberculosis and most protozoan parasites, are various sources; e.g., lead compounds investigated several
also present in multiple di erent states. decades ago at Hoffmann La-Roche are currently beingff 29
Persister organisms pose another challenge.23 Persisters are studied. There are also a number of species of soil-transmitted
usually de ned as forms that are less susceptible to drug action, helminths (STH) that cause human disease: principally Ascarisfi
often due to some form of quiescence. Persister bacteria are well lumbricoides, Trichuris trichiura, and hookworms (Necator
described in TB and divided into class I persisters, a small americanus and Ancylostoma duodenale). It is estimated that 1.5
population that occurs stochastically, and class II persisters, billion people are infected with one or more STH, and only
where themicroenvironment triggers the persister state in a local limited treatment options lacking a broad spectrum of efficacyare available.30 Similar screening cascades have been developed
population. Recreating class I in vitro may not be possible, but for these pathogens. As is the case with schistosomes, these are
under certain conditions (such as hypoxia or other conditions low throughput. Unfortunately, there is very limited drug
that induce a state of nonreplication), class II persisters can be
24 discovery ongoing against helminths, despite the immenseobtained and used in phenotypic screens. Recently, persister clinical need.
parasites were also identified in vitro for Trypanosoma cruzi, and Antibacterial drug discovery is proving very challenging, and
the ability to kill these appears to be the best in vitro predictor of
25,26 the growing problem of antimicrobial resistance (AMR) is wellin vivo efficacy, emphasizing the need to dedicate sufficient documented. Despite the rise in antimicrobial resistance, the
time and resources to developing appropriate in vitro assays. commercial drivers are low and most large pharmaceutical
Another issue is that, for many infectious organisms, companies have pulled out of antibacterial drug discovery. As in
phenotypic hits often target a small set of common targets. other areas of anti-infectives research, both target-based and
For example, for TB, cell wall inhibitors and Qcrb inhibitors are phenotypic approaches have been used. Target-based ap-
frequently identi ed,27fi whereas the ergosterol biosynthetic proaches have a poor track record for antibacterial drug
pathway enzyme CYP51 is a very frequent target in T. cruzi discovery, for several reasons. Compounds either are not
phenotypic screens. To contend with this, secondary assays need taken up by bacteria or are effluxed before they can exert their
to be put in place to quickly identify such hits as compounds effect. Second, use of a single target has a high resistance
with novel modes of action are more desirable. Alternatively, potential. The qualifications for a target are as follows:
primary screens can be adjusted to avoid finding hits for these • It must be essential for survival of the bacterium both in
promiscuous targets, especially those with a history of failure in vitro and in vivo.
clinical trials, such as T. cruzi CYP51.
Another consideration is that in vitro assays can never fully • It must be possible to achieve selectivity compared to the
reproduce the in vivo situation and that key differences may exist human homologue, if any.
between animal models and humans. In this respect, the in vivo • It must have a useful antibacterial spectrum of activity.
microenvironment plays a key role and can trigger alternative • The target must be druggable.
metabolic states, provide bypass metabolites, or change • There must be a very low frequency of resistance, and
expression of key targets. As such, a target that is essential for there must not be existing resistant mutants. Ideally,
growth in vitromay not be required by all disease-causing stages. targets should be selected where multiple mutations are
This brings us to the long-standing question of target versus required for resistance.
phenotypic-based drug discovery approaches. Phenotypic hits Single target antibacterials will almost certainly need to be
have some key advantages, in particular that polypharmacology combined with other agents as a fixed dose or standard
may drive their activity, potentially resulting in higher efficacy or combination. This brings challenges, such as matching PK
lower resistance potential. Target-based programs have the key between the individual agents, and it can be difficult to
advantage of allowing rational design of inhibitors but may yield demonstrate clinical benefit to the regulators. There are limited
compounds with a higher likelihood of resistance, although this models for testing resistance development; hollow fiber
risk is attenuated when using such compounds in combinations. infection models (HFIM) are useful but lack in vivo factors,
There is value in both approaches, and the choice between them while high inoculum animal models are very difficult to achieve.
will depend on many factors, including resistance generation Another approach is to prepare compounds that target more
potential of the infectious organism, the availability of disease- than one enzyme; however, this is very challenging from a design
8 DOI: 10.1021/acsinfecdis.9b00371
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perspective and is further complicated by issues such as • It allows identification of any human homologues that can
selectivity compared to human homologues. be used to optimize selectivity.
Another approach to finding chemical starting points is • It can be used to circumvent pharmacokinetic or
phenotypic screening. However, this tends to have a low hit rate, toxicological issues using techniques such as scaffold
probably due to the challenges associated with uptake and efflux. hopping.
It also appears that chemical libraries screened are not optimal
for the chemical space required for compounds active against • If multiple series work through the same molecular target,
Gram-negative antibacterials.31,32 it can assist in portfolio management by providing
The entry of compounds into bacteria is potentially complex information that allows the series to be prioritized.
and not well understood. The cell envelope contains lipo- • It can be used to deprioritize compound series that work
polysaccharides, which are hydrated and repel hydrophobic through less attractive mechanisms, allowing resource
molecules. Penetration across the cell membrane is more typical reassignment.
of diffusion across lipid bilayers, requiring a degree of • It can assist in the rational selection of compounds for
lipophilicity, which is contradictory to the properties for passage combination therapy.
through the porins. Therefore, diffusion is very limited. In some The MoA group uses high-throughput genetics, cellular
cases, active transport is found across the plasma membrane. A biology approaches, and proteomics approaches to determine
Trojan horse strategy has also been used, piggybacking onto a the molecular targets of compounds. Their experience
molecule for active or facilitated transport. Further efforts demonstrates that no one technique or methodology is sufficient
should focus on understanding antibacterial drug delivery to determine the mechanism of action of every compound or
routes. Compound efflux is another issue, which is poorly series; thus the use of multiple, orthogonal, and unbiased
understood and affects many compounds that penetrate the approaches is crucial.
bacterial envelope, impacting compound levels within bacteria. Jacquin Niles has been developing new genetic tools for target
Furthermore, there is a potential for metabolism of compounds identification in malaria. Chemical (recombinant) genetics
by bacteria, and this also needs to be investigated. approaches are proving very powerful for drug target
Other strategies are being investigated, for example, externally deconvolution in Plasmodium falciparum, despite challenges
exposed bacterial targets. Host driven strategies could be associated with low transfection efficiency and with high
another fruitful area for further development. genomic AT bias. An engineered translational switch system
TB in particular is a complicated disease, with the bacterium has been used to toggle gene expression up or down. The system
found in multiple states and with different tissue distributions.33 can be used to screen for essentiality and also to assist in target
There is a need for new drugs that can tackle problems such as deconvolution for phenotypic screening hits. tRNA synthetase
drug resistance and dramatically shorten the current treatment inhibitors provide a proof of concept for a synthetic hyper-
regimens, which is 6 months, even for drug sensitive TB. There sensitivity approach, and further compounds have been
are many similarities with drug discovery for Gram-negative confirmed as on-target with scaling and multiplexing currently
bacterial infections. Target-based approaches are problematic underway.
due to challenges associated with getting compounds into Natural products remain one of the best sources of anti-
bacteria, efflux, and in the case of TB, metabolism of the infective drug leads. They contain an enormous chemical
compounds. As with Gram-negative bacteria (and most diversity and often exhibit new mechanisms of action.35,36 A key
infectious diseases), there are very few highly validated drug challenge is identifying novel natural product scaffolds that are
targets. In contrast to Gram-negative bacteria, phenotypic likely to have a therapeutic effect and obtain those in adequate
screening has identified a number of different compound series;
34 amounts for further development. Jennifer Herrmann discussedhowever, target deconvolution is complicated. how Myxobacteria are a rich source of novel natural products,
which have diverse biological activities.37 Modern bioinformatic
■ NEW START POINTS AND TARGETS and genomic approaches contribute to finding and modifying
In the case of infectious diseases, there are very few robustly these new natural products. The pipeline at Helmholtz Institute
validated drug targets. For many of these diseases, it is also for Pharmaceutical Research Saarland includes screening,
challenging to identify quality chemical starting points. This is compound isolation and structure elucidation, mode of action,
exacerbated in some disease areas where millions of compounds resistance and biosynthesis studies, scale-up, and targeted
have been screened phenotypically, and accessing chemical modification of interesting candidates. Promising natural
matter for screening that occupies a differentiated chemical compound classes from Myxobacteria include antibacterial
space is becoming increasingly challenging. Cystobactamids exhibiting broad-spectrum activity against all
38,39
Susan Wyllie talked about the work of the Mode of Action major multidrug resistant pathogens, antifilarial Corallopyr-
(MoA) group in Dundee, who employ multiple orthogonal onins depleting Wolbachia endosymbionts of nematodes
40,41
techniques to determine the molecular targets of compounds through targeting RNA polymerase, and antimalarial
that are phenotypically active against kinetoplastid parasites. Chlorotonils exhibiting activity against all erythrocytic stages
Targets identified in the course of these studies can be of Plasmodium falciparum including stages responsible for
42
considered as high-value drug targets, since they are associated transmission. The application of multidisciplinary method-
with a compound known to kill the parasite. Knowledge of the ologies supports and accelerates the preclinical development of
molecular target of phenotypically active compounds can natural products in various ways. To name a few examples,
facilitate the drug discovery process: resistance genes and related mechanisms can be identified using
biosynthetic gene cluster analysis, assisting mode of action
• It opens up the possibility of target (structure)-based studies.43,44 Semisynthesis is a good route to diversify and
projects against better validated targets, derisking optimize the potency and pharmaceutical properties of
medicinal chemistry optimization of compounds. molecules. Heterologous expression systems can significantly
9 DOI: 10.1021/acsinfecdis.9b00371
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increase product titers to improve natural product supply for and pathogen screens. There is also a “practice” effect, since
further investigations.45 there are large differences between companies, when comparing
A group at Novartis Institute for Tropical Diseases has been the properties of compounds that are acting on the same target.
working on a program for human African trypanosomiasis, as This may be due to the culture within the company and how hits
outlined by Srinivasa Rao. Their starting point was a phenotypic are selected and optimized.49
screen of >2 million compounds, which gave rise to about 1000 For oral drugs, the ligand efficiency (LE) and lipophilic ligand
hits that targeted multiple trypanosomatids. These were further efficiency (LLE) values show wide variability. However, it is
prioritized through a series of assays. Tractable scaffolds were important to note that the LE and LLE values tend to be greater
identified through clustering and identifying clusters with a large for drugs than the mean value of published inhibitors for the
variation in potency (∼3 log units). Further triaging used disease target.50,51 This is true even for drugs and candidate drugs
relevant assays. These included: cidality assays, looking at the discovered using phenotypic screens such as antimalarials and
concentration of compound and time to kill; a wash-off assay to for those in the “beyond rule-of-five” space. In general, where
predict reversibility; early assessment in animal models of there is a low dose of compounds, the improvement of LE and
infection; the use of physicochemical properties to optimize LLE over the target mean is even higher.
brain penetration. They also found that early identification of John Overington discussed a number of issues around drug
the mode of action was important to ensure compounds remain discovery that have been analyzed using a chemoinformatic
“on-target”. approach. It is important to understand the molecular targets of
Fred Buckner from the University of Washington in Seattle drugs and compounds in the pipeline. Looking at antibacterial
contrasted target-based and phenotypic approaches. Analysis drug discovery, there are still relatively few different mechanisms
from Swinney and Anthony46 indicates that the majority of rst targeted.52fi While some drugs target enzymes that are present in
in class new medicines are discovered through a phenotypic the bacteria but absent in the human host, actually, it is possible
approach, while the follow-on compounds are more often to obtain very good levels of selectivity between bacterial
discovered through target-based programs. Starting with the enzymes and their human orthologues, and we need to be careful
Target Product Profile is key; in the case of human African not to discard potentially good targets just because there is a
trypanosomiasis (HAT), this includes blood brain penetration. human orthologue. While overall the physicochemical proper-
They triaged compounds from a screen of >700 000 compounds ties of antibacterial compounds are different from those of
against the bloodstream-form of T. brucei. Triaging included the human drugs, there is also an influence of the target; so, drugs
rule-of-five, as well as avoiding singletons. Exemplars from the that target the ribosome often have very different physicochem-
scaffolds identified were investigated for CNS penetration, ical properties from those that target a typical cytoplasmic
53
leaving nine series. Following hit expansion, three series were enzyme.
further progressed. Compound series were terminated due to There are a number of different approaches that can be used
flat SAR, pharmacokinetic issues, and static rather than cidal for developing drug combinations. From the target perspective,
mechanisms of action. This was contrasted with a program there can be two different active sites on the same target, two
targeting methionyl tRNA synthetase that had the distinct different enzymes in the same pathway, or two enzymes in
advantage of being structurally enabled, facilitating preparation different pathways. From the compound perspective, there can
of highly potent and selective compounds. A major challenge is be single drugs targeting single targets; single drugs targeting
the CNS penetration. multiple targets; multiple drugs targeting the same target;
A presentation from Richard Amewu at the University of multiple drugs targeting multiple targets.
Ghana highlighted the issues of coinfections that are often found There are a number of ways that “combinations” can be used
in patients, a particular issue in malaria/TB. He discussed to tackle resistance of a particular target. For example, in
approaches for the design of compounds that target both switched sequential therapy, there is a change to a drug that is
pathogens. active against the mutant protein. In “blended therapy”, drugs
An analysis of current drugs, by Paul Leeson, revealed some that are competitive for the same active site but are active against
important information about what constitutes a good drug different mutants are dosed simultaneously.
molecule.47 Over the last 70 years, the average molecular weight Finally by mining data from different types of assays
of oral drugs has been increasing. This is in contrast to the clogP, (biochemical, cell-based, functional, animal models, DMPK,
which has remained reasonably constant (∼2.5), although it has and human clinical data), we should be able to improve the
risen slightly over the past few years, possibly due to the decision making process for when to progress a compound to
development of inhibitors of protein−protein interactions. It is further assays and what are the appropriate assays.
also instructive to see how the properties of molecules change as
the dose in increased.48 It is generally reported that metabolism ■ SUMMARY
and toxicity problems are likely to increase as the MW increases Many common themes ran through the presentations. In short,
above 400 Da and the clogP above 4. Therefore, it is important infectious diseases represent a huge unmet medical need, which
to see how safety margins are maintained as the dose increases. has a much more significant effect in Low and Middle Income
With smaller doses (<300 mg), clogP is about 3.0; average MW Countries. There is a need for new medicines, and the current
is about 360 Da, and about 50% of compounds are basic, while investment is insufficient. Furthermore, many of the patients are
35% are neutral and 10% are acidic. However, for high dose malnourished and many have multiple diseases, for example, TB
compounds (>1000 mg), the average clogP is ∼1; the MW is and HIV coinfection. Many are also children. This complicates
about 250 Da, and 45% of compounds are neutral, while 31% are the clinical development and clinical trials and places a high bar
acidic and 16% are basic. on safety and efficacy.
Optimum physicochemical properties vary from target to There is a need for smarter clinical trials to give an earlier read
target, due to differences in the size of the binding site, the out of likely efficacy, ideally before being taken to some quite
interactions available, and also access to the target in phenotypic challenging environments currently used for clinical trials.
10 DOI: 10.1021/acsinfecdis.9b00371
ACS Infect. Dis. 2020, 6, 3−13
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Approaches include using imaging technologies and human Jennifer Keiser: 0000-0003-0290-3521
challenge models. There is also a need for biomarkers to assist in Paul D. Leeson: 0000-0003-0212-3437
clinical trials for diseases such as Chagas’ disease. Patient Ujjini H. Manjunatha: 0000-0002-7461-9303
recruitment is not to be underestimated, for example, in the case Timothy J. Miles: 0000-0001-7407-7404
of bacterial infections. In many cases, the majority of the patients Valerie Mizrahi: 0000-0003-4824-9115
are infants and children, and this needs to be factored into the
clinical development of compounds. John P. Overington: 0000-0002-5859-1064
Combination therapy is already standard in many disease Kevin D. Read: 0000-0002-8536-0130
areas, and in other areas, it is likely to become more important. Thomas Spangenberg: 0000-0002-5654-8919
Combination therapy can have multiple facets: improvement of Susan Wyllie: 0000-0001-8810-5605
efficacy, for example, to achieve a single dose cure; broadening Paul G. Wyatt: 0000-0002-0397-245X
the spectrum of efficacy as for the soil transmitted helminths; Ian H. Gilbert: 0000-0002-5238-1314
reducing the risk of resistance; dealing with pathogens with Notes
widespread tissue distribution and population heterogeneity. The authors declare the following competing financial
The selection of combination partners is very challenging. interest(s): M.J.L.-M. and T.J.M. are employees of GlaxoS-
Ideally, this requires matched PK and that there are no clinically
− mithKline. U.H.M. is an employee of Novartis Institutes forsignificant adverse drug drug interactions. It is also important BioMedical Research and a shareholder in Novartis. J.P. is an
to make sure that the modes of action of the individual employee of ViiV Healthcare and a shareholder in Glax-
compounds are compatible (for example, not significantly oSmithKline. T.S. is an employee of Ares Trading SA, an affiliate
antagonistic and, where there is population heterogeneity, that of Merck KGaA, Darmstadt, Germany. S.P.S.R. is an employee
modes of action cover all forms of the pathogen). of Novartis Institutes for BioMedical Research. W.V.V. is part
Understanding the PK/PD is critical and can help to owner of ParaTheraTech LLC., a company that is developing
understand what is required for a drug. Therefore, the bumped-kinase inhibitors for use in animal health.
establishment of animal models of infection that are relevant
to human disease is very important. Knowledge of where the
pathogens are located within the body determines the required ■ ACKNOWLEDGMENTS
distribution of the compound. Various imaging technologies, The conference was in part supported by a Wellcome Trust
including mass spectroscopy imaging, are under development, Centre Award 203134/Z/16/Z. We would also like to
which can address these issues. acknowledge the following for providing travel bursaries to
For many of these diseases, drug discovery pathways have not allow scientists from Low and Middle Income Countries to
been established. Therefore, the determination of disease attend the meeting: the Bill & Melinda Gates Foundation
relevant assays and screening cascades is critical. It is important (OPP1199837); Medicines for Malaria Venture; Novartis
to develop assays to understand issues such as the susceptibility Institutes for Biomedical Research (NIBR); Drug for Neglected
of persisters or slow growing pathogens to compounds and Diseases initiative. We would also like to acknowledge Dr.
compound uptake and efflux from pathogens. The speed of kill Catharine Goddard and Diane Purves for their huge efforts in
of the pathogens is also an important factor in many cases. The organizing the conference and the help from the Administration
development of resistance is also a major problem. It is not Team of the Division of Biological Chemistry and Drug
always clear what is the correlation between the development of Discovery during the conference.
resistance in the laboratory setting and that in the clinic and also
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