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Pre-clinical Antiviral Testing for Dengue Virus Infection in Mouse Models and Its Association with Clinical Studies Satoru Watanabe, Jenny Guek Hong Low, and Subhash G. Vasudevan ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00054 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Infectious Diseases

Pre-clinical Antiviral Testing for Dengue Virus Infection in Mouse Models and Its Association with Clinical Studies

Satoru Watanabea, Jenny Guek-Hong Lowa,b and Subhash G. Vasudevana*

a

Program in Emerging Infectious Diseases, Duke-NUS Medical School, 8-College Road, b

Singapore 169857. Department of Infectious Diseases, Singapore General Hospital, 20 College Road, Singapore 169856

*Correspondence to: Subhash G. Vasudevan, Program in Emerging Infectious Diseases, DUKE-NUS Medical School, 8 College Road, Singapore 169857, Phone: +65 6516 6718, Fax: +65 6221 2529 Email: [email protected]

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At present, there are no licensed antiviral drug against dengue virus (DENV) infection. Mouse models of DENV infection have been widely used for pre-clinical evaluation of antivirals. However, only in a few instances so far have the data obtained from pre-clinical mouse model testing been associated with data from clinical studies in humans. In this review, we focus on the antiviral drugs targeting viral replication that have been tested in animals/humans, and discuss how pre-clinical drug evaluation in suitable mouse/animal models may be more fruitfully used to inform early phase clinical testing.

Key

words:

dengue

virus, antiviral

drug,

mouse model,


clinical

trial,

celgosivir

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Dengue virus (DENV) infection with any of the 4 related viral serotypes (DENV1–4) causes a variety of clinical manifestations ranging from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), characterized by vascular leakage, thrombocytopenia, bleeding and elevated levels of cytokines 1-3. Dengue is emerging as a global public health threat with an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly 4.

The first preventative dengue vaccine, Dengvaxia (CYD-TDV, Sanofi

Pasteur), has only recently been licensed for clinical use in a few countries 5, 6, and there are still no approved antiviral drugs. Antiviral strategy for dengue aims to inhibit viral replication and promote faster viremia clearance in patients, which is expected to shorten the febrile period in DF and curtail the progression to more severe dengue diseases (DHF/DSS) 7. Although some antiviral compounds have been tested in dengue patients, none of them could provide significant beneficial effect on viremia reduction or clinical parameters (discussed in more detail in section. 2). Animal models are pre-clinical tools to evaluate the in vivo efficacy of antiviral compounds or vaccines, and an increasing number of compounds have been shown to be capable of reducing viremia or preventing the progression toward severe diseases in mouse models. In this review, we summarize the antiviral compounds that were tested in mice and/or humans, and describe how their efficacies were evaluated and also provide some possible prospects for better preclinical drug assessment.

1. Pre-clinical drug testing against DENV infection in animals Currently there are no animal models capable of fully reproducing the spectrum of human dengue disease. The details of the development of animal models of DENV infection can be


3


found in previous reviews

8-10

Page 4 of 32

. In particular an extensive coverage of the various DENV disease

models from mouse to man that have been developed for addressing the mechanistic aspects of severe dengue in humans is reviewed in Chan et al 10. Briefly, non-human primate (NHP) models have been used for the development of vaccine or Ab-based therapy for dengue

11

, however,

antiviral drugs have seldom been tested in NHPs. Issues such as high cost, difficulty in obtaining large numbers of animals and the absence of severe dengue symptoms may limit the use of NHPs for the screening of antivirals. On the other hand, mouse models have been widely used for testing in vivo drug efficacy against DENV. Wild-type immunocompetent mouse models are seldom used for antiviral evaluation due to the major shortcoming of low/transient systemic infection (viremia) and a lack of severe dengue disease symptoms. Currently, AG129 mice, lacking IFN-α/β and γ receptors, are widely used for drug testing against DENV, even though the interpretative limitation still remains due to their immune incompetence. Development of mouse-adapted DENV strains as well as the use of non-mouse-adapted DENVs that possess high virulence in AG129 mice have enabled the assessment of several disease manifestations relevant to human dengue infection such as high viremia, increased levels of pro-inflammatory cytokines, thrombocytopenia, elevated hematocrit (vascular leakage) and death

10

. Thus, in vivo drug

efficacy can be readily evaluated by scoring for viremia reduction and/or protection from mortality in AG129 mice. It is noteworthy that, although many small molecule compounds have been shown to possess antiviral activities in vitro as assessed by DENV infection in cell lines or high throughput screening (HTS) assays, not all the drugs have been demonstrated to be effective in vivo, such as a pyrimidine synthesis inhibitor

12

, a dopamine agonist (bromocriptine)

13

and

other different types of compound (our unpublished data). Table 1 summarizes the compounds that have shown to be effective in mouse models.


4

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1.1 α-glucosidase inhibitors Iminosugars, substrate mimics that inhibit the host alpha-glucosidase enzyme, have been found to be effective against DENV in vivo. An initial study reported that castanospermine, derived from the Moreton Bay chestnut tree 14, reduced mortality at doses of 10, 50, and 250 mg/kg once a day (QD) in A/J mice (defective in complement 5), a mouse model which succumbs to DENV infection with signs of neuro-related disease

15

. Subsequently, celgosivir, a pro-drug of

castanospermine, was shown to be effective in AG129 mice by reducing 88% of peak viremia on day 3 post-infection (pi) at a dose of 75mg/kg twice daily (BID) 16. Celgosivir was further tested in AG129 mice for lethal DENV infection 17, 18 and shown to completely protect mice from death with significant reduction of peak viremia at 50mg/kg BID for 5 days. Notably the mortality was still reduced even when treatment was delayed for 48 hrs

17

. The same team showed that BID

regimen of 10, 25 and 50mg/kg was more protective than a single daily dose of 100mg/kg, indicating that celgosivir, when divided into two doses, is more effective than giving the same total dose once a day

18

. In vivo potency of celgosivir appeared to be 2-fold higher than

castanospermine although the in vitro potency was more than 100-fold higher 18. Armed with this background information, celgosivir was tested in a small placebo-controlled clinical trial (discussed later in section 2). The efficacy of another type of iminosuger, 1-deoxynojirimycin (DNJ), found in Mulberry

19

,

has also been demonstrated against DENV in mice. The related N-nonyl-DNJ (NN-DNJ) was found to reduce peak viremia by 93% at a dose of 75mg/kg BID for 3 days in AG129 with similar efficacy to celgosivir

16

. N-butyl-DNJ (NB-DNJ), which exhibits lower cytotoxicity

compared to NN-DNJ, was also shown to be effective at a dose of 500mg/kg BID for 7 days by


5


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scoring for the reduction of mortality and tissue viral load in a lethal DENV model 20. UV-4 (N9-methoxy-nonyl-DNJ), another DNJ derivative, protected mice from lethal DENV infection at 2.5, 5, 10, and 100mg/kg three times daily (TID) for 7 days accompanied with about 100-fold reduction of viremia at 100mg/kg on day 4 pi 21. The treatment was still effective when delayed for 48 hrs, but not beneficial when delayed for 72 hrs 21. This study also showed that UV-4 has better in vivo efficacy than NB-DNJ and similar to NN-DNJ but less toxic in mice when treated at 100mg/kg BID for 7 days. UV-4B (UV-4 hydrochloride salt) was also shown to be protective at the concentrations of 10-100mg/kg TID for 7 days even after 48 hrs delay in a lethal mouse model

22

.

Yet

another

iminosugar

UV-12

[(2R,3R,4R,5S)-2-(hydroxymethyl)-1-(8-

(tetrahydrofuran)-2-yl)octyl)piperidine-3,4,5-triol], also completely protected AG129 mice from lethal DENV infection at 20 or 100mg/kg TID for 7 days

23

. Interestingly, this compound

significantly reduced viral load in kidney and small intestine but not in serum, liver and spleen, and reduced serum levels of several cytokine/chemokine that were tested contrast to UV-4

21

23

, suggesting that, in

, the efficacy of this drug is tissue-dependent. Two oxygenated alkyl

iminosugar derivatives, CM-9-78 and CM-10-18, reduced peak viremia (2.3-fold and 1.8-fold, respectively) in AG129 mice, and combination therapy with ribavirin significantly increased antiviral activity of CM-10-18 with 4.7-fold reduction of peak viremia although ribavirin alone failed to reduce viremia 24. In a subsequent study, CM-10-18 was shown to be protective against lethal DENV infection at 75 or 150mg/kg BID until day3 pi and prolong mouse survival at 325mg/kg BID until day3 pi 25.

1.2 Nucleoside analog


6

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Several nucleoside analogs that inhibit the viral RNA-dependent RNA polymerase (RdRp), have demonstrated their effectiveness against DENV in mice. 7-DMA (7-deaza-2’-C-methyladenosine) was first shown to be effective at a dose of 50mg/kg BID for 3 days with 70% reduction of peak viremia

16

. Subsequently, NITD008 (7-deaza-2’-C-acetylene-adenosine) was

shown to be protective against DENV lethal infection at 3-50mg/kg BID for 3 days accompanied by reduction in peak viremia (1.6- to 6-fold) and pro-inflammatory cytokines such as IL-6 and TNF-α 26. The reduction in peak viremia and serum NS1 was observed even when the treatment at 25mg/kg BID was delayed for 48 hours pi 26. Furthermore, treatment with a single dose of 75300mg/kg at 12 hours p.i. could achieve more than 10-fold reduction in peak viremia, and the single-dose treatment at 75mg/kg provided full protection against lethal infection

27

. Despite

good anti-viral potency in vivo, no-observable adverse-effect level (NOAEL) could not be achieved when rats (10mg/kg/day) and dogs (1mg/kg/day) were dosed daily for 2 weeks

26

.

Another adenosine nucleoside analog NITD203 (3’,5’-O-diisobutyryl-2’-C-acetylene-7-deaza-7carbamoyladenosine), a prodrug of NITD449 (2’-C-acetylene-7-deaza-7-carbamoyladenosine), was tested for its efficacy in viremia reduction in AG129 mice 28. The compound reduced peak viremia at 3, 10, and 25 mg/kg BID for 3 days (2.7-, 4.4-, and 30-fold, respectively), and was found to be 3-fold more potent than NITD008 in vivo. However, the compound could not reach a satisfactory NOAEL in rat at >30 mg/kg/day for 2 weeks 28.

1.3 Other inhibitors NITD451 was identified through a HTS campaign of the Novartis compound library and found to inhibit translation in a viral RNA sequence-independent manner 29. This compound drastically reduced peak viremia by about 40-fold in AG129 mice at 25mg/kg QD for 3 days, however, mice


7


treated with 75mg/kg QD exhibited adverse effects

Page 8 of 32

29

. Another compound ST-148 was

identified through a HTS using a focussed chemical compound library and suggested to be targeting the viral capsid protein 30. The compound could significantly reduce peak viremia (up to 52-fold) as well as lower tissue viral load and serum cytokines level in AG129 mice at 50mg/kg either QD, BID or TID for 3 days

30

. ST-610, identified from the same chemical

compound library and found to inhibit NS3 helicase activity, also reduced peak viremia (up to 5.2-fold) as well as tissue viral load in AG129 mice at 100mg/kg either QD or BID for 3 days 31. Interestingly, however, this compound increased serum levels of some inflammatory cytokines such as IL-6 and MCP-1 31. The synthetic retinoid, 4-hydroxyphenylretinamide (4-HPR) that has been tested in humans for the treatment of several cancers, was identified as an inhibitor of the interaction of DENV NS5 protein with host nuclear transport proteins Importin through an alphascreen based assay of FDA-approved drugs

32, 33

.4-HPR was active against all four serotypes of

DENV and could reduce mouse mortality at either 20mg/kg QD or BID for 5 days

32

, and the

compound formulated as an oral powder in Lym-X-Sorb lipid matrix could drastically reduce viremia by 50-fold on day 7 pi at 90mg/kg BID starting at 24 hours prior to infection

34

.

Lovastatin, a cholesterol synthesis inhibitor that was shown to inhibit DENV2 replication in human PBMCs 35, was tested in a DENV lethal encephalitis AG129 mouse model after a clinical trial was initiated to test its efficacy in dengue patients

36

(discussed later in section 2). Pre-

treatment of lovastatin with a single dose (24 hrs before infection) or three doses (72, 48 and 24 hrs before infection) at 100mg/kg prolonged mouse survival and only latter regimen resulted in a 22% decrease in peak viremia37. Interestingly, however, viremia was enhanced 1.2-fold when the treatment was delayed by 24 hrs and/or 48 hrs, although the survival rate still increased, suggesting that the effect of lovastatin on viremia depends on the timing of treatment


37

.

8

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Recently, the immunocompetent wild type C57BL/6 was used for the testing the anti-DENV efficacy of bortezomib, a FDA-approved reversible proteasome inhibitor

38

since the ubiquitin

proteasome pathway (UPP) has been implicated to be important for DENV replication in several studies

39-41

. Although not as robust as the immunocompromised mouse model, a single dose of

Bortezomib at 1mg/kg at 6 hrs pi reduced viral load in spleen and prevented some signs of dengue pathologies.

2. Antiviral therapy for DENV infection in clinical settings Since 2008, four clinical trials of antiviral drugs for dengue have been completed

36, 42-44

, and

one is currently underway (ivermectin: NCT02045069). Not included in this account is the testing of the corticosteroid prednisolone to counter the dysregulation of immune responses in severe dengue. It was tested in a randomized trial with 225 patients in three arms (75 patients in each arm) – placebo, 0.5mg/kg or 2mg/kg prednisolone, and was found to not impact the progression to severe diseases or show any association with prolonged viremia or cause other significant adverse effects

45

. From the perspective of antiviral drugs, chloroquine was the first

small molecule inhibitor that was tested in humans as a potential antiviral drug against DENV infection. Chloroquine is a well-tolerated 4-aminoquinoline class of drug that is widely used as an anti-malaria drug. The compound inhibits DENV replication in vitro modulating the acidic condition in endosomes

48

46, 47

presumably by

which is necessary for fusion. Amodiaquine

which also belongs to the same class of anti-malaria drugs has been reported to be more efficacious in vitro

49

by inhibiting autophagy

50

, which appears to be required for virus

replication 51. The effect of chloroquine against DENV infection in animal models has not been reported. However, our unpublished data showed that chloroquine treatment at 50mg/kg BID


9


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started at the time of infection did not suppress peak viremia in AG129 mice. A double-blind, randomized, placebo-controlled trial was conducted in 307 adults with suspected acute DENV infection

42

. Patients with illness of less than 72 hours duration were randomized to a 3-day

course of chloroquine (n=153) with 600mg on day1 and 2, and 300mg on day3, or placebo (n=154). The major endpoints were time to resolution of viremia, serum NS1 level and fever. The treatment showed a trend toward a longer duration of viremia but no difference in the time to resolution of serum NS1. There was also a trend that correlated the reduction in fever clearance time with lower incidence of DHF in patients treated with chloroquine probably due to its known immunomodulatory properties 48. However, differences in levels of T cell activation or serum cytokines between chloroquine and placebo treated groups could not explain the trend toward lowering fever or incidence of DHF

42

. A second clinical study conducted with 500mg

twice daily provided no significant impact on the duration of dengue disease or fever

52

.

However, patients treated with chloroquine experienced less pain and better performance of daily activities, suggesting chloroquine may have some benefits for relieving dengue symptoms. Balapiravir (R1626) is a prodrug of a nucleoside analogue (R1479) and was developed for the treatment of chronic hepatitis C Virus (HCV) infection

53

. The compound could inhibit DENV

replication in human cells with mean half maximal effective concentration (EC50) values of 1.9– 11µM 43. An exploratory, dose-escalating, randomized placebo-controlled trial was conducted in adult male patients with acute DENV infection within 48 hrs of fever onset

43

. Patients were

treated with 1500mg (n=10) or 3000mg (n=22) of balapiravir or placebo (n=32) every 12 hrs for 5 days (total 10 doses). Balapiravir treatment failed to result in any beneficial effect on the time to resolution of viremia, serum NS1 level and fever as well as hematological and immunological profiles. Further study suggested that balapiravir loses its antiviral potency in the cells pre-


10

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infected with DENV

54

, which may possibly explain its lack of efficacy in patients. Moreover,

balapiravir also failed to show a reduction in peak viremia and protection from lethality in AG129 mice even at a dose of 100mg/kg BID for 3 days

54

, suggesting that in vivo efficacy of

this compound against DENV infection is poor. Celgosivir was tested in a randomized, double-blind, placebo-controlled trial in 50 adult with acute dengue infection

44

. Pre-clinical study showed that celgosivir had low micromolar EC50

against all 4 DENV serotypes in in vitro cell-based assays 17. Furthermore in the AG129 animal model, the compound had 100% protective efficacy against lethal infection at 50mg/kg BID

18

,

which is comparable to a clinical dose (initial 400mg loading dose followed by 200mg twice daily for 5 days) 44 when translated to human equivalent dose (HED)

55.

Thus, celgosivir became

the first dengue antiviral drug to be tested in infected patients where the antiviral effect was preclinically evaluated in an animal model prior to the initiation of the Phase 1b clinical trial. In humans, mean virological log reduction (VLR) from baseline for days 2, 3, and 4 was greater in celgosivir group than placebo group, however, the difference was not statistically significant

44

.

Clearance of serum NS1 was faster in the celgosivir group than in the placebo group in patients with secondary infection, but the difference was also non-significant. There were also nonsignificant trends of effect on better platelet count and hematocrit in secondary dengue patients 56, but no trend toward beneficial effect on clinical parameters including fever 44. Further evaluation of the lack of efficacy of celgosivir in the clinical trial was carried out in the AG129 mouse model and is elaborated in Section 3. Lovastatin, a cholesterol synthesis inhibitor, can inhibit DENV replication in human cells

35, 57

.

In addition, statin is known to stabilize endothelial function by inhibiting the expression of proinflammatory cytokines and controlling leukocyte migration and proliferation


58, 59

. Thus the

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drug was hypothesized to prevent severe dengue caused by vascular leakage. A randomized, double-blind, placebo-controlled trial was conducted in 300 DENV NS1-positive adult patients presenting within 72 hrs of fever onset, over a 5-day course of 80mg lovastatin (n=149) or placebo (n=151)

36

. Lovastatin treatment could not show a beneficial effect on any of the pre-

defined clinical or virological parameters such as time to resolution of viremia and serum NS1. In a mouse model study, which was reported while the trial was in progress, viremia was found to be enhanced when the treatment was delayed by 24 hrs and/or 48 hrs 37, suggesting that the in vitro effect of lovastatin does not translate to in vivo efficacy, especially with delayed treatment.

3. Future directions for pre-clinical in vivo studies Although an increasing number of antiviral compounds have been shown to possess in vivo efficacy in the mouse model, only celgosivir has been trialed in humans after extensive evaluation in pre-clinical tests so far. This was a precious experience to know the association of the results of pre-clinical animal test with human clinical studies. Here we describe our recent animal studies that may guide possible future directions for pre-clinical tests.

3.1 Assessment of viremia reduction In past studies using mouse models of dengue, the levels of “peak viremia reduction” have been taken into account to assess the antiviral effect of the drugs (Table 1). The reduction can be observed even when the initiation of treatment was delayed for 48 hrs in some cases (Table 1). In contrast, most of human dengue patients exhibit peak viremia when they seek medical attention. This makes it challenging to use the reduction in peak viremia level by antiviral treatment as a clinical endpoint. Consequently, “time to resolution of viremia” has been considered as a major


12

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endpoint to assess the antiviral efficacy (Table 2). However, none of the compounds clearly induced faster reduction of viremia in humans. Consistent with this, a follow-up study for celgosivir using a non-lethal AG129 mouse model showed that twice daily dosing at 50mg/kg, as was used in the human trial

44

, significantly reduced peak viremia when dosing commenced on

day 0, however, it resulted in a poor outcome when treatment begun at the time of peak viremia (Fig.1), suggesting that in vivo antiviral efficacy becomes less prominent once viremia reaches the peak level

60

. This effect was also observed for NITD008, a potent directly acting antiviral,

indicating that it was not related to celgosivir being a host-targeted drug 60. Viremia clearance is associated with the immune status of the patients and virus serotype/genotype 61, 62. In the case of adult patients, the viremia reduction with secondary infection has been reported to be significantly greater than that for primary infection

44, 62

, most probably due to the rapid

reactivation of the acquired immunity. In addition to the difficulty in showing good drug efficacy during acute viremia phase as observed in mice, the wide variation in viremia reduction rate in humans also complicates comparison of mean VLR between celgosivir- and placebo-treated groups

44

. On the other hand, increasing the dosing frequency of celgosivir, to four-times-daily,

was found to be beneficial for faster viremia clearance even when the mice were viremic

60

,

suggesting that an optimization of dosing regimen may improve the drug efficacy in patients. Based on this data, a Phase II clinical trial to test the efficacy of celgosivir in adult dengue patients using a new dosing regimen has been approved in Singapore (NCT02569827), but the trial has not commenced. Results from this trial may provide valuable new insights that can shape the strategies for future antiviral drug development for DENV.

3.2 Assessment of disease severity/dengue-associated morbidities


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“Prevention of mortality” is another major parameter for assessing antiviral drugs in mice (Table 1). Lethal dengue AG129 mouse models have exhibited some of the human dengue disease manifestations such as high viremia, elevated levels of cytokines, vascular leakage, intestinal bleeding, and thrombocytopenia

10

, although the details of DENV pathogenesis in

humans remain largely unknown because of difficulty in obtaining human tissues biopsies. Recent study in AG129 mice showed that kinetics of viral load vary among different tissues and does not always correlate with serum viremia level 63. In this model, the magnitude of infection and cytokines production in small intestine seem to be responsible for disease severity which can be prevented by anti-TNF antibody

63

. In this aspect, it is conceivable that a drug that can

decrease tissue viral load which is directly associated with disease severity, may prevent progression to severe disease in patients even when serum viremia clearance is not significantly impacted. One of the interpretative limitations of the observations in the mouse model is its relevance to humans. It is worth noting that when patients are classified as having DHF/DSS, infectious virus is almost no longer detectable in blood 64-66. Different from human cases, AG129 mice die of DHF/DSS-like diseases only 1-2 days after reaching peak viremia levels

60

. This

makes it challenging to evaluate the drug efficacy especially on viremia clearance and its correlation to disease severity, which is one of the drawbacks of the current mouse models. Since only a small proportion of dengue patients develop DHF/DSS, a large patient cohort would be required to assess the antiviral efficacy in association with prevention of severity/morbidities.

3.3 Non-invasive imaging approach DENV infection is associated with increased pro-inflammatory cytokine production which is believed to be linked to severe dengue by disrupting the integrity of vascular endothelial lining


14

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and causing increased vascular permeability 67. In lethal AG129 mouse models, anti- TNF-α Ab can consistently protect mice from mortality without reduction in viremia

63, 68

, indicating that

enhancement of TNF-α production is responsible for disease severity in the mouse model.

18

F-

fluorodeoxyglucose (FDG)-PET is a widely used medical imaging probe to identify sites of abnormal glucose metabolism in the body, and is most often used for the diagnosis/staging of cancer 69. FDG-PET is also used clinically for the early detection of metabolically active cellular components in inflammatory diseases

70

, thus, FDG-PET may have the potential to identify

active sites of inflammation during acute DENV infection in patients. A recent study in AG129 mice model showed that FDG uptake was visualized most prominently in intestines that correlated with increased viral load and pro-inflammatory cytokines 71. Notably, FDG uptake in intestines is also responsive to celgosivir treatment 71, therefore, this approach may help to noninvasively define inflammation reduction as a surrogate biomarker for drug efficacy during preclinical development and possibly even early phase clinical validation.

Conclusion Although no animal model can mimic the course of human dengue infection, AG129 mice have been widely used for the evaluation of antiviral compounds. The lethal mouse model could be suitable for the initial drug evaluation to determine whether the candidates have potential in vivo efficacy by scoring for peak viremia reduction and protection from mortality. As a second step, based on the knowledge that dengue patients are already at peak viremia at the initiation of treatment, a non-lethal viremia model that enable one to follow up the kinetics of viremia clearance could be useful to inform appropriate dosing regimens for human trials. In addition, non-invasive imaging technology that can identify active sites of inflammation during the course


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of acute disease, such as FDG, may serve as a surrogate biomarker for assessing treatment response during therapeutic intervention trials not only for antivirals but also for immune modulators.

Acknowledgements The support of the National Medical Research Council of Singapore grant NMRC/CBRG/2016 to SGV and National Medical Research Council/ Open Fund - Young Investigator Research Grant/0003/2016 to SW is acknowledged. NMRC/CTGCoD-1001 grant to JGL is specifically acknowledged for ongoing dengue translational studies in collaboration with 60° Pharma PLC.

List of abbreviations DENV: dengue virus, DHF: dengue hemorrhagic fever, DSS: dengue shock syndrome, NHP: non-human primate, HTS: high throughput screening, pi: post-infection, hr(s): hour(s), QD: once a day, BID: twice a day, TID: three times a day, QID: four times a day, NOAEL: satisfactory noobservable adverse-effect level

Author Information Biography Satoru Watanabe ([email protected]) received his Ph.D. degree from Tokyo Medical and Dental University in the field of Medical Science. He is currently Principle Research Scientist of the EID Experimental Therapeutics Laboratory at Duke-NUS Medical School. His current research is mainly focussed on pathogenesis study of flaviviruses.


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Jenny Low ([email protected]) received her medical degree from the National University of Singapore and obtained Masters in Public Health training with the Johns Hopkin University (Bloomberg School of Public Health). She is currently an infectious diseases senior consultant with the Singapore General Hospital and regular rank Associate Professor at the Duke-NUS Medical School. Her current research focus is on host immunity against acute viral diseases and early phase clinical trials.

Subhash Vasudevan ([email protected]) received his Ph.D. degree in Biochemistry from The Australian National University (ANU). He was the founding Unit Head of Dengue Research at the Novartis Institute for Tropical Diseases and moved to Duke-NUS Medical School where he is presently a Professor and Lead Principal Investigator of the EID Experimental Therapeutics Laboratory. His research mainly focuses on therapeutics discovery for flaviviruses. All authors contributed to the preparation of this manuscript.

Conflict of Interest SGV has received grants from Janssen Pharma and support from 60° Pharma. JGL collaborates with 60° Pharma.


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Figure legend

Fig. 1. The efficacy of celgosivir against clinical DENV strains initiated either on day 0 or day 3 post-infection in AG129 mice. Clinical DENV2 strains were expanded ex vivo from sera of 3 patients (#031, #013 and #036) who participated in celgosivir treatment trial

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. AG129 mice were inoculated i.v. with DENV2

of #031 (2 x 107 pfu), #013 (1 x 107 pfu) or #036 (2 x 107 pfu). Celgosivir treatment at 50mg/kg BID was started either on day 0 or day 3 pi. Vehicle control mice (VC) received PBS with the same regimens. Serum samples were subjected to realtime RT-PCR to measure viral genome RNA copy number. The graphs show the average results with standard deviations. The VLR from day 3 to day 6 was also shown as the mean value and standard deviations. A P values were determined by 2-tailed Student t test analysis. The number of mice per group is 6. (Source: The data is adapted from Watanabe et al., Antiviral Research, 2016 59 and presented here with figure modifications).


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Table 1: Antiviral compounds shown to be effective on DENV infection in animal models Compound Animal /Virus Regimen Endpoints Reference s 10Mortality Whitby et Castanospermin A/J mouse Mouse250mg/kg/dose al (2005) e 15 neuroadapted i.p. QD until (α α-glucosidase DENV-2 (NGC) day10 pi from inhibitor) day0

Celgosivir (6-Obutanoyl castanospermine ) (α α-glucosidase inhibitor)

AG129 mice Mouse-adapted DENV-2 (S221)

50mg/kg/dose Mortality i.p. BID until day4 pi from day0

AG129 mice Non-adapted DENV-2 (TSV01)

7.5 or Peak viremia Schul et al 75mg/kg/dose (day3 pi) (2007) 16 p.o. BID until day3 pi from day0 or 1 pi


50mg/kg/dose Mortality i.p. BID until Viremia day5 pi from and 3 pi) day0, 1 or 2 pi


10100mg/kg/dose i.p. BID or QD for 5 days from day0 pi AG129 mice 50mg/kg/dose Mouse-adapted p.o. BID for 3 DENV-2 (S221) days from day0 or Non-adapted 3 pi DENV-1 and 50mg/kg/dose DENV-2 clinical p.o. QID until strains day6 pi from day2 or 3 pi AG129 mice 50mg/kg/dose Mouse-adapted p.o. QID for 2 DENV-2 (S221) days from day2 pi Non-adapted DENV-2 (EDEN3295) NN-DNJ (N- AG129 mice Non-adapted nonyl-DNJ)

Watanabe et al 18 (2012)

Rathore et (day1 al (2011) 17

Mortality Watanabe Peak viremia et al (day3 pi) (2012) 18

Mortality Watanabe al Viremia (day1-4 et 60 (2016) pi) Virological log reduction (VLR: day3-6 pi)

Mortality Viremia, tissue viral load and cytokines (day1-5 pi) PET imaging (18F-FDG uptake) 75mg/kg/dose Peak viremia p.o. BID for 3 Splenomegaly


Chacko et al (2017) 71

Schul et al (2007) 16

26

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and serum cytokines (day3 pi) 125Mortality Miller et al NB-DNJ (N- AG129 mice Mouse-adapted 500mg/kg/dose Peak viremia and (2012) 20 butyl-DNJ) DENV-2 (D2S10) i.p. BID for 7 tissue viral load (α α-glucosidase days from day0 pi (day3.5 pi) inhibitor) (α α-glucosidase inhibitor)

DENV-2 01)

(TSV- days from day0 pi

UV-4 (N-9- AG129 mice Mouse-adapted methoxy-nonylDENV-2 (S221) DNJ) (α α-glucosidase inhibitor)

2.5100mg/kg/dose i.g. BID or TID for 7 days from day0 or 1-3 pi

UV-4B (UV-4 AG129 mice Mouse-adapted hydrochloride DENV-2 (S221) salt) (α α-glucosidase inhibitor)

10Mortality 100mg/kg/dose Weight p.o. TID for 7 Clinical score days from day0, 1 or 2 pi

Warfield et al (2016)

20 or 100mg/kg/dose p.o. TID for 7 days from day0 pi

Warfield et al (2015)

Mortality Viremia, tissue viral load and serum cytokines (day3-4 pi) 75mg/kg/dose Peak viremia CM-9-78, CM- AG129 mice Non-adapted p.o. BID until (day3 pi) 10-18 DENV-2 (TSVday3 pi from day0 (α α-glucosidase 01) inhibitor) UV-12 (α α-glucosidase inhibitor)

CM-10-18 (α α-glucosidase inhibitor)

7-DMA (nucleoside analog)

NITD008 (nucleoside analog)


Mortality Perry et al Viremia, tissue (2013) 21 viral load and serum cytokines (day1-4 pi)

AG129 mice Mouse-adapted DENV-2 (D2S10) Non-adapted DENV-2 (D2Y98P) AG129 mice Non-adapted DENV-2 (TSV01)

3-150mg/kg/dose Mortality p.o. BID until day3 pi from day0

50mg/kg/dose Peak viremia p.o. BID for 3 Splenomegaly days from day0 pi and serum cytokines (day3 pi) Mortality AG129 mice 1-50mg/kg/dose Non-adapted p.o. BID until Peak viremia, DENV-2 (TSV- day3 pi from serum NS1 and day0, 1 or 2 pi serum cytokines 01) Mouse-adapted (day3 pi)


22

23

Chang et al (2011) 24

Chang et al (2011) 25

Schul et al (2007) 16

Yin et al (2009) 26

27


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DENV-2 (D2S10)

AG129 mice Non-adapted DENV-2 (TSV01) Mouse-adapted DENV-2 (D2S10)

NITD203 (nucleoside analog)

25Mortality Chen et al 300mg/kg/dose Peak viremia (day (2010) 27 p.o. Single dose at 3 pi) 12 hrs pi

AG129 mice 3-25mg/kg/dose Peak viremia Chen et al Non-adapted p.o. BID for 3 (day3 pi) (2010) 28 DENV-2 (TSV- days from day0 pi 01)

AG129 mice NITD451 (RNA translation Non-adapted DENV-2 (TSVinhibitor) 01)

25 or Peak viremia Wang et al 75mg/kg/dose (day3 pi) (2011) 29 s.c. QD for 3 days from day0 pi

ST-148 (capsid inhibitor)

AG129 mice Non-adapted DENV-2 (NGC)

50mg/kg/dose i.p. QD or BID for 3 days from day0 pi p.o. TID for 3 days from day0 pi

Peak viremia, Byrd et al tissue viral load (2013) 30 and serum cytokines (day3 pi)

ST-610 (helicase inhibitor)


3-100mg/kg/dose i.p. QD or BID for 3 days from day0 pi

Peak viremia, Byrd et al tissue viral load (2013) 31 and serum cytokines (day3 pi)

4-HPR [N-(4- AG129 mice hydroxyphenyl) Mouse-adapted DENV-2 (S221) retinamide] (nuclear transport inhibitor)

20mg/kg/dose Mortality Fraser et al p.o. QD or BID Serum cytokines (2014) 32 for 5 days from (day3 pi) day0 pi


28

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Carocci et al (2015)

AG129 mice Non-adapted DENV-2 (D2Y98P)

Mortality 90mg/kg/dose p.o. BID until day Viremia (day7 pi) 10 pi from day-1 pi

Lovastatin (cholesterol synthesis inhibitor)


200mg/kg/dose Mortality Martinezp.o. QD for pre- Peak viremia Gutierrez treatment (day-3 (day3 pi) et al to -1) or post(2014) 37 treatment (day1-6 pi)

Bortezomib (protease inhibitor)

C57BL/6 mice Non-adapted DENV-2 (EDEN3295)

1mg/kg/dose Tissue viral load Choy et al s.c. Single dose at Platelets and (2015) 38 6 hrs pi hematocrit Mast cell activation and tissue TNFα (day1, 2 and 3 pi)

34

pi- post-infection, hrs- hours i.p.- intraperitoneal, p.o.- oral, i.g.- intragastric, s.c.- subcutaneous QD- once a day, BID- twice a day, TID- three times a day, QID- four times a day


29


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Table 2: Clinical trials with antiviral drugs targeting viral replication Compound Regimen Major endpoints Results Chloroquine (anti-malaria)

Reference s 600mg on day1 Resolution of Trend toward a Tricou et and 2, 300mg on viremia, serum longer duration of al (2010) day3 NS1 level and viremia, reduction 42 fever in fever clearance time and a lower incidence of DHF

Balapiravir (nucleoside analog)

1500mg 3000mg days

or Resolution of for 5 viremia, serum NS1 level and fever Hematological profile

Celgosivir (α α-glucosidase inhibitor)

Initial 400 mg loading dose within 6 h, followed by 200 mg every 12 h (total of nine doses)

Lovastatin (cholesterol synthesis inhibitor)

80mg for 5days

No trend toward Nguyen et beneficial effect al (2012) on clinical or 43 virologic parameters

Resolution of viremia, serum NS1 level and fever Hematological profile

Trend toward a faster reduction of viremia, and higher platelet count, lower hematocrit, and faster serum NS1 clearance in secondary patients Resolution of No trend toward viremia, serum beneficial effect NS1 level and on clinical or fever virologic Hematological parameters profile Quality of life

Low et al (2014) 44 Sung et al (2016) 56

Whitehorn et al 36 (2016)

DHF- Dengue hemorrhage fever


30

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For Table of Contents Use Only


31


Fig. 1

From day 0

From day 3

Drug treatment

Day 0 Infection

Drug treatment

1

2

3

bleed

bleed

bleed

Day 0 1 2 3

4

Infection

9 7.8-fold (P=0.0002)

8

log (copies/ml)

log (copies/ml)

#013

7 2

bleed

bleed

bleed

VLR (day 3-6)

8

VC celgosivir

7

3

-2.22 ± 0.19 -2.32 ± 0.16 (P=0.395)

4

5

6

9

9 6.8-fold (P=0.0013)

8

log (copies/ml)

log (copies/ml)

6

9

3

10

7

8

VLR (day 3-6)

7

VC celgosivir

6

-2.37 ± 0.48 -2.63 ± 0.49 (P=0.409)

5 1

2

3

3

10

4

5

6

10

9

12.5-fold (P=0.0041)

8 7

log (copies/ml)

#036

5

6 1

#031

bleed

4

10

10

log (copies/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 32

9

VLR (day 3-6)

8

VC celgosivir

7 6

1

2

3

3

Days post infection

4

5

6

Days post infection

VC celgosivir

VC celgosivir

Data from Watanabe et al., Antiviral Research (2016) with figure modification


-1.87 ± 0.15 -2.14 ± 0.35 (P=0.160)

Pre-clinical Antiviral Testing for Dengue Virus ... - ACS Publications

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