The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis.

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg(-1) for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg(-1) than at 3 mg kg(-1) (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL.

Introduction

The repurposing of drugs and clinical candidates offers an attractive alternative to de novo drug discovery (Fischbach and Walsh, 2009; Cragg et al., 2014; Peters, 2013; Law et al., 2013; Novac, 2013; Aube, 2012), particularly in terms of reducing research and development costs for neglected diseases of poverty (Andrews et al., 2014). Visceral leishmaniasis (VL), a neglected tropical disease resulting from infection with the protozoan parasites Leishmania donovani or L. infantum is a case in point, with the two anti-leishmanial front-line therapies miltefosine and amphotericin B both originally developed for other indications (Stuart et al., 2008). In addition, the anti-trypanosomal clinical candidate fexinidazole was recently discovered to have potent activity in a murine VL model (Wyllie et al., 2012), resulting in a phase II proof of concept clinical trial (NCT01980199) against VL being conducted in Sudan.

There are approximately 50,000 reported cases of VL per year, with the vast majority of infections in South America, East Africa and the Indian subcontinent. However, the number of cases is likely to be vastly underreported, with the actual annual incidence estimated to be between 200,000 and 400,000 (Alvar et al., 2012). VL is fatal if untreated and, in the absence of effective vaccines and vector control methods, efficacious chemotherapy is required to combat the disease. Each of the currently available drugs has one or more drawbacks, including the need for hospitalization, prolonged therapy, parenteral administration, high cost, variable efficacy, severe toxic side-effects and resistance (Croft et al., 2006). Thus, there is an urgent need for better, safer efficacious drugs that are fit-for-purpose in resource-poor settings.

Given the success of repurposing fexinidazole for use in the treatment of VL (Wyllie et al., 2012), there is now a renewed interest in the anti-parasitic potential of nitroaromatic drugs. Recently, we demonstrated that the anti-tubercular clinical candidate (S)-PA-824 possesses moderate activity against L. donovani parasites both in vitro and in vivo (Patterson et al., 2013). Although (R)-PA-824, the enantiomer of the candidate showed superior activity, this compound has not entered pre-clinical development, precluding a rapid move to a VL clinical trial. In addition, a recently reported screen of anti-tubercular nitroimidazoles against L. donovani identified DNDI-VL-2098 as a suitable compound for further preclinical evaluation (Mukkavilli et al., 2014; Gupta et al., 2015). The high degree of structural similarity between delamanid (Deltyba, OPC-67683) and both (R)-PA-824 and DNDI-VL-2098 (Figure 1) prompted us to investigate this nitroimidazole, which has recently received conditional approval in Europe for the treatment of multidrug-resistant tuberculosis (Committee for Medicinal Products for Human Use, 2013; Ryan and Lo, 2014).

Figure 1.

Chemical structures of delamanid ((R)-OPC-67683) and the known anti-leishmanial nitroimidazoles DNDI-VL-2098, (R)-PA-824, fexinidazole and fexinidazole sulfone.

Synthetic schemes for the synthesis of delamanid and analogues are described in Figure 1—figure supplements 1–2.

DOI: http://dx.doi.org/10.7554/eLife.09744.003

Reagents and conditions; a) 2-bromo-4-nitro-1H-imidazole, DIPEA, EtOAc, 65°C, 20 hr; b) K2CO3, MeOH, room temp., 16 hr; c) MsCl, pyridine, CH2Cl2, 0°C→room temp., 16 hr; d) DBU, EtOAc, room temp., 16 hr; e) 6, NaH, DMF, 0→50°C, 1.5–4 hr.

DOI: http://dx.doi.org/10.7554/eLife.09744.004

Reagents and conditions; a) DIPEA, 2-nitroimidazole, EtOAc/MeCN, 77°C, 44 hr, 72%; K2CO3, MeOH, room temp., 20 hr, 83%; MsCl, pyridine, CH2Cl2, 0°C→room temp., 16 hr; DBU, EtOAc, room temp., 16 hr, 15% over 2 steps; NaH, DMF, 0→50°C, 5 h, 30%.

DOI: http://dx.doi.org/10.7554/eLife.09744.005

Figure 1—figure supplement 1.

Synthetic route towards delamanid (7) and (S)-delamanid (13).

Reagents and conditions; a) 2-bromo-4-nitro-1H-imidazole, DIPEA, EtOAc, 65°C, 20 hr; b) K2CO3, MeOH, room temp., 16 hr; c) MsCl, pyridine, CH2Cl2, 0°C→room temp., 16 hr; d) DBU, EtOAc, room temp., 16 hr; e) 6, NaH, DMF, 0→50°C, 1.5–4 hr.

DOI: http://dx.doi.org/10.7554/eLife.09744.004

Figure 1—figure supplement 2.

Synthetic route towards des-nitro-delamanid (18).

Reagents and conditions; a) DIPEA, 2-nitroimidazole, EtOAc/MeCN, 77°C, 44 hr, 72%; K2CO3, MeOH, room temp., 20 hr, 83%; MsCl, pyridine, CH2Cl2, 0°C→room temp., 16 hr; DBU, EtOAc, room temp., 16 hr, 15% over 2 steps; NaH, DMF, 0→50°C, 5 h, 30%.

DOI: http://dx.doi.org/10.7554/eLife.09744.005

Results

In vitro sensitivity of L. donovani to (S)- and (R)-delamanid

The life cycle of L. donovani alternates between a flagellated promastigote form residing in the alkaline midgut of the female sandfly vector and an amastigote form that multiplies intracellularly in acidic phagolysosomes of the mammalian host macrophages. Both stages can be cultured axenically; however, intra-macrophage cultures of amastigotes are a more suitable model of mammalian infection for drug discovery. The anti-tubercular drug delamanid and its corresponding S-enantiomer were synthesized (Appendix 1 and Figure 1—figure supplement 1) and assessed for anti-leishmanial activity. The potency of both compounds was determined in vitro against L. donovani (LdBOB) promastigotes and against intracellular amastigotes (LV9) in mouse peritoneal macrophages. The (S)-enantiomer of delamanid showed promising anti-leishmanial activity against both developmental stages of the parasite (EC50 values of 147 ± 4 and 1332 ± 106 nM against promastigotes and amastigotes, respectively). However, delamanid (the R-enantiomer) proved to be an order of magnitude more potent against promastigotes, axenic amastigotes and intracellular amastigotes with EC50 values of 15.5, 5.4 and 86.5 nM, respectively (Table 1). Both compounds were found to be inactive (EC50 >50 µM) in a counter screen against the mammalian cell line HepG2 (Table 1).

Table 1.

Activity of delamanid against laboratory and clinical isolates of L. donovani in vitro. EC90 values are calculated from the EC50, Hill slopes and the molecular weight of delamanid.

DOI: http://dx.doi.org/10.7554/eLife.09744.006

Species	Developmental stage	EC50, nM (Hill slope)	EC90, nM	EC90,ng ml-1
Leishmania donovani (LdBOB)	Promastigote	15.5 ± 0.07 (8.4)	20.2	10.8
Leishmania donovani (LdBOB)	Amastigote (axenic)	5.4 ± 0.05 (5.3)	8.2	4.4
Leishmania donovani (LV9)	Amastigote (in macrophage)	86.5 ± 1.7 (2.3)	225	120
Leishmania donovani (DD8)	Amastigote (in macrophage)	298 ± 13 (2.7)	672	359
Leishmania donovani (BHU1)	Amastigote (in macrophage)	230 ± 10 (4.1)	393	210
Leishmania donovani (SUKA001)	Amastigote (in macrophage)	259 ± 7 (3.6)	476	254
L. infantum (ITMAP263)	Amastigote (in macrophage)	940 ± 0.05 (3.4)	1790	955
Human (HepG2)	N/A	>5000	-	-

Future anti-leishmanial therapies will be required to demonstrate a broad spectrum of activity against different Leishmania strains and against drug resistant parasites (Patterson and Wyllie, 2014). With this in mind, L. donovani and L. infantum clinical isolates were assessed for their sensitivity to delamanid (Table 1). These included: the Indian WHO reference strain DD8; an Indian antimony resistant isolate BHU1; a recent Sudanese isolate SUKA 001; and the L. infantum strain ITMAP263 from Morocco. These clinical isolates were marginally less sensitive to delamanid than our laboratory strain LV9 from Ethiopia, but at the EC90 varied by only 3-fold (L. donovani) or 8-fold (L. infantum) (Table 1). Although not investigated further here, promastigotes of L. major Friedlin, a cause of cutaneous leishmaniasis, were also highly sensitive to delamanid (EC50 6.3 ± 0.11 nM, slope factor 2.2).

The corresponding des-nitro analogue was also synthesized (Appendix 1 and Figure 1—figure supplement 2) and assayed against L. donovani promastigotes. Des-nitro-delamanid was found to be inactive (EC50 >50 µM), which is consistent with the nitro group being involved in the mechanism of action or having a role in the binding of delamanid to its molecular target(s) in L. donovani.

Physicochemical properties of delamanid

The plasma protein binding of delamanid was measured and found to be high (Fu = 0.0045), in agreement with that reported previously (Committee for Medicinal Products for Human Use, 2013). A kinetic solubility assay demonstrated that delamanid possesses sufficient aqueous solubility (>250 µM in 2.5% DMSO) for use in in vitro assays.

Efficacy of delamanid in a murine model of visceral leishmaniasis

The efficacy of delamanid was assessed in a murine model of VL. Groups of infected BALB/c mice (seven days post infection with ex vivo L. donovani LV9 amastigotes) were dosed twice-daily, for five consecutive days with an oral formulation of delamanid (1, 3, 10, 30 or 50 mg kg-1). On day 14 post-infection, the parasite burdens in the livers of infected mice were determined and compared with those of control animals. The only current oral anti-leishmanial therapy miltefosine (30 mg kg-1, once-daily, 5 days) was included as a positive control. Both delamanid and miltefosine were well tolerated at these doses, with no mice displaying any overt signs of toxicity. An initial experiment showed that treatment with delamanid at 50 mg kg-1 effectively cured the study mice, with no detectable parasites in the liver smears, whereas control mice dosed with vehicle alone showed a high level of infection (Figure 2). A second in vivo study with mice dosed twice-daily at 30, 10 or 3 mg kg-1 suppressed infection in the murine model by 99.5%, 63.5% and 16.0%, respectively, establishing a dose-dependent anti-leishmanial effect within the range of 3–50 mg kg-1. These results give an estimated ED50 and ED90 of 7.3 and 21.5 mg kg-1, respectively (Figure 2—figure supplement 1). At 30 and 50 mg kg-1 delamanid compares favourably with miltefosine (98.8–99.8% suppression at 30 mg kg-1), which exemplifies the therapeutic potential of delamanid.

Figure 2.

Effects of drug treatment on the parasite burden of mice infected with L. donovani.

Groups of mice (five per group) infected with L. donovani (strain LV9) were dosed with drug vehicle (orally), miltefosine (orally) or delamanid (twice daily, orally) on day 7 post-infection and for a total of 5 or 10 days. Two days after the final dose, animals were humanely euthanized and parasite burdens were determined microscopically by examining Giemsa-stained liver smears. Grey bars, 5-day treatment; red bars, 10 day treatment. This graph shows the combined data from six individual animal studies; n = 9, or 10 for 1, 3 and 10 mg/kg dosing; for all other experiments n = 5. These data are available in tabular form in Figure 2—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.09744.007

DOI: http://dx.doi.org/10.7554/eLife.09744.008

Results are based on dosing in the range 3 to 50 mg kg-1 b.i.d. oral for 5 days.

DOI: http://dx.doi.org/10.7554/eLife.09744.009

Figure 2—figure supplement 1.

ED50 determination for delamanid in a mouse model of VL.

Results are based on dosing in the range 3 to 50 mg kg-1 b.i.d. oral for 5 days.

DOI: http://dx.doi.org/10.7554/eLife.09744.009

Efficacy and PK/PD data from all experiments.

A third in vivo study with a further reduced delamanid dose of 1 mg kg-1 resulted in a suppression of parasitaemia of 86.3% compared with control mice, proving unexpectedly superior to dosing at 3 or 10 mg kg-1 (Figure 2). A subsequent experiment encompassing a range of doses (10, 3, 1 mg kg-1, 5 days) in a single study showed a similar hormetic effect, with twice daily dosing at 1 mg kg-1 being more efficacious than 10 mg kg-1. However, this study also demonstrated that there is some variability in the efficacy of delamanid at lower doses (Figure 2—source data 1).

The hormetic effect was also observed in an extended dosing experiment in which delamanid was instead dosed twice-daily for 10 days at 10, 3 or 1 mg kg-1, with the suppression of infection being 92.3%, 24.3% and >99.9%, respectively. A second 10-day experiment with a broader range of doses (30, 10, 3, 1, 0.3 mg kg-1) further confirmed the hormetic effect. In addition, this study demonstrated that further reducing the delamanid dose (0.3 mg kg-1) resulted in a reduction in efficacy comparable to dosing at 3 mg kg-1, resulting in a biphasic dose response relationship (Figure 2).

Blood levels of orally dosed delamanid in a mouse model

It is important to understand the pharmacokinetic and pharmacodynamic (PK/PD) behaviour of delamanid in order to optimise the efficacious dosing regimen (Velkov et al., 2013). By measuring the change in drug concentration over time in L. donovani-infected mice, two standard PK parameters can be obtained: maximum concentration (Cmax) in blood; and the area under the curve (AUC), a measure of total drug exposure over time. The drug concentration over time is measured in order to determine whether the concentration of a drug exceeds the minimum inhibitory concentration (MIC, EC90 in this case) and, if so, for how long (time over MIC, T>MIC). Parameters such as Cmax/MIC, AUC/MIC and T>MIC are important for achieving drug efficacy in an in vivo model of disease. Both Cmax and AUC measure the total drug level in blood or plasma; however, only unbound drug molecules are able to bind to their targets (Bohnert and Gan, 2013). Therefore, the plasma protein binding level (expressed as the fraction unbound, Fu) of delamanid was also measured and used to calculate an adjusted EC90 (assay EC90 × 1/Fu) for comparison with blood concentration over time.

Accordingly, the blood levels of the drug were measured at intervals (up to 8 hr post dose) during the in vivo efficacy studies. Data for the first and ninth dose in a 5-day twice daily treatment experiment (Figure 3A,B) show a dose-dependent response with accumulation over time. A similar effect was noted in a 10-day study (1st and 19th dose; Figure 3C,D). More detailed analysis of the combined PK data from five experiments (including two 10-day treatment studies) shows a linear relationship between doses of 0.3–10 mg kg-1 and peak blood concentration (Cmax) or area under the curve (AUC(0-t)) with accumulation from day 1 through to day 10 (Figure 3E,F). The 10 and 30 mg kg-1 doses should provide adequate coverage over the EC90 (120 ng ml-1) as measured for the L. donovani isolate LV9 in macrophages over a 3 day exposure (Table 1). However, due to high protein binding, the free fraction (Fu = 0.0045) cannot account for biological activity in vivo at any dose. An explanation for why the free drug theory (Bohnert and Gan, 2013) is not applicable in this case is presented below.

Figure 3.

Pharmacokinetic behaviour of delamanid in infected mice.

(A and B) show the blood levels of delamanid following the first oral dose on day 1 (A) and the penultimate oral dose on day 5 (B) for 1, 3, 10 and 30 mg kg-1 b.i.d. (teal, black, red and blue symbols, respectively). Error bars are SEM (n = 3 for 30 mg kg-1, n = 6 all other doses). (C and D) show the blood levels of delamanid from a single VL PK/PD study following the first oral dose on day 1 (C) and the penultimate oral dose on day 10 (D) for 0.3, 1, 3, 10 and 30 mg kg-1 b.i.d. (grey, teal, black, red and blue symbols, respectively). Error bars are SEM (n = 5). (E and F) show the relationship between dose with Cmax or AUC(0-8 h), respectively, after the first oral dose on day 1 (black), or the penultimate dose on day 5 (red) or day 10 (blue). Error bars in (E) are SEM (n ranges from 3–8 depending upon day and dose – see Figure 2 – source data1). Lines in (E) and (F) are best fits by linear regression.

DOI: http://dx.doi.org/10.7554/eLife.09744.010

Delamanid-mediated cell killing

To determine whether delamanid was cytostatic or cytotoxic, mid-log promastigotes were incubated with drug concentrations equivalent to 10 times the EC50 value (Figure 4A). Growth of drug-treated cultures ceased almost immediately with cell numbers declining after 8 hr and no live parasites visible at 24 hr. To determine the actual point where treated cells lost viability, at defined intervals parasites were washed and sub-cultured without drug. No viable parasites could be recovered after 12 hr in the presence of drug, confirming that delamanid is rapidly leishmanicidal. In support of this apparent rapid mechanism of cell killing, EC50 values determined after 24, 48 and 72 hr were essentially identical (Figure 4B). In addition, the potency (EC50 value) of delamanid was found to be dependent on the initial cell density (Figure 4C) and on the assay serum concentration (Figure 4D).

Figure 4.

Effects of delamanid on L. donovani promastigotes.

(A) Delamanid causes rapid cell killing. Promastigotes were exposed to delamanid (10 times EC50) and samples removed at intervals to determine cell density and cell viability. Black symbols: no inhibitor; red symbols: plus drug; the point of irreversible drug toxicity. (B) Drug sensitivity is independent of exposure beyond 24 hr. Black, red and blue symbols are EC50 curves determined after 24, 48 and 72 hr, respectively. (C) Drug sensitivity is cell-density dependent. Black, red and blue symbols are EC50 curves determined after 72 hr, with initial seeding densities of 103, 104 and 105 cells ml-1, respectively. (D) Drug sensitivity is serum dependent. Black, red and blue symbols are EC50 curves determined after 72 hr in the presence of 5, 10 and 20% FCS, respectively.

DOI: http://dx.doi.org/10.7554/eLife.09744.011

Delamanid – mode of action studies

Many nitroheterocyclics require bio-activation of their nitro groups to become biologically active. In Mycobacterium tuberculosis, delamanid is assumed to be reductively activated by the same unusual deazaflavin (F420)-dependent nitroreductase (Ddn) known to activate the closely related nitroimidazo-oxazine drug PA-824 (Manjunatha et al., 2006; Singh et al., 2008; Manjunatha et al., 2009). In the absence of a Ddn homologue in Leishmania, we assessed whether the reduction of delamanid is catalysed by the NADH-dependent bacterial-like nitroreductase (NTR) already shown to activate the nitroimidazoles fexinidazole and nifurtimox in these parasites (Wyllie et al., 2012). The potency of delamanid was determined against parasites overexpressing NTR. Increased concentrations of NTR in these transgenic parasites were confirmed by a 13-fold increase in their sensitivity to nifurtimox (EC50 of 8.0 ± 0.2 and 0.61 ± 0.006 μM for WT and transgenic parasites, respectively Figure 5A), known to undergo two-electron reduction by NTR (Hall et al., 2011). However, overexpression of NTR in promastigotes did not significantly alter their sensitivity to delamanid (EC50 of 4.5 ± 0.004 and 4.1 ± 0.003 nM for WT and transgenic parasites, respectively) (Figure 5B). To confirm that the same was also true in the amastigote stage of these parasites, metacyclic promastigotes overexpressing NTR were used to infect mouse peritoneal macrophages. The resulting intracellular parasites were found to be just as sensitive to delamanid as WT parasites with EC50 values of 57.8 ± 2.1 and 55.2 ± 4.3 nM, respectively (Figure 5C). These findings indicate that NTR does not play a role in the activation of delamanid in L. donovani in either stage of the life cycle and that the mechanism of action of this nitroheterocyclic drug is different from that of fexinidazole.

Figure 5.

Mode of action of delamanid is distinct from nifurtimox.

(A, B and C) Susceptibility to nifurtimox (A) is increased in NTR-overexpressing promastigotes (red symbols), but not to delamanid (B) compared to WT cells (black symbols). Data are the mean of triplicate cultures from a single experiment. (C) The susceptibility of delamanid is not increased in NTR-overexpressing amastigotes in macrophages (red symbols) compared to WT (black symbols). Data are the mean of duplicate cultures from a single experiment.

DOI: http://dx.doi.org/10.7554/eLife.09744.012

Metabolism of delamanid in L. donovani

Given that NTR does not activate delamanid in L. donovani promastigotes and the requirement of the nitro group for biological activity, it was important to determine if the drug is metabolised in culture. To address this issue, the concentration of delamanid was determined by UPLC-MS/MS in cultures of promastigotes over a 24 hr period. Delamanid is known to be primarily metabolised in plasma by albumin (Shimokawa et al., 2015) and to a lesser extent by CYP3A4, CYP1A1, CYP2D6 and CYP2E1 (Sasahara et al., 2015). Thus, the concentration of delamanid in culture medium without parasites was measured over the same time period as a control. In the presence of medium alone, delamanid decreased linearly in a concentration-dependent manner (Figure 6A). However, in the presence of L. donovani promastigotes the rate of disappearance of delamanid was markedly increased, such that the drug had essentially disappeared by 6 hr (Figure 6B). The net amount of delamanid metabolised by parasites as a function of time is also linear and dependent on the initial concentration in the medium (Figure 6C). Linear regression of these data revealed that the rate of cell metabolism is not saturated up to the top concentration tested (Figure 6D). Analogous experiments using mouse peritoneal macrophages and THP-1 monocytes found no evidence of delamanid metabolism by these host cell lines. Elucidation of the chemical identity of the delamanid metabolite(s), their possible role in parasite killing and the enzyme(s) responsible for their biosynthesis will be the focus of future studies.

Figure 6.

Delamanid metabolism in L. donovani promastigotes.

(A) Medium plus delamanid alone and (B) cells incubated in medium plus delamanid. Delamanid concentrations added are 15, 45 and 150 nM (black, red and blue, respectively). The lines represent best fits by linear regression for all data points in (A) and 0 to 5 hr in (B). The dotted line in (B) is the best fit by non-linear regression to a single exponential decay. (C) Net metabolism of delamanid by cells was obtained by subtraction of (A) from (B). Data fitted by linear regression gave correlation coefficients of 0.996, 0.991 and 0.951 for delamanid concentrations of 15, 45 and 150 nM, respectively. (D) Rates of delamanid metabolism obtained from (C) are linear up to 150 nM (correlation coefficient 0.996, explicit errors used in fit).

DOI: http://dx.doi.org/10.7554/eLife.09744.013

Discussion

For diseases of poverty such as visceral leishmaniasis there is limited financial incentive to initiate expensive, high risk and time-consuming de novo drug discovery programmes. Consequently, the repurposing of existing drugs has become an attractive approach towards the identification of much needed new treatments for VL and other neglected parasitic diseases (Andrews et al., 2014; Wyllie et al., 2012). The recently approved anti-tubercular drug delamanid (Ryan and Lo, 2014) was deemed to be of particular interest as a number of other nitroimidazoles have been shown to also possess promising anti-leishmanial activities (Wyllie et al., 2012; Patterson et al., 2013; Gupta et al., 2015).

In the current study, we show that delamanid is highly active in vitro against intracellular L. donovani amastigotes (EC50 0.087 µM) with activity superior to that of both the current VL drug miltefosine (EC50 3.3 µM) and the active sulfone metabolite of the VL clinical candidate fexinidazole (EC50 5.3 µM) in the same assay (Wyllie et al., 2012). The in vitro anti-leishmanial activity of delamanid shows the same enantiomeric specificity as the delamanid analogue DNDI-VL-2098 in vivo (Gupta et al., 2015). Similarly, the R-enantiomer of an analogue of delamanid has been shown to be more potent against M. tuberculosis than its corresponding S-enantiomer (Sasaki et al., 2006). However, this contrasts with the closely related nitroimidazole PA-824, where the enantiomeric specificity for L. donovani and M. tuberculosis is opposite (Patterson et al., 2013). Delamanid is rapidly leishmanicidal with a cell-density dependent potency and as expected, demonstrated no observable toxicity in a mammalian cell assay. The observed shift in drug sensitivity with increasing serum concentration is likely due to increased metabolism by albumin to inactive metabolites, rather than changes in free drug concentration due to protein binding.

Delamanid is orally bioavailable, well tolerated, shows dose linearity up to 10 mg kg-1 and accumulation with repeated administration in agreement with previous pharmacokinetic (PK) studies in mice (Matsumoto et al., 2006). However, the pharmacokinetic / pharmacodynamic (PK/PD) relationship is not straightforward.

First, the PK/PD relationship does not fit with the free drug hypothesis, which states that, in the absence of energy-dependent transport processes, the extracellular and intracellular free drug concentrations are equal after steady-state equilibrium has been achieved, and that only the free drug is able to bind to the target to exert its pharmacological effect (Bohnert and Gan, 2013; Smith et al., 2010). Based on the in vitro intra–macrophage amastigote EC90 value (120 ng ml-1), the high PPB of delamanid (Fu 0.0045) results in an adjusted intra macrophage L. donovani EC90 of 26,700 ng ml-1, a concentration that is not achieved in whole blood at any point in the efficacy study. Therefore, the efficacy of delamanid in the VL animal model is unexpected. However, there are exceptions to the free drug theory, such as drugs that form active metabolites resulting in inactivation (covalent or otherwise) of multiple targets (Smith et al., 2010). The parasite-specific metabolism presented here is entirely consistent with this exception.

Second, the pronounced bi-phasic suppression of parasite burden at high and low doses of delamanid in vivo is highly unusual. This U-shaped dose-response curve is reminiscent of hormesis in toxicology (Calabrese and Baldwin, 2003; 2002), but, to our knowledge, is unprecedented in microbiology. This effect is not observed in dose response curves in vitro, so must be related to a physiological or metabolic threshold response in the infected drug-treated animal. In vivo delamanid is proposed to undergo primary metabolism with loss of the nitro group, mainly catalysed by albumin (Committee for Medicinal Products for Human Use, 2013; Shimokawa et al., 2015). Further metabolism to seven other metabolites is thought to occur via hydrolysis reactions and oxidation by CYP3A4 (Committee for Medicinal Products for Human Use, 2013; Sasahara et al., 2015). The cause of this U-shaped dose-response curve is currently not known. One possibility is that a drug metabolite of delamanid antagonises the bio-activation of delamanid, or antagonises the downstream effects in the leishmania parasite. The formation of a putative antagonist metabolite would have to show a saturable sigmoidal dose response, such that at higher concentrations, delamanid, or an active parasite-specific metabolite thereof, are able to displace the antagonist from the bio-activating enzyme, or proteins related to the downstream effect respectively. It should be noted that in the same VL animal model the related nitroimidazole (S)-PA-824 was also more efficacious at a lower dose (30 vs 100 mg kg-1) (Patterson et al., 2013). Further studies with (S)-PA-824 should be conducted to determine if this compound also displays a hormetic PK/PD relationship and establish if this is a chemotype-related characteristic.

Plots of Cmax versus parasite suppression and calculated AUC(0–24 hr) versus parasite suppression (Figure 7A,B) suggest that the delamanid blood levels required for cure in the VL model exceed those observed in TB patients receiving the drug. Increasing the dosing duration in the in vivo VL model from 5 to 10 days improved the mean parasite suppression at all investigated doses (Figure 2) and resulted in some mice with no detectable liver parasites when dosed at 1 mg kg-1 (Figure 2—source data 1). As it known that delamanid is tolerated for up to six months (Committee for Medicinal Products for Human Use, 2013) further extending the duration of the VL model beyond 10 days should be considered. The VL target product profile calls for a treatment regimen of <10 days. However, in the current VL therapy, miltefosine is dosed orally for 28 days, so extended dosing should be clinically acceptable. Note, the study mice dosed twice-daily at 1 mg kg-1 have drug levels lower than that achieved in human TB patients dosed once daily at 100 mg. Given that this low dose is more efficacious than any other dose below 30 mg kg-1, model studies of extended duration should focus around this dosing level.

Figure 7.

PK/PD relationships in mice.

(A and B) Mean suppression of parasite burden as a function of Cmax for the penultimate dose (panel A) and extrapolated AUC(0–24 hr) for the last day of the 10-day treatment regimen (panel B). The black dotted line in (A) is the EC90 value obtained for infected macrophages after 72 hr exposure (120 ng ml-1). The red dotted line in (A) and (B) represents the mean delamanid Cmax (375–400 ng ml-1) and mean AUC(0–24 hr) (7000–8000 h*ng ml-1) obtained in 144 TB patients after 14 days treatment with 100 mg, oral, once daily from day 14–56 (Sasahara et al., 2015). The data in this graph were derived from a single in vivo study; related aggregated data from previous studies is shown in Figure 7—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.09744.014

Open and closed symbols represent data combined from the 5- and 10-day studies, respectively. This graph represents the aggregated data from 3 separate in vivo studies. The black dotted line in (A) is the EC90 value obtained for infected macrophages after 72-hr exposure. The red dotted line in (A) and (B) represents the mean delamanid Cmax (375–400 ng ml-1) and mean AUC(0–24 hr) (7000–8000 h*ng ml-1) obtained in TB patients after 14 days treatment with 100 mg, oral, once daily (Sasahara et al., 2015).

DOI: http://dx.doi.org/10.7554/eLife.09744.015

Figure 7—figure supplement 1.

(A and B) Mean suppression of parasite burden as a function of Cmax for the penultimate dose and extrapolated AUC(0–24 hr) for the last day of the 5- and 10-day treatment regimens, respectively.

Open and closed symbols represent data combined from the 5- and 10-day studies, respectively. This graph represents the aggregated data from 3 separate in vivo studies. The black dotted line in (A) is the EC90 value obtained for infected macrophages after 72-hr exposure. The red dotted line in (A) and (B) represents the mean delamanid Cmax (375–400 ng ml-1) and mean AUC(0–24 hr) (7000–8000 h*ng ml-1) obtained in TB patients after 14 days treatment with 100 mg, oral, once daily (Sasahara et al., 2015).

DOI: http://dx.doi.org/10.7554/eLife.09744.015

Despite a wealth of pharmacokinetic data in patients and human volunteers (Committee for Medicinal Products for Human Use, 2013) the unusual PK/PD relationship hinders our ability to accurately predict the outcome of delamanid dosing in VL patients. Indeed, careful examination of parasite suppression versus Cmax (Figure 7—figure supplement 1) shows that the mean Cmax in delamanid-treated TB patients corresponds to an efficacy minimum in the VL model. Given that delamanid is rapidly metabolised by leishmania-infected macrophages in vitro, we examined the effect of delamanid with a 5 day exposure with daily drug and medium change. This gave an EC50 value of 28.0 ± 1.6 nM (slope 2.7) from which an EC90 could be calculated (62.7 nM or 33.5 ng ml-1). This concentration is lower than the lowest observed Cmax in the efficacy studies (Figure 7A). Thus, careful design of the dosing regimen for VL patients may avoid the risk that treatment will lack efficacy due to reaching a Cmax and AUC(0–24 hr) within the higher ineffective concentration range.

The nature of the parasite-specific metabolising / activating enzyme(s) is not known, but is clearly distinct from the deazaflavin-dependent nitroreductase in M. tuberculosis (Manjunatha et al., 2006) and the nitroreductase in leishmania involved in the activation of fexinidazole metabolites (Wyllie et al., 2012). The identification of this target, and the metabolites that it produces, are the focus of our current work. The cell-density dependent potency of delamanid is consistent with the formation of a putative reactive, covalent metabolite. In addition, the rapidly cytocidal activity of delamanid is consistent with the rapid rate of drug metabolism by L. donovani in culture. In terms of drug development the divergent modes of action for fexinidazole and delamanid are advantageous, as the likelihood of cross-resistance developing is reduced, and the potential for their co-administration as a combination therapy is retained.

The current practice in the pharmaceutical industry is to avoid developing compounds containing a nitro-aromatic group due to the known liabilities of this class, particularly potential mutagenicity and carcinogenicity. As a result, outside the anti-infectives there are relatively few nitro-aromatic drugs. Indeed, nitro-aromatic moieties are relatively common in drugs or clinical candidates for kinetoplastid diseases compared to chemotherapies for other indications. This over-representation is linked to the mechanism of action of nitro-drugs; selective bio-activation by parasitic bacterial-like NTRs leading to selective anti-parasitic activity. The studies presented herein are consistent with delamanid also being activated by a parasite-specific enzyme absent from host cells. Preclinical studies have demonstrated that delamanid is not mutagenic (Matsumoto et al., 2006). Moreover, repeated oral administration in mice or rats for up to 104 weeks showed no evidence of carcinogenicity (Committee for Medicinal Products for Human Use, 2013). Taken together these points alleviate some of the concerns ordinarily associated with the development of nitro-drugs, although long term safety can only be established after extensive clinical use in relevant populations.

While revising this manuscript, Thompson and co-workers reported the structure-activity relationships for an extensive series of bicyclic nitro-compounds, including delamanid (Thompson et al., 2016). These authors observed partial efficacy in L. infantum-infected hamsters dosed with delamanid, not inconsistent with our findings. The lower efficacy observed by these authors could be due to one or more of the following: inadequate dosing (once versus twice per day); different animal model (hamster versus mouse); or different species of leishmania (L. infantum is less sensitive than L. donovani). Although this paper found VL-2098 to have superior efficacy in a once daily treatment regimen, pre-clinical development of this compound has been abandoned due to testicular toxicity (http://www.dndi.org/diseases-projects/portfolio/completed-projects/vl-2098/ last accessed 3rd April 2016). Importantly, this reproductive toxicity has not been observed with delamanid (Committee for Medicinal Products for Human Use, 2013).

Delamanid meets many of the criteria specified in the target product profile (TPP) for VL [http://www.dndi.org]. It is rapidly cytocidal and thus potentially efficacious in immunocompromised patients such as those co-infected with HIV (Alvar et al., 2008) particularly since delamanid is not associated with any clinically relevant anti-retroviral drug-drug interactions (Ryan and Lo, 2014; Blair and Scott, 2015). Due to the prevalence of TB-VL coinfection in Ethiopia (Hurissa et al., 2010) and Sudan (El-Safi et al., 2004) the TPP also specifies that any new treatments should be compatible with TB medications, a stipulation met by delamanid (Blair and Scott, 2015). Delamanid can be administered orally, an important requirement for patients who have limited access to even the most basic of health care facilities, whereas liposomal amphotericin B (AmBisome) has to be administered intravenously and requires cold storage for stability. Perhaps the most challenging issue may be cost of goods. Assuming an efficacious dose of 100 mg once-daily for in excess of 10 days and the market cost of delamanid in developed countries (US$42 per 50 mg tablet) (Lessem, 2014), the predicted cost per patient would be over US$840, significantly higher than that specified in the VL TPP (

In summary, these data suggest that delamanid has the potential to be repurposed as a VL therapy. Additional VL animal model studies exploring the effect of extended delamanid dosing beyond 10 days should be investigated.

Materials and methods

Cell lines and culture conditions

All leishmania strains follow the WHO International Code designating the animal from which the parasite was isolated, country, date of isolation and strain designation (see International Leishmania Network http://leishnet.net/site/?q=node/5). The WHO designations and origins of each Leishmania isolate used are detailed in Table 2. The clonal Leishmania donovani cell line LdBOB was grown as promastigotes at 24°C in 10% FCS, as previously described (Goyard et al., 2003), except when investigating the effect of serum concentration on drug efficacy, in which case 5, 10, or 20% FCS was used. Transgenic LdBOB promastigotes expressing the L. major nitroreductase (LmjF.05.0660) enzyme (Wyllie et al., 2012) were cultured under identical conditions in the presence of nourseothricin (100 μg ml−1). L. major promastigotes (Friedlin strain) were grown in M199 medium (Caisson Laboratories, Logan, UT) with supplements, as previously described (Oza et al., 2005). L. donovani (LV9 strain) ex vivo amastigotes were used in both in vitro and in vivo drug sensitivity assays. Amastigotes were derived from hamster spleens, as previously described (Wyllie and Fairlamb, 2006). All other Leishmania clinical isolates (Table 2) were grown in RPMI 1640 (Sigma, UK) supplemented with 20% FCS, 100 µM adenine, 5 µM hemin, 20 mM MES, 3 µM 6-biopterin and 1 mM biotin. In all cases the FCS used was certified as mycoplasma free.

Table 2.

Leishmania isolates used in delamanid drug sensitivity studies.

DOI: http://dx.doi.org/10.7554/eLife.09744.016

Species	WHO Code	Laboratory Code	Origin	Year of Isolation	Provided by
L. donovani	MHOM/SD/62/1S CL2D a	LdBOB	Sudan	1962	via Professor Stephen Beverley, Washington University
L. donovani	MHOM/ET/67/HU3	LV9	Ethiopia	1967	via Professor Jennie Blackwell, Cambridge University
L. donovani	MHOM/IN/02/BHU1	BHU1	India	2002	via LSHTM, London, UK
L. donovani	MHOM/SU/09/SUKA001	SUKA001	Sudan	2009	via LSHTM, London, UK
L. donovani	MHOM/IN/80/DD8	DD8	India	1980	via LSHTM, London UK
L. infantum	MHOM/MA/67/ITMAP263	ITMAP263	Morocco	1967	via LSHTM, London, UK
L. major	MHOM/IL/81/Friedlin	Friedlin	Israel	1981	via LSHTM, London, UK

a Derived from this strain (Goyard et al., 2003).

Chemical synthesis of delamanid and analogues

Delamanid was prepared as previously described (Sasaki et al., 2006; Kiyokawa and Aki, 2005) (Figure 1—figure supplement 1). Modification of the delamanid synthetic route afforded (S)-delamanid and des-nitro-delamanid (Figure 1—figure supplements 1–2). Compound purity was determined by liquid chromatography-mass spectrometry, with all compounds found to be >95% pure. For in vivo experiments, delamanid was further analysed by ultra high-performance liquid chromatography-mass spectrometry (UPLC-MS), with all batches found to be of >98% purity. The optical rotation of delamanid was in close agreement to the published value (Sasaki et al., 2006), confirming the optical purity of the material used in this study. Detailed synthetic procedures and analysis of key compounds and intermediates are provided in Appendix 1.

In vitro drug sensitivity assays against promastigotes

To examine the effects of test compounds on growth, triplicate cultures were seeded with 1 × 105 parasites ml-1. Parasites were grown in the presence of drug for 72 hr, after which 50 μM resazurin was added to each well and fluorescence (excitation of 528 nm and emission of 590 nm) measured after a further 2 hr incubation (Jones et al., 2010). Data were processed using GRAFIT (version 5.0.13; Erithacus software) and fitted to a 2-parameter equation, where the data are corrected for background fluorescence, to obtain the effective concentration inhibiting growth by 50% (EC50):

In this equation [I] represents inhibitor concentration and m is the slope factor. Experiments were repeated at least two times and the data is presented as the weighted mean plus weighted standard deviation (Young, 1962). When investigating the speed of drug-mediated cell killing, parasites were grown in the presence of drug for 24, 48, or 72 hr in an otherwise identical assay. The same assay was used to investigate the effect of seeding density upon drug efficacy, except that the number of parasites used to seed the assays was varied to be either 103, 104 or 105 parasites ml-1.

Cytocidal effects of delamanid on L. donovani promastigotes

Delamanid was added to early-log cultures of LdBOB promastigotes (~1 × 106 ml-1) at concentrations equivalent to 10 times its EC50 value. At intervals, the cell density was determined, samples of culture (500 µl) removed, washed and resuspended in fresh culture medium in the absence of drug. The viability of drug-treated parasites was monitored for up to 24 hr and the point of irreversible drug toxicity determined by microscopic examination of subcultures after 5 days.

In vitro drug sensitivity assays in mouse macrophages and toxicity to HepG2 cells

In-macrophage drug sensitivity assays were carried out using starch-elicited mouse peritoneal macrophages and hamster-derived ex vivo amastigotes (Wyllie et al., 2012) or metacyclic promastigotes (Wyllie et al., 2013), where appropriate. Assays to determine the sensitivity of HepG2 cells to test compounds were carried out precisely as previously described (Patterson et al., 2013). HepG2 were obtained from ATCC and routinely tested for mycoplasma contamination by Mycoplasma Experience Ltd.

In vitro pharmacokinetic and biophysical properties

The PPB of delamanid was determined by the equilibrium dialysis method (Jones et al., 2010). The aqueous solubility of delamanid was measured using a laser nephelometry-based method (Patterson et al., 2013).

In vivo drug sensitivity

Groups of female BALB/c mice (5 per group) were inoculated intravenously (tail vein) with approximately 2 × 107 L. donovani LV9 amastigotes harvested from the spleen of an infected hamster (Wyllie and Fairlamb, 2006). From day 7 post-infection, groups of mice were treated with either drug vehicle only (orally), with miltefosine (30 mg kg-1 orally), or with delamanid (1, 3, 10, 30 or 50 mg kg-1 orally). Miltefosine was administered once daily for 5, or 10 days, with vehicle and delamanid administered twice daily over the same period. Drug dosing solutions were freshly prepared each day, and the vehicle for delamanid was 0.5% hydroxypropylmethylcellulose, 0.4% Tween 80, 0.5% benzyl alcohol, and 98.6% deionized water. On day 14 (for 5 day dosing experiments), or day 19 post-infection (for 10 day dosing experiments), all animals were humanely euthanized and parasite burdens were determined by counting the number of amastigotes/500 liver cells (Wyllie et al., 2012). Parasite burden is expressed in Leishman Donovan Units (LDU): mean number of amastigotes per 500 liver cells × mg weight of liver (Bradley and Kirkley, 1977). The LDU of drug-treated samples are compared to that of untreated samples and the percent inhibition calculated. ED50 values were determined using GRAFIT (version 5.0.13; Erithacus software) by fitting data to a 2-parameter equation, as described above.

Determination of delamanid exposure in infected mice after oral dosing

Blood samples (10 μl) from 3 of 5 infected mice (see in vivo drug sensitivity above) in each dosing group were collected from the tail vein and placed into Micronic tubes (Micronic BV) containing deionized water (20 μl). Samples were taken following the first dose on the first (day 7 post-infection) and last day of dosing (day 11, or 16 post-infection) at 0.5, 1, 2, 4 and 8 hr post-dose. Diluted blood samples were freeze-thawed three times prior to bioanalysis. The concentration of delamanid in mouse blood was determined by UPLC-MS/MS on a Xevo TQ-S (Waters, UK) by modification of that described previously for the analysis of fexinidazole (Sokolova et al., 2010) and PK parameters determined using PKsolutions software (Summit, USA). AUC(0–24 hr) was extrapolated from the calculated AUC(0-8 hr), with second daily dose administered at 8 hr post first daily dose.

Rate of delamanid metabolism in L. donovani promastigotes

Rate of metabolism studies were carried out at 15, 45 and 150 nM delamanid (equivalent to 1-, 3- and 10-times EC50) in culture medium alone and in the presence of wild type L. donovani promastigotes (1 × 107 parasites ml-1). At 0, 0.5, 1, 2, 4, 6, 8 and 24 hr aliquots were removed, precipitated by addition of a 3-fold volume of acetonitrile and centrifuged (1665 × g, 10 min, room temperature). The supernatant was diluted with water to maintain a final solvent concentration of 50% and stored at −20ºC prior to UPLC-MS/MS analysis, as described below.

UPLC-MS/MS was performed on a Waters Acquity UPLC interfaced to a Xevo TQ-S MS. Chromatographic resolution was achieved on a 2.1 × 50 mm Acquity BEH C18, 1.7 µm column which was maintained at 40ºC with an injection volume of 8 µl. The mobile phase consisted of A: deionized water plus 0.01% (v/v) formic acid and B: acetonitrile plus 0.01% (v/v) formic acid at a flow rate of 0.6 ml min-1. The initial gradient was 5% B held for 0.5 min before increasing to 95% B from 0.5–2 min, where it was held from 2–2.6 min before decreasing back to 5% B from 2.6–3 min. Mass spectra were obtained using electrospray ionization (ESI), in positive ion mode with the following conditions: capillary 3.5 kV; desolvation temperature 600ºC; source temperature 150°C; desolvation gas flow (nitrogen) 1000 l h-1 and collision gas (argon) gas of 0.15 ml min-1. Multiple reaction monitoring (MRM) was performed for delamanid using the transition 535.02 > 351.80 at a cone voltage of 16 V and collision energy of 33 V. Data was processed using the TargetLynx feature of Mass Lynx v4.1.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis" for peer review at eLife. Your submission has been favorably evaluated by Prabhat Jha as the Senior editor, Jon Clardy as Reviewing Editor, and two reviewers including Clifton Barry.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

Visceral leishmaniasis is a disease caused by protozoal parasites that imposes a significant burden of mortality and morbidity on impoverished tropical populations. All current therapies have significant liabilities. The authors have adopted a repurposing strategy to screen drugs for other indications for their potential to treat leishmaniasis, and through that approach they identified delamanid as a potential oral treatment. The manuscript addresses pharmacokinetic/pharmacodynamic (PK/PD) properties of delamanid and concludes that it could be a useful therapeutic agent.

Essential revisions:

All reviewers noted the importance of improved treatments for visceral leishmaniasis, appreciated the benefits of a drug-repurposing strategy, and enthused about the potency of delamanid. They also recommended publication with revisions focusing on the following points:

1) The authors and reviewers are experienced in drug development, but almost all eLife readers are not. It would be useful in revising the manuscript to keep this in mind and provide clues to what the various tests are trying to determine. For example, most readers will be unfamiliar with a term like 'area under the curve (AUC)’ in pharmacology, and adding a brief note that it measures the total dose of drug and is useful for comparing different formulations would help enormously. It's a challenge, but a challenge worth addressing. The report as written is informative to a specialist and almost unintelligible to anyone else.

2) It would be useful to add some clarification about different developmental stages of the parasite: the difference between a promastigote and amastigote, delamanid's efficacy against different stages, possibility of host metabolism of delamanid altering efficacy, and the possibility of different targets for different stages.

3) The presence of a nitro group, which is common in this field and quite familiar to the authors, should be addressed for the general reader. Why are nitro groups relatively rare in other drugs but (almost) indispensable in drugs for these parasites? What have the authors done to address concerns about the presence of nitro groups? A very informative review is referenced, but again just a few sentences would help.

4) The hormetic or U-shaped dose response curve is somewhat unusual, and the addition of blood concentration in Figure 3A and 3B might help. It's also possible that this issue could be left undiscussed as it doesn't seem to have any ready interpretation that would help the reader or provide any insights that would illuminate the main points of the article.

Reviewer #2:

This manuscript presents compelling data to support the conclusion that the tuberculosis drug delamanid-can be repurposed as an inhibitor of Leishmania donovani. The compound is very effective in vitro versus promastigotes. More importantly it is effective in vivo when given twice daily, orally, at 1 mg per kilogram for 10 days. I am not concerned with the U-shaped dose response curve or issues of drug cost. I agree with the authors that delamanid represents a potential oral treatment for visceral leishmaniasis but also conclude that the manuscript would be strengthened by attention to the following points:

1) The experiments illustrated in Figures 4 – 6 were carried out with Leishmania promastigotes. Did the authors also determine in vitro activity of the delamanid versus amastigotes within macrophages? Given the efficacy of this drug in the mouse model of infection one would expect efficacy in vitro as well versus intracellular amastigotes. The focus of this proposed experiment should be the effect of drug not against axenic amastigotes, but against amastigotes within macrophages.

2) Because metabolism of amastigotes differs from that of promastigotes, it would also be important to reevaluate whether the mechanism of action of delamanid is distinct from fexinidazole in amastigotes harvested from macrophages. This is important because the macrophage itself may modify the drug as well as the parasite.

3) A concern commonly voiced in the pharmaceutical industry with any nitro containing drug is the potential for genotoxicity and carcinogenesis. Was an Ames test carried out on delamanid? While this drug has been approved for use against tuberculosis, its potential for genotoxicity or cancer induction will not really be known until it has been used extensively in different populations "post market". While the TPP for leishmaniasis dictates a relatively short course of the drug, the length of treatment is only one variable and again, unfortunately, only extensive use clinically will reveal if delamanid is truly safe. The authors should at least comment on this possibility in the Discussion.

Also in this regard it should be noted that proponents of fexinidazole for the treatment of sleeping sickness have argued that although fexinidazole is positive in the Ames test, it is negative in mammalian genotoxic tests. They also argue that the nitroreductases necessary to produce free radicals from fexinidazole were not present in some bacteria. Nevertheless, one will not be certain that the positive Ames test is of the concern until fexinidazole has been used in several clinical settings. A major concern is the difference in the microbiome between a patient in India, Africa, and Europe. One cannot assume that the absence of an enzyme like a nitroreductase in one microbiome means it will be absent in all. Again this is not a criticism of the current manuscript, but at least a warning or recognition of this potential issue should be stated in the Discussion.

Reviewer #3:

The manuscript describes in vitro and in vivo activity against leishmania of a nitroimidazole-containing TB drug that has recently been EMA approved for the treatment of MDR tuberculosis. The most important and exciting finding is that delamanid is 30-50 times more active in vivo than either the current standard of care or one of the most promising clinical candidates for the treatment of VL. The authors have reported on analogs of this class of molecule before, with the rather disappointing finding that the other TB drug candidate in this class (PA-824) had only moderate activity. PA-824 is a chiral molecule with an S configuration on the oxazine ring; the surprising finding was that the opposite stereoisomer (which is not being developed for clinical use) was considerably more active. Additional screening revealed another molecule with similar stereospecificity that was similar to delamanid and the crucial insight in this work was that delamanid, (R)-PA-824, and the screening hit all presented similar stereochemical configurations suggesting delamanid might be potent against VL.

On balance the work is well done, carefully and thoughtfully presented and convincing that delamanid should be a strong candidate for repurposing for the treatment of VL – which is an extremely important bit of news in this disease.

There are a couple of things that could be improved upon particularly around the discussion of the "hormetic" dose-response curve which is very confusing and needs to be clarified – specifically I would suggest that they add the blood-concentration curve for the 1 mg kg-1dose to Figure 3A and B and show that this is not due to differences in accumulation rates for the lower dose. This is a really confusing piece of information and it is not clear if they have looked for this effect in vitro at the relevant concentration ranges in vitro to see if this is metabolism by the organism or metabolism by the host (if in fact their explanation of the antagonistic metabolite is correct).

There have been two (very) recent publications on the in vivo metabolism of delamanid (Shimokawa et al., Drug Metabol Dispos 2015 Aug;43(8)1277-83 and Sasahara et el in the same issue pp 1267-76) that should be cited and discussed. It seems at least plausible that the antagonistic metabolite is the molecule referred to as M1 in those publications that is formed by cleavage of the nitroimidazooxazole. They describe a simple looking procedure for producing M1 from human serum albumin that might allow the authors to more definitively address the plausibility of this pharmacodynamic mechanism.

My only other complaint is in the subsection “Delamanid – mode of action studies” where they discuss the lack of cross-resistance between fexinidazole resistant LV9 promastigotes and delamanid but state that the data will be published elsewhere. Either show the data or take this out of the Results section. Personally I don't think this is necessary as the lack of hypersensitivity of the NTR overexpressing parasite is already pretty convincing evidence that the drugs act by different mechanisms.

1) The authors and reviewers are experienced in drug development, but almost all eLife readers are not. It would be useful in revising the manuscript to keep this in mind and provide clues to what the various tests are trying to determine. For example, most readers will be unfamiliar with a term like 'area under the curve' (AUC) in pharmacology, and adding a brief note that it measures the total dose of drug and is useful for comparing different formulations would help enormously. It's a challenge, but a challenge worth addressing. The report as written is informative to a specialist and almost unintelligible to anyone else.

An explanatory paragraph to address this has been added to the beginning of the “Blood levels of orally dosed delamanid in a mouse model” Results section.

2) It would be useful to add some clarification about different developmental stages of the parasite: the difference between a promastigote and amastigote, delamanid's efficacy against different stages, possibility of host metabolism of delamanid altering efficacy, and the possibility of different targets for different stages.

A) Explanatory text has been added to the “in vitro sensitivity of L. donovani to (S)- and (R)-delamanid” section of the Results to clarify the difference between the developmental stages. The comparative efficacy against these stages is reported in Table 1.

B) We have carried out additional experiments on mouse macrophages and THP-1 cells and found no evidence for further metabolism by these host cell lines. This has been added to the text in the Results section “Metabolism of delamanid in L. donovani”.

C) The target enzyme(s) involved in activation of delamanid for each stage is not known. However, we have carried out an additional experiment (Figure 5C) showing that overexpression of NTR does not affect the efficacy of delamanid against the intra macrophage amastigote. See Figure 5C and Results section “Delamanid – mode of action studies”. Thus NTR is not involved in either stage of the life cycle.

3) The presence of a nitro group, which is common in this field and quite familiar to the authors, should be addressed for the general reader. Why are nitro groups relatively rare in other drugs but (almost) indispensable in drugs for these parasites?

There are a number of potential explanations for the “over representation” of nitroaromatic-containing drugs/clinical candidates for kinetoplastid diseases relative to other indications: 1) as for other drugs against these diseases both nifurtimox and benznidazole were developed >40 years ago before there was a general (justifiable) aversion to the development of nitro drugs by the pharma industry; 2) the Drugs for Neglected Diseases initiative made a strategic decision to investigate the efficacy of nitroaromatic-containing compound collections against kinetoplastid parasites (leading to the development of fexinidazole and DNDI-VL-2098); and 3) the kinetoplastids possess a bacterial-like NTR which activates nitroaromatic prodrugs. Bacterial-like and mammalian-like NTRs have different substrate specificities; therefore, there is the potential for nitro drugs to be selectively toxic to parasites over mammalian cells.

A paragraph discussing this question is included in the Discussion (seventh paragraph).

What have the authors done to address concerns about the presence of nitro groups? A very informative review is referenced, but again just a few sentences would help.

We have not conducted any experiments of our own to specifically address concerns relating to the nitro functionality of delamanid. However, reference to the work of Matsumoto et al. is cited in the eighth paragraph of the Discussion, showing that there is no evidence for mutagenicity based on a comprehensive series of bacterial reverse mutation experiments.

4) The hormetic or U-shaped dose response curve is somewhat unusual, and the addition of blood concentration in Figure 3A and 3B might help.

See response to reviewer #3 below.

It's also possible that this issue could be left undiscussed as it doesn't seem to have any ready interpretation that would help the reader or provide any insights that would illuminate the main points of the article. Although we do not know why the U-shaped dose response curve is observed, we feel that it is so unusual that it merits some level of discussion, including hypotheses as to why it might occur. Therefore, we propose to leave the discussion as it is. Note, as our additional studies showed some variation in the efficacy at lower delamanid doses we have amended the abstract to reflect that doses of 30 mg/kg (5 days) are curative and those at 1 mg/kg (10 days) on average suppress parasite burden by 79%.

Reviewer #2:

1) The experiments illustrated in Figures 4 – 6 were carried out with Leishmania promastigotes. Did the authors also determine in vitro activity of the delamanid versus amastigotes within macrophages? Given the efficacy of this drug in the mouse model of infection one would expect efficacy in vitro as well versus intracellular amastigotes. The focus of this proposed experiment should be the effect of drug not against axenic amastigotes, but against amastigotes within macrophages.

Yes, see Table 1, showing delamanid is efficacious against intra macrophage amastigotes in vitro. The additional study displayed in Figure 5C using intracellular amastigotes overexpressing NTR shows that NTR does not activate delamanid in amastigotes either. For practical reasons the studies displayed in Figures 4 & 6 have not been repeated using intra macrophage amastigotes.

2) Because metabolism of amastigotes differs from that of promastigotes, it would also be important to reevaluate whether the mechanism of action of delamanid is distinct from fexinidazolein amastigotes harvested from macrophages. This is important because the macrophage itself may modify the drug as well as the parasite.

The new experiment displayed in Figure 5C (see point 1 above) shows that the mode of activation of delamanid is distinct from fexinidazole and nifurtimox in intracellular amastigotes.

We have established that the macrophage does not metabolise delamanid – see Essential revisions, point 2B above.

3) A concern commonly voiced in the pharmaceutical industry with any nitro containing drug is the potential for genotoxicity and carcinogenesis. Was an Ames test carried out on delamanid?

The results of a delamanid bacterial reverse mutagenesis test (Ames test) are reported in the literature, see Matsumoto et al.(2006) PloS Med. 3(11): e466. Delamanid (then called OPC-67683) was found not to be mutagenic. Prolonged (104 week) exposure of rats or mice to delamanid showed no evidence of carcinogenicity (EMA Assessment report 2013). These points have been added to the Discussion.

While this drug has been approved for use against tuberculosis, its potential for genotoxicity or cancer induction will not really be known until it has been used extensively in different populations "post market". While the TPP for leishmaniasis dictates a relatively short course of the drug, the length of treatment is only one variable and again, unfortunately, only extensive use clinically will reveal if delamanid is truly safe. The authors should at least comment on this possibility in the Discussion.

Text to cover the concerns associated with the potential mutagenicity of nitro drugs has been inserted in the Discussion (eighth paragraph).

Also in this regard it should be noted that proponents of fexinidazole for the treatment of sleeping sickness have argued that although fexinidazole is positive in the Ames test, it is negative in mammalian genotoxic tests. They also argue that the nitroreductases necessary to produce free radicals from fexinidazole were not present in some bacteria. Nevertheless, one will not be certain that the positive Ames test is of the concern until fexinidazole has been used in several clinical settings. A major concern is the difference in the microbiome between a patient in India, Africa, and Europe. One cannot assume that the absence of an enzyme like a nitroreductase in one microbiome means it will be absent in all. Again this is not a criticism of the current manuscript, but at least a warning or recognition of this potential issue should be stated in the Discussion.

Text has been inserted into the manuscript to highlight some of the known liabilities of nitro drugs (see above), specifically “although long term safety can only be established after extensive clinical use in relevant populations.”

Reviewer #3:

On balance the work is well done, carefully and thoughtfully presented and convincing that delamanid should be a strong candidate for repurposing for the treatment of VL – which is an extremely important bit of news in this disease.

There are a couple of things that could be improved upon particularly around the discussion of the "hormetic" dose-response curve which is very confusing and needs to be clarified – specifically I would suggest that they add the blood-concentration curve for the 1 mg kg-1dose to Figure 3A and B and show that this is not due to differences in accumulation rates for the lower dose.

The blood concentration data for the 1 mg kg-1 b.i.d. 5 day study has been added to Figures 3A & B. In addition the delamanid blood concentration data for a new 0.3, 1, 3, 10 and 30 mg kg-1 b.i.d. 10 day study has been added (Figure 3C & D). This data demonstrates that the U-shaped dose-response curve is not due to non-linear drug accumulation when dosed at 1 mg kg-1.

This is a really confusing piece of information and it is not clear if they have looked for this effect in vitro at the relevant concentration ranges in vitro to see if this is metabolism by the organism or metabolism by the host (if in fact their explanation of the antagonistic metabolite is correct).

We did not perform any specific experiments to look for the hormetic effect in vitro. However, as mentioned in the Discussion, analysis of the EC50 curves from assaying delamanid against promastigotes and intra macrophage amastigotes showed no hormetic effect at the drug concentration range used in the assays.

There have been two (very) recent publications on the in vivo metabolism of delamanid (Shimokawa et al., Drug Metabol Dispos 2015 Aug;43(8)1277-83 and Sasahara et el in the same issue pp 1267-76) that should be cited and discussed. It seems at least plausible that the antagonistic metabolite is the molecule referred to as M1 in those publications which is formed by cleavage of the nitroimidazooxazole. They describe a simple looking procedure for producing M1 from human serum albumin that might allow the authors to more definitively address the plausibility of this pharmacodynamic mechanism.

We thank the reviewer for highlighting the relevance of these recent publications to this study, and we have included citations to them in the manuscript. As per the reviewer’s suggestion, we do intend to investigate the effect of M1 on the metabolism of delamanid. However, the suggested experiments will probably take some time to complete, and so we intend to report them in an additional publication focussing on the metabolism of delamanid/mechanism of action, rather than be appended to the current manuscript.

My only other complaint is in the subsection “Delamanid – mode of action studies” where they discuss the lack of cross-resistance between fexinidazole resistant LV9 promastigotes and delamanid but state that the data will be published elsewhere. Either show the data or take this out of the Results section. Personally I don't think this is necessary as the lack of hypersensitivity of the NTR overexpressing parasite is already pretty convincing evidence that the drugs act by different mechanisms.

The following text has been removed as suggested, “In addition, in studies to be published elsewhere, LV9 promastigotes which had been selected for resistance to fexinidazole retained sensitivity to delamanid (3.24 ± 0.04 and 3.09 ± 0.03 nM for WT and resistant lines, respectively).”

Other alterations not suggested by the referees, or editor:

An additional affiliation has been added for some of the authors.

We have conducted two additional delamanid VL animal model studies since the original submission of this manuscript. The relevant efficacy and DMPK data from these studies has been aggregated with that included originally. This has resulted in small changes to the mean Cmax and AUC values throughout, which is reflected by minor alterations to some figures.

The unusual U-shaped dose response is still visible despite changes in the mean parasite suppression. The only significant finding is that dosing at 1 mg kg-1 for 10 days is not curative in every instance (n has doubled from 5 to 10 mice) giving a new mean parasite suppression of 79%. Due to this observation, we have removed any writing which states that this dose is curative, and any resultant extrapolation as to what a curative human dose of delamanid might be. For example; 3 sentences have been removed from the Discussion; “However, plots of Cmax versus […] observed in humans”.

Discussion, tenth paragraph. As we are no longer predicting that once daily 100 mg kg-1 dosing of delamanid will be efficacious, we have amended the treatment cost per patient to be >US$840.

Our summary statement at the end of the Discussion has been amended to reflect other changes in the Discussion section – see above.

As a substantial amount of data from six individual animal studies is now included in this manuscript we have produced a spreadsheet which shows detailed efficacy and DMPK data for each individual animal. This is to ensure the transparent communication of this large set of results. This spreadsheet is reported as a supplementary file.

Whilst this manuscript was under revision a paper describing the in vivo and in vitro activity of delamanid and analogues against Leishmania spp. has been published (Thompson et al. J Med Chem 2016). A paragraph has been added (Discussion, ninth paragraph to discuss the relevance of their results in relation to our own. We have also added an additional EC50 determination using the L. infantum strain used in the study of Thompson et al. to Table 1.

We have added the details of the software used to calculate AUC values to the manuscript. We apologise for failing to include this in the original submission.