Bacterial cell wall synthesis

Mycobacterium tuberculosis complex's cell wall is made up primarily of mucolic acids, which are inhibited by delamanid[1].
When it comes to M. tuberculosis strains that are both drug-susceptible and drug-resistant, delamanid exhibits more potent antibacterial activity[2].
The coadministration of delamanid results in approximately 25% higher ethambutol AUCτ and Cmax values; however, delamanid does not affect the exposure to pyrazinamide, rifampin, or isoniazid[3].

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Intense search has been made in the discovery of newer anti-TB drugs to tackle the issues such as drug resistance, HIV co-infection and risk of drug-drug interactions in the management of TB. Delamanid, a newer mycobacterial cell wall synthesis inhibitor, received a conditional approval from European medicines agency (EMA) for the treatment of MDR-TB. Preclinical and clinical studies have shown that delamanid has high potency, least risk for drug-drug interactions and better tolerability. [2]

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In vitro sensitivity of L. donovani to (S)- and (R)-Delamanid [4]
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).

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.

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Physicochemical properties of Delamanid [4]
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.

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Delamanid-mediated cell killing [4]
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).

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Delamanid – mode of action studies [4]
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.

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Metabolism of Delamanid in L. donovani [4]
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.

In a mouse model of VL, delamanid (oral administration; 30 mg/kg; 5 days) causes sterile cures[4].

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Delamanid is a medicinal product approved for treatment of multidrug-resistant tuberculosis. Three studies were conducted to evaluate the potential drug-drug interactions between delamanid and antiretroviral drugs, including ritonavir, a strong inhibitor of CYP3A4, and selected anti-TB drugs, including rifampin, a strong inducer of cytochrome P450 (CYP) isozymes. Multiple-dose studies were conducted in parallel groups of healthy subjects. Plasma samples were analyzed for delamanid, delamanid metabolite, and coadministered drug concentrations, and pharmacokinetic (PK) parameters were determined. The magnitude of the interaction was assessed by the ratio of the geometric means and 90% confidence intervals. Coadministration of delamanid with tenofovir or efavirenz did not affect the PK characteristics of delamanid. Coadministration of Kaletra (lopinavir/ritonavir) with delamanid resulted in an approximately 25% higher delamanid area under the concentration-time curve from time 0 to the end of the dosing interval (AUCτ). Tenofovir, efavirenz, lopinavir, and ritonavir exposure were not affected by delamanid. Coadministration of delamanid with the TB drugs (ethambutol plus Rifater [rifampin, pyrazinamide, and isoniazid]) resulted in lower delamanid exposures (47 and 42% for the AUCτ and Cmax [maximum concentration of a drug in plasma] values, respectively), as well as decreased exposure of three primary metabolites (approximately 30 to 50% lower AUCτ values). Delamanid did not affect rifampin, pyrazinamide, and isoniazid exposure; the ethambutol AUCτ and Cmax values were about 25% higher with delamanid coadministration. The lack of clinically significant drug-drug interactions between delamanid and selected antiretroviral agents (including the strong CYP inhibitor ritonavir) and a combination of anti-TB drugs was demonstrated. Although there was a decrease in the delamanid concentrations when coadministered with ethambutol plus Rifater, this is likely related to decreased delamanid absorption and not to CYP induction. [3]

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Efficacy of Delamanid in a murine model of visceral leishmaniasis [4]
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.
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).

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Blood levels of orally dosed Delamanid in a mouse model [4]
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.

In vitro drug sensitivity assays against promastigotes [4]
To examine the effects of test compounds such as Delamanid 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.

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Cytocidal effects of Delamanid on L. donovani promastigotes [4]
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.

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In vitro drug sensitivity assays in mouse macrophages and toxicity to HepG2 cells [4]
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.

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In vitro pharmacokinetic and biophysical properties [4]
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 [4]
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.

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Determination of Delamanid exposure in infected mice after oral dosing [4]
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.

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Rate of Delamanid metabolism in L. donovani promastigotes [4]
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.

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(i) Study 1. [3]
Study 1 was a phase 1, randomized, double-blind, placebo-controlled, drug-drug interaction study following multiple once daily oral doses in three parallel groups of clinic-confined healthy subjects receiving either (i) Delamanid, (ii) ethambutol plus Rifater (ethambutol-Rifater), or (iii) delamanid plus ethambutol-Rifater. Rifater is a combination tablet of rifampin, isoniazid, and pyrazinamide. The study was conducted at PPD Development, LP, in Austin, TX.

(ii) Study 2. [3]
Study 2 was a phase 1, randomized, open-label, oral multiple-dose drug interaction study in seven parallel groups of clinic-confined healthy subjects. Delamanid (twice-daily dosing), tenofovir, efavirenz, or Kaletra (lopinavir/ritonavir) were administered alone, and delamanid was also coadministered with tenofovir, efavirenz, or Kaletra for 14 days. The study was conducted at PPD Development, LP, in Austin, TX. The efavirenz arms (alone and with delamanid) were discontinued midstudy due to adverse events (AEs) and a revised design tested in study 3.

(iii) Study 3. [3]
Study 3 was a phase 1, randomized, open-label, modified sequential, oral multiple-dose drug interaction study in two parallel groups of clinic-confined healthy subjects. Subjects were administered either efavirenz for 10 days (group 1) or Delamanid twice daily for 7 days, followed by delamanid twice daily plus efavirenz for 10 days (group 2). The study was conducted at Covance Clinical Research Unit in Evansville, IN.

Absorption, Distribution and Excretion
Following a single oral dose administration of 100 mg Delamanid, the peak plasma concentration was 135 ng/mL. Steady-state concentration is reached after 10-14 days. Delamanid plasma exposure increases less than proportionally with increasing dose. In animal models (dog, rat, mouse), the oral bioavailability of delamanid was reported to be 35%–60%. The absolute oral bioavailability in humans is estimated to range from 25 to 47%. Oral bioavailability in humans is enhanced when administered with a standard meal, by about 2.7 fold compared to fasting conditions because delamanid exhibits poor water solubility.
Delamanid is excreted primarily in the stool, with less than 5% excretion in the urine.
The apparent volume of distribution (Vz/F) is 2,100 L. Pharmacokinetic data in animals have shown excretion of delamanid and/or its metabolites into breast milk. In lactating rats, the Cmax for delamanid in breast milk was 4-fold higher than that of the blood.

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Metabolism / Metabolites
Delamanid predominantly undergoes metabolism by albumin and to a lesser extent, CYP3A4.. The metabolism of delamanid may also be mediated by hepatic CYP1A1, CYP2D6, and CYP2E1 to a lesser extent [31966]. Four major metabolites (M1–M4) have been identified in plasma in patients receiving delamanid where M1 and M3 accounts for 13%–18% of the total plasma exposure in humans. While they do not retain significant pharmacological activity, they may still contribute to QT prolongation. This is especially true for the main metabolite of delamanid, M1 (DM-6705). Delamanid is predominantly metabolized by serum albumin to form M1 (DM-6705) via hydrolytic cleavage of the 6-nitro-2,3-dihydroimidazo[2,1-b] oxazole moiety. The formation of this major metabolite is suggested to be a crucial starting point in the metabolic pathway of delamanid. M1 (DM-6705) can be further catalyzed by three pathways. In the first metabolic pathway, DM-6705 undergoes hydroxylation of the oxazole moiety to form M2 ((4RS,5S)-DM-6720), followed by CYP3A4-mediated oxidation of hydroxyl group and tautomerization of oxazole to an imino-ketone metabolite, M3 ((S)-DM-6718). The second metabolic pathway involves the hydrolysis and deamination of the oxazole amine to form M4 (DM-6704) followed by hydroxylation to M6 ((4R,5S)-DM-6721) and M7 ((4S,5S)-DM-6722) and oxidation of oxazole to another ketone metabolite, M8 ((S)-DM-6717). The third pathway involves the hydrolytic cleavage of the oxazole ring to form M5 (DM-6706).

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Biological Half-Life
The half life ranges from 30 to 38 hours.

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Pharmacokinetic results.[3]
The Delamanid plasma concentration-versus-time profiles after multiple dosing for delamanid alone or with each coadministered drug (by study) are shown in Fig. 1. Key PK parameters for delamanid, the number of PK evaluable subjects, and statistical evaluations of potential drug-drug interactions (by study) are presented in Table 2. PK parameters of coadministered drugs and statistical evaluation of potential drug-drug interactions (by study) are presented in Table 3.

(i) Study 1.[3]
Delamanid concentrations reached steady state by day 15, the last day of dosing, following 200-mg once-daily dosing of delamanid alone or with ethambutol-Rifater. As expected, given the long half-life of metabolites (19), delamanid metabolite concentrations did not yet reach steady state with the 15-day duration of the study. Based on the criteria of Williams et al., nonequivalence of steady-state delamanid Cmax and AUCτ was documented when coadministered with ethambutol-Rifater (Cmax geometric mean ratio [GMR] = 0.577 [90% CI = 0.492 to 0.676] and AUCτ GMR = 0.525 [90% CI = 0.439 to 0.628]) (Table 2). The concentrations of the primary and most prevalent metabolites of delamanid in this study (DM-6704, DM-6705, and DM-6706) were also about 30 to 50% lower (based on the AUC) when delamanid was coadministered with ethambutol-Rifater (Table 4). The mean day 15 AUC ratio of metabolite DM-6704 to delamanid and the ratio of metabolite DM-6705 to delamanid were similar between treatments. This observation, coupled with the overall lower concentrations of metabolites, suggests that induction of CYP3A4 by rifampin does not play a major role in the observed lower delamanid exposure with combination treatment and that reduced bioavailability of delamanid may occur when delamanid is coadministered with ethambutol-Rifater under the conditions of this study. CYP2C9 genotype had no effect on delamanid PK (unpublished results).

With regard to ethambutol concentrations, following coadministration of ethambutol-Rifater with Delamanid, equivalence was suggested (Table 3). After the coadministration of ethambutol-Rifater with delamanid, equivalence was suggested for rifampin and pyrazinamide exposures when compared to Rifater given alone (Table 3). With regard to isoniazid, as expected, NAT2 genotype had a profound effect on isoniazid exposure, with slow acetylators having ∼2-fold-higher isoniazid concentrations than intermediate/rapid acetylators. Since the two groups were not matched for genotype, the prospective statistical analysis for isoniazid AUCτ was not interpretable. From visual inspection of individual AUCτ values for slow acetylators compared to intermediate/rapid acetylators, delamanid had no effect on isoniazid PK (Fig. 2).

(ii) Study 2.[3]
Delamanid concentrations reached steady-state by day 14, the last day of dosing, following 100-mg twice-daily dosing of delamanid alone or with either 300 mg of tenofovir once daily or 400 mg of lopinavir plus 100 mg of ritonavir (Kaletra) twice daily, as indicated by the individual day 12 through day 14 predose delamanid plasma concentrations. Table 2 provides the summary PK data and a statistical evaluation for delamanid. Equivalence in the steady-state exposure of delamanid was documented when coadministered with tenofovir. Equivalence was suggested for delamanid exposure after lopinavir/ritonavir coadministration. When coadministered with delamanid, equivalence was documented for steady-state exposure of lopinavir and suggested for tenofovir and ritonavir (Table 3).

(iii) Study 3.[3]
Steady-state was reached for Delamanid (7 days of 100-mg twice-daily dosing) and efavirenz (10 days of once-daily 600 mg dosing in the evening) concentrations as indicated by the individual predose plasma concentrations. As shown in Table 2 and Fig. 1, efavirenz did not affect the steady-state exposure of delamanid, and delamanid did not affect efavirenz plasma concentrations (Table 3). The efavirenz plasma exposure was in agreement with CYP2B6* genotype (unpublished results).

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Delamanid is not approved for marketing in the United States by the U.S. Food and Drug Administration, but is available in other countries. No information is available on the clinical use of delamanid during breastfeeding. Preliminary evidence indicates that delamanid and its active metabolite are present in milk at low levels. Delamanid is usually given with several other drugs for resistant tuberculosis, so the clinical importance of these small amounts is unclear.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Delamanid highly binds to all plasma proteins with a binding to total proteins of ≥99.5%.

[1]. Delamanid (OPC-67683) for treatment of multi-drug-resistant tuberculosis. Expert Rev Anti Infect Ther. 2015 Mar;13(3):305-15.

[2]. Delamanid: A new armor in combating drug-resistant tuberculosis. J Pharmacol Pharmacother. 2014 Jul;5(3):222-4

[3]. Delamanid Coadministered with Antiretroviral Drugs or Antituberculosis Drugs Shows No Clinically Relevant Drug-Drug Interactions in Healthy Subjects. Antimicrob Agents Chemother. 2016 Sep 23;60(10):5976-85.

[4]. The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis. Elife. 2016 May 24;5:e09744.

Delamanid is a member of piperidines.
Delamanid is an anti-tuberculosis agent derived from the nitro-dihydro-imidazooxazole class of compounds that inhibits mycolic acid synthesis of bacterial cell wall. It is used in the treatment of multidrug-resistant and extensively drug-resistant tuberculosis (TB) in a combination regimen. Emergence of multidrug-resistant and extensively drug-resistant tuberculosis creates clinical challenges for patients, as the disease is associated with a higher mortality rate and insufficient therapeutic response to standardized antituberculosis treatments as [DB00951] and [DB01045]. Multidrug-resistant tuberculosis may also require more than 2 years of chemotherapy and second-line therapies with narrow therapeutic index. In a clinical study involving patients with pulmonary multidrug-resistant tuberculosis or extensively drug-resistant tuberculosis, treatment of delamanid in combination with WHO-recommended optimised background treatment regimen was associated with improved treatment outcomes and reduced mortality rate. Spontaneous resistance to delamanid was observed during treatment, where mutation in one of the 5 F420 coenzymes responsible for bioactivation of delamanid contributes to this effect. Delamanid is approved by the EMA and is marketed under the trade name Deltyba as oral tablets. It is marketed by Otsuka Pharmaceutical Co., Ltd (Tokyo, Japan).
Delamanid is a nitro-dihydro-imidazooxazole derivative, with antimycobacterial activity. Upon oral administration, delamanid, a prodrug, is activated via the mycobacterial F420 coenzyme system to form a reactive intermediate metabolite that inhibits the synthesis of the mycobacterial cell wall components methoxy-mycolic and keto-mycolic acid. This leads to the depletion of these cell wall components and destruction of mycobacteria.

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Drug Indication
Indicated for use as part of an appropriate combination regimen for pulmonary multi-drug resistant tuberculosis (MDR-TB) in adult patients when an effective treatment regimen cannot otherwise be composed for reasons of resistance or tolerability.
Deltyba is indicated for use as part of an appropriate combination regimen for pulmonary multi-drug resistant tuberculosis (MDR-TB) in adults, adolescents, children and infants with a body weight of at least 10 kg when an effective treatment regimen cannot otherwise be composed for reasons of resistance or tolerability (see sections 4. 2, 4. 4 and 5. 1). Consideration should be given to official guidance on the appropriate use of antibacterial agents.
Treatment of multi-drug-resistant tuberculosis

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Mechanism of Action
Delamanid is a prodrug that requires biotransformation via via the mycobacterial F420 coenzyme system, including the deazaflavin dependent nitroreductase (Rv3547), to mediate its antimycobacterial activity against both growing and nongrowing mycobacteria. Mutations in one of five coenzyme F420 genes, _fgd, Rv3547, fbiA, fbiB, and fbiC_ has been proposed as the mechanism of resistance to delamanid. Upon activation, the radical intermediate formed between delamanid and desnitro-imidazooxazole derivative is thought to mediate antimycobacterial actions via inhibition of methoxy-mycolic and keto-mycolic acid synthesis, leading to depletion of mycobacterial cell wall components and destruction of the mycobacteria. Nitroimidazooxazole derivative is thought to generate reactive nitrogen species, including nitrogen oxide (NO). However unlike isoniazid, delamanid does not alpha-mycolic acid.

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Pharmacodynamics
The minimum inhibitory concentrations (MIC) of delamanid against _Mycobacterium tuberculosis_ isolates ranges from 0.006 to 0.024 g/mL. Among non-tuberculosis mycobacteria, delamanid has _in vitro_ activity against _M. kansasii_ and _M. bovis_. Delamanid has no in vitro activity against Gram negative or positive bacterial species and does not display cross-resistance to other anti-tuberculosis drugs. In murine models of chronic tuberculosis, the reduction of _M. tuberculosis_ colony counts by delamanid was demonstrated in a dose-dependent manner. Repeated dosing of delamanid may cause QTc-prolongation via inhibition of cardiac potassium channel (hERG channel), and this effect is mostly contributed by the main metabolite of delamanid, DM-6705. Animal studies indicate that delamanid may attenuate vitamin K-dependent blood clotting, increase prothrombin time (PT), and activated partial thromboplastin time (APTT).

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The research and development of delamanid was carried out by Otsuka Pharmaceutical Development and Commercialization (Osaka, Tokyo, Japan). It belongs to the group of nitroimidazoles. It inhibits the synthesis of mycolic acids, crucial component of the cell wall of the Mycobacterium tuberculosis complex. It is insoluble in water and its activity was proven in several in vitro and in vivo studies. Its market approval was obtained in April 2014 in Europe. Its bactericidal activity was demonstrated in individuals with drug-susceptible and drug-resistant tuberculosis (MDR- and XDR-TB). The safety and tolerability profile was good; the notified increased QT interval was not clinically relevant. It was approved for adults but ongoing clinical trials and clinical experiences have been proving its efficacy in the pediatric population.[1]

C25H25F3N4O6

534.4844

534.172

C, 56.18; H, 4.71; F, 10.66; N, 10.48; O, 17.96

681492-22-8

Delamanid-d4

6480466

Off-white to yellow solid powder

1.5±0.1 g/cm3

653.7±65.0 °C at 760 mmHg

349.1±34.3 °C

0.0±2.0 mmHg at 25°C

1.611

4.75

0

11

7

38

795

1

FC(OC1C([H])=C([H])C(=C([H])C=1[H])OC1([H])C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C([H])C=2[H])OC([H])([H])[C@@]2(C([H])([H])[H])C([H])([H])N3C([H])=C([N+](=O)[O-])N=C3O2)C([H])([H])C1([H])[H])(F)F

XDAOLTSRNUSPPH-XMMPIXPASA-N

InChI=1S/C25H25F3N4O6/c1-24(15-31-14-22(32(33)34)29-23(31)38-24)16-35-18-4-2-17(3-5-18)30-12-10-20(11-13-30)36-19-6-8-21(9-7-19)37-25(26,27)28/h2-9,14,20H,10-13,15-16H2,1H3/t24-/m1/s1

(2R)-2-Methyl-6-nitro-2-[(4-{4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl}phenoxy)methyl]-2,3-dihydroimidazo[2,1-b][1,3]oxazole

OPC-67683; Delamanid; 681492-22-8; OPC-67,683; deltyba; Delamanid [USAN]; OPC 67,683; 8OOT6M1PC7; Deltyba (TN);OPC 67683; OPC67683; trade name Deltyba

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL ( ~187.09 mM)
Ethanol : ~2 mg/mL (~3.74 mM)

Solubility in Formulation 1: 2.5 mg/mL (4.68 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (4.68 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)