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]
\nGroups 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.\n

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\n\nDetermination of Delamanid exposure in infected mice after oral dosing [4]
\nBlood 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.\n

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\n\nRate of Delamanid metabolism in L. donovani promastigotes [4]
\nRate 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.\n

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\n\n(i) Study 1. [3]
\nStudy 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.\n

\n(ii) Study 2. [3]
\nStudy 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.\n

\n(iii) Study 3. [3]
\nStudy 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 of 100 mg delamani, the peak plasma concentration is 135 ng/mL. Steady-state concentrations are reached after 10–14 days. Plasma exposure to delamani does not increase proportionally with increasing dose. In animal models (dogs, rats, mice), the oral bioavailability of delamani is 35%–60%. The estimated absolute oral bioavailability in humans is 25%–47%. Due to its poor water solubility, oral bioavailability is approximately 2.7-fold higher when taken with a standard meal compared to an empty stomach. Delamani is primarily excreted in feces, with less than 5% excreted in urine. The apparent volume of distribution (Vz/F) is 2100 L. Animal pharmacokinetic data show that delamani and/or its metabolites are secreted into breast milk. In lactating rats, the peak plasma concentration (Cmax) of delamani in breast milk is four times that in blood.

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Metabolism/Metabolites
Dramani is primarily metabolized via albumin, and secondarily via CYP3A4. Hepatic CYP1A1, CYP2D6, and CYP2E1 may also be involved in the metabolism of delamani, but to a lesser extent [31966]. Four major metabolites (M1-M4) have been identified in the plasma of patients receiving delamani treatment, with M1 and M3 accounting for 13%-18% of total plasma exposure. Although these metabolites do not possess significant pharmacological activity, they may still contribute to QT interval prolongation. This is particularly true of the major delamani metabolite M1 (DM-6705). Delamani is primarily metabolized via serum albumin, through the hydrolytic cleavage of the 6-nitro-2,3-dihydroimidozolo[2,1-b]oxazole moiety to generate metabolite M1 (DM-6705). The generation of this major metabolite is considered a key starting point in the delamani metabolic pathway. M1 (DM-6705) can be further catalyzed via three pathways. In the first metabolic pathway, the oxazole moiety of DM-6705 undergoes hydroxylation to generate metabolite M2 ((4RS,5S)-DM-6720), followed by CYP3A4-mediated hydroxylation and oxazole tautomerization to generate the iminoketone metabolite M3 ((S)-DM-6718). The second metabolic pathway involves the hydrolysis and deamination of oxazolamide to form M4 (DM-6704), which is then hydroxylated to generate M6 ((4R,5S)-DM-6721) and M7 ((4S,5S)-DM-6722), and oxazole is oxidized to generate another ketone metabolite, M8 ((S)-DM-6717). The third pathway involves the hydrolytic cleavage of the oxazol ring to generate M5 (DM-6706).

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

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Pharmacokinetic Results.
[3] Figure 1 shows the plasma concentration-time curves of delamani after multiple administrations alone or in combination with each drug (by study). Table 2 lists the key pharmacokinetic parameters of delamani, the number of subjects for which pharmacokinetic assessments were performed, and the statistical assessment of potential drug interactions (by study). Table 3 lists the pharmacokinetic parameters of combination therapy and the statistical assessment of potential drug interactions (by study). (i) Study 1. [3] Steady-state concentrations of delamani were reached on day 15 (the last day of administration). 200 mg of delamani was administered once daily, alone or in combination with ethambutol-Riford. As expected, due to the long half-life of the metabolite (19), steady-state concentrations of the delamani metabolite were not reached during the 15-day study period. According to Williams et al.'s criteria, steady-state Cmax and AUCτ of delamani were not equivalent when used in combination with ethambutol-rifotaxel (Cmax geometric mean ratio [GMR] = 0.577 [90% CI = 0.492 to 0.676], AUCτ GMR = 0.525 [90% CI = 0.439 to 0.628]) (Table 2). In this study, the concentrations of delamani's major and most common metabolites (DM-6704, DM-6705, and DM-6706) were also reduced by approximately 30% to 50% (based on AUC) when delamani was used in combination with ethambutol-rifapine (Table 4). The mean AUC ratio of metabolite DM-6704 to delamani and the ratio of metabolite DM-6705 to delamani were similar across treatment groups on day 15. This observation, combined with the decrease in overall metabolite concentration, suggests that rifampin-induced CYP3A4 is not the primary cause of the reduced delamani exposure during combination therapy, and that under the conditions of this study, the bioavailability of delamani may be reduced when it is combined with ethambutol-rifampin. The CYP2C9 genotype had no effect on the pharmacokinetics of delamani (unpublished results). Regarding ethambutol concentration, the concentrations of ethambutol-rifampin and delamani were comparable after combination therapy (Table 3). The exposures of rifampin and pyrazinamide were also comparable after combination therapy compared to rifampin alone (Table 3). Regarding isoniazid, as expected, the NAT2 genotype had a significant effect on isoniazid exposure, with isoniazid concentrations in slow acetylated individuals being approximately twice that in moderate/rapid acetylated individuals. Due to the genotype mismatch between the two groups, a prospective statistical analysis of isoniazid AUCτ was not possible. Visual examination of individual AUCτ values in slow-acetylated and intermediate/rapid-acetylated individuals revealed that delamani had no effect on the pharmacokinetics of isoniazid (Figure 2).
(ii) Study 2.[3]
Delamani concentrations reached steady state on day 14 (the last day of administration), determined based on plasma concentrations of delamani prior to administration from day 12 to day 14. Results of delamani monotherapy or in combination with tenofovir (300 mg once daily) or lopinavir (400 mg twice daily) plus ritonavir (caletra twice daily) were presented. Table 2 provides a summary of pharmacokinetic data and statistical evaluation of delamani. Steady-state exposure of delamani was comparable to that of tenofovir when used in combination with tenofovir. Exposure of delamani was considered equivalent to that of lopinavir/ritonavir when used in combination. When used in combination with delamani, steady-state exposures of lopinavir were confirmed to be equivalent, as were steady-state exposures of tenofovir and ritonavir (Table 3).
(iii) Study 3.[3]
Based on individual pre-dose plasma concentrations, steady-state concentrations of delamani (100 mg twice daily for 7 days) and efavirenz (600 mg once daily at night for 10 days) were reached. As shown in Table 2 and Figure 1, efavirenz did not affect the steady-state exposure of delamani, nor did delamani affect the plasma concentration of efavirenz (Table 3). Efavirenz plasma exposure was consistent with the CYP2B6 genotype (unpublished results).

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Dramani has not yet received marketing approval from the U.S. Food and Drug Administration (FDA), but it is available in other countries. There is currently no information regarding the clinical use of deramani during lactation. Preliminary evidence suggests that deramani and its active metabolites are present in low concentrations in breast milk. Deramani is often used in combination with several other drugs to treat drug-resistant tuberculosis; therefore, the clinical significance of these small amounts of medication is unclear.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
Dramani is highly bound to all plasma proteins, with a total protein binding rate ≥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 belongs to the piperidine class of compounds. Delamanid is an anti-tuberculosis drug belonging to the nitrodihydroimidazole-oxazole class of compounds that inhibits the synthesis of mycolic acid in bacterial cell walls. It is often used in combination with other drugs to treat multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (DR-TB). The emergence of MDR-TB and DR-TB presents clinical challenges for patients due to their high mortality rates and poor response to standard anti-tuberculosis therapies (such as [DB00951] and [DB01045]). Treatment of MDR-TB may require more than two years of chemotherapy and second-line therapy with a narrow treatment index. In a clinical study of patients with pulmonary MDR-TB or DR-TB, delamanid, combined with an optimized background treatment regimen recommended by the World Health Organization, improved treatment outcomes and reduced mortality. Spontaneous resistance to delamani was observed during treatment, with mutations in one of the five F420 coenzymes responsible for delamani bioactivation being the cause. Delamani has been approved by the European Medicines Agency (EMA) and marketed under the brand name Deltyba as an oral tablet. It is marketed by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Delamani is a nitrodihydroimidazole-oxazole derivative with anti-mycobacterial activity. After oral administration, the prodrug delamani is activated via the mycobacterial F420 coenzyme system, forming an active intermediate metabolite that inhibits the synthesis of mycobacterial cell wall components methoxymycolic acid and ketomycolic acid. This leads to the depletion of these cell wall components and the destruction of mycobacteria. Indications: This drug is indicated for adult patients with multidrug-resistant tuberculosis (MDR-TB) and may be used as part of an appropriate combination therapy when an effective treatment regimen cannot be established due to resistance or tolerance.
Deltyba is indicated for adults, adolescents, children, and infants weighing at least 10 kg. It may be used as part of an appropriate combination therapy regimen in patients with multidrug-resistant tuberculosis (MDR-TB) when an effective treatment regimen cannot be established due to resistance or tolerance (see Sections 4.2, 4.4, and 5.1). Official guidelines for the rational use of antimicrobial agents should be considered.
Treatment of Multidrug-Resistant Tuberculosis

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Mechanism of Action
Deltyba is a prodrug that requires biotransformation via the mycobacterial F420 coenzyme system (including desoxyflavin-dependent nitroreductase Rv3547) to exert its antimycobacterial activity against both vegetative and non-vegetative mycobacteria. Mutations in one of the five F420 coenzyme genes (fgd, Rv3547, fbiA, fbiB, and fbiC) are considered a mechanism of resistance to deltyba. Upon activation, the free radical intermediate formed by denitroimidazole oxazole derivatives is thought to mediate anti-mycobacterial activity by inhibiting the synthesis of methoxymycolic acid and ketomycolic acid, leading to the depletion of mycobacterial cell wall components and ultimately destroying the mycobacteria. Nitroimidazole oxazole derivatives are thought to generate reactive nitrogen species, including nitric oxide (NO). However, unlike isoniazid, denitroimidazole does not produce α-mycolic acid.

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Pharmacodynamics
The minimum inhibitory concentration (MIC) of denitroimidazole against Mycobacterium tuberculosis isolates ranges from 0.006 to 0.024 μg/mL. Among non-tuberculous mycobacteria, denitroimidazole exhibits in vitro activity against Mycobacterium kansas and Mycobacterium bovis.
Denitroimidazole has no in vitro activity against either Gram-negative or Gram-positive bacteria and does not exhibit cross-resistance with other anti-tuberculosis drugs. In a mouse model of chronic tuberculosis, denitroimidazole dose-dependently reduces Mycobacterium tuberculosis colony counts. Repeated administration of delamani may lead to QTc interval prolongation by inhibiting cardiac potassium channels (hERG channels), an effect primarily caused by delamani's major metabolite, DM-6705. Animal studies have shown that delamani may reduce vitamin K-dependent blood clotting and prolong prothrombin time (PT) and activated partial thromboplastin time (APTT). Delamani was developed by Otsuka Pharmaceutical Development & Commercialization Co., Ltd. (Tokyo, Osaka, Japan). It belongs to the nitroimidazole class of drugs. It inhibits the synthesis of mycolic acid, a key component of the cell wall of the Mycobacterium tuberculosis complex. It is insoluble in water, and its activity has been confirmed in numerous in vitro and in vivo studies. The drug received marketing authorization in Europe in April 2014. Its bactericidal activity has been demonstrated in patients with drug-sensitive and drug-resistant tuberculosis (MDR-TB and XDR-TB). The drug has a good safety and tolerability; the reported QT interval prolongation is not clinically significant. The drug is approved for use in adults, but ongoing clinical trials and clinical experience have demonstrated its effectiveness in children as well. [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.)