Development of a HPLC Method for Analysis of a Combination of Clofazimine, Isoniazid, Pyrazinamide, and Rifampicin Incorporated into a Dermal Self-Double-Emulsifying Drug Delivery System.

We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths-254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.

Single and combination treatment of Toxoplasma gondii infections with a bumped kinase inhibitor and artemisone in vitro and with artemiside in experimentally infected mice.

In previous studies, the artemisinin derivatives artemisone, its pro-drug artemiside and the bumped-kinase inhibitor BKI-1748 were effective against T. gondii via different modes of action. This suggests that they may act synergistically resulting in improved efficacies in vitro and in vivo. To test this hypothesis, the compounds were applied alone and in combination to T. gondii infected human fibroblast host cells in order to determine their inhibition constants and effects on cellular ultrastructure. In addition, the efficacy of either single- or combined treatments were assessed in an acute TgShSp1-oocyst infection model based on CD1 outbred mice. Whereas the IC50 of the compounds in combination (42 nM) was close to the IC50 of BKI-1748 alone (46 nM) and half of the IC50 of artemisone alone (92 nM), the IC90 of the combination was half of the values found with the single compounds (138 nM vs. ca. 270 nM). Another indication for synergistic effects in vitro were distinct alterations of the cellular ultrastructure of tachyzoites observed in combination, but not with the single compounds. These promising results could not be reproduced in vivo. There was no decrease in number of T. gondii positive brains by either treatment. However, the levels of infection in these brains, i. e. the number of tachyzoites, was significantly decreased upon BKI-1748 treatment alone, and the combination with artemiside did not produce any further decrease. The treatment with artemiside alone had no significant effects. A vertical transmission model could not be established since artemiside strongly interfered with pregnancy and caused abortion. These results show that is difficult to extrapolate from promising in vitro results to the situation in vivo.

The Development of Dermal Self-Double-Emulsifying Drug Delivery Systems: Preformulation Studies as the Keys to Success.

Self-emulsifying drug delivery systems (SEDDSs) are lipid-based systems that are superior to other lipid-based oral drug delivery systems in terms of providing drug protection against the gastrointestinal (GI) environment, inhibition of drug efflux as mediated by P-glycoprotein, enhanced lymphatic drug uptake, improved control over plasma concentration profiles of drugs, enhanced stability, and drug loading efficiency. Interest in dermal spontaneous emulsions has increased, given that systems have been reported to deliver drugs across mucus membranes, as well as the outermost layer of the skin into the underlying layers. The background and development of a double spontaneous emulsion incorporating four anti-tubercular drugs, clofazimine (CFZ), isoniazid (INH), pyrazinamide (PZY), and rifampicin (RIF), are described here. Our methods involved examination of oil miscibility, the construction of pseudoternary phase diagrams, the determination of self-emulsification performance and the emulsion stability index of primary emulsions (PEs), solubility, and isothermal micro calorimetry compatibility and examination of emulsions via microscopy. Overall, the potential of self-double-emulsifying drug delivery systems (SDEDDSs) as a dermal drug delivery vehicle is now demonstrated. The key to success here is the conduct of preformulation studies to enable the development of dermal SDEDDSs. To our knowledge, this work represents the first successful example of the production of SDEDDSs capable of incorporating four individual drugs.

The Science of Selecting Excipients for Dermal Self-Emulsifying Drug Delivery Systems.

Self-emulsification is considered a formulation technique that has proven capacity to improve oral drug delivery of poorly soluble drugs by advancing both solubility and bioavailability. The capacity of these formulations to produce emulsions after moderate agitation and dilution by means of water phase addition provides a simplified method to improve delivery of lipophilic drugs, where prolonged drug dissolution in the aqueous environment of the gastro-intestinal (GI) tract is known as the rate-limiting step rendering decreased drug absorption. Additionally, spontaneous emulsification has been reported as an innovative topical drug delivery system that enables successful crossing of mucus membranes as well as skin. The ease of formulation generated by the spontaneous emulsification technique itself is intriguing due to the simplified production procedure and unlimited upscaling possibilities. However, spontaneous emulsification depends solely on selecting excipients that complement each other in order to create a vehicle aimed at optimizing drug delivery. If excipients are not compatible or unable to spontaneously transpire into emulsions once exposed to mild agitation, no self-emulsification will be achieved. Therefore, the generalized view of excipients as inert bystanders facilitating delivery of an active compound cannot be accepted when selecting excipients needed to produce self-emulsifying drug delivery systems (SEDDSs). Hence, this review describes the excipients needed to generate dermal SEDDSs as well as self-double-emulsifying drug delivery systems (SDEDDSs); how to consider combinations that complement the incorporated drug(s); and an overview of using natural excipients as thickening agents and skin penetration enhancers.

In Vitro versus in Mice: Efficacy and Safety of Decoquinate and Quinoline-O-Carbamate Derivatives against Experimental Infection with Neospora caninum Tachyzoites.

The effects of decoquinate (DCQ) and three O-quinoline-carbamate-derivatives were investigated using human foreskin fibroblasts (HFF) infected with Neospora caninum tachyzoites. These compounds exhibited half-maximal proliferation inhibition (IC50s) from 1.7 (RMB060) to 60 nM (RMB055). Conversely, when applied at 5 (DCQ, RMB054) or 10µM (RMB055, RMB060), HFF viability was not affected. Treatments of infected cell cultures at 0.5µM altered the ultrastructure of the parasite mitochondrion and cytoplasm within 24 h, most pronounced for RMB060, and DCQ, RMB054 and RMB060 did not impair the viability of splenocytes from naïve mice. Long-term treatments of N. caninum-infected HFF monolayers with 0.5µM of each compound showed that only exposure to RMB060 over a period of six consecutive days had a parasiticidal effect, while the other compounds were not able to kill all tachyzoites in vitro. Thus, DCQ and RMB060 were comparatively assessed in the pregnant neosporosis mouse model. The oral application of these compounds suspended in corn oil at 10 mg/kg/day for 5 d resulted in a decreased fertility rate and litter size in the DCQ group, whereas reproductive parameters were not altered by RMB060 treatment. However, both compounds failed to protect mice from cerebral infection and did not prevent vertical transmission/pup mortality. Thus, despite the promising in vitro efficacy and safety characteristics of DCQ and DCQ-derivatives, proof of concept for activity against neosporosis could not be demonstrated in the murine model.

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation.

The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 µM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium.

Combining computational and experimental evidence on the activity of antimalarial drugs on papain-like protease of SARS-CoV-2: A repurposing study.

The development of inhibitors that target the papain-like protease (PLpro) has the potential to counteract the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent causing coronavirus disease 2019 (COVID-19). Based on a consideration of its several downstream effects, interfering with PLpro would both revert immune suppression exerted by the virus and inhibit viral replication. By following a repurposing strategy, the current study evaluates the potential of antimalarial drugs as PLpro inhibitors, and thereby the possibility of their use for treatment of SARS-CoV-2 infection. Computational tools were employed for structural analysis, molecular docking, and molecular dynamics simulations to screen antimalarial drugs against PLpro, and in silico data were validated by in vitro experiments. Virtual screening highlighted amodiaquine and methylene blue as the best candidates, and these findings were complemented by the in vitro results that indicated amodiaquine as a µM PLpro deubiquitinase inhibitor. The results of this study demonstrate that the computational workflow adopted here can correctly identify active compounds. Thus, the highlighted antimalarial drugs represent a starting point for the development of new PLpro inhibitors through structural optimization.

Efficacies and ADME properties of redox active methylene blue and phenoxazine analogues for use in new antimalarial triple drug combinations with amino-artemisinins.

Efforts to develop new artemisinin triple combination therapies effective against artemisinin-tolerant strains of Plasmodium falciparum based on rational combinations comprising artemisone or other amino-artemisinins, a redox active drug and a third drug with a different mode of action have now been extended to evaluation of three potential redox partners. These are the diethyl analogue AD01 of methylene blue (MB), the benzo [α]phenoxazine PhX6, and the thiosemicarbazone DpNEt. IC50 values in vitro against CQ-sensitive and resistant P. falciparum strains ranged from 11.9 nM for AD01-41.8 nM for PhX6. PhX6 possessed the most favourable pharmacokinetic (PK) profile: intrinsic clearance rate CLint was 21.47 ± 1.76 mL/min/kg, bioavailability was 60% and half-life was 7.96 h. AD01 presented weaker, but manageable pharmacokinetic properties with a rapid CLint of 74.41 ± 6.68 mL/min/kg leading to a half-life of 2.51 ± 0.07 h and bioavailability of 15%. DpNEt exhibited a half-life of 1.12 h and bioavailability of 8%, data which discourage its further examination, despite a low CLint of 10.20 mL/min/kg and a high Cmax of 6.32 µM. Efficacies of AD01 and PhX6 were enhanced synergistically when each was paired with artemisone against asexual blood stages of P. falciparum NF54 in vitro. The favourable pharmacokinetics of PhX6 indicate this is the best partner among the compounds examined thus far for artemisone. Future work will focus on extending the drug combination studies to artemiside in vitro, and conducting efficacy studies in vivo for artemisone with each of PhX6 and the related benzo[α]phenoxazine SSJ-183.

The evaluation of ADME and pharmacokinetic properties of decoquinate derivatives for the treatment of malaria.

The emergence of Plasmodium falciparum (Pf) parasite strains tolerant of the artemisinin component and resistant to the other drug component in artemisinin combination therapies (ACTs) used for treatment now markedly complicates malaria control. Thus, development of new combination therapies are urgently required. For the non-artemisinin component, the quinolone ester decoquinate (DQ) that possesses potent activities against blood stage Pf and acts on a distinct target, namely the Pf cytochrome bc 1 complex, was first considered. However, DQ has poor drug properties including high lipophilicity and exceedingly poor aqueous solubility (0.06 µg/ml), rendering it difficult to administer. Thus, DQ was chemically modified to provide the secondary amide derivative RMB005 and the quinoline O-carbamate derivatives RMB059 and RMB060. The last possesses sub-nanomolar activities against multidrug resistant blood stages of Pf, and P. berghei sporozoite liver stages. Here we present the results of ADME analyses in vitro and pharmacokinetic analyses using C57BL/6 mice. The amide RMB005 had a maximum mean whole blood concentration of 0.49 ± 0.02 µM following oral administration; however, the area under the curve (AUC), elimination half-life (t1/2) and bioavailability (BA) were not significantly better than those of DQ. Surprisingly, the quinoline O-carbamates which can be recrystallized without decomposition were rapidly converted into DQ in human plasma and blood samples. The maximum concentrations of DQ reached after oral administration of RMB059 and RMB060 were 0.23 ± 0.05 and 0.11 ± 0.01 µM, the DQ elimination half-lives were 4.79 ± 1.66 and 4.66 ± 1.16 h, and the DQ clearance were 19.40 ± 3.14 and 21.50 ± 3.38 respectively. Under these assay conditions, the BA of DQ could not be calculated Overall although RMB059 and -060 are labile in physiological medium with respect to the DQ parent, the potential to apply these as prodrugs is apparent from the current data coupled with their ease of preparation.

Antimalarial and antitumour activities of the steroidal quinone-methide celastrol and its combinations with artemiside, artemisone and methylene blue.

Artemisinin, isolated from the traditional Chinese medicinal plant qing hao (Artemisia annua) and its derivatives are used for treatment of malaria. With treatment failures now being recorded for the derivatives and companion drugs used in artemisinin combination therapies new drug combinations are urgently required. The amino-artemisinins artemiside and artemisone display optimal efficacies in vitro against asexual and sexual blood stages of the malaria parasite Plasmodium falciparum and are active against tumour cell lines. In continuing the evolution of combinations of the amino-artemisinins with new drugs, we examine the triterpenoid quinone methide celastrol isolated from the traditional Chinese medicinal plant léi gong téng (Tripterygium wilfordii). This compound is redox active, and has attracted considerable attention because of potent biological activities against manifold targets. We report that celastrol displays good IC50 activities ranging from 0.50-0.82 µM against drug-sensitive and resistant asexual blood stage Pf, and 1.16 and 0.28 µM respectively against immature and late stage Pf NF54 gametocytes. The combinations of celastrol with each of artemisone and methylene blue against asexual blood stage Pf are additive. Given that celastrol displays promising antitumour properties, we examined its activities alone and in combinations with amino-artemisinins against human liver HepG2 and other cell lines. IC50 values of the amino-artemisinins and celastrol against HepG2 cancer cells ranged from 0.55-0.94 µM. Whereas the amino-artemisinins displayed notable selectivities (SI > 171) with respect to normal human hepatocytes, in contrast, celastrol displayed no selectivity (SI < 1). The combinations of celastrol with artemiside or artemisone against HepG2 cells are synergistic. Given the promise of celastrol, judiciously designed formulations or structural modifications are recommended for mitigating its toxicity.

In Vitro Activity of the Arylaminoartemisinin GC012 against Helicobacter pylori and Its Effects on Biofilm.

This study evaluated the in vitro activity of the arylaminoartemisinin GC012, readily obtained from dihydroartemisinin (DHA), against clinical strains of Helicobacter pylori (H. pylori) with different antibiotic susceptibilities in the planktonic and sessile state. The activity was assessed in terms of bacteriostatic and bactericidal potential. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. After treatment with GC012, all bacterial strains showed significantly lower MIC and MBC values compared to those of DHA. The effect of combination of GC012 with antibiotics was examined using the checkerboard method. GC012 displayed synergistic interactions with metronidazole, clarithromycin, and amoxicillin in all the strains. The antibiofilm activity was evaluated via crystal violet staining, AlamarBlue® assay, colony-forming unit count, and fluorescence microscopy. At ½ MIC and » MIC concentration, both GC012 and DHA inhibited biofilm formation, but only GC012 showed a minimal biofilm eradication concentration (MBEC) on mature biofilm. Furthermore, both compounds induced structural changes in the bacterial membrane, as observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is thereby demonstrated that GC012 has the potential to be efficacious against H. pylori infection.

Adapting Clofazimine for Treatment of Cutaneous Tuberculosis by Using Self-Double-Emulsifying Drug Delivery Systems.

Although chemotherapeutic treatment regimens are currently available, and considerable effort has been lavished on the development of new drugs for the treatment of tuberculosis (TB), the disease remains deeply intractable and widespread. This is due not only to the nature of the life cycle and extraordinarily disseminated habitat of the causative pathogen, principally Mycobacterium tuberculosis (Mtb), in humans and the multi-drug resistance of Mtb to current drugs, but especially also to the difficulty of enabling universal treatment of individuals, immunocompromised or otherwise, in widely differing socio-economic environments. For the purpose of globally eliminating TB by 2035, the World Health Organization (WHO) introduced the "End-TB" initiative by employing interventions focusing on high impact, integrated and patient-centered approaches, such as individualized therapy. However, the extraordinary shortfall in stipulated aims, for example in actual treatment and in TB preventative treatments during the period 2018-2022, latterly and greatly exacerbated by the COVID-19 pandemic, means that even greater pressure is now placed on enhancing our scientific understanding of the disease, repurposing or repositioning old drugs and developing new drugs as well as evolving innovative treatment methods. In the specific context of multidrug resistant Mtb, it is furthermore noted that the incidence of extra-pulmonary TB (EPTB) has significantly increased. This review focusses on the potential of utilizing self-double-emulsifying drug delivery systems (SDEDDSs) as topical drug delivery systems for the dermal route of administration to aid in treatment of cutaneous TB (CTB) and other mycobacterial infections as a prelude to evaluating related systems for more effective treatment of CTB and other mycobacterial infections at large. As a starting point, we consider here the possibility of adapting the highly lipophilic riminophenazine clofazimine, with its potential for treatment of multi-drug resistant TB, for this purpose. Additionally, recently reported synergism achieved by adding clofazimine to first-line TB regimens signifies the need to consider clofazimine. Thus, the biological effects and pharmacology of clofazimine are reviewed. The potential of plant-based oils acting as emulsifiers, skin penetration enhancers as well as these materials behaving as anti-microbial components for transporting the incorporated drug are also discussed.

The Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside.

Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5-2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

A Drug Repurposing Approach for Antimalarials Interfering with SARS-CoV-2 Spike Protein Receptor Binding Domain (RBD) and Human Angiotensin-Converting Enzyme 2 (ACE2).

Host cell invasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by the interaction of the viral spike protein (S) with human angiotensin-converting enzyme 2 (ACE2) through the receptor-binding domain (RBD). In this work, computational and experimental techniques were combined to screen antimalarial compounds from different chemical classes, with the aim of identifying small molecules interfering with the RBD-ACE2 interaction and, consequently, with cell invasion. Docking studies showed that the compounds interfere with the same region of the RBD, but different interaction patterns were noted for ACE2. Virtual screening indicated pyronaridine as the most promising RBD and ACE2 ligand, and molecular dynamics simulations confirmed the stability of the predicted complex with the RBD. Bio-layer interferometry showed that artemisone and methylene blue have a strong binding affinity for RBD (KD = 0.363 and 0.226 µM). Pyronaridine also binds RBD and ACE2 in vitro (KD = 56.8 and 51.3 µM). Overall, these three compounds inhibit the binding of RBD to ACE2 in the µM range, supporting the in silico data.

Intracellular Accumulation of Novel and Clinically Used TB Drugs Potentiates Intracellular Synergy.

The therapeutic repertoire for tuberculosis (TB) remains limited despite the existence of many TB drugs that are highly active in in vitro models and possess clinical utility. Underlying the lack of efficacy in vivo is the inability of TB drugs to penetrate microenvironments inhabited by the causative agent, Mycobacterium tuberculosis, including host alveolar macrophages. Here, we determined the ability of the phenoxazine PhX1 previously shown to be active against M. tuberculosis in vitro to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. We also investigated the extent of permeation into uninfected and M. tuberculosis-infected human macrophage-like Tamm-Horsfall protein 1 (THP-1) cells directly and by comparing to results obtained in vitro in synergy assays. Our data indicate that PhX1 (4,750 ± 127.2 ng/ml) penetrates more effectively into THP-1 cells than do the clinically used anti-TB agents, rifampin (3,050 ± 62.9 ng/ml), moxifloxacin (3,374 ± 48.7 ng/ml), bedaquiline (4,410 ± 190.9 ng/ml), and linezolid (770 ± 14.1 ng/ml). Compound efficacy in infected cells correlated with intracellular accumulation, reinforcing the perceived importance of intracellular penetration as a key drug property. Moreover, we detected synergies deriving from redox-stimulatory combinations of PhX1 or clofazimine with the novel prenylated amino-artemisinin WHN296. Finally, we used compound synergies to elucidate the relationship between compound intracellular accumulation and efficacy, with PhX1/WHN296 synergy levels shown to predict drug efficacy. Collectively, our data support the utility of the applied assays in identifying in vitro active compounds with the potential for clinical development. IMPORTANCE This study addresses the development of novel therapeutic compounds for the eventual treatment of drug-resistant tuberculosis. Tuberculosis continues to progress, with cases of Mycobacterium tuberculosis (M. tuberculosis) resistance to first-line medications increasing. We assess new combinations of drugs with both oxidant and redox properties coupled with a third partner drug, with the focus here being on the potentiation of M. tuberculosis-active combinations of compounds in the intracellular macrophage environment. Thus, we determined the ability of the phenoxazine PhX1, previously shown to be active against M. tuberculosis in vitro, to differentially penetrate murine compartments, including plasma, epithelial lining fluid, and isolated epithelial lining fluid cells. In addition, the extent of permeation into human macrophage-like THP-1 cells and H37Rv-infected THP-1 cells was measured via mass spectrometry and compared to in vitro two-dimensional synergy and subsequent intracellular efficacy. Collectively, our data indicate that development of new drugs will be facilitated using the methods described herein.

Toward New Transmission-Blocking Combination Therapies: Pharmacokinetics of 10-Amino-Artemisinins and 11-Aza-Artemisinin and Comparison with Dihydroartemisinin and Artemether.

As artemisinin combination therapies (ACTs) are compromised by resistance, we are evaluating triple combination therapies (TACTs) comprising an amino-artemisinin, a redox drug, and a third drug with a different mode of action. Thus, here we briefly review efficacy data on artemisone, artemiside, other amino-artemisinins, and 11-aza-artemisinin and conduct absorption, distribution, and metabolism and excretion (ADME) profiling in vitro and pharmacokinetic (PK) profiling in vivo via intravenous (i.v.) and oral (p.o.) administration to mice. The sulfamide derivative has a notably long murine microsomal half-life (t1/2 > 150 min), low intrinsic liver clearance and total plasma clearance rates (CLint 189.4, CLtot 32.2 ml/min/kg), and high relative bioavailability (F = 59%). Kinetics are somewhat similar for 11-aza-artemisinin (t1/2 > 150 min, CLint = 576.9, CLtot = 75.0 ml/min/kg), although bioavailability is lower (F = 14%). In contrast, artemether is rapidly metabolized to dihydroartemisinin (DHA) (t1/2 = 17.4 min) and eliminated (CLint = 855.0, CLtot = 119.7 ml/min/kg) and has low oral bioavailability (F) of 2%. While artemisone displays low t1/2 of <10 min and high CLint of 302.1, it displays a low CLtot of 42.3 ml/min/kg and moderate bioavailability (F) of 32%. Its active metabolite M1 displays a much-improved t1/2 of >150 min and a reduced CLint of 37.4 ml/min/kg. Artemiside has t1/2 of 12.4 min, CLint of 673.9, and CLtot of 129.7 ml/kg/min, likely a reflection of its surprisingly rapid metabolism to artemisone, reported here for the first time. DHA is not formed from any amino-artemisinin. Overall, the efficacy and PK data strongly support the development of selected amino-artemisinins as components of new TACTs.

Varying degrees of homostructurality in a series of cocrystals of antimalarial drug 11-azaartemisinin with salicylic acids.

The X-ray structures of three new 1:1 pharmaceutical cocrystals of 11-azaartemisinin (11-Aza; systematic name: 1,5,9-trimethyl-14,15,16-trioxa-11-azatetracyclo[10.3.1.04,13.08,13]hexadecan-10-one, C15H23NO4) with bromo-substituted salicylic acids [namely, 5-bromo- (5-BrSalA, C7H5BrO3), 4-bromo- (4-BrSalA, C7H5BrO3) and 3,5-dibromosalicylic acid (3,5-Br2SalA, C7H4Br2O3)] are reported. All the structures are related to the parent 11-Aza:SalA cocrystal (monoclinic P21) reported previously. The 5-BrSalA analogue is isostructural with the parent, with lattice expansion along the c axis. The 4-BrSalA and 3,5-Br2SalA cocrystals retain the highly preserved 21 stacks of the molecular pairs, but these pack with a varying degree of slippage with respect to neighbouring stacks, altering the close contacts between them, and represent two potential alternative homostructural arrangements for the parent compound. Structure redeterminations of the bromosalicylic acids 5-BrSalA, 4-BrSalA and 3,5-Br2SalA at 100 K show that the packing efficiency of the cocrystals need not be higher than the parent coformers, based on specific-volume calculations, attributable to the strong O-H...O=C hydrogen bonds of 2.54 Šin the cocrystals.

Anti-Melanoma Activities of Artemisone and Prenylated Amino-Artemisinins in Combination With Known Anticancer Drugs.

The most frequently occurring cancers are those of the skin, with melanoma being the leading cause of death due to skin cancer. Breakthroughs in chemotherapy have been achieved in certain cases, though only marginal advances have been made in treatment of metastatic melanoma. Strategies aimed at inducing redox dysregulation by use of reactive oxygen species (ROS) inducers present a promising approach to cancer chemotherapy. Here we use a rational combination of an oxidant drug combined with a redox or pro-oxidant drug to optimize the cytotoxic effect. Thus we demonstrate for the first time enhanced activity of the amino-artemisinin artemisone and novel prenylated piperazine derivatives derived from dihydroartemisinin as the oxidant component, and elesclomol-Cu(II) as the redox component, against human malignant melanoma cells A375 in vitro. The combinations caused a dose dependent decrease in cell numbers and increase in apoptosis. The results indicate that oxidant-redox drug combinations have considerable potential and warrant further investigation.

Accumulation of TB-Active Compounds in Murine Organs Relevant to Infection by Mycobacterium tuberculosis.

Tuberculosis (TB), the leading cause of death due to an infectious agent, requires prolonged and costly drug treatments. With the rise in incidence of MDR and XDR TB, newer more efficacious treatments which are better able to permeate into the deeper recesses of the human lung where bacteria reside are urgently required. To this end, two new promising drug candidates, the decoquinate derivative RMB041 and the phenoxazine PhX1, were assessed for their abilities to permeate into specific murine organs. In particular, PhX1 permeation into the lungs and heart was notably efficient, as reflected in the high relative AUC values of 9669 ± 120.2 min/nmol/mg and 12450 ± 45.2 min/nmol/mg for lung and heart tissue, respectively. However, neither compound maintained a free concentration in the lung which exceeded the compound's respective MIC90 values, indicating the importance of correcting for organ specific binding.