Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

The antibiotic sorangicin A inhibits promoter DNA unwinding in a Mycobacterium tuberculosis rifampicin-resistant RNA polymerase.

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (RifR) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of RifR RNAPs, including the most prevalent clinical RifR RNAP substitution found in Mtb infected patients (S456>L of the ß subunit). Here, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

Rifampicin nanocrystals: Towards an innovative approach to treat tuberculosis.

Tuberculosis (TB) is one of the top ten causes of death worldwide and a leading cause of death in HIV patients. Rifampicin (Rif), a low water-soluble drug, is a critical first-line treatment and the most effective drug substance for therapy of drug-susceptible TB. However, Rif has high interindividual pharmacokinetic variability, mainly due to its highly variable absorption caused by its poor solubility. Drug nanocrystals are a promising technology to overcome this variability by increasing the surface area. This strategy allows for increasing the dissolution rate and improving the bioavailability of this BCS class II drug. In this study, Rif nanocrystals were prepared by a wet-bead milling method. A 3-factor, 3-level Box-Behnken design was used to investigate the independent variables: the concentration of rifampicin, the concentration of the stabilizing agent (Povacoat® type F), and the mass of zirconia beads. Two optimized formulations, F1-Rif and F2-Rif, were characterized by determining their particle size and size distribution, morphology, crystal properties, and antimicrobial activity. Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) revealed that rifampicin's polymorph II crystal structure was unchanged. The reduced particle size of <500 nm (100-fold decrease) increased the saturation solubility and dissolution rate up to 1.74-fold. The novel polymer, Povacoat®, demonstrated to be a suitable stabilizer to maintain the physical stability of nanosuspensions over two years. The Rif nanocrystals showed antimicrobial activity (0.25 µg/mL) not significantly different from standard rifampicin powder. However, the low cytotoxicity of the nanosuspensions in HepG2 cells was determined. When compared to the commercial product, the nanosuspension increased the rifampicin concentration 2-fold. In conclusion, the Rif nanosuspension allows half the needed volume of administration, which might increase compliance among children and elderly patients throughout the long-term treatment of TB.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Targeted protein degradation in antibacterial drug discovery?

Drug induced degradation of a target protein is a novel concept in drug discovery. Traditionally drugs modulate activity, as opposed to abundance, of their targets. Degradation inducing ligands act catalytically. Thus, one advantage of target degradation over the classical on-target mechanism is that lower drug concentration may be sufficient to cause the desired cellular effects. The first promoters of target degradation were discovered unintentionally: it turned out that some drugs 'accidently' promote degradation of their target by the cellular proteolytic machinery. Elegant methods were developed to target specific proteins of interest for degradation, thus enabling the rational discovery of degradation inducers. The application of targeted degradation has so far been limited to human cells. Recently, we discovered that an antibacterial drug, the anti-tuberculosis antibiotic pyrazinamide, functions as a promotor of degradation of its bacterial target. Increasing antimicrobial resistance makes the discovery of novel antibiotics more urgent than ever. Can rational target degradation be applied for the discovery of anti-bacterials? Here, we first discuss briefly some historic examples and then recent approaches in rational target degradation for human diseases. Then, we describe how the first anti-bacterial target degradation promoter pyrazinamide triggers removal of its target. Efforts are under way to exploit this specific mechanistic knowledge for the discovery of next generation pyrazinamide. We end with the big - and open - question whether targeted protein degradation as an approach to anti-bacterial drug discovery can be generalized, similar to what has been achieved in the area of drug discovery for human diseases.

Imaging of hepatic drug transporters with [131I]6-ß-iodomethyl-19-norcholesterol.

We examined whether [131I]6-ß-iodomethyl-19-norcholesterol (NP-59), a cholesterol analog, can be used to measure function of hepatic drug transporters. Hepatic uptake of NP-59 with and without rifampicin was evaluated using HEK293 cells expressing solute carrier transporters. The stability of NP-59 was evaluated using mouse blood, bile, and liver, and human liver S9. Adenosine triphosphate-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles expressing multidrug resistance protein 1, multidrug resistance-associated protein (MRP)1-4, breast cancer resistance protein (BCRP), or bile salt export pump with and without MK-571 and Ko143. Single photon emission computed tomography (SPECT) was performed in normal mice injected with NP-59 in the presence or absence of Ko143. Uptake of NP-59 into HEK293 cells expressing organic anion transporting polypeptide (OATP)1B1 and OATP1B3 was significantly higher than that into mock cells and was inhibited by rifampicin. NP-59 was minimally metabolized in mouse blood, bile, and liver, and human liver S9 after 120 min of incubation. In vesicles, NP-59 was transported by MRP1 and BCRP. Excretion of NP-59 into bile via BCRP was observed in normal mice with and without Ko143 in the biological distribution and SPECT imaging. NP-59 can be used to visualize and measure the hepatic function of OATP1B1, OATP1B3, and BCRP.

Antimicrobial Lavandulylated Flavonoids from a Sponge-Derived Streptomyces sp. G248 in East Vietnam Sea.

Three new lavandulylated flavonoids, (2S,2''S)-6-lavandulyl-7,4'-dimethoxy-5,2'-dihydroxylflavanone (1), (2S,2''S)-6-lavandulyl-5,7,2',4'-tetrahydroxylflavanone (2), and (2''S)-5'-lavandulyl-2'-methoxy-2,4,4',6'-tetrahydroxylchalcone (3), along with seven known compounds 4-10 were isolated from culture broth of Streptomyces sp. G248. Their structures were established by spectroscopic data analysis, including 1D and 2D nuclear magnetic resonance (NMR), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The absolute configurations of 1-3 were resolved by comparison of their experimental and calculated electronic circular dichroism spectra. Compounds 1-3 exhibited remarkable antimicrobial activity. Whereas, two known compounds 4 and 5 exhibited inhibitory activity against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) values of 6.0 µg/mL and 11.1 µg/mL, respectively.

Identification of 4-Amino-Thieno[2,3-d]Pyrimidines as QcrB Inhibitors in Mycobacterium tuberculosis.

Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex.IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.

A Spray-Dried Combination of Capreomycin and CPZEN-45 for Inhaled Tuberculosis Therapy.

Tuberculosis (TB) remains the single most serious infectious disease attributable to a single-causative organism. A variety of drugs have been evaluated for pulmonary delivery as dry powders: capreomycin sulfate has shown efficacy and was safely delivered by inhalation at high doses to human volunteers, whereas CPZEN-45 is a new drug that has also been shown to kill resistant TB. The studies here combine these drugs-acting by different mechanisms-as components of single particles by spray-drying, yielding a new combination drug therapy. The spray-dried combination powder was prepared in an aerodynamic particle size range suitable for pulmonary delivery. Physicochemical storage stability was demonstrated for a period of 6 months. The spray-dried combination powders of capreomycin and CPZEN-45 have only moderate affinity for mucin, indicating that delivered drug will not be bound by these mucins in the lung and available for microbicidal effects. The pharmacokinetics of disposition in guinea pigs demonstrated high local concentrations of drug following direct administration to the lungs and subsequent systemic bioavailability. Further studies are required to demonstrate the in vivo efficacy of the combination to confirm the therapeutic potential of this novel combination.

Solidified SNEDDS for the oral delivery of rifampicin: Evaluation, proof of concept, in vivo kinetics, and in silico GastroPlusTM simulation.

The present investigation was performed to develop a rifampicin (RIF)-loaded solidified self-nanoemulsifying drug delivery system (SNEDDS) (solidified RIF-OF1) for in vitro and in vivo evaluations. Optimized formulations were tested for their powder flow characteristics, loading efficiency, and in vitro dissolution (at pH-1.2, 6.8 and 7.4). Compatibility studies were also performed. The formulations were also tested for hemocompatibility, intestinal permeation, histopathological effects, and in vivo pharmacokinetics. Additionally, an in silico simulation study using GastroPlus was performed. At different varied pH values, we observed immediate release (T85% within 15 min) based on the dissolution profile. This could be due to labrasol-assisted RIF solubilization. In vitro hemolysis study of the reconstituted RIF-OF1 revealed normal architecture of erythrocytes compared to the positive control (lysed and fragmented). Through in vivo permeation and biopsy studies, a rationale for facilitated intestinal permeation of RIF with components deemed physiological safe (normal anatomy of mucosal membrane evidenced from biopsy study) could be established. The in vitro-in vivo correlation (IVIVC) plus module of GastroPlusTM simulation showed a good IVIVC between in vitro release and in vivo absorption with a predicted systemic absorption of ∼96.5%. Solidified SNEDDS showed improved pharmacokinetic profiles compared to RIF suspension. Solid RIF-SNEDDS was demonstrated to be a suitable carrier for enhanced intestinal permeation and oral bioavailability. Hence, it may serve as a suitable alternative to conventional delivery systems for tuberculosis treatment.

Sensitive Ultra-performance Liquid Chromatography Tandem Mass Spectrometry Method for Determination of Cycloserine in Plasma for a Pharmacokinetics Study.

A simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry method has been developed and validated for the analysis of cycloserine in patients' plasma. Using methanol, cyloserine and propranolol (internal standard (IS)) was extracted from plasma by protein precipitation procedure. The chromatographic separation was successfully achieved on Phenomenex KinetexTM PFP C18 (2.1 mm × 100 mm, 2.6 µm) reversed-phase column. Acidified with 0.1% formic acid, water and acetonitrile were used as mobile phases for gradient elution. Cycloserine and IS were detected by Xevo® TQ MS triple quadrupole tandem mass spectrometer. The transition of protonated precursor to product ion were monitored at 103 → 75 m/z and 260.2 → 183 m/z for cycloserine and IS, respectively. The lower limit of quantification was 0.01 µg/mL. The method was linear over the concentration range 0.01-50 µg/mL with average coefficient of determination of 0.9994. The within-run and between-run precision and accuracy were in the range 3.7-19.3% (RSD) and 98.7-117.3%, respectively. Processed cycloserine sample was stable for 48 hours at 8°C and after three freeze-thaw cycles. The extraction efficiency ranged between 88.7 and 91.2%. The method was successfully applied in a pharmacokinetic study for the determination of cycloserine in plasma of patients with drug-resistant tuberculosis.

Drug delivery of rifampicin by natural micelles based on inulin: Physicochemical properties, antibacterial activity and human macrophages uptake.

This work aims at designing a drug delivery system for rifampicin (RIF) to be used for the therapy of infections from mycobacterium tuberculosis or other lung-colonizing bacteria. We are proposing, in particular, the delivery of RIF by micelles based on inulin functionalized with vitamin E (INVITE). We previously demonstrated that INVITE micelles are formed from the self-assembling sustained by the interaction, within the hydrophobic core, of aromatic groups belonging to vitamin E. It points on the effectiveness of these biocompatible systems in incorporating aromatic-group-bearing hydrophobic drug such as RIF. The succinilated derivative of INVITE, namely INVITESA, was further studied. Other than a full physicochemical characterization, the obtained micelles containing RIF were tested for their antibacterial activity against Gram- or Gram+bacteria including mycobacterium smegmatis. Furthermore, uptake studies on human alveolar macrophages and MTT studies were performed.

Promising Chitosan-Coated Alginate-Tween 80 Nanoparticles as Rifampicin Coadministered Ascorbic Acid Delivery Carrier Against Mycobacterium tuberculosis.

The aim of this study was to design a nanocarrier system for inhalation delivery of rifampicin (RIF) in combination with ascorbic acid (ASC), namely constituted of sodium alginate coated with chitosan and Tween 80 (RIF/ASC NPs) as a platform for the treatment of pulmonary tuberculosis infection. A Box-Behnken experimental design and response surface methodology (RSM) were applied to elucidate and evaluate the effects of several factors on the nanoparticle properties. On the other hand, it was found that RIF/ASC NPs were less cytotoxic than the free RIF, showing a significantly improved activity against nine clinical strains of Mycobacterium tuberculosis (M. tb) in comparison with the free drug. RIF/ASC NPs had an average particle size of 324.0 ± 40.7 nm, a polydispersity index of 0.226 ± 0.030, and a zeta potential of - 28.52 ± 0.47 mV and the surface was hydrophilic. The addition of sucrose (1% w/v) to the nanosuspension resulted in the formation of a solid pellet easily redispersible after lyophilization. RIF/ASC NPs were found to be stable at different physiological pH values. In summary, findings of this work highlight the potential of the RIF/ASC NP-based formulation development herein to deliver RIF in combination with ASC through pulmonary route by exploring a non-invasive route of administration of this antibiotic, increasing the local drug concentrations in lung tissues, the primary infection site, as well as reducing the risk of systemic toxicity and hence improving the patient compliance.

Ultrafast dynamics of the antibiotic Rifampicin in solution.

Rifampicin (Rif) is an effective antibiotic against mycobacterial infections and a wide range of Gram-positive and Gram-negative bacteria. The geometry, conformation and the intramolecular H-bond network of Rif can affect its antibacterial efficiency. In this work, we report on the excited-state dynamics of Rif in sodium phosphate buffer and dichloromethane solutions using femtosecond time-resolved spectroscopic methods. The femtosecond UV-Vis-nearIR transient absorption and fluorescence up-conversion experiments reveal an ultrafast (<100 fs) Franck-Condon relaxation with a partial charge transfer character in S1, and a short-lived emission (τ∼ 6 ps) due to a non-radiative relaxation to the ground state, associated with the stretching of the vibrational mode of the Rif internal H-bond network. The large Stokes-shifted emission (∼6800 cm-1) indicates a significant electronic change in the excited-state. In deuterated potassium phosphate buffer, the decay time becomes longer (∼20 ps). The large kinetic isotope effect (KIE) of ∼4 on the decay rate indicates that the stretching modes of the internal H-bond network are slowed by the H/D isotope substitution. The results provide new information on the dynamics of Rif structures and the related processes in aqueous solutions, showing that the internal H-bonding interactions are the ones that govern the ground and excited state properties of Rif but water molecules exert additional stabilization of its zwitterionic form through intermolecular H-bonds, which is responsible for its high antimicrobial activity.

Newly synthesized surfactants for surface mannosylation of respirable SLN assemblies to target macrophages in tuberculosis therapy.

The present study reports about new solid lipid nanoparticle assemblies (SLNas) loaded with rifampicin (RIF) surface-decorated with novel mannose derivatives, designed for anti-tuberculosis (TB) inhaled therapy by dry powder inhaler (DPI). Mannose is considered a relevant ligand to achieve active drug targeting being mannose receptors (MR) overexpressed on membranes of infected alveolar macrophages (AM), which are the preferred site of Mycobacterium tuberculosis. Surface decoration of SLNas was obtained by means of newly synthesized functionalizing compounds used as surfactants in the preparation of carriers. SLNas were fully characterized in vitro determining size, morphology, drug loading, drug release, surface mannosylation, cytotoxicity, macrophage internalization extent and ability to bind MR, and intracellular RIF concentration. Moreover, the influence of these new surface functionalizing agents on SLNas aerodynamic performance was assessed by measuring particle respirability features using next generation impactor. SLNas exhibited suitable drug payload, in vitro release, and more efficient ability to enter macrophages (about 80%) compared to bare RIF (about 20%) and to non-functionalized SLNas (about 40%). The involvement of MR-specific binding has been demonstrated by saturating MR of J774 cells causing a decrease of RIF intracellular concentration of about 40%. Furthermore, it is noteworthy that the surface decoration of particles produced a poor cohesive powder with an adequate respirability (fine particle fraction ranging from about 30 to 50%). Therefore, the proposed SLNas may represent an encouraging opportunity in a perspective of an efficacious anti-TB inhaled therapy.

Mucoadhesive guargum hydrogel inter-connected chitosan-g-polycaprolactone micelles for rifampicin delivery.

Natural polymer guar gum has one of the highest viscosities in water solution and hence, these are significantly used in pharmaceutical applications. Guar gum inter-connected micelles as a new carrier has been developed for poor water soluble rifampicin drug. The hydrogel inter-connected micelle core was formulated as a hydrophilic inner and hydrophobic outer core by using guar gum/chitosan/polycaprolactone and the carrier interaction with rifampicin was confirmed by FT-IR. The morphological observations were carried out through TEM, SEM and AFM analysis. The encapsulation efficiency and in-vitro drug release behavior of prepared hydrogel based micelle system was analyzed by UV-vis spectrometry. The anti-bacterial activity against K. pneumoniae and S. aureus was studied by observing their ruptured surface by SEM. The cytotoxicity study reveals that the pure polymeric system has no toxic effect whereas drug loaded ones showed superior activity against THP-1 cells. From the cell apoptosis analyses, the apoptosis was carried out in a time dependent manner. The cell uptake behavior was also observed in THP-1 cells which indicate that the hydrogel based micelle system is an excellent material for the mucoadhesive on intracellular alveolar macrophage treatment.

Structures of DPAGT1 Explain Glycosylation Disease Mechanisms and Advance TB Antibiotic Design.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Alternative Pharmaceutical Formulation for Oral Administration of Rifampicin.

Tuberculosis (TB) is considered an emergency global public health, mainly due to the TB-HIV co-infection, bacillus dormancy stage, and emergence of resistant strains. In addition, the therapeutic toxicity and its pharmacokinetic interactions with other drugs may influence treatment non-compliance, low serum concentration of drugs, and, consequently, treatment failure. Strategies using nanotechnology represent a new tool for the therapy, since they are effective delivery systems due to the possibility of solubilization of hydrophobic compounds, enable the production of formulations for oral use, and, in addition, increase bioavailability of drugs. This study aimed to develop a nanoemulsion (NE) containing rifampicin (RIF-NE) and evaluate its in vitro antimycobacterial activity using Resazurin Microtiter Assay against three Mycobacterium tuberculosis strains: two susceptible and a multidrug-resistant. Using the hot solvent diffusion method associated with phase inversion technique was possible to develop a liquid formulation containing 500 µg/mL rifampicin (RIF), which is a hydrophobic compound, of average size 25 nm. The results showed that the minimum inhibitory concentration of the encapsulated RIF was equal to the free form of RIF, indicating that the process of production of NEs did not affect the activity of the compound. Thus, RIF-NE could be a promising alternative for oral administration of RIF, being considered a child-friendly pharmaceutical formulation. Its application could avoid the administration of unknown and/or non-ideal concentrations, being functional in the regimes of prevention and treatment of TB and, in addition, in the fight against drug resistance.

Development of Novel Octanoyl Chitosan Nanoparticles for Improved Rifampicin Pulmonary Delivery: Optimization by Factorial Design.

A novel hydrophobic chitosan derivative, octanoyl chitosan (OC) with improved organic solubility was synthesized, characterized, and employed for the preparation of rifampicin (Rif) encapsulated nanoparticle formulations for pulmonary delivery. OC was characterized to confirm acyl group substitution and cytotoxicity in A549 epithelial lung cells. OC nanoparticles were produced by the double emulsion solvent evaporation technique without cross-linking and characterized for particle size distribution, morphology, crystallinity, thermal stability, aerosol delivery, and drug release rate. OC was successfully synthesized with substitution degree of 44.05 ± 1.75%, and solubility in a range of organic solvents. Preliminary cytotoxicity studies of OC showed no effect on cell viability over a period of 24 h on A549 cell lines. OC nanoparticles were optimized using a 32 full factorial design. An optimized batch of OC nanoparticles, smooth and spherical in morphology, had mean hydrodynamic diameter of 253 ± 19.06 nm (PDI 0.323 ± 0.059) and entrapment efficiency of 64.86 ± 7.73% for rifampicin. Pulmonary deposition studies in a two-stage impinger following aerosolization of nanoparticles from a jet nebulizer gave a fine particle fraction of 43.27 ± 4.24%. In vitro release studies indicated sustained release (73.14 ± 3.17%) of rifampicin from OC nanoparticles over 72 h, with particles demonstrating physical stability over 2 months. In summary, the results confirmed the suitability of the developed systems for pulmonary delivery of drugs with excellent aerosolization properties and sustained-release characteristics.