Determination of bactericidal activity against 3HC-2-Tre-labelled Mycobacterium abscessus (Mycobacteroides abscessus) by automated fluorescence microscopy.

The minimum bactericidal concentration (MBC) of antibiotics is an important parameter for the potency of a drug in eradicating a bacterium as well as an important measure of the potential of a drug candidate in research and development. We have established a fluorescence-based microscopy method for the determination of MBCs against the non-tuberculous mycobacterium Mycobacterium abscessus (Mycobacteroides abscessus) to simplify and accelerate the performance of MBC determination compared to counting colony forming units on agar. Bacteria are labelled with the trehalose-coupled dye 3HC-2-Tre and analysed in a 96-well plate. The results of the new method are consistent with MBC determination by plating on agar. The method was used to evaluate the bactericidality of the antibiotics rifabutin, moxifloxacin, amikacin, clarithromycin and bedaquiline. Bactericidal effects against M. abscessus were observed, which are consistent with literature data.

Rifabutin central nervous system concentrations in a rabbit model of tuberculous meningitis.

Tuberculous meningitis (TBM) has a high mortality, possibly due to suboptimal therapy. Drug exposure data of antituberculosis agents in the central nervous system (CNS) are required to develop more effective regimens. Rifabutin is a rifamycin equivalently potent to rifampin in human pulmonary tuberculosis. Here, we show that human-equivalent doses of rifabutin achieved potentially therapeutic exposure in relevant CNS tissues in a rabbit model of TBM, supporting further evaluation in clinical trials.

Population pharmacokinetics of rifabutin among HIV/TB co-infected children on lopinavir/ritonavir-based antiretroviral therapy.

In adults requiring protease inhibitor (PI)-based antiretroviral therapy (ART), replacing rifampicin with rifabutin is a preferred option, but there is lack of evidence to guide rifabutin dosing in children, especially with PIs. We aimed to characterize the population pharmacokinetics of rifabutin and 25-O-desacetyl rifabutin (des-rifabutin) in children and optimize its dose. We included children from three age cohorts: (i) <1-year-old cohort and (ii) 1- to 3-year-old cohort, who were ART naïve and received 15- to 20-mg/kg/day rifabutin for 2 weeks followed by lopinavir/ritonavir (LPV/r)-based ART with 5.0- or 2.5 mg/kg/day rifabutin, respectively, while the (iii) >3-year-old cohort was ART-experienced and received 2.5-mg/kg/day rifabutin with LPV/r-based ART. Non-linear mixed-effects modeling was used to interpret the data. Monte Carlo simulations were performed to evaluate the study doses and optimize dosing using harmonized weight bands. Twenty-eight children were included, with a median age of 10 (range 0.67-15.0) years, a median weight of 11 (range 4.5-45) kg, and a median weight-for-age z score of -3.33 (range -5.15 to -1.32). A two-compartment disposition model, scaled allometrically by weight, was developed for rifabutin and des-rifabutin. LPV/r increased rifabutin bioavailability by 158% (95% confidence interval: 93.2%-246.0%) and reduced des-rifabutin clearance by 76.6% (74.4%-78.3%). Severely underweight children showed 26% (17.9%-33.7%) lower bioavailability. Compared to adult exposures, simulations resulted in higher median steady-state rifabutin and des-rifabutin exposures in 6-20 kg during tuberculosis-only treatment with 20 mg/kg/day. During LPV/r co-treatment, the 2.5-mg/kg/day dose achieved similar exposures to adults, while the 5-mg/kg/day dose resulted in higher exposures in children >7 kg. All study doses maintained a median Cmax of <900 µg/L. The suggested weight-band dosing matches adult exposures consistently across weights and simplifies dosing.

Rifabutin loaded inhalable ß-glucan microparticle based drug delivery system for pulmonary TB.

Inhalable microparticle-based anti TB drug delivery systems are being investigated extensively for Tuberculosis [TB] treatment as they offer efficient and deep lung deposition with several advantages over conventional routes. It can reduce the drug dose, treatment duration and toxic effects and optimize the drug bioavailability. Yeast derived ß-glucan is a ß-[1-3/1-6] linked biocompatible polymer and used as carrier for various biomolecules. Due to presence of glucan chains, particulate glucans act as PAMP and thereby gets internalized via receptor mediated phagocytosis by the macrophages. In this study, ß-glucan microparticles were prepared by adding l-leucine as excipient, and exhibited 70% drug [Rifabutin] loading efficiency. Further, the sizing and SEM data of particles revealed a size of 2-4 µm with spherical dimensions. The FTIR and HPLC data confirmed the ß-glucan composition and drug encapsulations efficiency of the particles. The mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD] data indicated that these particles are inhalable in nature and have better thermal stability as per DSC thermogram. These particles were found to be non-toxic upto a concentration of 80 µg/ml and were found to be readily phagocytosed by human macrophage cells in-vitro as well as in-vivo by lung alveolar macrophage. This study provides a framework for future design of inhalable ß-glucan particle based host-directed drug delivery system against pulmonary TB.

Efficacy of rifapentine and other rifamycins against Coxiella burnetii in vitro.

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii. Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii. Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL-1. Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized, and rifapentine, not previously reported against C. burnetii. These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL-1. In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL-1. Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL-1 and should be further explored as alternative Q fever treatments given their efficacy in vitro. IMPORTANCE: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii. Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro. Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages.

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.

Genome-Driven Discovery of Hygrocins in Streptomyces rapamycinicus.

Ansamycins, represented by the antituberculosis drug rifamycin, are an important family of natural products. To obtain new ansamycins, Streptomyces rapamycinicus IMET 43975 harboring an ansamycin biosynthetic gene cluster was fermented in a 50 L scale, and subsequent purification work led to the isolation of five known and four new analogues, where hygrocin W (2) belongs to benzoquinonoid ansamycins, and the other three hygrocins, hygrocins X-Z (6-8), are new seco-hygrocins. The structures of ansamycins (1-8) were determined by the analysis of spectroscopic (1D/2D NMR and ECD) and MS spectrometric data. The Baeyer-Villiger enzyme which catalyzed the ester formation in the ansa-ring was confirmed through in vivo CRISPR base editing. The discovery of these compounds further enriches the structural diversity of ansamycins.

Antibiotic Adjuvant 4-Hexylresorcinol Enhances the Efficiency of Antituberculosis Drugs.

We studied the possibility of using 4-hexylresorcinol to increase the efficiency of anti-mycobacterial chemotherapy. In an in vitro experiment, 4-hexylresorcinol increased the efficiency of rifampicin, kanamycin, and isoniazid against Mycobacterium smegmatis by 3-5 times. Experiments in sanitation of BALB/c mice infected with M. smegmatis showed the best efficacy of the isoniazid and 4-hexylresorcinol combination in comparison with isoniazid monotherapy. The growth-inhibiting activity of the combination of antibiotic rifabutin with 4-hexylresorcinol was shown on 6 strains of M. tuberculosis. A 2-fold decrease in the minimum inhibitory concentration of this antibiotic in the presence of half-minimum inhibitory concentration of 4-hexylresorcinol was demonstrated for monoresistant strain M. tuberculosis 5360/42Hr. On the mouse model of experimental tuberculosis caused by M. tuberculosis H37Rv, a 5-fold decrease in lung contamination and more rapid complete cure were achieved in animals treated with the combination of rifabutin and 4-hexylresorcinol in comparison with rifabutin monotherapy.

Rifabutin but not rifampicin can partly out-balance P-glycoprotein induction by concurrent P-glycoprotein inhibition through high affinity binding to the inhibitory site.

Physiology-based pharmacokinetic modeling suggests that rifabutin can out-balance P-glycoprotein (P-gp) induction by concurrent P-gp inhibition. However, clinical or experimental evidence for this Janus-faced rifabutin effect is missing. Consequently, LS180 cells were exposed to a moderately (2 µM) and strongly (10 µM) P-gp-inducing concentration of rifampicin or rifabutin for 6 days. Cellular accumulation of the fluorescent P-gp substrate rhodamine 123 was evaluated using flow cytometry, either without (induction only) or with adding rifamycin drug to the cells during the rhodamine 123 efflux phase (induction + potential inhibition). Rhodamine 123 accumulation was decreased similarly by both drugs after 6-day exposure (2 µM: 55% residual fluorescence compared to non-induced cells, P < 0.01; 10 µM: 30% residual fluorescence compared to non-induced cells, P < 0.001), indicating P-gp induction. Rhodamine 123 influx transporters mRNA expressions were not affected, excluding off-target effects. Acute re-exposure to rifabutin, however, considerably re-increased rhodamine 123 accumulation (2 µM induction: re-increase by 55%, P < 0.01; 10 µM induction: 49% re-increase, P < 0.001), suggesting P-gp inhibition. In contrast, rifampicin only had weak effects (2 µM induction: no re-increase; 10 µM induction: 16% re-increase; P < 0.05). Molecular docking analysis eventually revealed that rifabutin has a higher binding affinity to the inhibitor binding site of P-gp than rifampicin (ΔG (kcal/mol) = -11.5 vs -5.3). Together, this study demonstrates that rifabutin can at least partly mask P-gp induction by P-gp inhibition, mediated by high affinity binding to the inhibitory site of P-gp.

Molecular characterization of rifabutin-resistance in refractory Helicobacter pylori infection in Taiwan.

OBJECTIVES: To explore the molecular characteristics of rpoB, encoding ß-subunit of DNA-directed RNA polymerase, and unravel the link to rifabutin-resistance in patients with refractory Helicobacter pylori infection. METHODS: From January 2018-March 2021, a total of 1590 patients were screened for eligibility to participate in the study. Patients with refractory H. pylori infection were confirmed by using the (13C)-urea breath assay. All enrolled patients underwent esophagogastroduodenoscopy, and biopsies were taken for H. pylori culture and antibacterial susceptibility testing. Sequence analysis of rpoB was conducted for all rifabutin-resistant isolates. RESULTS: In total, 70 patients were diagnosed with refractory H. pylori infection, and 39 isolates were successfully cultured. Amongst, 10 isolates were identified as rifabutin-resistance and nine isolates exhibited at least one amino acid substitution in RpoB. Isolates with a minimal inhibitory concentration >32 mg/l displayed a higher number of mutational changes in RpoB than the others. Additionally, more amino acid substitutions in RpoB correlated with developing a higher minimal inhibitory concentration for H. pylori rifabutin-resistance. CONCLUSION: Our findings highlight the relationship between rifabutin-resistance in refractory H. pylori infection and specific mutations in RpoB, which will aid the clinical selection of appropriate antibacterial agents with better therapeutic effects.

The differences in drug disposition gene induction by rifampicin and rifabutin are unlikely due to different effects on important pregnane X receptor (NR1I2) splice variants.

Rifampicin and rifabutin can activate the pregnane X receptor (PXR, NR1I2), thereby inducing pharmacokinetically important genes/proteins and reducing exposure to co-administered drugs. Because induction effects vary considerably between these antibiotics, differences could be due to unequal rifamycin-induced activation or tissue expression of the three major NR1I2 splice variants, PXR.1 (NM_003889), PXR.2 (NM_022002), and PXR.3 (NM_033013). Consequently, PXR activation (PXR reporter gene assays) and mRNA expression levels of total NR1I2, PXR.1, PXR.2, and PXR.3 were investigated by polymerase chain reaction in colon and liver samples from eleven surgical patients, in LS180 cells, and primary human hepatocytes. Compared to the colon, total NR1I2 mRNA expression was higher in the liver. Both tissues showed similar expression levels of PXR.1 and PXR.3, respectively. PXR.2 was not quantifiable in the colon samples. Rifampicin and rifabutin similarly enhanced PXR.1 and PXR.2 activity when transfected into LS180 cells, while PXR.3 could not be activated. In LS180 cells, rifampicin (10 µM) reduced total NR1I2 and PXR.3 expression 2-fold after 24 h, while rifabutin (10 µM) increased total NR1I2, PXR.1, PXR.2, and PXR.3 mRNA by approx. 50% after 96-h exposure. In primary human hepatocytes, rifampicin (10 µM) suppressed total NR1I2, PXR.1, and PXR.3 after 48-h exposure, and rifabutin (10 µM) had no significant impact on total NR1I2 or any of the splice variants studied. In conclusion, both antibiotics activated the studied PXR splice variants similarly but modified their expression differently. While rifampicin can suppress mRNA of PXR forms, rifabutin rather increases their expression levels.

[In vitro Activity of Rifabutin and Clofazimine to Macrolide-Resistant Mycobacterium abscessus Complex Clinical Isolates]. / Rifabutin ve Klofaziminin Makrolidlere Dirençli Mycobacterium abscessus Kompleks Klinik Izolatlarina In vitro Etkinligi.

Mycobacterium abscessus complex (MABSC) is one of the most resistant bacteria against antimicrobial agents. The number of agents that can be used by oral route, such as macrolides, is limited in antimicrobial therapy. In recent years, rifabutin and clofazimine have gained importance as they can be administered by oral route and have shown synergistic effects with macrolides and aminoglycosides. The aim of this study was to determine the in vitro activity of rifabutin and clofazimine against clinical isolates of MABSC resistant to macrolides. A total of 48 MABSC isolates obtained from respiratory tract and other clinical samples in the Tuberculosis Laboratories of the Faculty of Medicine of Manisa Celal Bayar and Ege Universities were included in the study. Subspecies differentiation and aminoglycoside and macrolide resistance of the isolates were determined by GenoType NTM-DR test. Rifabutin and clofazimine susceptibilities were determined by standard broth microdilution method. Of the MABSC isolates 42 were identified as M.abscessus subsp. abscessus, three as M.abscessus subsp. bolletii and three as M.abscessus subsp. massiliense. None of the isolates exhibited rrs and rrl mutations indicating acquired macrolide resistance and aminoglycoside resistance. However, the erm(41) T28 genotype which is associated with inducible macrolide resistance was detected in 41 (85%) of the strains. All M.abscessus subsp. massiliense isolates were found to be genotypically susceptible to macrolides. The minimum inhibitory concentration (MIC) range values for rifabutin were 0.0625 to 32 µg/mL, while for clofazimine, the range was 0.0625 to 1 µg/mL. Rifabutin MIC values were significantly higher (mean 5.98 µg/mL vs 0.5 µg/mL, p= 0.026) in the isolates with macrolide resistance. There was no correlation between macrolide resistance and clofazimine MIC values (mean 0.25 µg/mL vs. 0.214 µg/mL, p= 0.758). The MIC50 and MIC90 values for rifabutin were 1 and 8 µg/mL, respectively, while for clofazimine they were 0.25 and 0.5 µg/mL. Macrolide resistance was found to be higher in isolates with rifabutin MIC values above the MIC50 value (p= 0.045). In conclusion, the determination of higher rifabutin MIC values in isolates resistant to macrolides suggested that susceptibility testing should be performed before adding rifabutin to the treatment regimen. The low MIC values of clofazimine in all strains indicated that it may be used as a first choice in the combination therapy. However, further studies using a larger number of clinical isolates and applying genotypic and phenotypic susceptibility tests are needed to determine threshold MIC values to assist clinicians in making treatment decisions.

Successful Anticoagulation With Warfarin After Switching From Rifampin to Rifabutin.

PURPOSE: A case of a patient receiving warfarin for pulmonary embolism (PE) concomitantly with rifampin for treatment of active pulmonary tuberculosis (PTB) is presented. A successful clinical intervention whereby the patient achieved therapeutic anticoagulation after switching to an alternative rifamycin antibacterial, rifabutin, is described. SUMMARY: The drug-drug interaction between warfarin and rifampin is well known and documented. However, to our knowledge, no case reports of the interaction between warfarin and rifabutin have been published, and literature describing this interaction is lacking. We describe the case of a 27-year-old African American female referred to a pharmacist-managed anticoagulation clinic for treatment of PE with warfarin. The patient was also being treated for active tuberculosis with rifampin, isoniazid, pyrazinamide, and ethambutol. Warfarin was initiated and over the course of 1 month was continuously increased to a total weekly dose (TWD) of 140 mg without ever achieving the target international normalized ratio (INR) of 2 to 3. In an attempt to reach the target INR, rifampin was switched to rifabutin to minimize the drug-drug interaction with warfarin. Six days after this switch, the target INR was achieved with a lower warfarin TWD of 115 mg. Rifabutin interacts with warfarin to a lesser degree than rifampin and may be considered as an alternative in patients taking warfarin who require treatment with a rifamycin. CONCLUSION: For patients in whom therapeutic anticoagulation with warfarin has been difficult, the use of rifabutin may be considered in place of rifampin when the concomitant use of a rifamycin is required.

ADP-ribosylation-resistant rifabutin analogs show improved bactericidal activity against drug-tolerant M. abscessus in caseum surrogate.

Necrotic lesions and cavities filled with caseum are a hallmark of mycobacterial pulmonary disease. Bronchocavitary Mycobacterium abscessus disease is associated with poor treatment outcomes. In caseum surrogate, M. abscessus entered an extended stationary phase showing tolerance to killing by most current antibiotics, suggesting that caseum persisters contribute to the poor performance of available treatments. Novel ADP-ribosylation-resistant rifabutin analogs exhibited bactericidal activity against these M. abscessus persisters at concentrations achievable by rifamycins in caseum.

Respirable konjac glucomannan microparticles as antitubercular drug carriers: Effects of in vitro and in vivo interactions.

Pulmonary delivery of drugs is potentially beneficial in the context of lung disease, maximising drug concentrations in the site of action. A recent work proposed spray-dried konjac glucomannan (KGM) microparticles as antitubercular drug (isoniazid and rifabutin) carriers to treat pulmonary tuberculosis. The present work explores in vitro and in vivo effects of these microparticles, focusing on the ability for macrophage uptake, the exhibited antibacterial activity and safety issues. Efficient uptake of KGM microparticles by macrophages was demonstrated in vitro, while the antitubercular activity of the model drugs against Mycobacterium bovis was not affected by microencapsulation in KGM microparticles. Despite the good indications provided by the developed system, KGM is not yet approved for pulmonary applications, which is a limiting characteristic. To reinforce the available data on the performance of the material, safety parameters were evaluated both in vitro and in vivo, showing promising results. No significant cell toxicity was observed at concentrations considered realistic for lung delivery approaches (up to 125 µg/mL) when lung epithelial cells and macrophages were exposed to KGM microparticles (both drug-loaded and unloaded). Finally, no signs of systemic or lung inflammatory response were detected in mice after receiving 10 administrations of unloaded KGM microparticles.

Defining the minimum inhibitory concentration of 22 rifamycins in iron limited, physiologic medium against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates.

Recently, we reported rifabutin hyper-activity against Acinetobacter baumannii. We sought to characterize if any additional rifamycins (n = 22) would also display hyper-activity when tested in iron-limited media against A. baumannii, K. pneumoniae, and E. coli. MICs were determined against representative clinical isolates using the iron-limited media RPMI-1640. Only rifabutin was hyperactive against A. baumannii.

Using Dynamic Oral Dosing of Rifapentine and Rifabutin to Simulate Exposure Profiles of Long-Acting Formulations in a Mouse Model of Tuberculosis Preventive Therapy.

Administration of tuberculosis preventive therapy (TPT) to individuals with latent tuberculosis infection is an important facet of global tuberculosis control. The use of long-acting injectable (LAI) drug formulations may simplify and shorten regimens for this indication. Rifapentine and rifabutin have antituberculosis activity and physiochemical properties suitable for LAI formulation, but there are limited data available for determining the target exposure profiles required for efficacy in TPT regimens. The objective of this study was to determine exposure-activity profiles of rifapentine and rifabutin to inform development of LAI formulations for TPT. We used a validated paucibacillary mouse model of TPT in combination with dynamic oral dosing of both drugs to simulate and understand exposure-activity relationships to inform posology for future LAI formulations. This work identified several LAI-like exposure profiles of rifapentine and rifabutin that, if achieved by LAI formulations, could be efficacious as TPT regimens and thus can serve as experimentally determined targets for novel LAI formulations of these drugs. We present novel methodology to understand the exposure-response relationship and inform the value proposition for investment in development of LAI formulations that have utility beyond latent tuberculosis infection.

Cascade Transformation of the Ansamycin Benzoquinone Core into Benzoxazole Influencing Anticancer Activity and Selectivity.

The metal-free cascade transformation of geldanamycin benzoquinone core is proposed at relatively mild conditions. This approach yields new benzoxazole ansamycin antibiotics and enables their functionalization in an atom-economic manner, irrespective of the type of amine used. The analysis of the heterocyclization course reveals the dependence of its rate on the nature of the para-substituent within the benzylamine moiety (EDG/EWG) and the strength of the base. The reduction of the ansamycin core enables an increase in anticancer potency and selectivity.

Comprehensive in vitro analysis evaluating the variable drug-drug interaction risk of rifampicin compared to rifabutin.

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug-drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin's concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.