ABSTRACT
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.
Subject(s)
Adenosine Triphosphate/metabolism , Antitubercular Agents/therapeutic use , Mycobacterium tuberculosis/drug effects , Pyridines/therapeutic use , Quinine/analogs & derivatives , Tuberculosis/drug therapy , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Ethers/chemistry , Ethers/pharmacokinetics , Ethers/pharmacology , Ethers/therapeutic use , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/pharmacology , Quinine/chemistry , Quinine/pharmacokinetics , Quinine/pharmacology , Quinine/therapeutic use , Tuberculosis/metabolismABSTRACT
There are currently 18 drug classes for the treatment of tuberculosis, including those in the development pipeline. An in silico simulation enabled combing the innumerably large search space to derive multidrug combinations. Through the use of ordinary differential equations (ODE), we constructed an in silico kinetic platform in which the major metabolic pathways in Mycobacterium tuberculosis and the mechanisms of the antituberculosis drugs were integrated into a virtual proteome. The optimized model was used to evaluate 816 triplets from the set of 18 drugs. The experimentally derived cumulative fractional inhibitory concentration (∑FIC) value was within twofold of the model prediction. Bacterial enumeration revealed that a significant number of combinations that were synergistic for growth inhibition were also synergistic for bactericidal effect. The in silico-based screen provided new starting points for testing in a mouse model of tuberculosis, in which two novel triplets and five novel quartets were significantly superior to the reference drug triplet of isoniazid, rifampin, and ethambutol (HRE) or the quartet of HRE plus pyrazinamide (HREZ).
Subject(s)
Antitubercular Agents/therapeutic use , Ethambutol/therapeutic use , Isoniazid/therapeutic use , Rifampin/therapeutic use , Tuberculosis/drug therapy , Animals , Mice , Mice, Inbred BALB C , Microbial Sensitivity TestsABSTRACT
Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.
Subject(s)
Antitubercular Agents/pharmacology , Piperidines/pharmacology , Spiro Compounds/pharmacology , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacokinetics , Bacteria/drug effects , Drug Resistance, Bacterial/genetics , Genome, Bacterial , High-Throughput Screening Assays , Hypoxia , Lipids/chemistry , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 13/genetics , Mice , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacokinetics , Structure-Activity RelationshipABSTRACT
The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.
Subject(s)
Antimalarials/pharmacology , Plasmodium falciparum/drug effects , Pyrimidines/pharmacology , Amines/pharmacology , Animals , Drug Evaluation, Preclinical , Drug Resistance, Microbial , Guinea Pigs , Half-Life , RatsABSTRACT
From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.
Subject(s)
Aminopyridines/chemistry , Antimalarials/chemistry , Benzimidazoles/chemistry , Aminopyridines/pharmacokinetics , Aminopyridines/pharmacology , Animals , Antimalarials/pharmacokinetics , Antimalarials/pharmacology , Benzimidazoles/pharmacokinetics , Benzimidazoles/pharmacology , Hepatocytes/metabolism , Humans , Malaria/drug therapy , Malaria/mortality , Mice, SCID , Microsomes, Liver/metabolism , Plasmodium berghei/drug effects , Plasmodium falciparum/drug effects , Rats , Structure-Activity RelationshipABSTRACT
Type II topoisomerases are well conserved across the bacterial species, and inhibition of DNA gyrase by fluoroquinolones has provided an attractive option for treatment of tuberculosis (TB). However, the emergence of fluoroquinolone-resistant strains of Mycobacterium tuberculosis (Mtb) poses a threat for its sustainability. A scaffold hopping approach using the binding mode of novel bacterial topoisomerase inhibitors (NBTIs) led to the identification of a novel class of benzimidazoles as DNA gyrase inhibitors with potent anti-TB activity. Docking of benzimidazoles to a NBTI bound crystal structure suggested that this class of compound makes key contacts in the enzyme active site similar to the reported NBTIs. This observation was further confirmed through the measurement of DNA gyrase inhibition, and activity against Mtb strains harboring mutations that confer resistance to aminopiperidines based NBTIs and Mtb strains resistant to moxifloxacin. Structure-activity relationship modification at the C-7 position of the left-hand side ring provided further avenue to improve hERG selectivity for this chemical series that has been the major challenges for NBTIs.
ABSTRACT
New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.
Subject(s)
Antitubercular Agents/therapeutic use , Indoles/therapeutic use , Pyridines/therapeutic use , Tuberculosis, Pulmonary/drug therapy , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacokinetics , Dogs , Drug Therapy, Combination , Female , Humans , Indoles/chemical synthesis , Indoles/pharmacokinetics , Male , Mice , Mice, Inbred BALB C , Pyridines/chemical synthesis , Pyridines/pharmacokinetics , RatsABSTRACT
DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.
Subject(s)
Antitubercular Agents/chemical synthesis , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Piperidines/chemical synthesis , Topoisomerase II Inhibitors/chemical synthesis , Acute Disease , Administration, Oral , Animals , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Availability , Chronic Disease , DNA Gyrase/genetics , DNA Gyrase/metabolism , Drug Resistance, Bacterial , ERG1 Potassium Channel , Fluoroquinolones/pharmacology , Humans , Macrophages/drug effects , Macrophages/microbiology , Mice, Inbred BALB C , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutation , Mycobacterium tuberculosis/enzymology , Piperidines/pharmacokinetics , Piperidines/pharmacology , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Stereoisomerism , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacokinetics , Topoisomerase II Inhibitors/pharmacology , Tuberculosis, Pulmonary/drug therapyABSTRACT
In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.
Subject(s)
Antitubercular Agents/chemical synthesis , Indoles/chemical synthesis , Alcohol Oxidoreductases , Animals , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Disease Models, Animal , Humans , Indoles/pharmacokinetics , Mice , Mycobacterium tuberculosis/drug effects , Oxidoreductases/antagonists & inhibitors , Rats , Structure-Activity RelationshipABSTRACT
4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of â¼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.
Subject(s)
Amides/chemistry , Antitubercular Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Oxidoreductases/antagonists & inhibitors , Piperidines/chemistry , Quinolones/chemistry , Alcohol Oxidoreductases , Amides/pharmacokinetics , Amides/pharmacology , Animals , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Catalytic Domain , Cell Line, Tumor , Drug Resistance, Bacterial , Genome, Bacterial , Humans , Kinetics , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Piperidines/pharmacokinetics , Piperidines/pharmacology , Protein Binding , Quinolones/pharmacokinetics , Quinolones/pharmacology , Rats, Wistar , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Robust and physiologically relevant infection models are required to investigate pharmacokinetic-pharmacodynamic (PK/PD) correlations for anti-tuberculosis agents at preclinical discovery. We have validated an inhalation-based rat infection model of tuberculosis harbouring mycobacteria in a replicating state, that is suitable for investigating pharmacokinetics and drug action of anti-tubercular agents. A reproducible and actively replicating lung infection was established in Wistar rats by inhalation of a series of graded inocula of Mycobacterium tuberculosis. Following an initial instillation of â¼10(5) log10 CFU/lung, M. tuberculosis grew logarithmically for the first 3 weeks, and then entered into a chronic phase with no net increase in pulmonary bacterial loads. Dose response of front-line anti-TB drugs was investigated following pharmacokinetic measurements in the plasma of infected rats. Rifampicin, Isoniazid, and Ethambutol dosed per orally exhibited bactericidality and good dose response with maximal effect of 5.66, 4.66, and 4.80 log10 CFU reductions in the lungs, respectively. In contrast, Pyrazinamide was merely bacteriostatic with 1.92 log10 CFU/lung reduction and did not reduce the bacterial burden beyond the initial bacterial loads present at beginning of treatment in spite of high Pyrazinamide blood levels. Rat infection model with actively replicating bacilli provides a physiologically distinct and pharmacologically relevant model that can be exploited to distinguish investigational compounds in to bacteriostatic or bactericidal scaffolds. We propose that this rat infection model though need more drug substance, can be used in early discovery settings to investigate pharmacology of novel anti-tubercular agents for the treatment of active pulmonary tuberculosis.
Subject(s)
Antitubercular Agents/pharmacokinetics , Tuberculosis, Pulmonary/drug therapy , Animals , Antitubercular Agents/administration & dosage , Bacterial Load/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Male , Mycobacterium tuberculosis , Rats, Wistar , Treatment OutcomeABSTRACT
Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 µg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 µg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was â¼10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.
Subject(s)
Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Mycobacterium tuberculosis/pathogenicity , Tuberculosis/drug therapy , Animals , Cell Line , Humans , Mice , Mycobacterium tuberculosis/drug effects , Structure-Activity RelationshipABSTRACT
We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Indoles/chemical synthesis , Mycobacterium tuberculosis/drug effects , Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases , Animals , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/therapeutic use , Drug Discovery , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Indoles/pharmacokinetics , Indoles/pharmacology , Indoles/therapeutic use , Mice , Rats , Tuberculosis, Multidrug-Resistant/drug therapyABSTRACT
A pharmacophore-based search led to the identification of thiazolopyridine ureas as a novel scaffold with antitubercular activity acting through inhibition of DNA Gyrase B (GyrB) ATPase. Evaluation of the binding mode of thiazolopyridines in a Mycobacterium tuberculosis (Mtb) GyrB homology model prompted exploration of the side chains at the thiazolopyridine ring C-5 position to access the ribose/solvent pocket. Potent compounds with GyrB IC50 ≤ 1 nM and Mtb MIC ≤ 0.1 µM were obtained with certain combinations of side chains at the C-5 position and heterocycles at the C-6 position of the thiazolopyridine core. Substitutions at C-5 also enabled optimization of the physicochemical properties. Representative compounds were cocrystallized with Streptococcus pneumoniae (Spn) ParE; these confirmed the binding modes predicted by the homology model. The target link to GyrB was confirmed by genetic mapping of the mutations conferring resistance to thiazolopyridine ureas. The compounds are bactericidal in vitro and efficacious in vivo in an acute murine model of tuberculosis.
Subject(s)
Antitubercular Agents/pharmacology , DNA Gyrase/metabolism , Mycobacterium tuberculosis/drug effects , Pyridines/pharmacology , Topoisomerase II Inhibitors/pharmacology , Tuberculosis/drug therapy , Urea/pharmacology , Animals , Antitubercular Agents/administration & dosage , Antitubercular Agents/chemistry , Disease Models, Animal , Dose-Response Relationship, Drug , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Mycobacterium tuberculosis/enzymology , Pyridines/administration & dosage , Pyridines/chemistry , Structure-Activity Relationship , Topoisomerase II Inhibitors/administration & dosage , Topoisomerase II Inhibitors/chemistry , Urea/analogs & derivatives , Urea/chemistryABSTRACT
Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.
Subject(s)
Amoxicillin-Potassium Clavulanate Combination , Anti-Bacterial Agents , Clavulanic Acid , Mycobacterium tuberculosis/drug effects , Thienamycins , Tuberculosis, Pulmonary/drug therapy , beta-Lactams , Amoxicillin-Potassium Clavulanate Combination/administration & dosage , Amoxicillin-Potassium Clavulanate Combination/pharmacokinetics , Amoxicillin-Potassium Clavulanate Combination/pharmacology , Amoxicillin-Potassium Clavulanate Combination/therapeutic use , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Clavulanic Acid/administration & dosage , Clavulanic Acid/pharmacokinetics , Clavulanic Acid/pharmacology , Clavulanic Acid/therapeutic use , Disease Models, Animal , Drug Therapy, Combination , Female , Humans , Meropenem , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests/standards , Mycobacterium tuberculosis/growth & development , Streptomycin/pharmacology , Thienamycins/administration & dosage , Thienamycins/pharmacokinetics , Thienamycins/pharmacology , Thienamycins/therapeutic use , Treatment Outcome , Tuberculosis, Pulmonary/microbiology , beta-Lactams/administration & dosage , beta-Lactams/pharmacokinetics , beta-Lactams/pharmacology , beta-Lactams/therapeutic useABSTRACT
The discovery of novel therapeutics for the treatment of tuberculosis involves routine testing in a mouse model over four weeks of daily dosing with test compounds. In this model, daily oral administration of rifampin (10 mg/kg) showed significantly lower plasma exposure on day 5 compared to day 1. The absence of PXR-mediated induction of mouse Cyp3a isoforms was confirmed in the present study by incubating liver microsomes prepared from control and rifampin treated mice with probe substrates of CYP3A. To test whether the reduction in exposure was due to Pgp-mediated efflux, verapamil, a known Pgp inhibitor, was dosed to the rifampin pre-treated mice which led to an increase in exposure to that obtained after a single dose of rifampin, suggesting the role of Pgp induction in reducing exposure to rifampin. To further confirm Pgp induction in rifampin treated mice, digoxin, a known substrate of Pgp, was administered to the rifampin pre-treated mice, and a significant drop in the digoxin exposure was observed compared to the control group. Collectively, our results show that repeated administration of rifampin in mice leads to a reduction in oral exposure due to induction of Pgp-mediated efflux of rifampin, and not via induction of CYP3A isoforms.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Microsomes, Liver/drug effects , Rifampin/administration & dosage , Rifampin/pharmacokinetics , Administration, Intravenous , Administration, Oral , Animals , Cytochrome P-450 Enzyme System/metabolism , Mice , Mice, Inbred BALB C , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Rifampin/blood , Time FactorsABSTRACT
We describe a rapid screening methodology for performing pharmacokinetic (PK) studies in mice called Fast PK. In this Fast PK method, two mice were used per compound and four blood samples were collected from each mouse. The sampling times were staggered (sparse sampling) between the two mice, thus yielding complete PK profile in singlicate across eight time points. The plasma PK parameters from Fast PK were comparable to that obtained from conventional PK methods. This method has been used to rapidly screen compounds in the early stages of drug discovery and about 600 compounds have been profiled in the last 3 years, which has resulted in reduction in the usage of mice by 800 per year in compliance with the 3R principles of animal ethics. In addition, this Fast PK method can also help in evaluating the PK parameters from the same set of animals used in safety/toxicology/efficacy studies without the need for satellite groups.
Subject(s)
Drug Evaluation, Preclinical/methods , Pharmacokinetics , Administration, Intravenous , Administration, Oral , Animals , Male , Mice , Mice, Inbred BALB C , Pharmaceutical Preparations/metabolism , Rifampin/administration & dosage , Rifampin/blood , Rifampin/pharmacokineticsABSTRACT
Coadministration of moxifloxacin and rifampin was evaluated in a murine model of Mycobacterium tuberculosis pulmonary infection to determine whether the finding of antagonism documented in a hollow-fiber infection model could be recapitulated in vivo. Colony counts were followed in a no-treatment control group, groups administered moxifloxacin or rifampin monotherapy, and a group administered a combination of the two agents. Following 18 days of once-daily oral administration to mice infected with M. tuberculosis, there was a reduction in the plasma exposure to rifampin that decreased further when rifampin was coadministered with moxifloxacin. Pharmacodynamic analysis demonstrated a mild antagonistic interaction between moxifloxacin and rifampin with respect to cell kill in the mouse model for tuberculosis (TB). No emergence of resistance was noted over 28 days of therapy, even with monotherapy. This was true even though one of the agents in the combination (moxifloxacin) induces error-prone replication. The previously noted antagonism with respect to cell kill shown in the hollow-fiber infection model was recapitulated in the murine TB lung model, although to a lesser extent.