Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, a novel semisynthetic series of spectinomycin analogs was generated with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross-resistance with existing tuberculosis therapeutics, activity against MDR/XDR-tuberculosis, and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target based tuberculosis drug discovery.

The antibiotic spectinomycin is a potent inhibitor of bacterial protein synthesis with a unique mechanism of action and an excellent safety index, but it lacks antibacterial activity against most clinically important pathogens. A novel series of N-benzyl substituted 3'-(R)- 3'-aminomethyl-3'-hydroxy spectinomycins was developed based on a computational analysis of the aminomethyl spectinomycin binding site and structure guided synthesis. These compounds had ribosomal inhibition values comparable to spectinomycin but showed increased potency against common respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Moraxella catarrhalis as well as the sexually transmitted bacteria Neisseria gonorrhoeae and Chlamydia trachomatis. Non-ribosome binding 3'-(S) isomers of the leads demonstrated weak inhibitory activity in in vitro protein translation assays and poor antibacterial activity, indicating that the antibacterial activity of the series remains on target. In addition to improved antibacterial potency, compounds also demonstrated no mammalian cytotoxicity, improved microsomal stability, and favorable pharmacokinetic properties in rats. The lead compound from the series, compound 1, exhibited excellent chemical stability, which was superior to spectinomycin and had no significant interaction with a panel of human receptors and drug metabolism enzymes suggesting low potential for adverse reactions or drug-drug interactions in vivo. Compound 1 was active in vitro against a panel of penicillin, macrolide, and cephalosporin resistant S. pneumoniae clinical isolates and cured mice of fatal pneumococcal pneumonia and sepsis at a dose of 5 mg/kg. Together, these studies indicate N-benzyl aminomethyl spectinomycins possess suitable properties for further development as novel antibacterial agents to treat drug resistant respiratory tract and sexually transmitted bacterial infections.

A series of tetracyclic nitrofuran isoxazoline antituberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent antituberculosis activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, structural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and metabolically blocking functional groups. The compounds generated were evaluated for their in vitro antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and protein binding. Pharmacokinetic profiles for the most promising candidates were then determined. Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most potent antituberculosis agents in the series. These compounds retained a narrow antibacterial spectrum of activity, with weak anti-gram positive and no gram negative activity, as well as good activity against non-replicating M. tuberculosis in a low oxygen model. Overall, the addition of solubilizing and metabolically blocked outer rings did improve solubility and decrease protein binding as designed. However, the metabolic stability for compounds in this series was generally lower than desired. The best three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with one notable compound showing a significantly longer half-life and good tolerability supporting its further advancement.

Spectinamides are promising new semisynthetic anti-tubercular agents that are modified with a pyridyl side chain, which blocks native efflux from the tuberculosis cell. This study, describes the stability of an advanced panel of spectinamide analogs, with varying substitutions to the pyridyl side chain, to Phase-II conjugative metabolism by glucuronosyl transferase, sulfotransferase and glutathione-S-transferase enzymes using both human and rat S9 enzyme fractions. All solely 5-substituted pyridyl spectinamides exhibited complete stability towards Phase II conjugative enzymes. However, 4-chloro substituted pyridyl spectinamides were susceptible to glutathione conjugation with rates dependent on other substitutions to the pyridine ring. Electron donating 5-substitutions increased the propensity for glutathione conjugation and conversely the introduction of an electron withdrawing 5-fluoro group blocked all observed glutathione conjugation. Based on these Phase II metabolism studies, lead spectinamides 1329, 1445, 1599, 1661 and 1810 were found to have favorable properties for potential lead compounds with no Phase II liabilities.

The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as 9a). In the current study, we report the synthesis and antimicrobial properties of 9a and panel of 9a analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of Mycobacterium tuberculosis with favorable selectivity indices (>150) and a narrow spectrum of activity. In vivo, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity in vivo, 9a remained the series lead. 9a exerted a lengthy post antibiotic effect and was highly active against nonreplicating M. tuberculosis grown under hypoxia. 9a showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that 9a is a nanomolar inhibitor of actively growing as well as nonreplicating M. tuberculosis.

The in vivo biodistribution and pharmacokinetics of 1329, a novel spectinamide antibiotic with anti-tubercular activity, were studied during intravenous administration of an tritium-labeled compound for nine consecutive, 12-hourly doses to rats. Serial blood samples were collected after the first and the eighth dose, and major organs and tissues were collected 1 h after the ninth dose. Urinary and fecal excretion was monitored throughout the dosing period. Radioactivity in the collected samples was assessed by scintillation counting. During the course of treatment, 86.6% of the administered radioactivity was recovered in urine, feces, organs, and muscle tissue. Urinary excretion was the major route of elimination, with 70% of radioactivity recovered from urine and 12.6% from feces. The time profiles of radioactivity in serum after the first and the eighth dose were identical for the first 2 h post-dose, with similar Cmax (3.39 vs. 3.55 mCi/L) and AUC0-τ (5.08 vs. 5.17 mCi • h/L), indicating no substantial accumulation of 1329 during multiple dosing. Radioactivity in major target organs for pulmonary tuberculosis infection, the lungs and spleen, was 2.79- and 3.06-fold higher than in the blood. Similarly, the intracellular uptake of 1329 into macrophages was sixfold higher than for streptomycin. Overall, these observations suggest biodistribution properties favorable for targeting pulmonary tuberculosis infections.

Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I‐III Regulatory, Quality, ManufacturingPostmarketing Phase IV The efficacy of the prodrug C2E5, the pentaethyl ester of diethylenetriaminepentaacetic acid (DTPA), as an orally administered chelator of americium was evaluated in rats. C2E5 enhanced 241Am decorporation over vehicle‐only controls (187 μmol/kg, 14.8 ± 4.2% vs control, 7.1 ± 1.4% injected 241Am, P < 0.05) when administered 1 h after 241Am nitrate contamination by intramuscular (i.m.) injection. Reduction in liver burden was also achieved (375 μmol/kg, 13.1 ± 2.0% vs control, 17.3 ± 2.4% 241Am). Under these conditions, a single C2E5 dose did not match the efficacy of intravenous (i.v.) Ca‐DTPA (26.8 μmol/kg; decorporation 24.5 ± 3.5% and liver burden 10.2 ± 2.7% 241Am). Dividing the 375 μmol/kg C2E5 dose enhanced efficacy (4 × 94.5 μmol/kg C2E5; decorporation 19.7 ± 4.5% and liver burden 11.5 ± 2.8% 241 Am; P < 0.001, P < 0.05 vs control), approaching parity with i.v. Ca‐DTPA. When the contamination model was changed to i.v. or i.m. 241Am citrate injection, C2E5 (75 μmol/kg) enhanced decorporation and reduced liver burden. Higher doses of C2E5 (375 μmol/kg) were comparable with i.v. Ca‐DTPA (26.8 μmol/kg), with liver burdens at 3.2 ± 1.4% and 7.4 ± 2.7%, and decorporation of 43.5 ± 8.9% and 37.4 ± 3.9% i.m. 241Am citrate, respectively. The differences observed in oral C2E5 efficacy relative to i.v. Ca‐DTPA between contamination models may be related to the kinetics of the injected americium complex. C2E5 proved to be an effective decorporation agent for 241Am when orally administered and may be improved by optimized treatment strategies.

Spectinamides are a novel class of antibiotics under development for the treatment of MDR-and XDR-tuberculosis, with 1599 and 1445 as early lead candidates within this group. In order to evaluate and differentiate the pharmacological properties of these compounds and assist in candidate selection and design of optimal dosing regimens in animal model of Mtb infection, time kill curve assessments were performed in a previously established in vitro PK/PD model system. The performed studies and subsequent pharmacometric analysis indicate that the antimycobacterial activity of 1599 exhibits concentration-dependent killing whereas 1445 shows timedependent killing. These findings are supported by the fact that the PKPD index that best describes bacterial killing is T>MIC for 1445, but fCmax/AUC for 1599. The differential killing behavior among the lead candidates can be rationalized by the differences in post-antibiotic effect: 15.7 h for 1445 compared the 133 h for 1599. Overall, the PK/PD based analysis of the in vitro pharmacologic killing profile of spectinamides 1599 and 1445 on mycobacteria provided valuable insights that contributed to lead candidate selection and preclinical development of these compounds.