Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) bacterial pathogen of acute clinical significance. Resistance to current standard-of-care antibiotics, such as vancomycin and linezolid, among nosocomial and community-acquired MRSA clinical isolates is on the rise. This threat to global public health highlights the need to develop new antibiotics for the treatment of MRSA infections. Here, we describe a new benzamide FtsZ inhibitor (TXH9179) with superior antistaphylococcal activity relative to earlier-generation benzamides like PC190723 and TXA707. TXH9179 was found to be 4-fold more potent than TXA707 against a library of 55 methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, including MRSA isolates resistant to vancomycin and linezolid. TXH9179 was also associated with a lower frequency of resistance relative to TXA707 in all but one of the MSSA and MRSA isolates examined, with the observed resistance being due to mutations in the ftsZ gene. TXH9179 induced changes in MRSA cell morphology, cell division, and FtsZ localization are fully consistent with its actions as a FtsZ inhibitor. Crystallographic studies demonstrate the direct interaction of TXH9179 with S. aureus FtsZ (SaFtsZ), while delineating the key molecular contacts that drive complex formation. TXH9179 was not associated with any mammalian cytotoxicity, even at a concentration 10-fold greater than that producing antistaphylococcal activity. In serum, the carboxamide prodrug of TXH9179 (TXH1033) is rapidly hydrolyzed to TXH9179 by serum acetylcholinesterases. Significantly, both intravenously and orally administered TXH1033 exhibited enhanced in vivo efficacy relative to the carboxamide prodrug of TXA707 (TXA709) in treating a mouse model of systemic (peritonitis) MRSA infection. Viewed as a whole, our results highlight TXH9179 as a promising new benzamide FtsZ inhibitor worthy of further development.

Brensocatib is a novel, oral, selective, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), which activates several neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) in the bone marrow during the early stage of neutrophil maturation. These NSPs are associated with pathogen destruction and inflammatory mediation; their dysregulated activation can result in excess secretion of active NSPs causing damaging inflammation and contributing to neutrophil-mediated inflammatory and autoimmune diseases. Pharmacological inhibition of DPP1 in the bone marrow could therefore represent an attractive strategy for these neutrophil-driven diseases. A completed Phase 2 trial in non-cystic fibrosis bronchiectasis patients (ClinicalTrials.gov number NCT03218917; EudraCT number: 2017-002533-32) indeed demonstrated that administration of brensocatib attenuated the damaging effects of chronic inflammation by inhibiting the downstream activation of NSPs. To support a range of preclinical programs and further understand how rodent species and strains may affect brensocatib’s pharmacokinetic (PK) profile and its pharmacodynamic (PD) effects on NE, PR3, and CatG, an extensive naïve dosing study with brensocatib at different dosing levels, frequencies, and durations was undertaken. Dose-dependent PK exposure responses (AUC and Cmax) were observed regardless of the rodent species and strain. Overall, mice showed greater reduction in NSP activities compared to rats. Both mice and rats dosed once daily (QD) had equivalent NSP activity reduction compared to BID (twice a day) dosing when the QD dose was 1.5-times the BID daily dose. For both mouse strains, CatG activity was reduced the most, followed by NE, then PR3; whereas, for both rat strains, PR3 activity was reduced the most, followed by CatG, and then NE. Maximum reduction in NSP activities was observed after ∼7 days and recoveries were nearly symmetrical. These results may facilitate future in vivo brensocatib study dosing considerations, such as the timing of prophylactic or therapeutic administration, choice of species, dosage and dosing frequency.

Oxacillin is a first-line antibiotic for the treatment of methicillin-sensitive Staphylococcus aureus (MSSA) infections but is ineffective against methicillin-resistant S. aureus (MRSA) due to resistance. Here we present results showing that co-administering oxacillin with the FtsZ-targeting prodrug TXA709 renders oxacillin efficacious against MRSA. The combination of oxacillin and the active product of TXA709 (TXA707) is associated with synergistic bactericidal activity against clinical isolates of MRSA that are resistant to current standard-of-care antibiotics. We show that MRSA cells treated with oxacillin in combination with TXA707 exhibit morphological characteristics and PBP2 mislocalization behavior similar to that exhibited by MSSA cells treated with oxacillin alone. Co-administration with TXA709 renders oxacillin efficacious in mouse models of both systemic and tissue infection with MRSA, with this efficacy being observed at human-equivalent doses of oxacillin well below that recommended for daily adult use. Pharmacokinetic evaluations in mice reveal that co-administration with TXA709 also increases total exposure to oxacillin. Viewed as a whole, our results highlight the clinical potential of repurposing oxacillin to treat MRSA infections through combination with a FtsZ inhibitor.

Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.

Combination therapy of bacterial infections with synergistic drug partners offers distinct advantages over monotherapy. Among these advantages are (i) a reduction of the drug dose required for efficacy, (ii) a reduced potential for drug-induced toxicity, and (iii) a reduced potential for the emergence of resistance. Here, we describe the synergistic actions of the third-generation oral cephalosporin cefdinir and TXA709, a new, FtsZ-targeting prodrug that we have developed with improved pharmacokinetics and enhanced in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA) relative to earlier agents. We show that the active product of TXA709 (TXA707) acts synergistically with cefdinir in vitro against clinical isolates of MRSA, vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), and linezolid-resistant S. aureus (LRSA). In addition, relative to TXA707 alone, the combination of TXA707 and cefdinir significantly reduces or eliminates the detectable emergence of resistance. We also demonstrate synergy in vivo with oral administration of the prodrug TXA709 and cefdinir in mouse models of both systemic and tissue (thigh) infections with MRSA. This synergy reduces the dose of TXA709 required for efficacy 3-fold. Viewed as a whole, our results highlight the potential of TXA709 and cefdinir as a promising combination for the treatment of drug-resistant staphylococcal infections.

ABSTRACT The clinical development of FtsZ-targeting benzamide compounds like PC190723 has been limited by poor drug-like and pharmacokinetic properties. Development of prodrugs of PC190723 (e.g., TXY541) resulted in enhanced pharmaceutical properties, which, in turn, led to improved intravenous efficacy as well as the first demonstration of oral efficacy in vivo against both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Despite being efficacious in vivo , TXY541 still suffered from suboptimal pharmacokinetics and the requirement of high efficacious doses. We describe here the design of a new prodrug (TXA709) in which the Cl group on the pyridyl ring has been replaced with a CF 3 functionality that is resistant to metabolic attack. As a result of this enhanced metabolic stability, the product of the TXA709 prodrug (TXA707) is associated with improved pharmacokinetic properties (a 6.5-fold-longer half-life and a 3-fold-greater oral bioavailability) and superior in vivo antistaphylococcal efficacy relative to PC190723. We validate FtsZ as the antibacterial target of TXA707 and demonstrate that the compound retains potent bactericidal activity against S. aureus strains resistant to the current standard-of-care drugs vancomycin, daptomycin, and linezolid. These collective properties, coupled with minimal observed toxicity to mammalian cells, establish the prodrug TXA709 as an antistaphylococcal agent worthy of clinical development.

The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamide PC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despite its initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We describe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivo efficacy. The N-Mannich base derivative (TXY436) is ∼100-fold more soluble than PC190723 in an acidic aqueous vehicle (10 mM citrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting to PC190723 with a conversion half-life of 18.2 ± 1.6 min. Pharmacokinetic analysis following intravenous administration of TXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respectively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Using a mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus, we show that TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not efficacious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentrations at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 a worthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.

The benzamide derivative PC190723 was among the first of a promising new class of FtsZ-directed antibacterial agents to be identified that exhibit potent antistaphylococcal activity. However, the compound is associated with poor drug-like properties. As part of an ongoing effort to develop FtsZ-targeting antibacterial agents with increased potential for clinical utility, we describe herein the pharmacodynamics, pharmacokinetics, in vivo antistaphylococcal efficacy, and mammalian cytotoxicity of TXY541, a novel 1-methylpiperidine-4-carboxamide prodrug of PC190723. TXY541 was found to be 143-times more soluble than PC190723 in an aqueous acidic vehicle (10mM citrate, pH 2.6) suitable for both oral and intravenous in vivo administration. In staphylococcal growth media, TXY541 converts to PC190723 with a half-life of approximately 8h. In 100% mouse serum, the TXY541-to-PC190723 conversion was much more rapid (with a half-life of approximately 3min), suggesting that the conversion of the prodrug in serum is predominantly enzyme-catalyzed. Pharmacokinetic analysis of both orally and intravenously administered TXY541 in mice yielded a half-life for the PC190723 conversion product of 0.56h and an oral bioavailability of 29.6%. Whether administered orally or intravenously, TXY541 was found to be efficacious in vivo in mouse models of systemic infection with both methicillin-sensitive and methicillin-resistant S. aureus. Toxicological assessment of TXY541 against mammalian cells revealed minimal detectable cytotoxicity. The results presented here highlight TXY541 as a potential therapeutic agent that warrants further pre-clinical development.

Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound 10b (WYE-114152) had low nanomolar hNET potency (IC(50) = 15 nM) and good selectivity for hNET over hSERT (>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated efficacy in rat models of acute, inflammatory, and neuropathic pain.

Corticotropin-releasing factor (CRF) plays a major role in controlling the body's response to stress. Because painful conditions are inherently stressful, we hypothesize that CRF may act via CRF-1 receptors to contribute to the pain experience. Studies were designed to investigate whether blocking CRF-1 receptors with selective antagonists or reducing their expression with CRF-Saporin, would attenuate ulcer, inflammatory- and neuropathic-like pain. Five experimental designs were undertaken. In experiment 1, ulcer pain was induced in mice following oral administration of indomethacin, while in experiments 2 and 3, inflammatory pain was induced in rats with either carrageenan or FCA, respectively. For these studies, animals were dosed with CP-154,526 (3, 10, 30 mg/kg) and NBI 27914 (1-30 mg/kg) 1 h prior to the assessment of tactile, thermal or mechanical hypersensitivity, respectively. In experiment 4, neuropathic pain was induced. Twenty-one days following spinal nerve ligation (SNL), animals received CRF-Saporin or control. Three weeks later tactile allodynia was assessed. Similarly, in experiment 5, a separate set of rats received CRF-Saporin or control. Twenty-one days later, mechanical hyperalgesia was assessed following intraplantar carrageenan. Results from the antagonist studies showed that CP-154,526 and NBI 27914 either fully or partially reversed the referred ulcer pain with minimal effective doses (MED) equal to 3 and 10 mg/kg, respectively. Similarly, both NBI 27914 and CP-154,526 reversed the thermal and mechanical hypersensitivity elicited by carrageenan and FCA with MEDs </= 5 and 10 mg/kg, respectively. Findings from the two CRF-Saporin studies determined that pre-treatment with this toxin significantly attenuated SNL- and carrageenan-induced tactile hypersensitivity. Together, these findings suggest that CRF-1 receptors mediate pain and implicate CRF in this regard.

Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-α (DAGLα) and -β (DAGLβ) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show ∼80% reductions in 2-AG levels in the brain and spinal cord in DAGLα−/− mice and a 50% reduction in the brain in DAGLβ−/− mice. In contrast, DAGLβ plays a more important role than DAGLα in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLβ−/− mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLα−/− animals and relatively unaffected in DAGLβ−/− animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLα−/− and/or DAGLβ−/− mice. These findings provide the first evidence that DAGLα is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Sequential structural modifications of the aryloxypropanamine template (e.g., atomoxetine, 2) led to a novel series of 1-(3-amino-2-hydroxy-1-phenyl propyl)-1,3-dihydro-2H-benzimidazol-2-ones as selective norepinephrine reuptake inhibitors (NRIs). In general, this series of compounds potently blocked the human norepinephrine transporter (hNET) while exhibiting selectivity at hNET against both the human serotonin (hSERT) and dopamine transporters (hDAT). Numerous compounds (e.g., 19-22) had low nonamolar hNET potency with IC(50) values of 7-10 nM and excellent selectivity (>500 fold) at hNET over hSERT and hDAT. Several compounds, such as 20 and 22, were tested in a telemetric rat model of ovariectomized-induced thermoregulatory dysfunction and were efficacious at oral doses of 3 mg/kg in reducing the tail skin temperature. In addition, compound 20 was also studied in the rat hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain, respectively, and was orally efficacious at doses of 3-10 mg/kg.

Voltage-gated Na(+) channels may play important roles in establishing pathological neuronal hyperexcitability associated with chronic pain in humans. Na(+) channel blockers, such as carbamazepine (CBZ) and lamotrigine (LTG), are efficacious in treating neuropathic pain; however, their therapeutic utility is compromised by central nervous system side effects. We reasoned that it may be possible to gain superior control over pain states and, in particular, a better therapeutic index, by designing broad-spectrum Na(+) channel blockers with higher potency, faster onset kinetics, and greater levels of state dependence than existing drugs. 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) is a novel structural analog of the state-dependent Na(+) channel blocker V102862 [4-(4-fluorophenoxy)benzaldehyde semicarbazone]. Tested on recombinant rat Na(v)1.2 channels and native Na(+) currents in cultured rat dorsal root ganglion neurons, PPPA was approximately 1000 times more potent, had 2000-fold faster binding kinetics, and > or =10-fold higher levels of state dependence than CBZ and LTG. Tested in rat pain models against mechanical endpoints, PPPA had minimal effective doses of 1 to 3 mg/kg p.o. in partial sciatic nerve ligation, Freund's complete adjuvant, and postincisional pain. In all cases, efficacy was similar to clinically relevant comparators. Importantly, PPPA did not produce motor deficits in the accelerating Rotarod assay of ataxia at doses up to 30 mg/kg p.o., indicating a therapeutic index >10, which was superior to CBZ and LTG. Our experiments suggest that high-potency, broad-spectrum, state-dependent Na(+) channel blockers will have clinical utility for treating neuropathic, inflammatory, and postsurgical pain. Optimizing the biophysical parameters of broad-spectrum voltage-gated Na(+) channel blockers may lead to improved pain therapeutics.