Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis ( Mtb ). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are no orally available medications for prophylaxis for those exposed to SARS-CoV-2 and limited therapeutic options for those who develop COVID-19. We evaluated the antiviral activity of sulforaphane (SFN), a naturally occurring, orally available, well-tolerated, nutritional supplement present in high concentrations in cruciferous vegetables with limited side effects. SFN inhibited in vitro replication of four strains of SARS-CoV-2 as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN is a promising treatment for prevention of coronavirus infection or treatment of early disease.

Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a Fast, Luminescent, and Affordable Sensor of Hip1 (FLASH) for the diagnosis and monitoring of drug sensitivity of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that when processed, produces visible light that can be measured with a high signal to noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other non-tuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.

Background Pretomanid is used in combination with bedaquiline and linezolid (BPaL regimen) in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, the penetration of pretomanid in privileged sites remain unknown. Antimicrobial pharmacokinetic (PK) parameters are traditionally derived from clinical samples (blood and cerebrospinal fluid), which may not accurately represent the intralesional tissue PK, affected by drug properties, vascular supply, barrier permeability, and the microenvironment. Methods We developed 18F-pretomanid (chemically identical to pretomanid) for in vivo multi-compartment PK by positron emission tomography (PET). Dynamic 18F-pretomanid PET was used to obtain cross species pretomanid concentration-time profiles in animal models of TB (mice and rabbits) to quantify penetration into pulmonary and brain lesions. A subset of animals underwent PET/CT imaging with 18F-py-albumin and 18F-FDG to assess vascular supply and inflammation. Postmortem 18F-pretomanid autoradiography (high-resolution) and mass spectrometry were performed in infected tissues. A mouse model of TB meningitis was used to evaluate the bactericidal activity of the BPaL regimen (Figure 1). Figure 1. Experimental schematics. (A) A new synthetic approach was developed to obtain radiofluorinated pretomanid (18F-pretomanid), which is chemically identical to pretomanid and therefore undergoes identical PK and metabolism in vivo. Dynamic 18F-pretomanid PET/CT imaging was performed in validated preclinical models of tuberculosis following intravenous administration of 18F-pretomanid. (B) PET signal was quantified in multiple compartments to generate time activity curves (TACs) used to calculate area under the curve (AUC) over 0-60 minutes. A subset of animals also underwent PET/CT imaging of 18F-py-albumin to assess vascular supply to lung and brain lesions, and with 18F-FDG to confirm the presence of neuroinflammation in the mouse and rabbit models of TB meningitis. Tissue resection post-mortem was used to visualize the intralesional retention of 18F-pretomanid using high-resolution (10 µm) autoradiography. The efficacy of the BPaL regimen in TB meningitis was compared to that of standard treatment with rifampin, isoniazid, and pyrazinamide in the mouse model. Mass spectrometry was performed following oral administration of BPaL to determine brain drug levels. (C) These data provide multicompartment PK analysis, intralesional levels of pretomanid, and insights into the mechanism that govern pretomanid tissue distribution. Results 18F-Pretomanid PET provided detailed concentration-time profiles in infected tissues demonstrating excellent lung and brain tissue penetration (AUC ratio to plasma > 1) in both animal species, which was spatially compartmentalized, likely due to differential vascular supply (18F-py-albumin PET) (Figure 2). Brain lesions (identified by 18F-FDG PET) demonstrated localized leakiness on 18F-py-albumin PET. Autoradiography and mass spectrometry corroborated the imaging findings. The efficacy of the BPaL regimen in TB meningitis was substantially lower than standard TB treatment (Figure 3), likely due to restricted penetration of bedaquiline and linezolid into the brain parenchyma. Figure 2. Spatial heterogeneity of 18F-Pretomanid penetration and vascular supply to pulmonary TB lesions. (A) A novel synthetic was devised to obtain 18F-pretomanid, which is chemically identical to pretomanid. (B) Maximum intensity projection (MIP) of 18F-Pretomanid PET/CT in M.tb.-infected mice over 3 hrs shows hepatobiliary and renal excretion, high uptake into brown fat, brain, and lungs. (C) Resection of infected lungs 30 minutes post intravenous administration of 18F-pretomanid shows heterogenous distribution of 18F-pretomanid into the lungs visible by high resolution autoradiography. Areas of pneumonia are identifiable by hematoxylin and eosin (H&E) staining of the same tissue section used for autoradiography. (D) Time-activity curves of 18F-Pretomanid in infected mouse lung (0-3 hours) and derived area under the curve (AUC) ratios to plasma (E) in infected mouse lung. Representative MIP of 18F-pretomanid (F) and 18F-py-albumin (H) PET/CT in a rabbit with cavitary TB and quantification of the AUC ratios to plasma show reduced penetration into lung lesions and cavitary wall compared to areas of unaffected lung (G and I). Data are represented as median ± interquartile range, n=3-4 group. Figure 3. Exposure levels of 18/19F-pretomanid in models of TB meningitis. (A) Experimental timeline used to assess the penetration of pretomanid into infected mouse brain before and during treatment with antimicrobials bedaquiline (B), pretomanid (Pa), and linezolid (L), and corticosteroid dexamethasone (D). (B) Representative three-dimensional MIP of 18F-pretomanid PET/CT in the CNS-TB model, 10 min post-injection, and transverse section showing high and heterogeneous brain uptake. (C) High-resolution autoradiography was performed to confirm heterogeneous penetration of 18F-pretomanid into infected brain lesions in the mouse. (D). 8F-pretomanid AUC ratios of tissue to plasma in mouse brain before (day 0) and two weeks into treatment show a reduction in penetration at week 2. (E). Pretomanid concentrations (µg/mL) in mouse plasma and brain, at day 0 and two weeks into treatment, measured by mass spectrometry and derived concentration ratios of brain to plasma (F) suggest drug accumulation due to the long half-life. (G) While 18F-py-albumin and 18F-FDG PET/CT show vascular leakage and neuroinflammation in the rabbit model of TB meningitis, the penetration of 18F-pretomanid is heterogeneous and reduced at the lesion site (indicated by white arrow). (H) Quantification of the PET signal shows variability within the same animal. Data are represented as median ± interquartile range, n=3-5 group. Figure 4. Evaluation of a pretomanid-containing regimen in TB meningitis. (A) Mice with experimentally induced TB meningitis were treated with Bedaquiline (25 mg/day), Pretomanid (100 mg/day), Linezolid (100 mg/day), and Dexamethasone (2 mg/day) or Rifampin (10 mg/day), Isoniazid (10 mg/day), Pyrazinamide (150 mg/day) and Dexamethasone (2mg/day) for 8 weeks. Treatment efficacy was determined based on the brain bacterial burden after 2, 4, 6, and 8 weeks of treatment. (B) The penetration of 76Br-bedaquiline, 18F-linezolid, and 18F-pretomanid into the brain parenchyma was measured non-invasively by PET and revealed low penetration of 76Br-bedaquiline (AUC radio to plasma 0.15) and 18F-linezolid (AUC radio to plasma 0.3). (C) Mass spectrometry analysis was performed to confirm the brain penetration of bedaquiline, linezolid, and pretomanid following oral administration. Conclusion Dynamic 18F-pretomanid PET provided holistic data on pretomanid exposures showing excellent penetration into infected lung and brain tissues. The BPaL regimen was inferior to standard TB treatment for TB meningitis. Thus, new pretomanid-containing regimens need to be developed for the treatment of MDR-TB meningitis. Disclosures Charles A. Peloquin, Pharm.D., Nothing to disclose Alvaro A. Ordonez, MD, Cubresa (Consultant)Fujirebio Diagnostics (Research Grant or Support) Sanjay K. Jain, MD, Fujirebio Diagnostics, Inc., USA (Research Grant or Support)Novobiotic LLC, USA (Research Grant or Support)T3 Pharma, Switzerland (Research Grant or Support) Sanjay K. Jain, MD, Fujirebio Diagnostics, Inc., USA (Individual(s) Involved: Self): Research Grant or Support; Novobiotic LLC, USA (Individual(s) Involved: Self): Research Grant or Support; T3 Pharma, Switzerland (Individual(s) Involved: Self): Research Grant or Support

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Rationale: Despite a long history of use in synaptic physiology, the lobster has been a neglected model for behavioral pharmacology. A restauranteur proposed that exposing lobster to cannabis smoke reduces anxiety and pain during the cooking process. It is unknown if lobster gill respiration in air would result in significant Δ9-tetrahydrocannabinol (THC) uptake and whether this would have any detectable behavioral effects. Objective: The primary goal was to determine tissue THC levels in the lobster after exposure to THC vapor. Secondary goals were to determine if THC vapor altered locomotor behavior or nociception. Methods: Tissue samples were collected from muscle, brain and hemolymph of Homarus americanus (N=3 per group) following 30 or 60 minutes of exposure to vapor generated by an e-cigarette device using THC (100 mg/mL in a propylene glycol vehicle). Separate experiments assessed locomotor behavior and hot water nociceptive responses following THC vapor exposure. Results: THC vapor produced duration-related THC levels in all tissues examined. Locomotor activity was decreased (distance, speed, time-mobile) by 30 min inhalation of THC. Lobsters exhibit a temperature-dependent withdrawal response to immersion of tail, antennae or claws in warm water; this is novel evidence of thermal nociception for this species. THC exposure for 60 minutes had only marginal effect on nociception under the conditions assessed. Conclusions: Vapor exposure of lobsters, using an e-cigarette based model, produces dose-dependent THC levels in all tissues and reduces locomotor activity. Hot water nociception is temperature dependent in the lobster, but no clear effects of THC inhalation were confirmed.