(H-298) Doxycycline as Prophylactic Treatment for Prevention of Severe COVID-19 Induced Symptoms and Other Inflammatory Diseases

COVID-19 is an infectious disease caused by SARS-CoV-2 virus that has brought about a global pandemic and drastically affected the economy worldwide [1]. Since its emergence, COVID-19 has taken more than 6.9 million lives. Despite COVID-19 is no longer a public health emergency, SAR-CoVs continuously mutates and over 200,000 new cases are still being reported globally [2]. It is best we prepare ourselves against the next spread of harmful pathogens utilizing the information gathered from this recent pandemic. COVID-19 immuno-pathogenesis involves viral driven damage and prolific host inflammatory response towards the virus. Without treatment, the flu-like symptoms may develop into acute lung injury, multiple organ failure and mortality. Doxycycline is an FDA-approved antibiotic with minimal and rarely occurring side effects.  The drug also possesses anti-viral and anti-inflammatory properties [3] rendering it a suitable candidate as a prophylactic treatment for COVID-19 infection and other inflammatory related diseases. Our research group has developed a novel platform for anti-inflammatory drug testing and a nanoparticle-based drug delivery system capable of targeting and delivering Doxycycline to damaged elastin in the lungs. Our results from in vivo and in vitro models indicate that Doxycycline nanoparticles significantly lessened leukocyte recruitment to lung tissue, reduce inflammation, and protects epithelial and endothelial integrity, reduce local proinflammatory cytokine secretion, and thereby reduces inflammation. The long-term goal is to utilize this novel drug screening platform and targeted drug delivery system to provide treatment for the COVID-19 infection and other localized inflammatory diseases.

Doxycyclinehyclate (25 mg) (Sigma Aldrich, St. Louis, MO) and 100 mg of BSA (Seracare, MA, USA) was dissolved in 2 mL Deionized water and left to stir for 30 minutes, 500 rpm. 4 mL of ethanol and 8% glutaraldehyde (40μg/mg BSA) (EMS, PA, USA) was then added at the rate 1 ml/min. The reaction vessel was sealed and left to stir for 2 hours in the dark, 500 rpm, room temperature. Nanoparticle size was measured in deionized water using 90 Plus Particle Size Analyzer (Brookhaven Instruments Co, Holtsville, NY), yield was calculated by measuring nanoparticle dry weight, drug loading percentage was calculated by measuring the amount of doxycycline in supernatant via HPLC, Agilent 1100 (Wilmington, DE, USA).

Five weeks old C57BL/6j mice (Jackson Laboratory, ME) were divided into: (1) control, (2) COVID-19 Like Inflammation model (CLI), and (3) Doxycycline treatment. (Day 1), CLI and treatment groups received porcine pancreatic elastase (PPE) intratracheal instillation while the control group were instilled with saline. (Day 6), the control and COVID-19 model groups were injected intravenously with saline while the treatment group were injected with Doxycycline nanoparticles. (Day 7) control group mice were instilled with saline, and both CLI and treatment group were instilled with 1 mg/kg LPS (List Labs, Campbell, CA, USA). All mice were euthanized on day 8. Blood serum and Broncho-Alveolar Lavage Fluid were harvested for cytokine measurement and inflammatory cell analysis.  Heart, lung, liver, kidney, and spleen of each mouse were harvested and stored in 4%-paraformaldehyde for histological analysis.

Our COVID-19 Like Inflammation model (CLI) model is adapted from the work of Satoshi et al [4]. C57BL/6J mice were instilled with PPE and kept for one week to generate emphysema-like conditions followed by LPS instillation to mimic bacterial infection, an exacerbation to the already damaged lungs.  As neutrophils are the host first line of defense, their presence in the alveolar space and the interstitium are the major determinants of lung injury, the leading cause of mortality in SAR-CoV2 patients [5]. We followed the guidelines established by American Thoracic Society [6] to determine the inflammation level by examining the evidence of tissue injury, the elevation of inflammatory cytokines and lung infiltrates, and the alteration of the alveolar capillary barrier.

Our preliminary data showed that mice in the CLI model group, as compared to the control group, exhibited relevant features of acute lung injury (ALI) demonstrating significant host inflammatory response shown histologically and through cellular analysis using cell sorter and (Ly6-c+, Ly6-g-, CD11B+) antibody to identify the lung infiltrates presence within BALF samples, in addition to an alteration of the alveolar capillary barrier by analyzing BALF total protein concentrations  which increased from 0.287±0.01 mg/mL in the control group to 0.935±16 mg/mL in the COVID-19 group. These results suggest that this animal model is efficient to serve as an anti-inflammatory drug testing platform.

To demonstrate that Doxycycline is capable of preventing inflammation induced lung injury, we injected mice with Doxycycline nanoparticles of 254±69 nm average diameter, exhibiting an average drug loading of 17.4%, prior to inducing lung damage as described above. Twenty percent (20%) of injected Doxycycline nanoparticles were delivered and localized in the lung. Data from protein concentration analysis, cell sorting and histological analysis showed that Doxycycline reduced inflammation induced lung permeability by 30% as compared to non-treated group. Our results confirm that Doxycycline shows potential as a prophylactic treatment to mitigate inflammation, the process central to ARDS related injuries, the main cause of death in COVID-19 infection.

[1] Malek, AE, et al. "Doxycycline as a Potential Partner of COVID-19 Therapies." IDCases 21 (2020): e00864.

[2] WHO COVID-19 Dashboard. Geneva: World Health Organization, 2020. Available online: https://covid19.who.int/

[3] Soccal PM, et al. Matrix metalloproteinases correlate with alveolar-capillary permeability alteration in lung ischemia-reperfusion injury. Transplantation 70(7) (2000): 998-1005.

[4] Kobayashi, S, et al.  American Journal of Respiratory Cell and Molecular Biology 49(6) (2013): 971-977.

[5] Ruan Q, et al. Intensive Care Medicine, 46(5) (2020): 846-848.

[6] Hey, Ying-Ying, et al. Frontiers in immunology 6 (2016): 652.