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The pH Dependence of Niclosamide Solubility, Dissolution and Morphology Motivates a Potentially More Bioavailable Mucin-Penetrating Nasal and Throat Spray for COVID19, it’s Contagious Variants, and Other Respiratory Viral Infections

Motivation: With the coronavirus pandemic still raging, prophylactic nasal and early treatment throat sprays that “put the virus in lockdown”, could help prevent infection and reduce viral load. Niclosamide has the potential to treat a broad range of viral infections if local bioavailability is optimized as mucin-penetrating solutions instead of microparticles that cannot penetrate the mucin. Experimental: pH-dependence of supernatant concentrations and dissolution rates of niclosamide were measured in buffered solutions by Nanodrop-UV/Vis-spectroscopy for niclosamide from different suppliers, as precipitated material and as cosolvates. Data was compared to predictions from Henderson Hasselbalch and precipitation pH models. Optimal microscopy was used to observe the morphologies of precipitated and converted niclosamide. Results: Supernatant-concentrations of niclosamide increased with increasing pH: from 1.77uM at pH 3.66 to 30uM at pH 8; more rapidly from 90uM at pH8.5 to 300uM at pH9.1, reaching 641uM at pH 9.5. Logarithmic rates for dissolution increased by ~3x for pHs 8.62 to 9.44. However, when precipitated from supersaturated solution, niclosamide equilibrated to much lower final supernatant concentrations, reflective of more stable polymorphs at each pH that were also apparent for niclosamide from other suppliers and cosolvates. Conclusions: Niclosamide is not niclosamide is not niclosamide. A low dose (20uM) prophylactic solution of niclosamide at a nasally safe pH of 7.9 and a (up to 300uM) throat spray at pH 9.1 would be one of the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, with no cold chain. It now needs testing. Executive Summary Motivation: With the coronavirus pandemic still raging, more contagious (delta and delta+) variants already in circulation (1), a world-wide average of only 15.4% fully vaccinated (2), and many parts of the world with <1% vaccinated (3), there is still an urgent need for additional mitigation efforts to support current and potential vaccinations. A prophylactic nasal spray and an early treatment throat spray that “put the virus in lockdown”, could help to prevent infection and reduce viral load in the early stages for unvaccinated populations, as well as vaccinated spreaders where break through infections are now occurring. Niclosamide has emerged from multiple drug screens with potential to treat cancer and many other diseases and conditions (4), and now in a broad range of viral infections (5–20). At the cellular level, niclosamide can inhibit three of the six stages of viral infection by: preventing uncoating and RNA release from the endosome (18); inhibiting viral replication by reducing the amount of ATP available from mitochondria needed for ATP-dependent viral transcription and translation (10); and interfering with capsid assembly in the Golgi that then promotes the secretion of non-competent virions (21). The main issue for nose and throat formulations, is how to optimize local bioavailability for a drug that has intrinsic low solubility in aqueous solution (1-2uM) compared to its cellular efficacy (2-3uM) and the fact that the traditional choice for nasal sprays of microparticles cannot penetrate the mucin. It is here that we have focused on what it takes to provide a mucin-penetrating niclosamide solution. We have found a way to increase the amount of niclosamide in aqueous buffered solution and therefore its bioavailability to infectable and infected epithelial cells, just by slightly increasing the pH (22). Experimental: Experimentation focused on a series of specific aims that measured the pH-dependence of equilibrium supernatant concentrations and dissolution rates of niclosamide in buffered solutions. We compared niclosamide from different suppliers as well as precipitated niclosamide and presumed cosolvates of acetone and ethanol. Nanodrop UV/Vis spectroscopy was used to quantify niclosamide concentrations in supernatant solutions and data was compared to predictions from Henderson Hasselbalch and precipitation pH models. Optimal microscopy was used to observe the morphologies of precipitated and converted niclosamide. Results: When excess powdered niclosamide was dissolved in pH buffers, the supernatant concentration of niclosamide increased slowly with pH from 1.77uM at pH 3.66 to 30uM at pH 8. It then showed a more rapid rise in concentration to 90uM at 8.5 to 300uM at 9.1, reaching 641uM at pH 9.5. In dissolution experiments, the logarithmic rate for niclosamide dissolution over a pH range from 8.62 to 9.44 increased by a factor of just over 3x. A more optimized stirring gave a further increased rate of niclosamide dissolution as expected from dissolution models. However, when precipitated from supersaturated solution, niclosamide equilibrated to much lower final supernatant niclosamide concentrations, reflective of more stable polymorphs at each pH. While niclosamide from one supplier (AKSci), readily dissolved and remained as a stable solution, even in contact with the excess solid, that from another supplier (Sigma) converted to a much lower solubility polymorph over a period of the next several hours. Conclusions: In the preparation of any formulation of niclosamide, especially for local nasal and throat delivery, niclosamide is not niclosamide is not niclosamide; the source and nature of niclosamide can dramatically influence the solubility, amount in solution, bioavailability, and pharmaceutical performance of the drug compound. A low dose (20uM) prophylactic solution of niclosamide at a nasally safe pH of 7.9 and a (up to 300uM) throat spray at pH 9.1 would be one of the simplest and potentially most effective formulations from both an efficacy standpoint as well as manufacturing and distribution, with no cold chain. It now needs testing.