Safety and Pharmacokinetics of Intranasally Administered Heparin

PurposeIntranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. MethodsDouble-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 g) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. ResultsUFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 hours, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 hours. ConclusionsUFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 hours after dosing.

Preferential recognition and antagonism of SARS-CoV-2 spike glycoprotein binding to 3-O-sulfated heparan sulfate

The COVID-19 pandemic caused by SARS-CoV-2 is in immediate need of an effective antidote. Although the Spike glycoprotein (SgP) of SARS-CoV-2 has been shown to bind to heparins, the structural features of this interaction, the role of a plausible heparan sulfate proteoglycan (HSPG) receptor, and the antagonism of this pathway through small molecules remain unaddressed. Using an in vitro cellular assay, we demonstrate HSPGs modified by the 3-O-sulfotransferase isoform-3, but not isoform-5, preferentially increased SgP-mediated cell-to-cell fusion in comparison to control, unmodified, wild-type HSPGs. Computational studies support preferential recognition of the receptor-binding domain of SgP by 3-O-sulfated HS sequences. Competition with either fondaparinux, a 3-O-sulfated HS-binding oligopeptide, or a synthetic, non-sugar small molecule, blocked SgP-mediated cell-to-cell fusion. Finally, the synthetic, sulfated molecule inhibited fusion of GFP-tagged pseudo SARS-CoV-2 with human 293T cells with sub-micromolar potency. Overall, overexpression of 3-O-sulfated HSPGs contribute to fusion of SARS-CoV-2, which could be effectively antagonized by a synthetic, small molecule.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH and 6-O-desulfated enoxaparin with an IC50 of 5.99 {micro}g/L, 1.08 mg/L, 1.77 {micro}g/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

Effective Screening of SARS-CoV-2 Neutralizing Antibodies in Patient Serum using Lentivirus Particles Pseudotyped with SARS-CoV-2 Spike Glycoprotein

Pseuodotyped particles have significant importance and use in virology as tools for studying the biology of highly pathogenic viruses in a lower biosafety environment. The biological, chemical, and serological studies of the recently emerged SARS-CoV-2 will be greatly aided by the development and optimization of a suitable pseudotyping system. Here, we pseudotyped the SARS-CoV-2 Spike glycoprotein (SPG) on a retroviral (MMLV) as well as a third generation lentiviral (pLV) vector and tested the transduction efficiency in several mammalian cell lines expressing SARS-CoV-2 receptor hACE2. While MMLV pseudotyped the vesicular stomatitis virus G glycoprotein (VSV-G) efficiently, it could not pseudotype SPG. In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titers of pLV-G particles were produced. Among all the tested mammalian cells, 293Ts expressing hACE2 were most efficiently transduced using the pLV-S system. The pLV-S particles were efficiently neutralized by diluted serum (>:640) from a recently recovered COVID-19 patient who showed high SARS-CoV-2 specific IgM and IgG levels. In summary, pLV-S pseudotyped virus provides a valid screening tool for the presence of anti SARS-CoV-2 specific neutralizing antibodies in convalescent patient serum. Significance StatementSARS-CoV-2 has emerged as one of the biggest threats in the history of humankind and is comparable to medieval plague, 1918 Spanish Flu, as well as world wars. Investigations into the biology of SARS-CoV-2 are partially hindered by the highly transmissible and pathogenic nature of this virus, which requires biosafety level 3 containment in a laboratory for investigation. The study here describes a pseudotyping system which mimics the surface properties of SARS-CoV-2 and can be used in lower biosafety level laboratory for the purpose of vaccine studies, drug inhibition studies, and serological screening to determine the status of herd immunity.